CN111777686A

CN111777686A - FOLR1-MSLN dual targeting CAR-T cells, chimeric antigen receptors, and vectors for treating ovarian cancer

Info

Publication number: CN111777686A
Application number: CN202010677570.XA
Authority: CN
Inventors: 梁振; 梁志清; 蔡雄伟; 王延洲
Original assignee: First Affiliated Hospital of PLA Military Medical University
Current assignee: First Affiliated Hospital of PLA Military Medical University
Priority date: 2020-07-13
Filing date: 2020-07-13
Publication date: 2020-10-16
Anticipated expiration: 2040-07-13
Also published as: CN111777686B

Abstract

The invention provides a chimeric antigen receptor, which comprises a single-chain antibody segment, an extracellular hinge region, a transmembrane region, an intracellular immune receptor tyrosine activation motif and an IL-12 gene region which are connected in sequence; wherein the single chain antibody segment is selected from one of a folate receptor alpha (FOLR1) single chain antibody, a Mesothelin (MSLN) single chain antibody or a folate receptor alpha single chain antibody-mesothelin single chain antibody tandem complex. The invention also provides a plasmid vector and a lentivirus vector containing the chimeric antigen receptor, and three CAR-T cells. The FOLR1-CAR, the MSLN-CAR and the Tandem-CAR based on the invention can obviously secrete IL-12 and inhibit the growth of tumors, wherein the Tandem-CAR effect is most obvious.

Description

FOLR1-MSLN dual targeting CAR-T cells, chimeric antigen receptors, and vectors for treating ovarian cancer

Technical Field

The invention belongs to the field of immunology and molecular biology, and particularly relates to a FOLR1-MSLN dual-targeting CAR-T cell, a chimeric antigen receptor and a vector for treating ovarian cancer.

Background

The mortality rate of ovarian cancer is among the first three malignancies of the female reproductive system, with 90% being Epithelial Ovarian Cancer (EOC), which is insidious and 70% of patients are diagnosed with advanced stages with distant metastasis. 55-75% of patients who have remission after first-line therapy relapse within 2 years, resulting in a five-year survival rate of less than 40%, and therefore, the search for a new treatment method is of great significance.

Gross first proposed the concept of "chimeric receptors" in 1989, in which the variable region of the T cell receptor was replaced with a single chain antibody, so that the modified T cell had antigen targeting properties. The design of chimeric antigen receptor T cells (CAR-T) is born by this, and CAR-T can directionally recognize tumor cells and has specific killing function. The CAR gene consists of extracellular single-chain variable region (scFv) and intracellular activation motif, and the T cell modified in this way solves the problem of immune escape caused by the loss of tumor cell MHC (major histocompatibility complex) class I molecules, thus having great application prospect. Through the development of over thirty years, the second generation CAR-T developed in 2011 achieves nearly 90% of complete remission rate in the treatment of acute lymphoblastic leukemia, opens a new era of human anti-tumor history, and brings hope for malignant tumor patients.

However, CAR-T therapy suffers from difficulties in solid tumors, and the major bottlenecks to be met are: 1) heterogeneity of solid tumors, recurrence after single target CAR-T treatment (antigen escape); 2) immunosuppression microenvironments of solid tumors (hypoxia, low pH, Treg cells, indoleamine 2, 3-dioxygenase) result in insufficient T cell activation and/or T cell suppression; 3) insufficient persistence and insufficient expansion of T cells; 4) solid tumors have envelopes, and T cells are difficult to infiltrate and home.

Disclosure of Invention

Aiming at the problems in the prior art, the invention firstly selects two tumor-associated antigens (TAA) with high expression in EOC through screening of early bioinformatics and verification of clinical samples: folate receptor alpha (FOLR1) and Mesothelin (MSLN), and further constructing a Tandem CAR-T (Tandem CAR) which simultaneously targets FOLR1 and MSLN, and designing the Tandem CAR-T (Tandem CAR-T) which simultaneously targets FOLR1 and MSLN on the basis, thereby enabling CAR-T cells to have wider killing coverage rate. Meanwhile, the immune response of T cells in a tumor microenvironment is further enhanced through the immune activation effect of IL-12 (such as activating cytotoxic T cells, recruiting B cells, NK cells, macrophages and the like), and the survival and expansion capacity of CAR-T cells are further improved, so that the infiltration and activation problems of CAR-T are solved.

The technical scheme provided by the invention is as follows:

a chimeric antigen receptor comprising, in order, a single chain antibody segment, an extracellular hinge region (hind area), a transmembrane region, and an intracellular Immunoreceptor Tyrosine Activation Motif (ITAM); wherein,

the single chain antibody segment is selected from one of a folate receptor 1 single chain antibody (FOLR1scFV), a mesothelin single chain antibody (MSLNscFV) or a folate receptor 1 single chain antibody (FOLR1scFV) -mesothelin single chain antibody (MSLNscFV) tandem complex. In vivo imaging results confirmed by SNU119 cell transplantation tumor model show that FOLR1-CAR, MSLN-CAR and Tandem-CAR (CAR prepared by using FOLR1scFV-MSLN scFV Tandem complex as chimeric antigen receptor) three kinds of CAR-T can obviously inhibit tumor growth compared with Mock-T group (P < 0.0001).

In one embodiment according to the present invention, the intracellular Immunoreceptor Tyrosine Activation Motif (ITAM) is composed of a CD28 peptide fragment, a 4-1BB peptide fragment, and a CD3 zeta chain peptide fragment, which are linked in this order. The activation capability and the persistence of the CAR-T cell are stronger due to the inclusion of two costimulatory molecules of CD28 and 4-1 BB.

Preferably, the CD3 zeta chain peptide is followed by an IL-12 peptide. CAR-T cells expressing IL-12 and secreting high levels of cytokines can significantly contribute to anti-tumor effects.

In one embodiment according to the present invention, the amino acid sequence of the folate receptor 1 single chain antibody (FOLR1scFV) is SEQ ID NO 1;

the amino acid sequence of the mesothelin single-chain antibody (MSLN scFV) is SEQ ID NO 2;

the folate receptor 1 single-chain antibody (FOLR1scFV) -mesothelin single-chain antibody (MSLN scFV) tandem complex is characterized in that the folate receptor 1 single-chain antibody (FOLR1scFV) and the mesothelin single-chain antibody (MSLN scFV) are connected through an Llinker peptide segment, and the amino acid sequence of the Linker peptide segment is SEQ ID NO. 3.

The invention also provides a plasmid vector for expressing the chimeric antigen receptor, which comprises

A gene encoding a single-chain antibody segment, a gene encoding an intracellular immunoreceptor tyrosine activation motif, and a lentiviral plasmid; wherein the encoding gene of the single-chain antibody segment is selected from one of the encoding gene of a folate receptor 1 single-chain antibody (FOLR1scFV), the encoding gene of a mesothelin single-chain antibody (MSLN scFV) or the encoding gene of a folate receptor 1 single-chain antibody (FOLR1scFV) -mesothelin single-chain antibody (MSLN scFV) tandem complex. The lentiviral plasmid is preferably pCDH.

In one embodiment according to the present invention, the nucleotide sequence of the gene encoding the intracellular immunoreceptor tyrosine activation motif is SEQ ID NO 4.

In one embodiment according to the present invention, the nucleotide sequence of the gene encoding the folate receptor 1 single chain antibody (FOLR1scFV) is SEQ ID No. 5;

the nucleotide sequence of the coding gene of the mesothelin single-chain antibody (MSLN scFV) is SEQ ID NO. 6.

In one embodiment according to the present invention, the nucleotide sequence of the encoding gene of the folate receptor 1 single chain antibody (FOLR1scFV) and mesothelin single chain antibody (MSLN scFV) tandem complex is SEQ ID NO. 7. Preferably, the amino acid sequence of the duplex is SEQ ID NO 8.

The invention also provides a lentiviral vector which comprises a coding gene for the chimeric antigen receptor or is formed by packaging the plasmid vector after transfecting cells.

The invention further provides a CAR-T cell for use in the treatment of ovarian cancer, which CAR-T cell is obtained by infection of a CD3 positive T cell with the lentiviral vector described above.

The technical scheme provided by the invention has the following beneficial effects:

1) the lentiviral vectors of Tandem-CAR, FOLR1-CAR and MSLN-CAR are successfully constructed, the sequencing verification proves that the lentiviral vectors are correct, the lentiviruses are packaged and infect CD3 cells, and different types of CAR-T are established.

2) LDH experiments show that compared with a control group and two single-target CAR-T cells, the Tandem-CAR cells can obviously kill the SNU119 cells with FOLR1 and MSLN simultaneously highly expressed, and the killing rate of the A2780 cells with FOLR1 and MSLN low expression is lower.

3) The flow cytometry detection IL-2, IL-12, TNF-alpha and IFN-gamma levels show that after the co-culture with SNU119 cells, the positive proportion of released cytokines of Tandem-CAR is the highest, three CAR-T successfully express IL-12, and Mock-T cells (T cells without transfected virus) hardly express IL-12. The proportion of IL-2, TNF-alpha and IFN-gamma positive cells expressed by the three CAR-Ts is obviously higher than that of a control group (Mock-T).

4) Mouse experiments show that compared with the Mock-T group, the three CAR-T can effectively inhibit the growth of tumors, the treatment effect of the MSLN-CAR is characterized in that the tumors shrink first and then grow, and the fluorescence intensity of the FOLR1-CAR and the TANDEm-CAR treatment groups is continuously reduced. The therapeutic effect of Tandem-CAR was most pronounced, with regression of tumors occurring in 1 of the mice.

Drawings

FIG. 1 is a schematic diagram of a single CAR and a Tandem-CAR;

FIG. 2 is a diagram showing the results of transfection in 48h under a fluorescence microscope, wherein the magnification is X100 times;

FIG. 3 is a schematic diagram of flow cytometry titers 72h after infection of 293T cells with three viruses;

FIG. 4 is a schematic of sorting CD3 from PBMC by immunomagnetic beads;

FIG. 5 shows F (ab')₂Schematic diagram of detecting expression rates of different CARs;

FIG. 6 is a schematic view of infected CD3 cells observed under a fluorescence microscope at magnification X100;

FIG. 7 is a graph showing the expression levels of target antigens of three different tumor cells;

fig. 8 is a graph showing the killing rate of four T cells at different effective target ratios (E/T), wherein P <0.05, P <0.01, P <0.001, P < 0.0001;

figure 9 is a schematic of the ratios of four cytokine positive cells after co-culture of T cells with SNU119 tumor cells, wherein P <0.05, P <0.01, P <0.001, P < 0.0001;

FIG. 10 is a schematic comparison of the in vivo therapeutic effect of CAR-T after tumor-bearing SNU119 ovarian cancer cells;

fig. 11 is a graph showing comparison of fluorescence values in vivo imaging, wherein P <0.05, P <0.01, P <0.001, P < 0.0001.

Detailed Description

The following examples are intended to illustrate the present application but are not intended to limit the scope of the present application.

Specific embodiments of the present application will be described in more detail below. These embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the present application, but is made for the purpose of illustrating the general principles of the application and not for the purpose of limiting the scope of the application. The protection scope of the present application shall be subject to the definitions of the appended claims.

Unless otherwise specified, the reagents used in the present invention are commercially available.

Cell and reagent consumable

Laboratory apparatus

Related reagent configuration

1) LB liquid medium: taking 10g of NaCl, tryptone and yeast extract respectively, adding 1L of ddH2O, fully dissolving until the solution is clear and transparent, subpackaging into 100ml per conical flask, and labeling and sealing at 4 ℃ at 121 ℃ under high temperature and high pressure for 30 min.

2) LB solid medium: adding 1% Agar into 100ml LB liquid culture medium, sterilizing, placing in 55 deg.C water bath, adding 0.1% Amp after temperature is reduced, shaking thoroughly, pouring 10 plates rapidly, solidifying, sealing with sealing glue, and storing at 4 deg.C.

3) Humanized FOLR1-scFv (amino acid sequence is SEQ ID NO:1, nucleotide sequence is SDQ ID NO:5), MSLN-scFv (amino acid sequence is SEQ ID NO:2, nucleotide sequence is SEQ ID NO:6), Tandem-scFv sequence (amino acid sequence is SEQ ID NO:8, nucleotide sequence is SEQ ID NO:7) are biosynthesized and verified by Beijing Okagaku department through sequencing, wherein a folic acid receptor 1 single-chain antibody (FOLR1scFV) and a mesothelin single-chain antibody (MSLN scFV) are connected through an Llinker peptide segment, and the amino acid sequence of the Linker peptide segment is SEQ ID NO: 3; tyrosine Activation Motif (ITAM) amino acid sequence SEQ ID NO. 4.

Example 1 Lentiviral vector construction

Firstly, construction and verification of plasmid vector

1. The construction process of the recombinant plasmids FOLR1-CAR, MSLN-CAR and Tandem-CAR: a gene synthesis method of PCR bypass is utilized to artificially synthesize a CD8 signal peptide, a single-chain antibody (FOLR1-scFv or MSLN-scFv or Tandem-scFv), a CD8a hinge region, a CD28-41BB-CD3z CAR structure and an IL-12 gene sequence, an XbaI enzyme cutting site is introduced into the upstream of the gene synthesis method, a NotI enzyme cutting site is inserted into the downstream of the gene synthesis method, the gene synthesis method is consigned to Beijing Ongshigaku biological company, the gene synthesis method is loaded into a pCDH vector (purchased from Addge company) which is subjected to double enzyme cutting by XbaI and NotI,

PCDH。

2. large quantity extracted plasmid pCDH

1) A small amount of the frozen glycerol strain is inoculated on an LB solid culture plate containing Amp resistance and is placed in an incubator at 37 ℃ for overnight culture.

2) The next afternoon, the cells were inoculated into 150ml LB liquid medium containing Amp, and placed in a shaker at 37 ℃ and 260rpm for 18 hours.

3) On the third day, plasmid was extracted using the Tiangen kit, and the elution buffer TB was first preheated in a 66 ℃ water bath.

4) Subpackaging the bacteria liquid into a clean 50ml centrifuge tube, marking, centrifuging at 8000rpm for 3min to collect bacteria, discarding the supernatant, repeating for three times, and then sucking the liquid on the tube wall with absorbent paper.

5) Pipette 10ml of solution P1 (added RNase A) into the centrifuge tube, reverse the tube 2 times and quickly place on a vortex apparatus to mix the pellet.

6) 10ml of the P2 solution is added into a centrifuge tube, the upside is reversed for 10 times, and the mixture is placed at room temperature for 5min to ensure that the thalli are fully cracked.

7) 10ml of the P4 solution was added to a centrifuge tube, and the tube was inverted 10 times to mix the bacterial solution, and a large amount of white flocculent precipitate was observed after standing at room temperature for 15 min. After centrifugation at 8,200rpm for 35min, a white precipitate was visible at the bottom of the tube, and the entire solution was filtered by adding it to filter CS1 using a pipette gun and collecting the filtrate in a clean 50ml centrifuge tube.

8) 3ml of ER solution (endotoxin removed) was added to the centrifuge tube and turned upside down until the solution appeared uniformly yellow.

9) 9ml of isopropanol were added to the centrifuge tube and the solution was inverted upside down to mix well, 10ml of liquid each time was added to the adsorption column CP6 (equilibration solution was treated). After centrifugation at 8,200rpm for 2min at room temperature, the waste solution was discarded, and the whole was carefully passed through the column in succession, at which time the plasmid DNA was adsorbed to the column of CP 6.

10) 10ml of ED buffer was added to the adsorption column CP6, centrifuged at 8,200rpm for 2min at room temperature, the waste was discarded, the adsorption column was returned to the collection tube and the operation was repeated once.

11) 10ml of the rinsing solution PW (absolute ethanol added) is added into an adsorption column CP6, the solution is centrifuged at 8,200rpm for 2min at room temperature, the waste solution is discarded, and the adsorption column is placed back into the collection tube and the operation is repeated once.

12) The adsorption column CP6 was returned to the collection tube and centrifuged at 8,200rpm for 5min, and the lid of the adsorption column CP6 was opened and left at room temperature for 5min to allow the residual rinse solution on the adsorption membrane to be sufficiently dried.

13) A clean 50ml centrifuge tube was taken and the adsorption column CP6 was placed in, 1ml of the previously preheated elution buffer TB was aspirated, carefully added dropwise to each site of the adsorption membrane and left for 6min, and then centrifuged at 8,200rpm for 6 min. And dripping the liquid in the tube on the adsorption film for repeated elution once again, transferring the liquid in the centrifugal tube to a sterilized 1.5ml centrifugal tube, subpackaging and storing at-20 ℃ for a long time.

3. Enzyme digestion plasmid skeleton

1) The enzyme digestion is carried out for 2h at 37 ℃ by adopting the following system

2) Gel electrophoresis: 100ml of 1 XTAE was taken, 2g of agar sugar powder was added, the mixture was heated in a microwave oven until the agarose was completely melted, 3ul of GoldView was added after the temperature dropped to 55 ℃, and the gel was poured and inserted into a comb. And after the gel is solidified, putting the gel into an electrophoresis tank, pouring 1 XTAE electrophoresis Buffer solution, adding no sample into a first hole, adding 3ul of DNA Marker into a second hole, adding a mixed solution of a digestion product and a DNA loading Buffer into a third hole, carrying out 120V electrophoresis for 30min, observing the size of a strip in a gel scanning imager after the electrophoresis is finished, and judging the position of the fragment for gel cutting and recovery.

4. Recovery of plasmid backbone fragments

1) The gel was placed under an ultraviolet lamp and the desired fragment of interest was cut rapidly into 1.5ml EP tubes.

2) Weighing the gel block, adding 3 times of GDPBuffer into an EP tube according to the weight of 100 μ l of solution per 100mg of gel block, dissolving in a 55 deg.C water bath for 15min, and turning 3 times every 5min to accelerate the dissolution of the gel block.

3) The EP tube was centrifuged at 12,000rpm for 1min to collect droplets on the tube wall at the bottom, the recovery column was placed in a clean 1.5ml EP tube, the liquid was transferred to the column, centrifuged at 12,000rpm for 1min and the filtrate was discarded.

4) The column was returned to the EP tube, 300. mu.l of GDP Buffer was carefully added to the column and left for 1min, centrifuged at 12,000rpm for 1min, and the filtrate was discarded.

5) The column was placed back in the EP tube and 600. mu.l DW2Buffer (to which absolute ethanol had been added) was added to the column. The above operation was repeated once after centrifugation at 12,000rpm for 1 min.

6) The filtrate was decanted, the column was returned to the EP tube and centrifuged at 12,000rpm for 3min and the filtrate was discarded.

7) A clean 1.5ml EP tube was taken and the column was placed in it, 15. mu.l of an Elution Buffer (preheated) was carefully dropped onto the membrane of the column using a pipette, left to stand at room temperature for 3min and centrifuged at 12,000rpm for 1min, the column was discarded and the DNA at the bottom of the tube was stored at-20 ℃.

5. Connection of

The target fragment and the vector were mixed according to 4:1, T4DNA ligase was added for ligation and ligation was performed overnight at 4 ℃.

6. Transformation of competent cells

1) The competent cells Stbl3 were taken out from the freezer at-80 ℃ and thawed on ice, the ligation mixture was added, gently mixed with a tip, and then placed on ice for 30min to stand.

2) Heat shock at 42 ℃ for 60 seconds: the EP tube was allowed to stand in a water bath at 42 ℃ for 60 seconds, then quickly cooled on ice for 2min, taking care not to shake the EP tube.

3) 600ul of sterilized LB medium (containing no antibiotics) was added into an EP tube, mixed uniformly with a pipette, and put into a shaker (37 ℃, 150rpm) for shaking culture for 60min to recover the thallus.

4) Adding the bacterial liquid to an LB solid culture medium plate containing Amp under the aseptic condition, uniformly spreading cells by using an aseptic bacterial L rod, inverting the plate after the liquid in the plate is completely absorbed, and carrying out culture at 37 ℃ for 16 h.

7. Miniaspirate plasmid, restriction enzyme, gel electrophoresis, sequencing

1) 5 monoclonal colonies were picked from LB plates and added to 5 labeled 15ml centrifuge tubes, 2ml of LB liquid medium containing Amp was added to each tube, and shaking cultured at 280rpm and 37 ℃ for 14 hours.

2) Adding the bacterial liquid into a 1.5ml EP tube, centrifuging at 12,000rpm for 2min to collect the bacteria, then discarding the supernatant, and absorbing the liquid on the tube wall by using absorbent paper.

3) 500ul of P1 solution (RNase A was added) was added to an EP tube containing the pellet, and after reversing the tube 2 times, the tube was vortexed on a vortexer to mix the pellet.

4) 500ul of the P2 solution was added to the centrifuge tube and turned upside down 10 times to ensure the cells were lysed sufficiently.

5) 700ul of the P3 solution was added to a centrifuge tube, and the tube was inverted 10 times to mix the bacterial solution, and a large amount of white flocculent precipitate was observed after standing at room temperature for 3 min. After centrifugation at 1,400rpm for 15min, a white precipitate at the bottom of the tube was observed.

6) The supernatant was fractionated into CS filtration columns using a pipette and centrifuged at 14,000rpm for 2 min.

7) The collected filtrate was carefully added to a CP4 adsorption column (activated with equilibration fluid), centrifuged at 14,000rpm for 1min and the waste liquid discarded, and the CP4 adsorption column was replaced with the collection tube.

8) 500ul of PD (deproteinized solution) was added to the adsorption column CP4, and the mixture was centrifuged at 14,000rpm for 1min, and then the waste liquid was discarded.

9) 600ul of the rinsing solution PW (absolute ethanol added) was added to the adsorption column CP4, centrifuged at 14,000rpm for 2min at room temperature, the waste solution was discarded, and the adsorption column was returned to the collection tube and the operation was repeated once.

10) The adsorption column CP4 was returned to the collection tube and centrifuged at 14,000rpm for 5min, and the lid of the adsorption column CP4 was opened and placed at room temperature for 3min to allow the residual rinse solution on the adsorption membrane to be sufficiently dried.

11) A clean 1.5ml centrifuge tube was taken, the adsorption column CP4 was placed in, 100ul of TB (elution buffer) was slowly added dropwise to each part of the adsorption membrane, and after standing for 3min, the tube was centrifuged at 14,000rpm for 2min to obtain a plasmid DNA solution.

12) The concentration was measured by micro-spectrophotometry.

13) The plasmid is digested by the same steps as the previous steps.

14) And (5) analyzing the size of the band by gel electrophoresis, and sending to sequencing verification after primary judgment.

Second, packaging and concentration of lentivirus for titer determination

1. And (5) extracting plasmids in a large scale, wherein the concentration of the plasmids is ensured to be more than 1000 ng/ul.

2. Recovering 293T, taking out 293T cells from liquid nitrogen, quickly placing the 293T cells in a 37 ℃ water bath for unfreezing the cells, transferring the cells to a 10ml centrifuge tube (sterilized) in a sterile operating platform, adding 1ml of 10% DMEM complete culture medium into the centrifuge tube, centrifuging at a low speed of 1000rpm for 5min, discarding supernatant, resuspending the cells in 1ml of culture medium, lightly blowing the cells by using a pipette gun, inoculating the cells into a 10cm culture dish, marking the date, the type, the passage number and the like, and culturing in a 37 ℃ incubator.

3. Transfection of 293T

1) Cells were counted overnight before transfection, and 5 × 10 was taken⁶Laying a 10cm plate to ensure that the cell density is about 80% in the next morning and the cells are not too dense.

2) The next morning the cells were replaced with fresh complete medium before transfection and 2 1.5ml EP tubes, labeled A, B, respectively, were taken. Add 500ul of serum-reduced medium (Opti-MEM) and 21.7ul of lip3000 to tube A and mix well by vortexing for 2-3 seconds. 500ul Opti-MEM was added to tube B, followed by 16.5ug of two packaging plasmids psPAX2, pMD2G, and master plasmid, and 48ul of P3000 reagent was added to tube B, and mixed well.

3) And adding the liquid in the tube B into the tube A, uniformly mixing, incubating at room temperature for 15min, adding the mixture into 293T cell culture medium, slightly and uniformly mixing by a cross method, and photographing after 48h to check fluorescence.

4. Concentrating the virus

1) The virus was harvested at 48h, centrifuged at 1500rpm, cell debris removed, and the supernatant filtered through a 0.22um filter tip, following a 1: 3, adding Lenti-x Concentrator lentivirus concentrated solution, refrigerating overnight at 4 ℃, and centrifuging at 1500g for 45min at 4 ℃ the next morning.

2) The obtained white precipitate is virus concentrated product, removing supernatant, dissolving virus in 100ul DMEM, subpackaging 10ul each tube, spraying liquid nitrogen, quick freezing, and storing in refrigerator at-80 deg.C.

5. Measurement of viral titre

1) Day 0 digestion of 293T cells in good growth status was counted and 1 × 10 cells per well were used⁵And 12-hole plates are paved.

2) Day 1: adding virus concentrate 0.0001ul, 0.001ul, 0.01ul, 0.1ul, 1ul and 10ul into 12-well plate according to gradient dilution method, setting 1 negative control without virus, adding Polybrene 8ug/ml, mixing by cross method, adding 5% CO at 37 deg.C₂Culturing in an incubator for 72 h.

3) Day 4: and (4) observing under a fluorescence microscope, detecting the infection rate by flow and calculating the titer.

FIG. 2 is a schematic diagram of transfection results observed under a fluorescence microscope for 48h, wherein the magnification is × 100 times, FIG. 3 is a schematic diagram of titer measurement after 293T cells are infected by three viruses for 72h, as shown in FIG. 2 and FIG. 3, the transfection rate for 48h is more than eighty percent, and the titer measurement results of packaging, concentration and titer measurement of three kinds of lentiviruses are that the titer of FOLR1-CAR, MSLN-CAR and Tandem-CAR after concentration is respectively 3.4 × 10⁸、3.1×10⁸、3.5×10⁸TU/ml。

Example 2 separation of PBMC and CD3

1. Isolated PBMC

1) Peripheral blood from healthy volunteers was collected in 10ml, immediately placed in an ice box, taken back between the laboratory cells, washed three times with PBS, and 10ml of PBS + 2% FBS buffer was added.

2) Density gradient centrifugation: adding 15ml of Ficoll solution of Stemcell, centrifuging at 1500rpm and 19 ℃ for 30 minutes, and separating into 4 layers in a visible tube after centrifugation: the uppermost layer is plasma containing part of platelets; the second layer is a thin leucocyte layer which is mainly mononuclear cells and also mixed with a small amount of platelets; the third layer is a separation liquid layer; the fourth layer is granulocytes and erythrocytes, which sink to the bottom of the tube, and granulocytes cling to the packed erythrocytes and present a thin white membrane.

3) Carefully sucking the leucocyte, adding 5 times of PBS into the obtained PBMC, gently beating the PBMC evenly by using a capillary pipette to avoid generating small bubbles, enabling the height of a liquid column to be not more than 2/3 of a centrifuge tube, centrifuging the mixture at the speed of 1500r/min for 10min after evenly mixing, discarding the supernatant, repeatedly washing the mixture for 2 times, re-suspending the mixture by using 10ml of RPMI 1640 culture medium containing 10% FBS, counting the cells by using a machine, and enabling the total number of the cells to be 1.38 × 10⁷And (4) respectively.

2. Isolation of CD3 cells in PBMC

1)40um nylon mesh filtration to remove cell clumps that may clog the column. Buffer (PBScontaining 2% FBS and 1mM EDTA): 100ml of buffer 0.2ml of 0.5M EDTA +98ml of PBS +2ml of FBS was prepared, and the mixture was sterilized by filtration.

2) The number of cells was determined. Buffer was resuspended and the number of cells was adjusted to 5X 107 cells/ml.

3)5ml sterile flow tube preparation, add cells 0.25-2ml to the flow tube.

4) Cocktail 50ul/ml was added to the cells, mixed well and incubated for 5 minutes at room temperature.

5) Vortex the reagent Rapd Spheres for 30 seconds to ensure uniform particles.

6) Rapd Spheres was added to the cells at a ratio of 40ul/ml and mixed well.

7) Buffer was added, the total volume of liquid was made up to 2.5ml, the tube (without lid) was placed in a magnet and incubated for 3 minutes at room temperature.

8) The magnet and flow tube were picked up and the liquid in the centrifuge tube was poured directly into a clean fresh centrifuge tube, in which was separated CD3, and the machine counted the cells.

9) Flow identification of CD3 by adjusting cell concentration to 107/ml, taking 100ul of each tube, setting blank control group, single tube staining group, and total staining group, adding three flow antibodies of CD3-PE/CY7, CD8-APC/CY7, and CD4-percp-CY5.5, incubating on ice in dark for 30min, resuspending the rest cells with RP1640 MI medium containing 10% FBS and IL-2100U/ml, adjusting cell concentration to 1 × 106/ml, adding 25ul ImmunoCult into each 1ml cell suspension^TMThe cell activator of Human CD3/CD28T is prepared by culturing culture plate at 37 deg.c in 5% CO 2 incubator48h。

Sorting CD3 cells from PMBC showed that sorted CD3 was as high as 99.6% pure (fig. 4), with CD4 and CD8 in the proportions of 74.4% and 17.4%, respectively,

example 3 Lentiviral infection with CD3 and determination of infection results

1. The multiplicity of infection (MOI) was 10 infection of CD3 cells per well, and 8ug/ml of pluybrene was added for infection.

2. APC-F (ab')₂And dyeing and detecting the infection rate on a flow type computer.

3. Results of the experiment

The structural schematic diagram of the 4 th generation FOLR1-CAR plasmid, the MSLN-CAR plasmid and the Tandem-CAR plasmid is shown in figure 1, and the constructed plasmids are subjected to enzyme digestion and nucleic acid gel electrophoresis analysis to meet the size of a target gene fragment. Meanwhile, the three plasmids are subjected to sequencing verification, and the sequences are correct, so that three lentivirus plasmids containing two costimulatory molecules CD28 and 4-1BB and IL-12 genes are successfully constructed.

CD3 cells were infected at MOI 10 for 5 days and flow results showed CAR expression of 40%, 36%, 32%, respectively (fig. 5), and infected CD3 cells were seen to clump together and fluoresce green under a fluorescence microscope (fig. 6).

Example 4 Targeted killing of ovarian cancer cells by different CAR-T cells in vitro and in vivo

First, detection of cell line surface antigen

(1) Collecting A2780, SKOV3 and SNU119 ovarian cancer cells in logarithmic phase, digesting the cells, centrifuging to collect the cells, discarding the supernatant, resuspending 1ml of ice-cold staining Buffer, and washing twice.

(2) Cells were counted and made into a 1X 107/ml suspension using a cell staining Buffer. 100ul of cell suspension was added to the flow tube for use.

(3) Add flow antibody 5ul, tube 1: blank, tube 2: APC-MSLN, tube 3: PE-FOLR1, pipe 4: shuangyang, 4 deg.C, 30min, and protection from light.

(4) Adding 5ml of cell staining Buffer to resuspend the cells, centrifuging the cell suspension at 1000rpm for 5min, discarding the supernatant, washing twice, adding 200ul of cell staining Buffer to resuspend the cells, filtering with a 40um screen, and detecting and analyzing with a flow cytometer.

By using flow analysis of the expression levels of FOLR1 and MSLN antigens on the surfaces of three human ovarian cancer cell lines, namely A2780, SKOV3 and SNU119, the results show that the expression level of FOLR1 is SNU119> SKOV3> A2780 (figure 7), and the expression level of MSLN is SNU119> SKOV3> A2780 (figure 7), so that SNU119 cells can be used as ideal target cells of Tandem-CAR, and therefore, SNU119 cells are taken as a main research model for in vivo and in vitro killing.

Second, CAR-T cell killing experiment

1. LDH Release test

1) The experimental principle is as follows:

normally, cells contain abundant Lactate Dehydrogenase (LDH), when the cell membrane is intact, the cell membrane cannot pass through the cell membrane, when the cells are damaged or dead, the cell membrane is broken, the LDH is released to the outside of the cells, the extracellular LDH is stable in a certain time, so the number of cell death is in direct proportion to the LDH content in supernatant, and the killing efficiency of CAR-T can be calculated by measuring the LDH content.

2) Target cells are plated, A2780, SKOV3 and SNU119 cells in logarithmic growth phase are taken, digestion counting is carried out, and the concentration is adjusted to be 10⁵And/ml, taking three new 96-well plates, adding 5000 tumor cells into each well, adding 100ul PBS into the blank wells at the periphery to prevent the water of the intermediate cells from evaporating, and putting the 96-well plates back into an incubator to culture overnight after the tumor cells are laid.

3) Adding three CAR-T cells and one Mock T cell in proportion the next day, leaving blank control wells (targetSpontanoous and Effector Spontanoous) and simple culture Medium wells (Medium), putting a 96-pore plate back into an incubator for culturing for 18h, 45min before killing is finished, adding 10ul of 10 × Lysis Buffer into the maximum Lysis well, culturing for 45min, then centrifuging the 96-pore plate at the speed of 250g for 5min, transferring 50ul of supernatant of each well into a new 96-pore plate, taking out 12ml of detection Buffer (Assay Buffer), adding 50ul of Assay Buffer into each well, incubating for 30min at room temperature, keeping the whole process out of the sun, and adding 50ul of stop solution into each well after 30 min.

4) Enzyme-linked immunosorbent assay 490nm detection absorbance value

5) The target cell lysis rate was calculated as follows:

the killing rate is (Experimental-Target Spontaneous)/(Target Maximum-Target Spontaneous) × 100%.

The experimental results are as follows:

the results of comparison of killing efficacy of four effector cells (Tandem-CAR, FOLR1-CAR, MSLN-CAR and Mock-T) and three tumor cells (SNU119, SKOV3 and A2780) after co-culture for 18h by using an LDH cytotoxicity detection kit show that the killing efficacy of the Tandem-CAR is obviously higher than that of the Mock-T group (P2780) except for the A2780 group (E/T is 2:1)<0.0001). The kill rate of TANDEm-CAR was also significantly higher than the FOLR1-CAR and MSLN-CAR groups (E/T ═ 8:1, P)<0.0001). And the specific lethality of the Tandem-CAR is dependent on the expression level of two antigens, FOLR1 and MSLN, as shown in FIG. 8, SNU119 (F) highly expressed by FOLR1 and MSLN antigen^HM^H) Tumor cells, the killing ability of effector cells is: Tandem-CAR>FOLR1-CAR>MSLN-CAR>Mock-T, when the E/T ratio is 8: the highest killing efficacy of Tandem-CAR reached 79.96% at 1. When the target cell is SKOV3 (F)^HM^L) The killing efficacy of the Tandem-CAR and FOLR1-CAR was significantly higher than that of the MSLN-CAR and Mock-T (P)<0.0001), no statistical difference in killing ability between MSLN-CAR and Mock-T. When the target cell is A2780 (F)^LM^L) At this time, the highest killing rate of Tandem-CAR was only 30.9%, and there was a small statistical difference in killing ability between MSLN-CAR and Mock-T (E/T4: 1, E/T8: 1, P0.0110, P0.0102), indicating that Tandem-CAR can specifically recognize and kill tumor cells.

Flow detection of cytokine release

(1) Day 0: target cell SNU119 is plated, a 96-well plate is taken, and cell tumor cells 10 are added into each well⁴Cells were allowed to adhere overnight in culture.

(2) Day 1: according to the condition that E/T is 2:1, adding Mock-T, FOLR1-CAR, MSLN-CAR and Tandem-CAR cells, setting three multiple wells in each group, co-culturing for 14h, adding Ionomycin (Ionomycin)500ng/ml and PMA (phorbol ester) 50ng/ml for stimulation for 4h, and finally adding Golgi blocker for cytokine staining.

(3) Centrifuging to collect cells, resuspending the cells at 50ul, taking out 5ul of the cells from 50ul of the cells, mixing the cells together to obtain 150ul of mixed Solution, taking 10ul of the mixed Solution to stain surface Marker CD3, CD4 and CD8 respectively, centrifuging the rest 120ul, discarding the supernatant, adding 250ul of Fixation/Permeabilization Solution, and adding 200ul of Wash Buffer to resuspend the cells after centrifugation.

(4) 30ul of the sample was used as Blank, and 170ul of the sample was left to be stained with single color (IL-2, IL-12, TNF-. alpha., IFN-. gamma.) in 4 tubes.

(5) 50ul of pooled antibody (CD3, CD4, CD8) was added to each 45ul of samples, stained for 30min, and washed twice with Wash Buffer.

(6)250ul of Fixation/Permeabilization Solution resuspended cells, fixed for 15min and washed twice, 55ul of cytokine-mixed antibody was added, incubated on ice for 30min and washed twice with Wash Buffer.

(7) And (6) performing detection on the machine.

The experimental results are as follows:

the secretion of cytokines such as IL-2, FN-gamma, IL-12, TNF-alpha and the like is an important mark for the killing efficacy of the CAR-T cells, so the killing ability of different T cells can be effectively judged by utilizing the secretion level of the cytokines after the co-culture of four T cells and SNU119 target cells through flow detection, as shown in figure 9, the cytokine secretion ability of Tandem-CAR is strongest, the cytokine secretion level of three CAR-Ts is obviously higher than that of a Mock-T cell group P <0.0001),

and because Mock-T is not infected with virus, the secretion level of IL-12 is very low, which indicates that the four generations of CAR-T constructed by us are successfully infected and express IL-12. There was no statistical difference in the proportion of TNF- α positive cells between the three CAR-T groups (P >0.05), and no statistical difference in the secretion capacity of the four cytokines (P >0.05) for FOLR1-CAR and MSLN-CAR.

Model of mouse transplantation tumor

(1) 24 5 week old severe immunodeficiency mice (B-NDG) were purchased and randomized into four groups of 6 mice each, and labeled with ear tags.

(2) A number of luciferase-expressing SNU119 ovarian carcinoma cells (SNU119-Luc) were amplified, and each mouse was inoculated subcutaneously at the root of the thigh with 5 × 10⁶The SNU119-Luc cell of (1) was used as a biopsy instrument after reserving the biopsy instrument for 10 days, and the observation of the tumor formation was noted.

(3) After 10 days, the mice were observed to have had tumors, the living body was photographed, 5 mice were selected from each group according to the fluorescence intensity, and four groups of mice were administered with 1 × 10 via tail vein⁷The Mock-T, FOLR1-CAR, MSLN-CAR, and Tandem-CAR cells of (1), were scored as day 0, and were visualized by in vivo imaging every 7 days thereafter.

The experimental results are as follows:

as shown in FIGS. 10 and 11, the results of treatment of B-NDG mice with Mock-T, FOLR1-CAR, MSLN-CAR, and Tandem-CAR cells 10 days after tumor loading showed that FOLR1-CAR, MSLN-CAR, and Tandem-CAR all significantly inhibited tumor growth (P <0.0001) compared to the control group (Mock-T), and two mice in the Mock-T group died at 21 days and 28 days, respectively. The tumors of the MSLN-CAR group decreased first and then increased, the tumors of FOLR1-CAR and Tandem-CAR group continued to decrease, the tumor suppression effect of the Tandem-CAR group was most significant, significantly less than that of the MSLN-CAR group (P <0.0001), and one mouse had tumor regression. There was no statistical difference between the Tandem-CAR group and FOLR1-CAR group, and the therapeutic effect of FOLR1-CAR group was better than that of MSLN-CAR group (P ═ 0.0153).

It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.

Sequence listing

<110> first subsidiary hospital of China civil liberation army, military and medical university

<120> FOLR1-MSLN dual targeting CAR-T cells, chimeric antigen receptors and vectors for the treatment of ovarian cancer

<141>2020-07-13

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Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr

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

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

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Val Thr Val Ser Ser

500

Claims

1. A chimeric antigen receptor comprising, in order, a single chain antibody segment, an extracellular hinge region (hindarea), a transmembrane region, and an intracellular Immunoreceptor Tyrosine Activation Motif (ITAM); wherein,

the single chain antibody segment is selected from one of a folate receptor 1 single chain antibody (FOLR1scFV), a mesothelin single chain antibody (MSLNscFV) or a folate receptor 1 single chain antibody (FOLR1scFV) -mesothelin single chain antibody (MSLNscFV) tandem complex.

2. The chimeric antigen receptor according to claim 1, wherein the intracellular Immunoreceptor Tyrosine Activation Motif (ITAM) consists of a CD28 peptide stretch, a 4-1BB peptide stretch, and a CD3 zeta chain peptide stretch joined in sequence.

3. The chimeric antigen receptor according to claim 2, wherein an IL-12 peptide is further linked after the CD3 zeta chain peptide.

4. The chimeric antigen antibody of claim 1, wherein the amino acid sequence of the folate receptor 1 single chain antibody (FOLR1scFV) is SEQ ID NO 1;

5. A plasmid vector for expressing the chimeric antigen receptor of any one of claims 1-4, comprising:

a gene encoding a single-chain antibody segment, a gene encoding an intracellular immunoreceptor tyrosine activation motif, and a lentiviral plasmid; wherein the encoding gene of the single-chain antibody segment is selected from one of the encoding gene of a folate receptor 1 single-chain antibody (FOLR1scFV), the encoding gene of a mesothelin single-chain antibody (MSLN scFV) or the encoding gene of a folate receptor 1 single-chain antibody (FOLR1scFV) -mesothelin single-chain antibody (MSLN scFV) tandem complex.

6. The plasmid vector of claim 5 wherein the amino acid sequence of said intracellular immunoreceptor tyrosine activation motif is SEQ ID NO 4.

7. The plasmid vector of claim 5, wherein the nucleotide sequence of the gene encoding the folate receptor 1 single chain antibody (FOLR1scFV) is SEQ ID NO: 5;

the nucleotide sequence of the coding gene of the mesothelin single-chain antibody (MSLN scFV) is SEQ ID NO. 6;

8. the plasmid vector of claim 5, wherein the nucleotide sequence of the gene encoding the folate receptor 1 single chain antibody (FOLR1scFV) -mesothelin single chain antibody (MSLN scFV) tandem complex is SEQ ID NO. 7.

9. A lentiviral vector comprising a gene encoding the chimeric antigen receptor of any one of claims 1-3 or packaged after transfection of cells with the plasmid vector of claims 5-8.

10. A CAR-T cell for use in the treatment of ovarian cancer, wherein the CAR-T cell is obtained by infecting a CD3 positive T cell with the lentiviral vector of claim 8.