CN110559430B - Anti-lymphoma CAR-T medicine and application thereof - Google Patents

Abstract

The invention discloses an anti-lymphoma CAR-T drug and application thereof, wherein the anti-lymphoma CAR-T drug is a CD30CAR-T drug, and the CD30CAR-T drug can express a human CD30 antibody. The CD30CAR-T medicine prepared by the invention can effectively inhibit tumor growth and has higher tumor degeneration effect, the survival rate of peripheral T cell lymphoma patients is obviously prolonged by CD30CAR-T administration, and the TNF-alpha, INF-gamma and G-CSF levels of lymphoma patients are improved.

Description

Anti-lymphoma CAR-T medicine and application thereof

Technical Field

The invention belongs to the technical field of CAR-T therapy, and particularly relates to an anti-lymphoma CAR-T drug and application thereof.

Background

Chimeric antigen receptor T cell immunotherapy (Chimeric Antigen Receptor T-Cell Immunotherapy, CAR-T) is a novel accurate targeted cell therapy for treating tumors, and is a novel tumor cell immunotherapy method which is very promising, can accurately, rapidly and efficiently treat cancers, and can possibly cure the cancers. T lymphocytes, which are one of the human leukocytes, are derived from bone marrow hematopoietic stem cells, mature in the thymus and then distribute to human blood, lymph and surrounding tissue organs, exerting immune functions.

The first CAR-T treatment for cancer was approved by the us FDA in 2017, tisaponine for the treatment of patients with relapse and drug resistant ALL in children and young adults. Axicabtagene ciloleucel is a U.S. FDA approved CAR T cell therapy for specific classes of non-Hodgkin lymphomas in adults, including diffuse large B cell lymphomas, primary mediastinal B cell lymphomas, transformed follicular lymphomas.

The Chimeric Antigen Receptor (CAR) of CAR-T cells consists of an extracellular antigen binding region, a transmembrane region, and an intracellular signaling region. Wherein the chimeric antigen receptor is directed against a tumor-associated antigen, thus having high specificity, and is not limited by Major Histocompatibility Complex (MHC), thus the tumor antigen is not limited by species, and includes gangliosides, organosaccharide antigens, etc., in addition to the selectable protein polypeptides.

Current CAR-T drugs are mainly directed to B cell lymphomas, whereas CAR-T drugs directed to T cell lymphomas are rarely studied. The treatment of peripheral T cell lymphoma with difficult treatment is still a difficult problem to overcome, so that the development of a therapeutic drug for peripheral T cell lymphoma with difficult treatment is a technical problem to be solved.

Disclosure of Invention

This section is intended to outline some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description summary and in the title of the application, to avoid obscuring the purpose of this section, the description summary and the title of the invention, which should not be used to limit the scope of the invention.

The present invention has been made in view of the above technical drawbacks.

Accordingly, as one aspect of the invention, the invention provides an anti-lymphoma CAR-T medicament.

An anti-lymphoma CAR-T drug, wherein: the anti-lymphoma CAR-T drug is a CD30CAR-T drug, the CD30CAR-T drug being capable of expressing a human CD30 antibody.

As a preferred embodiment of the anti-lymphoma CAR-T drug of the present invention: the CD30 antibody is a CD30 monoclonal antibody, the CD30 monoclonal antibody is derived from hybridoma cells, and the preservation number of the CD30 monoclonal antibody is CCTCC NO: C201861.

as a preferred embodiment of the anti-lymphoma CAR-T drug of the present invention: the anti-lymphoma CAR-T drug is obtained by infecting T cells with a CD30CAR lentiviral expression vector.

As a preferred embodiment of the anti-lymphoma CAR-T drug of the present invention: the CD30CAR lentiviral expression vector comprises a CD30 scFv gene fragment formed by connecting a heavy chain and a light chain of a CD30 antibody with a single-chain variable region fragment scFv, wherein the sequence of the CD30 scFv gene fragment is shown in SEQ ID NO:1 is shown in the specification; the CD30 scFv gene fragment is connected with CD8aSP, a 4-1BB intracellular signal region and a CD3 zeta gene sequence in series to obtain a CD30CAR gene sequence; connecting the CD30CAR gene sequence with a pCDH-CMV-SMC-EF1a-GFP vector to obtain the CD30CAR lentiviral expression vector, wherein the gene sequence of the CD30CAR lentiviral expression vector is shown in SEQ ID NO: 2.

As a preferred embodiment of the anti-lymphoma CAR-T drug of the present invention: also included is packaging CD30CAR lentivirus by adding 3 μg pMD2G, 6 μg psPAX and 7.5 μg of the CD30CAR lentivirus expression vector into 150 μl OPTI-MEM, mixing, adding into 500 μl OPTI-MEM containing 25 μl Lipo 2000, standing at 37deg.C for 15min, adding into a culture dish，5％CO ₂ After 12h of culture in an incubator of (2) and the transfected 293T cells were expanded and lentiviruses were collected to obtain lentivirus Lenti-2G.CAR-CD30.

As a preferred embodiment of the anti-lymphoma CAR-T drug of the present invention: the preparation method of the anti-lymphoma CAR-T medicine comprises the steps of mixing the lentivirus Lenti-2G.CAR-CD30 with T cells, adding 8 mug/mL polybrene, and incubating in an incubator with 5% CO2 at 37 ℃ for 0.5-2 hours; t cell culture broth was prepared, 50. Mu.g/mL human serum albumin, 1000U/mL IL-2, 100U/mL OKT3 and CD28 were added to the GT-T551H3 broth, and the mixture was added to the incubated cells, and cultured in a 5% CO2 incubator at 37℃for 14 days.

As a preferred embodiment of the anti-lymphoma CAR-T drug of the present invention: the amount of CD30CAR lentivirus required to infect T cells was calculated as MOI 5.

As another aspect of the invention, the invention provides the use of said CAR-T drug against lymphoma, wherein: the anti-lymphoma CAR-T medicine can inhibit the growth of lymphoma cells, has tumor inhibition effect, and prolongs the survival rate.

As a preferred scheme for the use of the anti-lymphoma CAR-T drug of the present invention: the lymphomas include human anaplastic large cell lymphomas and peripheral T cell lymphomas.

The invention has the beneficial effects that: the CD30CAR-T medicine prepared by the invention can effectively inhibit tumor growth and has higher tumor degeneration effect, the survival rate of peripheral T cell lymphoma patients is obviously prolonged by CD30CAR-T administration, and the TNF-alpha, INF-gamma and G-CSF levels of lymphoma patients are improved.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:

FIG. 1 is a schematic diagram of the structure of a CD30CAR lentiviral expression vector.

FIG. 2 is a diagram of the identification of the cleavage of a CD30CAR lentiviral expression vector.

FIG. 3 is a graph showing CD30 CAR-T/T cell proliferation and cell surface molecule expression following lentiviral infection.

FIG. 4 is a graph showing the expression of CD30 on the surface of T cells after lentiviral infection by flow cytometry.

Figure 5 is a graph of LDH release experiments detecting the killing rate of three CD30CAR-T cells.

Figure 6 is an in vitro killing ability profile of RTCA assay for CD30CAR-T cells.

FIG. 7 is an inhibitory effect of CD30CAR-T on peripheral T cell lymphomas in vivo and the effect of CD30CAR-T on TNF- α, INF- γ and GM-CSF levels in vivo.

Detailed Description

In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.

Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

Example 1:

acquisition of anti-CD 30 single chain antibody scFv sequence and CD30CAR lentiviral expression vector construction and expression:

experimental materials:

experimental cell lines: hybridoma cells (preservation number is CCTCC NO: C201861, which is a published cell strain).

Tool cell 293T cell (preservation of laboratory)

Coli: e.coli DH 5. Alpha. Preserved in this laboratory

And (3) a carrier: pMD18-T Vector (purchased from Nanjing PPL company)

Preparing a solution:

preparation of lentiviral transfection reagents:

preparation of 0.25M CaCl ₂ Solution: 1.47g CaCl ₂ Dissolving the powder in deionized water, fixing the volume to 40mL, and sterilizing;

preparing 5M NaCl solution: 14.61g NaCl powder is dissolved in deionized water, and the volume is fixed to 50mL;

preparing a BES solution of 1M;

preparation of 1M Na ₂ HPO ₄ A solution;

preparation of 2×bbs solution: 5.6mL of 5M NaCl solution, 5.0mL of 1M BES solution, 150. Mu.L of 1M Na ₂ HPO ₄ The solution was added to 80mL of water, the pH was adjusted to 6.95.+ -. 0.02 with NaOH, deionized water was set to 100mL, and the solution was filtered to remove bacteria.

The experimental method comprises the following steps:

1. extraction of hybridoma cell RNA:

the QIAGEN RNeasy Mini Kit was used:

adding hybridoma cells (CCTCC NO: C201861) into PBS solution, and centrifuging for 10min at 300 g;

discarding the supernatant, and adding 700 mu L of RLT to obtain a cell lysate;

sucking 350 mu L of suspension from the cell lysate and vibrating;

adding 350 mu L of 70% ethanol, and uniformly mixing;

centrifuging for 15s by using 300g of an upper column, and discarding waste liquid;

700. Mu.L of Buffer RW1 was added to the column and centrifuged at 300g for 15s;

500. Mu.L of Buffer RPE was added to the column and centrifuged at 300g for 15s;

then 500 mu L Buffer RPE is added into the column, and 300g is centrifuged for 2min;

300g idle for 1min; RNA was dissolved by adding 50. Mu.L of DEPC water.

2. cDNA synthesis:

cDNA was extracted using Roche company Transcriptor First Strand cDNA Synthesis Kit.

(1) The reaction system:

denaturation at 65℃for 10min

The reaction system:

component (A)	Volume of
		Total RNA of cells	5μL
oligodT	1μL
		dNTPs Mix	1μL
DEPC water	Make up to 10 mu L

(2) Preparing a mixed solution:

mixed liquid component system:

component (A)	Volume of
		10×RT buffer	2μL
0.1M DTT	2μL
		RNaseOUT(40U/μL)	1μL
25mM MgCl 2	4μL
		SuperScriptⅢ	1μL

Heating in a water bath at 50 ℃ for 50min;

heating in a water bath at 85 ℃ for 5min;

ice bath and preservation at-20 ℃.

3. PCR amplification of the heavy chain of monoclonal antibody CD30 (V _H ) And light chain (V) _L )：

(1) Using 10. Mu.g of the cDNA prepared as described above as a template, the heavy chain (V _H ) And light chain (V) _L )：

PCR amplification system:

PCR amplification system:

heavy chain amplification primers:

MVH-F:GGCCAGTGGATAGTCAGATG

MVH-R:GAGGTGCAGCTGAAGGAGTC

light chain amplification primers:

MVK-F:GGATACAGTTGGTGCAGCATC

MVK-R:GATGTTTTGATGACCCAAACT

PCR reaction conditions:

the PCR product was detected by 1% agarose gel electrophoresis, and then subjected to rubber cutting recovery (QIAGEN rubber recovery kit).

(2) Heavy chain of clone antibody CD30 (V _H ) And light chain (V) _L ) Is determined by the method comprising the steps of (1) cloning and sequencing:

the heavy chain of the clone antibody CD30 recovered by cleavage (V _H ) And light chain (V) _L ) DNA is respectively connected with a cloning Vector pMD18-T Vector, and the reaction is carried out for 5min at room temperature; the reaction system is as follows:

and (3) connecting a reaction system:

component (A)	Volume of
		V L /V H	4μL
pMD18-T Vector	1μL
		SolutionⅠ	5μL

Respectively converting the connection products into competent cells DH5 alpha, and selecting monoclonal colonies on the next day for sequencing;

and cloning and extracting plasmids after the sequencing result is correct.

(3) Heavy chain of clone antibody CD30 (V _H ) And light chain (V) _L ) Ligation and amplification of Gene fragments (using the mouse antibody ScFv Gene amplification kit from PPL Co):

the reaction system is as follows:

the reaction system:

the PCR conditions were as follows:

and (3) carrying out agarose gel electrophoresis, and recovering the band with the size of about 330bp by tapping.

(4) Heavy chain of CD30 (V _H ) And light chain (V) _L ) Ligation was performed to construct the CD30 scFv fragment:

the reaction system (50. Mu.L system) was prepared according to the following composition

And (3) connecting a reaction system:

component (A)	Volume of
		scFv Mix (scFv ligation amplification System)	45μL
DNA Polymerase	0.3μL
		V H	1μL
V L	1μL
		ddH 2 O	2.7μL

The PCR conditions were as follows:

after passing through 2% agar, the PCR product is recovered to a band with the size of about 700bp, and after tapping recovery, cloning is carried out; cloning the target strip to a pMD18-TVECTOR vector after recovering;

the reaction system was configured as follows:

target band and carrier connection system:

component (A)	Volume of
		V L Or V H Gene fragment	4μL
pMD18-T Vector	1μL
		SolutionⅠ	5μL

The recovery fragments of each PCR are subjected to configuration of a connection system according to the volumes, and the reaction is carried out for 5min at room temperature; transforming DH5 alpha cells; ampicillin resistant plates were smeared and the next day the selection was sent to a monoclonal for sequencing.

Design and construction of Lenti-2G. CAR-CD30:

structural design and construction of CD30CAR gene sequence:

the CD30 scFv was concatenated with the CD8a hinge region-transmembrane region of Genebank (NCBI), the intracellular signaling region of CD8aSP (Signal peptide), 4-1BB and the CD3 zeta gene sequence.

Ligation of the CD30CAR Gene sequence with vector pCDH-CMV-SMC-EF1 a-GFP:

cloning the constructed CD30CAR gene sequence into a pCDH-CMV-SMC-EF1a-GFP lentiviral expression vector through XbaI and BamHI endonucleases to obtain a CD30CAR lentiviral expression vector of the invention; the CD30CAR lentiviral expression vector profile is shown in figure 1;

mu.g of pMD2G, 6. Mu.g of psPAX and 7.5. Mu.g of the CD30CAR lentiviral expression vector were added to 150. Mu.L of OPTI-MEM, mixed well, then added to 500. Mu.L of OPTI-MEM containing 25. Mu.L of Lipo 2000, left standing at room temperature for 15min, added to a petri dish, cultured in a 5% CO2 incubator at 37℃for 12 hours, and then transfected 293T cells were expanded and lentivirus was collected, and the resulting lentivirus was designated Lenti-2G.CAR-CD30.

(1) Plasmid pCDH-CMV-SMC-EF1a-GFP was digested with XbaI and BamHI, digested overnight at 37℃and reacted as follows:

plasmid enzyme digestion system:

name of the name	Volume of
		10×Buffer M	2μL
XbaI	1μL
		BamHI	1μL
pCDH-CMV-SMC-EF1a-GFP	1μL
		ddH 2 O	Make up to 20 mu L

(2) Adding 10×loading Buffer 5 μl, performing electrophoresis identification on 1% agarose gel, and recovering target band in the gel;

(3) Double cleavage of the synthesized pMD18-T Vector containing the CD30CAR gene sequence with XbaI and BamHI;

(4) The pCDH-CMV-SMC-EF1a-GFP fragment after digestion and recovery and the digestion products of the CD30CAR gene sequence are connected, and the specific connection reaction system is as follows:

and (3) connecting a reaction system:

component (A)	Volume of
		10×Buffer	1μL
Enzyme section of pCDH-CMV-SMC-EF1a-GFP	1μL
		CD30CAR gene sequence	6.4μL
T4 ligase	6.4μL
		PEG 4000	1μL

Reaction conditions: after gentle mixing, 16 ℃ overnight.

And (3) identification:

(5) Conversion of ligation products

The overnight-ligated mixture was directly transformed into Stbl3 competent cells. And (3) adding the transformed Stbl3 competent cells into an LB liquid culture medium for shaking, coating an LB plate, and culturing the cells in an incubator at 37 ℃ for overnight in an inverted mode.

(6) Amplification and extraction of recombinant plasmids

After 16h of transformation, 3 single bacterial colonies are selected and respectively inoculated into 5mL of LB ampicillin resistant medium, the final concentration of ampicillin in the culture solution is 100 mug/mL, shaking culture is carried out at 37 ℃ overnight, and plasmids are extracted the next day.

(7) DNA sequencing identification

The extracted plasmid was entrusted to Nanjing plasmid and protein sharing library (PPL) company for DNA sequencing identification. And obtaining the lentiviral expression vector containing the gene fragment of the CD30CAR after identification meets the requirements.

Experimental results:

amplification and identification of CD30CAR lentiviral expression vectors:

double digestion verification is carried out on the lentiviral expression vector according to the invention through double digestion sites of XbaI and BamHI, and the result shows that: the fragment size of the CD30CAR sequence, including the scFv fragment, was identical to that expected, about 1000bp (fig. 2).

CD30 heavy chain (V) _H ) Light chain (V) _L ) Sequencing results of the bands:

the research of the invention discovers that from hybridoma cell strain CCTCC NO: of the CD30 antibodies secreted by C201861, a number of different heavy chains of CD30 antibodies were identified (V _H ) And light chain (V) _L )：

V of CD30 antibody _H The sequence analysis results were as follows:

V _H (heavy chain) specific sequences

	CDR1	CDR2	CDR3
				V H -1	GFSLTSHG	IWSDGSTA	AREPPTTYVCL
V H -2	GFSLTSYG	IWSDGSTA	AREPPTTYVCL
				V H -3	GYTFTNYD	IYPGDGTA	ARGDGFDY
V H -4	GFSLSRYS	IWGGGIT	ARKYGLDYDGAMDY

V of CD30 antibody _L Sequence analysis:

V _L (light chain) specific sequences

	CDR1	CDR2	CDR3
				V L -1	KSVSTSGYSY	LVS	QHIRELTR
V L -2	KSVSTSGYSY	LVS	QHIRELTL
				V L -3	DNVHTY	GAS	GQSYRYPPT
V L -4	DNVHTY	GAS	GQSYRYPLT

CD30CAR gene sequence:

the Lenti-2G.CAR-CD30 was constructed from a combination of different heavy and light chains of the CD30 antibody:

heavy chain is V _H -4, light chain is V _L -3 the combined CD30 antibody was designated 9C11-1 and the heavy chain was V _H -4, light chain is V _L The CD30 antibody of the-4 combination was designated 9C11-2 and the heavy chain was V _H -4, light chain is V _L The CD30 antibody of the-1 combination was designated 9C11-3. The heavy and light chain combinations of the remaining CD30 antibodies were excluded as a result of experiments demonstrating lower antibody titers.

The sequences in the lentiviral expression vectors of the present invention are as follows:

(1)Signal peptide(SP)：

atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg

(2)CD8αTM

accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc

(3)41BB

aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg

(4)CD3ζ

agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc

(5)CD30 scFv(9C11-1)

gaggtgaaactgcagcagtctggacctggcctggtggcaccctcacagagcctgtccatcacatgcactgtctctgggttctcattatccagatatagtgtacactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtggtggaatcacagactataattcagctctcaaatccagactgagcatcaacaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatatactactgtgccagaaagtatgggttggattacgacggtgctatggactactggggccaagggaccacggtcaccgtctcctcaggtggtggtggttctggtggtggtggttctggcggcggcggctccgacatccagatgacccagtctcccaaatccatgtccatgtcagtaggagagagggtcaccttgagctgcaaggccactgacaatgtgcatacttatgtatcctggtatcaacaaaaaccagagcagtctcctaaactgctgatatacggggcatccaaccggtacactggggtccccgatcgcttcacaggcagtggatctgaaacagatttcactctgaccatcagcagtgtgcaggctgaagaccttgcagattatcactgtggacagagttacaggtatccgcccacgttcggtgctgggaccaagctggagctgaaa

(6) CD30 scFv (9C 11-2) (i.e. SEQ ID NO: 1)

gaggtgaaactgcagcagtctggacctggcctggtggcaccctcacagagcctgtccatcacatgcactgtctctgggttctcattatccagatatagtgtacactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtggtggaatcacagactataattcagctctcaaatccagactgagcatcaacaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatatactactgtgccagaaagtatgggttggattacgacggtgctatggactactggggccaagggaccacggtcaccgtctcctcaggtggtggtggttctggtggtggtggttctggcggcggcggctccgacatccagatgacccagtctcccaaatccatgtccatgtcagtaggagagagggtcaccttgagctgcaaggccactgacaatgtgcatacttatgtatcctggtatcaacaaaaaccagagcagtctcctaaactgctgatatacggggcatccaaccggtacactggggtccccgatcgcttcacaggcagtggatctgaaacagatttcactctgaccatcagcagtgtgcaggctgaagaccttgcagattatcactgtggacagagttacaggtatccgctcacgttcggtgctgggaccaagctggagctgaaa

(7)CD30 scFv(9C11-3)

gaggtgaaactgcagcagtctggacctggcctggtggcaccctcacagagcctgtccatcacatgcactgtctctgggttctcattatccagatatagtgtacactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtggtggaatcacagactataattcagctctcaaatccagactgagcatcaacaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatatactactgtgccagaaagtatgggttggattacgacggtgctatggactactggggccaagggaccacggtcaccgtctcctcaggtggtggtggttctggtggtggtggttctggcggcggcggctccgaaattgtgttgacgcagtctcctgcttccttagctgtatctctggggcagagggccaccatctcatacagggccagcaaaagtgtcagtacatctggctatagttatatgcactggaaccaacagaaaccaggacagccacccagactcctcatctatcttgtatccaacctagaatctggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatgctgcaacctattactgtcagcacattagggagcttacacgttcggaggggggaccaagctggaaatcaaa

(8) The lentiviral expression vector (9C 11-2) gene sequence (namely SEQ ID NO: 2) of the invention:

acgcgtgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgtatttaagtgcctagctcgatacaataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacctgaaagcgaaagggaaaccagagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccactgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcctcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagattggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggttaacttttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttatcgatactagtattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagattctagagccaccatggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaggtgaaactgcagcagtctggacctggcctggtggcaccctcacagagcctgtccatcacatgcactgtctctgggttctcattatccagatatagtgtacactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtggtggaatcacagactataattcagctctcaaatccagactgagcatcaacaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatatactactgtgccagaaagtatgggttggattacgacggtgctatggactactggggccaagggaccacggtcaccgtctcctcaggtggtggtggttctggtggtggtggttctggcggcggcggctccgacatccagatgacccagtctcccaaatccatgtccatgtcagtaggagagagggtcaccttgagctgcaaggccactgacaatgtgcatacttatgtatcctggtatcaacaaaaaccagagcagtctcctaaactgctgatatacggggcatccaaccggtacactggggtccccgatcgcttcacaggcagtggatctgaaacagatttcactctgaccatcagcagtgtgcaggctgaagaccttgcagattatcactgtggacagagttacaggtatccgctcacgttcggtgctgggaccaagctggagctgaaactcgagaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaaggatccgcggccgcgaaggatctgcgatcgctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaacgggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacagctgaagcttcgaggggctcgcatctctccttcacgcgcccgccgccctacctgaggccgccatccacgccggttgagtcgcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtctaggtaagtttaaagctcaggtcgagaccgggcctttgtccggcgctcccttggagcctacctagactcagccggctctccacgctttgcctgaccctgcttgctcaactctacgtctttgtttcgttttctgttctgcgccgttacagatccaagctgtgaccggcgcctacgctagacgccaccatggagagcgacgagagcggcctgcccgccatggagatcgagtgccgcatcaccggcaccctgaacggcgtggagttcgagctggtgggcggcggagagggcacccccaagcagggccgcatgaccaacaagatgaagagcaccaaaggcgccctgaccttcagcccctacctgctgagccacgtgatgggctacggcttctaccacttcggcacctaccccagcggctacgagaaccccttcctgcacgccatcaacaacggcggctacaccaacacccgcatcgagaagtacgaggacggcggcgtgctgcacgtgagcttcagctaccgctacgaggccggccgcgtgatcggcgacttcaaggtggtgggcaccggcttccccgaggacagcgtgatcttcaccgacaagatcatccgcagcaacgccaccgtggagcacctgcaccccatgggcgataacgtgctggtgggcagcttcgcccgcaccttcagcctgcgcgacggcggctactacagcttcgtggtggacagccacatgcacttcaagagcgccatccaccccagcatcctgcagaacgggggccccatgttcgccttccgccgcgtggaggagctgcacagcaacaccgagctgggcatcgtggagtaccagcacgccttcaagacccccatcgccttcgccagatcccgcgctcagtcgtccaattctgccgtggacggcaccgccggacccggctccaccggatctcgctaagtcgacaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaaaataagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagacttttgcagagacggcccaaattcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgccaagctg

CD30CAR-T cell preparation:

activation of T lymphocytes and CD30CAR T cell acquisition;

culture and activation of human T lymphocytes: regulating human peripheral blood mononuclear cell density to 5×10 ⁶ Individual cells/mL, human serum albumin at a concentration of 50. Mu.g/mL, and 1000U/mL of cytokines IL-2, 100U/mL of Anti-CD3 (OKT 3) and CD28 antibodies at 37℃in 5% CO ₂ Is cultured in an incubator for 48 hours;

lentivirus Lenti-2G. CAR-CD30 infected T lymphocytes: t cells were divided into CD30CAR group and T cell blank group; the amount of virus required for infection was calculated at an MOI of 5;

the calculation formula is as follows: moi= (viral titer x viral fluid volume)/T cell number

The virus solution was added to 500. Mu.L of GT-T551H3 medium containing a certain number of T cells, followed by 8. Mu.g/mL polybrene, mixed well and then added to 24 well plates at 37℃in 5% CO ₂ Is incubated in an incubator for 1 hour; the above medium was added to 9mL of GT-T551H3 medium containing 50. Mu.g/mL human serum albumin, 1000U/mL IL-2, 100U/mL OKT3 and CD28 at 37℃in 5% CO ₂ Culturing for 14 days. Cell counting and flow cytometry detect proliferation of CD30CAR T cells and CD30 expression levels.

The killing effect of CD30CAR-T cells on peripheral T cell lymphoma cell line (Karpas 299) was detected by LDH lactate dehydrogenase kit.

Calculating the killing rate:

killing rate = (experimental hole OD value-target cell natural release hole OD value)/(target cell maximum release tube OD value-target cell natural release tube OD value) ×100%.

Detecting the in vitro killing capacity of the CD30CAR-T cells by using a real-time label-free cell analysis (RTCA);

detecting the expression condition of cytokines in the supernatant by adopting a Luminex liquid chip technology;

cell membrane dye solution PKH26 and 7-AAD are combined to detect the killing effect of CD30CAR-T cells.

Experimental results:

flow cytometry detects CD3, CD4, CD8, CD30 expression on T cell surfaces after lentiviral infection:

the proportion of CD30CAR T cells was determined by examining the expression rate of CAR-expressed F a' b segment and T cell GFP, CD3, CD4, CD8 molecules after T cell transfection with lentivirus, and the experimental results are shown in FIG. 4, on day 1, day 3, day 5, day 7, day 9, day 11, day 13, the expression of CD3, CD4, CD8 molecules was increased with time, on day 13, CD3 ⁺ T cells, CD4 ⁺ T cells, CD8 ⁺ The proportion of T cells in total cells is 88.9%,52.9%,38.4% and the positive expression rate of CD30 reaches 53.3%.

In vitro proliferation of CD30CAR-T cells:

after the human peripheral blood T lymphocytes are extracted and cultured in vitro, the NC CAR-T cells and the CD30CAR-T cells with irrelevant sequences are counted on the 0 th day and the 13 th day respectively, proliferation of the cells before and after lentivirus transfection is recorded, and the result is that the CD30CAR-T cells are amplified about 10 times compared with the 0 th day on the 13 th day as shown in figure 4.

LDH lactate dehydrogenase kit detects killing effect of CD30CAR-T cells:

as shown in fig. 5, 3 different CD30CAR-T cells had killing effect on CD30 positive peripheral T cells Karpas299 with a target ratio of 40:1, the 9C11-2 cells are the strongest in killing, 1.8 times that of 9C11-3, 1.98 times that of 9C 11-1. 9C11-2 CD30CAR-T (hereinafter CD30 CAR-T) cells were selected for subsequent study.

With increasing effective target ratio, it can be found that at 40: under the condition of 1, compared with T cells, the killing effect of CD30CAR-T cells infected by slow viruses on target cells is obviously improved, the killing rate of the CD30CAR-T cells is 88.04 +/-1.41, and the difference is statistically significant (P < 0.01). The LDH lactate dehydrogenase release experiment shows that the CD30CAR-T cells have obvious killing effect on target cells Karpas299, and the killing effect is improved along with the effective target ratio within a certain effective target ratio range.

RTCA cell proliferation assay detects the in vitro killing ability of CD30CAR-T cells:

after 14 days of in vitro expansion of T cells, the following effect target ratios 20:1 and 10:1, co-culturing target cells Karpas299 and CD30CAR-T cells, and observing whether the killing effect of the T cells and the CD30CAR-T cells on the target cells is changed under the two effective target ratios. The results are shown in FIG. 6, at an effective target ratio of 20:1 and 10:1, the killing effect of the CD30CAR-T cells is obviously higher than that of NC CAR-T cells (P < 0.01), and the killing effect is increased along with the improvement of the effective target ratio within a certain range.

The Luminex liquid chip technology detects the expression of cytokines TNF-alpha, IL-2 and INF-r in the supernatant:

the killing effect of CD30CAR-T cells on target cells was shown by detecting the contents of cytokines TNF-alpha, IL-2 and INF-r in the T supernatant, and the target ratios were set to be 40:1,20: 1,10: 1, effective target ratio 40:1 and 20:1, the cytokines TNF- α, IL-2 and INF-r are present in the supernatant at approximately the same level, with a relatively efficient target ratio of 10:1, and the release amount of the cytokines is gradually increased within a certain range along with the increase of the effective target ratio.

PKH26 dye and 7-AAD combined detection of killing effects of CD30CAR-T cells:

target cells Karpas299 were stained with PKH26 dye, after 24 hours of co-culture with CD30CAR-T cells, the killing effect of CD30CAR-T cells was observed at 40 by labeling apoptotic target cells with 7-AAD flow antibody: 1,20: 1,10: the killing effect of the CD30CAR-T cells is higher than that of NC CAR-T cells under the effective target ratio of 1, and the killing rates are 78.3 percent, 43.7 percent and 22.6 percent respectively, which are statistically significant (P < 0.01). Wherein, 40: the effect of the experimental group 1 is most remarkable, and meanwhile, the higher the effective target ratio is, the more obvious the killing effect is in a certain range consistent with the previous experimental result.

In conclusion, through the LDH lactate dehydrogenase release experiment, RTCA proliferation curve, luminex liquid phase chip technology detection of TNF-alpha, IL-2 and INF-r expression levels in supernatant and PKH26 and 7-AAD combined method, the killing effect of CD30CAR-T cells on target cells is obviously improved compared with that of T cells.

Inhibition of peripheral T cell lymphoma in CD30 CAR-T:

to study the in vivo inhibition of CD30CAR-T cells on CD30 positive lymphomas, 2X 10 was taken ⁶ karpas299 cells were mixed with 100. Mu.L of PBS and 100. Mu.L of matrigel (Life sciences, USA), and injected into NOD-Prkdc ^em26Cd52 Il2rg ^em26Cd22 Right back of/Nju mice (NCG, gemPharmatech), a mouse xenograft model was constructed when tumor size reached 50mm ³ At this time, mice were injected 1X 10 intravenously ⁷ CD30CAR-T cells, or 1X 10 ⁷ NC CAR-T cells or normal saline. The tumor size was monitored with calipers and the tumor volume was calculated as: tumor long diameter x tumor wide diameter ² /2. After 30 days, mice were sacrificed for tumor tissue analysis. Determination by immunohistochemical methodLevels of TNF- α, INFγ and G-CSF in tumor tissue.

CD30CAR-T cells were effective in inhibiting tumor growth and had high tumor inhibition (fig. 7). TNF-alpha, INF-gamma and G-CSF levels were significantly higher in the CD30CAR-T group than in the NC CAR T group and the saline group, and we also analyzed the survival rate of the CD30CAR T, NC CAR T, saline group mice, and the results showed that the CD30CAR-T group mice significantly prolonged survival rate.

It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and 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 the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered in the scope of the claims of the present invention.

Sequence listing

<110> Jiangsu province tumor Hospital

<120> an anti-lymphoma CAR-T drug and uses thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 726

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

gaggtgaaac tgcagcagtc tggacctggc ctggtggcac cctcacagag cctgtccatc 60

acatgcactg tctctgggtt ctcattatcc agatatagtg tacactgggt tcgccagcct 120

ccaggaaagg gtctggagtg gctgggaatg atatggggtg gtggaatcac agactataat 180

tcagctctca aatccagact gagcatcaac aaggacaact ccaagagcca agttttctta 240

aaaatgaaca gtctgcaaac tgatgacaca gccatatact actgtgccag aaagtatggg 300

ttggattacg acggtgctat ggactactgg ggccaaggga ccacggtcac cgtctcctca 360

ggtggtggtg gttctggtgg tggtggttct ggcggcggcg gctccgacat ccagatgacc 420

cagtctccca aatccatgtc catgtcagta ggagagaggg tcaccttgag ctgcaaggcc 480

actgacaatg tgcatactta tgtatcctgg tatcaacaaa aaccagagca gtctcctaaa 540

ctgctgatat acggggcatc caaccggtac actggggtcc ccgatcgctt cacaggcagt 600

ggatctgaaa cagatttcac tctgaccatc agcagtgtgc aggctgaaga ccttgcagat 660

tatcactgtg gacagagtta caggtatccg ctcacgttcg gtgctgggac caagctggag 720

ctgaaa 726

<210> 2

<211> 8996

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca 60

acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta 120

cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga 180

attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacaata aacgggtctc 240

tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 300

agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 360

ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg 420

cccgaacagg gacctgaaag cgaaagggaa accagagctc tctcgacgca ggactcggct 480

tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt 540

gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag 600

aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt 660

aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt 720

agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg 780

atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag 840

gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag 900

taagaccacc gcacagcaag cggccactga tcttcagacc tggaggagga gatatgaggg 960

acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag 1020

cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag 1080

ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcc tcaatgacgc 1140

tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga 1200

gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc 1260

aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg 1320

gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata 1380

aatctctgga acagattgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440

ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500

acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1560

ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620

agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 1680

tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740

tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggttaacttt 1800

taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc 1860

aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttatcga 1920

tactagtatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac 1980

gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga 2040

tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg 2100

ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg 2160

caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctcg tttagtgaac 2220

cgtcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag attctagagc 2280

caccatggcc ttaccagtga ccgccttgct cctgccgctg gccttgctgc tccacgccgc 2340

caggccggag gtgaaactgc agcagtctgg acctggcctg gtggcaccct cacagagcct 2400

gtccatcaca tgcactgtct ctgggttctc attatccaga tatagtgtac actgggttcg 2460

ccagcctcca ggaaagggtc tggagtggct gggaatgata tggggtggtg gaatcacaga 2520

ctataattca gctctcaaat ccagactgag catcaacaag gacaactcca agagccaagt 2580

tttcttaaaa atgaacagtc tgcaaactga tgacacagcc atatactact gtgccagaaa 2640

gtatgggttg gattacgacg gtgctatgga ctactggggc caagggacca cggtcaccgt 2700

ctcctcaggt ggtggtggtt ctggtggtgg tggttctggc ggcggcggct ccgacatcca 2760

gatgacccag tctcccaaat ccatgtccat gtcagtagga gagagggtca ccttgagctg 2820

caaggccact gacaatgtgc atacttatgt atcctggtat caacaaaaac cagagcagtc 2880

tcctaaactg ctgatatacg gggcatccaa ccggtacact ggggtccccg atcgcttcac 2940

aggcagtgga tctgaaacag atttcactct gaccatcagc agtgtgcagg ctgaagacct 3000

tgcagattat cactgtggac agagttacag gtatccgctc acgttcggtg ctgggaccaa 3060

gctggagctg aaactcgaga ccacgacgcc agcgccgcga ccaccaacac cggcgcccac 3120

catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc 3180

agtgcacacg agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg 3240

gacttgtggg gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa 3300

gaaactcctg tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga 3360

agatggctgt agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa 3420

gttcagcagg agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga 3480

gctcaatcta ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc 3540

tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca 3600

gaaagataag atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg 3660

caaggggcac gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc 3720

ccttcacatg caggccctgc cccctcgcta aggatccgcg gccgcgaagg atctgcgatc 3780

gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg 3840

gaggggtcgg caattgaacg ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg 3900

atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag 3960

tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag ctgaagcttc 4020

gaggggctcg catctctcct tcacgcgccc gccgccctac ctgaggccgc catccacgcc 4080

ggttgagtcg cgttctgccg cctcccgcct gtggtgcctc ctgaactgcg tccgccgtct 4140

aggtaagttt aaagctcagg tcgagaccgg gcctttgtcc ggcgctccct tggagcctac 4200

ctagactcag ccggctctcc acgctttgcc tgaccctgct tgctcaactc tacgtctttg 4260

tttcgttttc tgttctgcgc cgttacagat ccaagctgtg accggcgcct acgctagacg 4320

ccaccatgga gagcgacgag agcggcctgc ccgccatgga gatcgagtgc cgcatcaccg 4380

gcaccctgaa cggcgtggag ttcgagctgg tgggcggcgg agagggcacc cccaagcagg 4440

gccgcatgac caacaagatg aagagcacca aaggcgccct gaccttcagc ccctacctgc 4500

tgagccacgt gatgggctac ggcttctacc acttcggcac ctaccccagc ggctacgaga 4560

accccttcct gcacgccatc aacaacggcg gctacaccaa cacccgcatc gagaagtacg 4620

aggacggcgg cgtgctgcac gtgagcttca gctaccgcta cgaggccggc cgcgtgatcg 4680

gcgacttcaa ggtggtgggc accggcttcc ccgaggacag cgtgatcttc accgacaaga 4740

tcatccgcag caacgccacc gtggagcacc tgcaccccat gggcgataac gtgctggtgg 4800

gcagcttcgc ccgcaccttc agcctgcgcg acggcggcta ctacagcttc gtggtggaca 4860

gccacatgca cttcaagagc gccatccacc ccagcatcct gcagaacggg ggccccatgt 4920

tcgccttccg ccgcgtggag gagctgcaca gcaacaccga gctgggcatc gtggagtacc 4980

agcacgcctt caagaccccc atcgccttcg ccagatcccg cgctcagtcg tccaattctg 5040

ccgtggacgg caccgccgga cccggctcca ccggatctcg ctaagtcgac aatcaacctc 5100

tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc 5160

tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca 5220

ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg 5280

tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca 5340

ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg 5400

cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg 5460

acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg 5520

ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg 5580

accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc 5640

ctcagacgag tcggatctcc ctttgggccg cctccccgcc tggtaccttt aagaccaatg 5700

acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg 5760

ctaattcact cccaacgaaa ataagatctg ctttttgctt gtactgggtc tctctggtta 5820

gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa 5880

taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac 5940

tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtag tagttcatgt 6000

catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag tgagaggaac 6060

ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat 6120

aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat 6180

catgtctggc tctagctatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc 6240

atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat 6300

tccagaagta gtgaggaggc ttttttggag gcctagactt ttgcagagac ggcccaaatt 6360

cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 6420

acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 6480

cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 6540

attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt 6600

cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 6660

caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 6720

caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 6780

ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 6840

cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 6900

ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 6960

tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 7020

gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 7080

ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 7140

ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 7200

gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 7260

aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 7320

tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 7380

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 7440

tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 7500

aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 7560

tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa 7620

ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac 7680

gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa 7740

gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 7800

taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg 7860

tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 7920

ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg 7980

tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 8040

ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 8100

tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata 8160

ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 8220

aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 8280

actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 8340

aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 8400

tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 8460

aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 8520

ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga 8580

ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc 8640

cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg 8700

cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg 8760

tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc 8820

gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg 8880

cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg 8940

taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagctg 8996

Claims (4)

1. An anti-lymphoma CAR-T drug, characterized by: the anti-lymphoma CAR-T drug is a CD30CAR-T drug, the CD30CAR-T drug being capable of expressing a human CD30 antibody;

wherein, the CD30 antibody is derived from hybridoma cells, and the preservation number is CCTCC NO: c201861; the anti-lymphoma CAR-T medicine is obtained by infecting T cells with a CD30CAR lentiviral expression vector;

the CD30CAR lentiviral expression vector comprises connecting a heavy chain and a light chain of a CD30 antibody with a single-chain variable region fragment scFv to form a CD30 scFv gene fragment; wherein, the sequence of the CD30 scFv gene fragment is shown in SEQ ID NO:1 is shown in the specification;

the CD30 scFv gene fragment is connected with CD8aSP, a 4-1BB intracellular signal region and a CD3 zeta gene sequence in series to obtain a CD30CAR gene sequence; connecting the CD30CAR gene sequence with a pCDH-CMV-SMC-EF1a-GFP vector to obtain the CD30CAR lentiviral expression vector, wherein the gene sequence of the CD30CAR lentiviral expression vector is shown in SEQ ID NO:2 is shown in the figure;

wherein the lymphoma is peripheral T cell lymphoma cell strain Karpas299.

2. The anti-lymphoma CAR-T drug of claim 1, wherein: the CD30CAR lentiviral expression vector infects T cells, and further comprises packaging the CD30CAR lentiviral expression vector by adding 3 μg pMD2G, 6 μg psPAX and 7.5 μg of the CD30CAR lentiviral expression vector into 150 μl OPTI-MEM, mixing, adding into 500 μl OPTI-MEM containing 25 μl Lipo 2000, standing at room temperature for 15min, adding into a culture dish, adding into 37 ℃ and 5% CO ₂ After 12h of culture in an incubator of (2) and then the transfected 293T cells were expanded and lentiviruses were collected to obtain lentivirus Lenti-2G. CAR-CD30.

3. The anti-lymphoma CAR-T drug of claim 2, wherein: the CD30CAR lentiviral expression vector infected T cells further included mixing the lentiviral Lenti-2G CAR-CD30 with T cells, adding 8 μg/mL polybrene, and 5% CO at 37℃ ₂ Incubating for 0.5-2 hours in the incubator; preparing a T cell culture solution, adding 50 mug/mL human serum albumin, 1000U/mL IL-2, 100U/mL OKT3 and CD28 into the GT-T551H3 culture solution, addingIn the cells after incubation, at 37℃5% CO ₂ Is cultured in an incubator for 14 days.

4. The anti-lymphoma CAR-T drug of claim 1, wherein: the CD30CAR lentiviral expression vector infects T cells, wherein the amount of CD30CAR lentiviral required to infect T cells is calculated as MOI of 5.

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