Patent ID: 12187808

DETAILED DESCRIPTION

In order to further illustrate the technical measures adopted by the present application and the effects thereof, the technical solutions of the present application are further described below with reference to the accompanying drawings and specific embodiments, and however, the present application is not limited to the scope of the embodiments.

In the examples, techniques or conditions, which are not specifically indicated, are performed according to techniques or conditions described in the literature of the art, or according to product instructions. The reagents or instruments for use, which are not indicated with manufacturers, are conventional products that are commercially available from many sources.

Example 1: Construction of a Chimeric Antigen Receptor (I)

(1) The Secretory signal peptide, GD2 antigen-binding domain, CD28 extracellular and transmembrane domains, CD28 intracellular signaling domain and 4-1BB signaling domain, CD3ζ signaling domain, 2A sequence and caspase 9 domain as shown inFIG.1, i.e., Secretory-GD2scFv-CD28-4-1BB-CD3ζ-2A-FBKP.Casp9 was synthesized by whole gene synthesis.

The amino acid sequence (SEQ ID NO. 12) of the chimeric antigen receptor was as follows:

MLLLVTSLLLCELPHPAFLLIPQVQLVESGPGVVQPGRSLRISCAVSGFSVTNYGVHWVRQPPGKGLEWLGVIWAGGITNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAMYYCASRGGHYGYALDYWGQGTLVTVSSGSTSGSGKPGSSEGSTKGEIVMTQTPATLSVSAGERVTITCKASQSVSNDVTWYQQKPGQAPRLLIYSASNRYSGVPARFSGSGYGTEFTFTISSVQSEDFAVYFCQQDYSSFGQGTKLEIKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASGGGGSGGGGSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGGSGGGGSGGGGSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRTSGSGATNFSLLKQAGDVEENPGPMGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGGGGSGAMVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS.

Example 2: Construction of a Chimeric Antigen Receptor (II)

(1) The Secretory signal peptide, GD2 antigen-binding domain, CD28 extracellular and transmembrane domains, CD28 signaling domain and 4-1BB signaling domain, CD3ζ signaling domain, 2A sequence and Caspase 9 domain, i.e., Secretory-GD2scFv-CD28-4-1BB-CD3ζ-2A-FBKP.Casp9 was synthesized by whole gene synthesis.

The amino acid sequence (SEQ ID NO. 13) of the chimeric antigen receptor was as follows:

MLLLVTSLLLCELPEVQLVQSGAEVEKPGASVKISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSTSTAYMHLKSLRSEDTAVYYCVSGMEYWGQGTSVTVSSGSTSGSGKPGSSEGSTKGDVVMTQTPLSLPVTPGEPASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASGGGGSGGGGSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGGSGGGGSGGGGSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRTSGSGATNFSLLKQAGDVEENPGPMGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGGGGSGAMVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS.

Example 3: Construction of a Chimeric Antigen Receptor (III)

(1) The Secretory signal peptide, GD2 antigen-binding domain, CD28 extracellular and transmembrane domains, CD28 signaling domain and 4-1BB signaling domain, CD3ζ signaling domain, 2A sequence and Caspase 9 domain, i.e., Secretory-GD2scFv-CD28-4-1BB-CD3ζ-2A-FBKP.Casp9 was synthesized by whole gene synthesis.

The amino acid sequence (SEQ ID NO. 14) of the chimeric antigen receptor was as follows:

MLLLVTSLLLCELPAFLLIPEVKLVESGGGLVLPGDSLRLSCATSEFTFTDYYMTWVRQPPRKALEWLGFIRNRANGYTTEYNPSVKGRFTISRDNSQSILYLQMNTLRTEDSATYYCARVSNWAFDYWGQGTTLTVSSGSTSGSGKPGSSEGSTKGDVVMTQTPLSLPVSLGDQASISCRSSQSLLKNNGNTFLHWYLQKSGQSPKLLIYKVSNRLSGVPDRFSGSGSGTYFTLKISRVEAEDLGVYFCSQSTHIPYTFGGGTKLEIKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASGGGGSGGGGSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGGSGGGGSGGGGSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRTSGSGATNFSLLKQAGDVEENPGPMGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGGGGSGAMVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS.

Example 4: Lentiviral Packaging

(1) 293T cells can be cultured in a six-well plate at a density of 1×106cells/well for 17-18 hours;

(2) 600 μL/well of fresh DMEM containing 10% FBS was added;

(3) To a sterile centrifuge tube were added the following reagents: 75 μL of DMEM collected from each well, helper DNA mix (pNHP, pHEF-VSV-G), a GFP reporter plasmid, and the pTYF CAR DNA vector (from Example 1, 2 or 3), and then vortexed;

(4) 7 μL of Superfect was taken from the center of each well, added to the centrifuge tube, mixed by pipetting up and down for five times, and allowed to stand at room temperature for 7-10 minutes;

(5) To each culture well the DNA-Superfect mixture in the centrifuge tube was added dropwise, mixed by vortex;

(6) Incubated in an incubator at 37° C. with 3% CO2for 4-5 hours;

(7) The media was removed, the cells were rinsed with 1.5 mL of culture media, and then 1.5 mL of media was added for further incubation:

(8) The plate was placed back into the incubator with 5% CO2for overnight incubation. The next morning, transfection efficiency was observed based on GFP expression with a fluorescence microscope.

Example 5: Lentivirus Purification and Concentration

1) Virus Purification

Cell debris were removed by a centrifugation at 1000 g for 5 minutes to obtain virus supernatant. The virus supernatant was filtered with a low protein-binding filter, and then the virus was divided into small portions and stored at −80° C.;

Typically, lentiviral vectors at a titer of 106to 108transducing units can be produced by transfected cells per ml media.

2) Concentration of Lentivirus with a Filter

(1) In a biosafety cabinet, a filter tube was disinfected with alcohol and then washed with sterile PBS;

(2) virus supernatant was added to the filter tube, then centrifuged at 2500 g for 30 minutes or until the virus volume was reduced by 20-50 times;

(3) The filter tube was shaken, then centrifuged at 400 g for 2 minutes, and the concentrated virus was collected into a collection cup, and finally the virus in all the tubes was collected into one centrifuge tube.

Example 6: Transduction of CAR-T Cells

The activated T cells were inoculated with AIM-V media containing growth factors IL-2, IL-7 and IL-15, and 10 μg/mL of polybrene was added. The concentrated lentivirus comprising CAR gene was added, centrifuged at a centrifugal force of 100 g at room temperature for 100 minutes, then incubated at 37° C. for 24 hours. Culture media was then added and incubated for 4 days. Then the cells were harvested and counted, and cultured for 2 days before infusion to patients.

The effects on treating tumors as shown inFIG.2which indicated that the CAR-T cells effectively reduced the tumor, and it was safe. The effects were specifically verified by in vitro and in vivo assays.

Example 7: In Vitro Killing Assay with CAR-T Cells

(1) A GD2-positive tumor cell line was transduced with lentiviral vectors expressing a green fluorescent protein (GFP) and the GFP was stably expressed;

(2) Non-specific T cells or CAR-T cells different from the GD2 scFv were co-cultured with said tumor cells in an incubator at 37° C., 5% CO2for 24-72 h;

(3) The survival of tumor cells was observed by fluorescence microscopy. The in vitro killing efficiency of different GD2-CAR-T cells were evaluated based on the fact that dead tumor cells did not express green fluorescent protein. The results were shown inFIG.3;

It can be seen fromFIG.3that compared with the control T cells, the three types of ScFv GD2-CAR-T cells had obvious killing effects, wherein the Type 3 scFv showed the best effect, thus confirming that the vector material used in the present application quickly screened out effective CAR structures for subsequent clinical use.

Example 8: Therapeutic Effects of the CAR-T Cells

(1) The unstained tumor sections from patients with neuroblastoma were confirmed for positive GD2 expression by immunohistochemical staining. The results were as shown inFIG.4. Tumors with high GD2 expression can be distinguished betweenFIG.4(a)andFIG.4(b).

(2) White blood cells were collected from patients. Peripheral mononuclear lymphocytes were separated from the white blood cells by gradient density centrifugation with Ficoll and T cells were screened with CD3 magnetic beads. Anti-CD28 antibody was added into the T cells for T cell activation. The subsequent GD2-CART preparation was carried out at 1×106CART cells/kg body weight;

(3) Patients were pretreated with low-dose chemotherapy. The pretreatment regimen was administration of cyclophosphamide 250 mg/m2for 3 days and fludarabine 25 mg/m2for 3 days. The pretreatment was performed at 24 h before CART infusion and lasted for 3 days (the chemotherapy regimen can be modified according to the patient's condition and this example is only used as an enumeration);

(4) CAR T cells were reinfused via intravenous injection.

(5) After the infusion, the patients were monitored and evaluated for toxic response by the clinician. The results were shown inFIG.5.

The safety of the GD2-CAR T cells can be seen from the statistical results inFIG.5, the GD2-CAR T cells were safe. After the infusion, 24% of patients had no adverse reactions, 50% had Grade 1 adverse reactions, and 26% had Grade 2 adverse reactions. Those adverse reactions included fever, fatigue, rash and hypotension, etc., which can be effectively controlled in clinical.

(6) After the infusion, a small amount of peripheral blood was obtained from the patients, and the peripheral mononuclear lymphocytes were prepared for chromosomal DNA (gDNA) extraction. The CAR copy number in the peripheral blood was quantified by qPCR using specific primers. The results are shown inFIG.6.

As can be seen fromFIG.6, the CAR value in vivo peaked in the patient about 20 days after infusion, and maintained in the body for about half a year.

(7) The tumor size was evaluated by imaging after GD2-CART infusion, and the results were shown inFIG.7andFIG.8.

As can be seen fromFIG.7, at 28 days after GD2-CART infusion, the abdominal and mediastinal masses in the patient as detected by B-scan ultrasonography was reduced by about 95% compared with that before infusion, and maintained for half a year. So far, the patient's condition is still stable. As can be seen fromFIG.8(a)-FIG.8(b), the abdominal mass in another patient was scanned with enhanced CT two months after infusion, and the mass was reduced from the original size of 1.9 cm in length and 4.9 cm in width as shown inFIG.8(a)to the size of 1.9 cm in length and 3.2 cm in width as shown inFIG.8(b).

In summary, the GD2-CAR T cells of the present application had a better effect than other chimeric antigen receptors and other tumor antigens, and had safety and a long-lasting effect, and did achieve a good effect in patients with relapsed and refractory stage IV neuroblastoma.

The Applicant declares that detailed methods of the present application have been described through the above examples, and however, the present application is not limited to the above detailed methods. That is to say, it does not mean that the implementation of the present application must rely on the above detailed methods. Those skilled in the art should understand that any improvement on the present application, including the equivalent replacement of the raw materials or the addition of auxiliary components to the product of the present application, and the selection of specific methods, etc., falls within the protection scope and the disclosure scope of the present application.