阅读说明：本技术 靶向叶酸受体α嵌合抗原受体、其制备方法及其应用 (Targeted folate receptor alpha chimeric antigen receptor, preparation method and application thereof ) 是由 敖罗权 徐祥 敖翔 于 2021-09-30 设计创作，主要内容包括：本发明公开了一种靶向叶酸受体α嵌合抗原受体,属于生物技术领域,包括人CD8α信号肽、结合叶酸受体α的多肽、人免疫球蛋白G1 Fc段、人CD8α铰链区、人NKG2D跨膜段、人2B4胞内段、人CD3ζ胞内段。同时提供了该嵌合抗原受体的编码核苷酸分子、表达载体、宿主细胞以及该嵌合抗原受体的制备方法。本发明还涉及包括该嵌合抗原受体的药物及其在制备抗肿瘤药物中的应用。本发明中的靶向叶酸受体α嵌合抗原受体可增强NK细胞的特异性识别能力,并增强其对叶酸受体α阳性的结直肠癌细胞系的杀伤能力。(The invention discloses a targeted folate receptor alpha chimeric antigen receptor, which belongs to the technical field of biology and comprises a human CD8 alpha signal peptide, a polypeptide combined with folate receptor alpha, a human immunoglobulin G1 Fc segment, a human CD8 alpha hinge region, a human NKG2D transmembrane segment, a human 2B4 intracellular segment and a human CD3 zeta intracellular segment. Also provides the coding nucleotide molecule, the expression vector, the host cell and the preparation method of the chimeric antigen receptor. The invention also relates to a medicament comprising the chimeric antigen receptor and application thereof in preparing antitumor medicaments. The chimeric antigen receptor targeting folate receptor alpha can enhance the specific recognition capability of NK cells and enhance the killing capability of the NK cells on folate receptor alpha positive colorectal cancer cell lines.)

1. A folate receptor-targeted alpha chimeric antigen receptor characterized by: the polypeptide is connected with a human CD8 alpha signal peptide, a polypeptide combined with a folate receptor alpha, a human immunoglobulin G1 Fc segment, a human CD8 alpha hinge region, a human NKG2D transmembrane segment, a human 2B4 intracellular segment and a human CD3 zeta intracellular segment through peptide bonds in turn.

2. The folate receptor alpha targeting chimeric antigen receptor of claim 1, wherein: the amino acid sequence of the chimeric antigen receptor targeting the folate receptor alpha is shown in SEQ ID NO. 11.

3. The folate receptor alpha targeting chimeric antigen receptor according to any one of claims 1 or 2, wherein: the polypeptide combined with the folate receptor alpha is a single-chain variable region of an anti-folate receptor alpha antibody, and the C terminal of the single-chain variable region of the anti-folate receptor alpha antibody is connected with the N terminal of an Fc segment of human immunoglobulin G1; the single chain variable region of the anti-folate receptor alpha antibody comprises a heavy chain variable region and a light chain variable region.

4. The folate receptor alpha targeting chimeric antigen receptor of claim 3, wherein: the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 14; the heavy chain variable region sequence is shown in SEQ ID NO. 15.

5. A nucleotide molecule characterized by: the nucleotide molecule encodes the targeted folate receptor alpha chimeric antigen receptor.

6. The nucleotide molecule of claim 5, wherein: the nucleotide molecule sequence is shown as SEQ ID NO. 10.

7. An expression vector, characterized in that: containing the nucleotide molecule.

8. A host cell, characterized in that: contains the expression vector.

9. A preparation method of a targeted folate receptor alpha chimeric antigen receptor is characterized by comprising the following steps:

constructing an expression vector comprising a folate receptor alpha targeting chimeric antigen receptor encoding polynucleotide sequence of claim 1;

transforming the expression vector into a host cell to induce expression;

separating the target folate receptor alpha chimeric antigen receptor from the expression product.

10. A medicament comprising the folate receptor alpha chimeric antigen receptor targeted drug of claim 1.

11. The use of the folate receptor α -targeting chimeric antigen receptor of claim 1 for the preparation of an anti-tumor medicament.

Technical Field

The invention relates to a targeted folate receptor alpha chimeric antigen receptor, a preparation method and application thereof, belonging to the technical field of biology.

Background

With the social development, the environmental pollution is increasingly serious, the life style and the eating habits of people are changed, and the cancer is increasingly developed. Among them, colorectal cancer (CRC) is one of the most common malignant tumors in the digestive system, and the incidence and mortality rate are third and second. The treatment of CRC includes surgical resection, radiation therapy, chemotherapy, and the like, but these treatments not only cause adverse reactions but also have poor treatment effects to some extent. However, as immunotherapy achieves good curative effect in the comprehensive treatment of various tumors, especially the Chimeric Antigen Receptor (CAR) technology which has emerged in recent years achieves good curative effect in hematologic disease tumors, a new idea is provided for CRC treatment.

CARs generally comprise two major domains: single-chain variable regions (scFv) targeted to recognize tumor antigens are located extracellularly and different intracellular signaling domains are located intracellularly. This particular structure enables immune effector cells expressing the CAR to specifically kill tumor cells expressing a specific tumor antigen. At present, a plurality of clinical tests show that the CAR modified T cell (namely CAT-T) has excellent curative effect on leukemia, lymphoma and other hematologic malignancies. Three generations of CAR structures have been developed. The first generation CARs contained only CD3 ζ or fcepsilon RI γ signaling domains in their intracellular structure, and the reduced proliferation of engineered CAR-T cells eventually led to T cell apoptosis. To address this problem, second and third generation CARs incorporate co-stimulatory molecular domains such as CD28, CD134 and CD137 into the CAR based on the structure of the first generation CAR to stimulate T cell proliferation and survival. Wherein, the CAR structure comprises one costimulatory molecule domain which is a second generation CAR, and the CAR structure comprises two costimulatory molecule domains which are third generation CAR.

CAR-T has achieved great success in the use process, and has a good therapeutic effect. However, there is a disadvantage in that CAR-T can be prepared only using T cells obtained from an autologous source, and the preparation process takes a long time, thereby possibly delaying valuable treatment time. In addition, some patients are not suitable for their own reasons to take autologous T cells for the preparation of CAR-T, which further limits the use of CAR-T.

Based on this, the researchers have proposed the use of chimeric antigen receptor natural killer cells (CAR-NK) for the treatment of tumors. Probably NK cells are better cellular carriers of CARs than T cells: NK cells can be used for allogeneic body, so that the cells can be prepared and stored in advance, produced in batches and applied in batches, and can be used as needed. However, the CAR-NK prepared at present mainly refers to and adopts CAR-T CAR structure to be directly transplanted into NK cells, and the most main problems of the method are that: since CAR structures designed for T cells are not suitable for NK cells, the activation of NK cells is weak.

Disclosure of Invention

The invention provides a targeted folate receptor alpha chimeric antigen receptor which can enhance the specific recognition capability of NK cells and enhance the killing capability of the NK cells on folate receptor alpha positive colorectal cancer cell lines.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows: a chimeric antigen receptor targeting folate receptor alpha is connected with a human CD8 alpha signal peptide, a polypeptide binding folate receptor alpha, a human immunoglobulin G1 Fc segment, a human CD8 alpha hinge region, a human NKG2D transmembrane segment, a human 2B4 intracellular segment and a human CD3 zeta intracellular segment through peptide bonds in sequence.

Preferably, the amino acid sequence of the chimeric antigen receptor targeting folate receptor alpha is shown in SEQ ID No. 11.

Preferably, the polypeptide that binds folate receptor alpha is a single chain variable region of an anti-folate receptor alpha antibody, the C-terminus of the single chain variable region of the anti-folate receptor alpha antibody being linked to the N-terminus of the Fc fragment of human immunoglobulin G1; the single chain variable region of the anti-folate receptor alpha antibody comprises a heavy chain variable region and a light chain variable region.

Preferably, the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 14; the heavy chain variable region sequence is shown in SEQ ID NO. 15.

The invention provides a nucleotide molecule, wherein the nucleotide molecule codes the targeted folate receptor alpha chimeric antigen receptor.

Preferably, the nucleotide molecule sequence is shown as SEQ ID NO. 10.

An expression vector comprising said nucleotide molecule.

A host cell comprising said expression vector.

The invention provides a preparation method of a targeted folate receptor alpha chimeric antigen receptor, which comprises the following steps: constructing an expression vector comprising a folate receptor alpha targeting chimeric antigen receptor encoding polynucleotide sequence of claim 1; transforming the expression vector into a host cell to induce expression; separating the target folate receptor alpha chimeric antigen receptor from the expression product.

The invention also relates to a drug containing the targeted folate receptor alpha chimeric antigen receptor and application of the drug in preparation of antitumor drugs.

The CAR intracellular part costimulatory molecule structure domain designed aiming at NK cells adopts the 2B4 intracellular part, and the transmembrane region adopts the transmembrane region of NKG2D, so that a targeted folate receptor alpha chimeric antigen receptor is obtained, the targeted folate receptor alpha chimeric antigen receptor is over-expressed in the NK cells, the specific recognition capability of the NK cells can be enhanced, and the killing capability of the targeted folate receptor alpha chimeric antigen receptor on a colorectal cancer cell line with positive folate receptor alpha is enhanced. The method for preparing the chimeric antigen receptor modified NK cell has short time and high killing efficiency, and has great application potential in clinical treatment of alpha FR positive malignant tumors such as colorectal cancer.

Drawings

FIG. 1 is a schematic diagram of the structure of a chimeric antigen receptor targeting folate receptor alpha according to an embodiment of the present invention.

FIG. 2 is a graph showing the results of flow cytometry conducted three times.

FIG. 3 is a graph showing the results of the LDH release experiments in example three.

FIG. 4 is a graph showing the results of ELISA performed in the third embodiment.

Detailed Description

For a better understanding of the nature of the invention, its description is further set forth below in connection with the specific embodiments and the drawings.

The invention provides a targeted folate receptor alpha chimeric antigen receptor, which sequentially comprises the following structural components: a human CD8 a signal peptide, a folate receptor a binding polypeptide, a human immunoglobulin G1 Fc fragment, a human CD8 a hinge region, a human NKG2D transmembrane fragment, a human 2B4 intracellular fragment, and a human CD3 ζ intracellular fragment. Its transmembrane segment used NKG2D instead of the CD8 α transmembrane segment often used in CAR-T, and the intracellular segment used the intracellular segment of 2B4 as a costimulatory domain instead of the costimulatory molecules CD28, CD134 and CD137 often used in CAR-T. Compared with a structure for activating T cells by adopting CD8 alpha-CD 28-CD137-CD3 zeta and the like, the transmembrane segment and the intracellular segment adopt NKG2D-2B4-CD3 zeta to be more suitable for NK cells and can better activate the killing activity of the NK cells. In NK cells, the NKG2D transmembrane segment can transmit signals better than the CD8 alpha transmembrane segment; intracellular use of the 2B4 co-stimulatory molecule domain and CD3 zeta signaling domain confers stronger tumor killing activity, proliferative capacity and survival time to CAR-modified NK cells than intracellular use of CD28, CD134, CD137 and CD3 zeta.

Example one

Firstly, designing a chimeric antigen receptor sequence targeting folate receptor alpha.

As shown in fig. 1, in the chimeric antigen receptor targeting folate receptor alpha (abbreviated as α FR-CAR), the N-terminus of the polypeptide binding folate receptor alpha is linked to the C-terminus of the signal peptide of human CD8 α, the C-terminus thereof is linked to the N-terminus of the Fc fragment of human immunoglobulin G1, and the C-terminus of the Fc fragment of human immunoglobulin G1 is linked to the N-terminus of the hinge region of human CD8 α to form the extracellular domain of the chimeric antigen receptor. The C end of the intracellular segment of human 2B4 and the N end of the intracellular segment of human CD3 zeta form the intracellular segment of the chimeric antigen receptor. The N end of the transmembrane segment of human NKG2D is connected with the C end of the hinge region of human CD8 alpha, and the C end of the transmembrane segment of human NKG2D is connected with the N end of the intracellular segment of human 2B4 to form the whole chimeric antigen receptor targeting the folate receptor alpha.

Wherein, the specific sequence of each fragment of the alpha FR-CAR is as follows:

1. the coding nucleotide sequence of the human CD8 alpha signal peptide is shown as SEQ ID NO.1, and specifically comprises the following components:

ATGGCCCTCCCAGTTACCGCCCTTCTCCTGCCCCTGGCCCTGCTGCTGCACGCCGCCCGCCCC。

2. the polypeptide capable of binding folate receptor alpha is a single chain variable region (scFv) of an anti-folate receptor alpha (alpha FR) antibody comprising a light chain variable region (V)_L) And heavy chain variable region (V)_H). Light chain variable region (V)_L) The coding nucleotide sequence of (A) is shown as SEQ ID NO.2, and specifically comprises:

CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGAGCATCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGGAGTTATAACCTGGTCTCCTGGTACCAGCAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGAGGGCAGTAAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACGCCGCCTCCCTGACAATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCCAGTCCTATGACAGCAGCCTGAGTGTGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTC。

heavy chain variable region (V)_H) The coding nucleotide sequence of (A) is shown as SEQ ID NO.3, and specifically comprises:

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTGCAGCCAGGGCGGTCCCTGAGACTCTCCTGCACAACTTCTGGATTCACTTTTGGCGATTATGCTATGATCTGGGCCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTAGTAGTAGTTACATCTACTACGCAGACTCAGTGAAGGGCAGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCTGTGTATTACTGTGCCAGAGAACGGTACGATTTTTGGAGTGGAATGGACGTCTGGGGCAAAGGGACCACCGTCACCGTGAGCAGT。

the encoding nucleotide sequence of the single chain variable region (scFv) of the anti-folate receptor alpha (alpha FR) antibody is shown in SEQ ID NO.4, and specifically comprises the following steps:

CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGAGCATCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGGAGTTATAACCTGGTCTCCTGGTACCAGCAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGAGGGCAGTAAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACGCCGCCTCCCTGACAATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCCAGTCCTATGACAGCAGCCTGAGTGTGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTCGGAGGAGGCGGATCAGGCGGAGGAGGCTCTGGCGGAGGCGGAAGCCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTGCAGCCAGGGCGGTCCCTGAGACTCTCCTGCACAACTTCTGGATTCACTTTTGGCGATTATGCTATGATCTGGGCCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTAGTAGTAGTTACATCTACTACGCAGACTCAGTGAAGGGCAGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCTGTGTATTACTGTGCCAGAGAACGGTACGATTTTTGGAGTGGAATGGACGTCTGGGGCAAAGGGACCACCGTCACCGTGAGCAGT。

3. the coding nucleotide sequence of the Fc segment of the human immunoglobulin G1 is shown as SEQ ID NO.5, and specifically comprises the following components:

GAGCCCAAATCTAGCGACAAAACTCACACATGCCCACCTTGCCCAGCACCTGAGCTCCTGGGGGGACCTTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGGGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCTCGGGAGGAGCAGTACAACAGCACCTACAGAGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCAGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACCCAGAAGAGCCTCTCCCTGTCTCCTGGCAAA。

4. the coding nucleotide sequence of the human CD8 alpha hinge region is shown as SEQ ID NO.6, and specifically comprises the following steps:

GCCAAGCCCACCACTACACCCGCCCCACGCCCCCCCACCCCCGCCCCCACCATCGCCAGCCAGCCCCTGAGCCTGCGCCCCGAGGCCTGCCGCCCCGCCGCCGGCGGCGCCGTGCACACCCGCGGCCTGGACTTCGCCTGCGAC。

5. the coding nucleotide sequence of the human NKG2D transmembrane segment is shown as SEQ ID NO.7, and specifically comprises the following steps:

CCATTTTTTTTCTGCTGCTTCATCGCTGTAGCCATGGGAATCCGTTTCATTATTATGGTAACA。

6. the coding nucleotide sequence of the human 2B4 intracellular segment is shown as SEQ ID NO.8, and specifically comprises the following steps:

TGGAGGAGAAAGAGGAAGGAGAAGCAGTCAGAGACCAGTCCCAAGGAATTTTTGACAATTTACGAAGATGTCAAGGATCTGAAAACCAGGAGAAATCACGAGCAGGAGCAGACTTTTCCTGGAGGGGGGAGCACCATCTACTCTATGATCCAGTCCCAGTCTTCTGCTCCCACGTCACAAGAACCTGCATATACATTATATTCATTAATTCAGCCTTCCAGGAAGTCTGGATCCAGGAAGAGGAACCACAGCCCTTCCTTCAATAGCACTATCTATGAAGTGATTGGAAAGAGTCAACCTAAAGCCCAGAACCCTGCTCGATTGAGCCGCAAAGAGCTGGAGAACTTTGATGTTTATTCC。

7. the coding nucleotide sequence of the intracellular segment of human CD3 zeta is shown in SEQ ID NO.9, and specifically comprises the following components:

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA。

8. the coding nucleotide sequence of the whole alpha FR-CAR is shown as SEQ ID NO.10, and specifically comprises the following steps:

ATGGCCCTCCCAGTTACCGCCCTTCTCCTGCCCCTGGCCCTGCTGCTGCACGCCGCCCGCCCCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGAGCATCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGGAGTTATAACCTGGTCTCCTGGTACCAGCAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGAGGGCAGTAAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACGCCGCCTCCCTGACAATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCCAGTCCTATGACAGCAGCCTGAGTGTGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTCGGAGGAGGCGGATCAGGCGGAGGAGGCTCTGGCGGAGGCGGAAGCCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTGCAGCCAGGGCGGTCCCTGAGACTCTCCTGCACAACTTCTGGATTCACTTTTGGCGATTATGCTATGATCTGGGCCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTAGTAGTAGTTACATCTACTACGCAGACTCAGTGAAGGGCAGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCTGTGTATTACTGTGCCAGAGAACGGTACGATTTTTGGAGTGGAATGGACGTCTGGGGCAAAGGGACCACCGTCACCGTGAGCAGTGAGCCCAAATCTAGCGACAAAACTCACACATGCCCACCTTGCCCAGCACCTGAGCTCCTGGGGGGACCTTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGGGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCTCGGGAGGAGCAGTACAACAGCACCTACAGAGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCAGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACCCAGAAGAGCCTCTCCCTGTCTCCTGGCAAAGCCAAGCCCACCACTACACCCGCCCCACGCCCCCCCACCCCCGCCCCCACCATCGCCAGCCAGCCCCTGAGCCTGCGCCCCGAGGCCTGCCGCCCCGCCGCCGGCGGCGCCGTGCACACCCGCGGCCTGGACTTCGCCTGCGACCCATTTTTTTTCTGCTGCTTCATCGCTGTAGCCATGGGAATCCGTTTCATTATTATGGTAACATGGAGGAGAAAGAGGAAGGAGAAGCAGTCAGAGACCAGTCCCAAGGAATTTTTGACAATTTACGAAGATGTCAAGGATCTGAAAACCAGGAGAAATCACGAGCAGGAGCAGACTTTTCCTGGAGGGGGGAGCACCATCTACTCTATGATCCAGTCCCAGTCTTCTGCTCCCACGTCACAAGAACCTGCATATACATTATATTCATTAATTCAGCCTTCCAGGAAGTCTGGATCCAGGAAGAGGAACCACAGCCCTTCCTTCAATAGCACTATCTATGAAGTGATTGGAAAGAGTCAACCTAAAGCCCAGAACCCTGCTCGATTGAGCCGCAAAGAGCTGGAGAACTTTGATGTTTATTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA。

the designed polynucleotide of the alpha FR-CAR is synthesized by Shanghai and Yuan Biotechnology Limited, inserted into a lentivirus expression vector pLenti-EF1 alpha, and after the sequencing is correct, the plasmid is extracted and purified by using a plasmid purification kit of TIANGEN company, so that the high-quality plasmid of the recombinant expression vector is obtained. Then sequencing is carried out to verify whether the sequence is correct.

The full-length gene sequence of the alpha FR-CAR is verified to be exactly consistent with the designed sequence.

1. The amino acid sequence of the alpha FR-CAR is shown as SEQ ID NO.11, and specifically comprises the following components:

MALPVTALLLPLALLLHAARPQSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEGSKRPSGVSNRFSGSKSGNAASLTISGLQAEDEADYYCQSYDSSLSVVFGGGTKLTVLGGGGSGGGGSGGGGSQVQLVESGGGLVQPGRSLRLSCTTSGFTFGDYAMIWARQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARERYDFWSGMDVWGKGTTVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPGVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDPFFFCCFIAVAMGIRFIIMVTWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

2. the amino acid sequence of the human CD8 alpha signal peptide is shown as SEQ ID NO.12, and specifically comprises the following steps:

MALPVTALLLPLALLLHAARP。

3. the amino acid sequence of the single chain variable region (scFv) of the anti-folate receptor alpha (alpha FR) antibody is shown in SEQ ID No.13, and specifically comprises the following steps:

QSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEGSKRPSGVSNRFSGSKSGNAASLTISGLQAEDEADYYCQSYDSSLSVVFGGGTKLTVLGGGGSGGGGSGGGGSQVQLVESGGGLVQPGRSLRLSCTTSGFTFGDYAMIWARQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARERYDFWSGMDVWGKGTTVTVSS。

light chain variable region (V) of Single chain variable region (scFv) of anti-folate receptor alpha (alpha FR) antibody_L) The amino acid sequence of (A) is shown as SEQ ID NO.14, and specifically comprises:

QSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEGSKRPSGVSNRFSGSKSGNAASLTISGLQAEDEADYYCQSYDSSLSVVFGGGTKLTVL。

heavy chain variable region (V) of Single chain variable region (scFv) of anti-folate receptor alpha (alpha FR) antibody_H) The amino acid sequence of (A) is shown as SEQ ID NO.15, and specifically comprises:

QVQLVESGGGLVQPGRSLRLSCTTSGFTFGDYAMIWARQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARERYDFWSGMDVWGKGTTVTVSS。

4. the amino acid sequence of the Fc segment of the human immunoglobulin G1 is shown as SEQ ID NO.16, and specifically comprises the following steps:

EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPGVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

5. the amino acid sequence of the human CD8 alpha hinge region is shown as SEQ ID NO.17, and specifically comprises the following steps:

AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD。

6. the amino acid sequence of the transmembrane segment of human NKG2D is shown in SEQ ID NO.18, and specifically comprises the following steps:

PFFFCCFIAVAMGIRFIIMVT。

7. the amino acid sequence of the human 2B4 intracellular segment is shown as SEQ ID NO.19, and specifically comprises the following steps:

WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYS。

8. the amino acid sequence of the intracellular segment of human CD3 zeta is shown in SEQ ID NO.20, and specifically comprises the following steps:

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

example two

Obtaining the lentivirus comprising the chimeric antigen receptor targeted to folate receptor alpha. The method specifically comprises the following steps:

1. lentivirus preparation of overexpressed alpha FR-CAR, the equipment and materials are shown in Table 1 below.

TABLE 1 Equipment and materials required for overexpression of lentiviral vectors

1.2 packaging of lentiviruses:

day 0: substituting 293T cells with good state with pancreatin (TrypLE)^TMExpress), centrifuged, collected, cells were resuspended in DMEM medium (containing 10% FBS) to prepare a cell suspension, and the cell suspension concentration was counted. 293T cells at 4X 10⁶One was inoculated in a 10cm petri dish and cultured overnight.

Day1, when the cells reached 90% confluence, replacing fresh medium one hour earlier, transfecting as described in Lipofectamine 2000, adding 600. mu.l OPTI-MEM medium to two 1.5ml centrifuge tubes, adding 4. mu.g of alpha FR-CAR plasmid, 3. mu.g of pLP1 plasmid, 3. mu.g of pLP2 plasmid and 3. mu.g of pLP/VSVG plasmid to one tube, shaking gently, mixing, adding the other tubeMix gently 40. mu.l Lipofectamine 2000. Then, after gently mixing the two tubes, standing at room temperature for 5 minutes, adding the transfection complex into 293T cells, placing the cells in an incubator at 37 ℃ and 5% CO₂And (5) culturing.

Day 2: fresh DMEM complete medium was replaced.

Day 4: the supernatant of the 293T cell culture medium was collected, centrifuged at 2000rpm to remove cell debris and dead cells, and then filtered through a 0.45 μm filter for use.

1.3 concentration of lentivirus:

the collected viral supernatant was centrifuged at 25000rpm (82700g) at 4 ℃ for 2 hours, and then the supernatant was discarded, and 100. mu.l of fresh medium was added thereto and dissolved at 4 ℃ for 2 hours, followed by gentle shaking every 20 minutes. After dissolution, the virus suspension was concentrated and divided into 50. mu.l portions, which were stored at-80 ℃. Namely, the lentivirus with the overexpressed alpha FR-CAR is obtained.

EXAMPLE III

The host cell may be one of NK-92 cells, human primary NK cells, human primary T cells, human primary macrophages, or cytokine-induced killer cells. The construction of NK-92 cells overexpressing alpha FR-CAR is illustrated by NK-92 cells.

1. The desired materials are shown in table 2 below:

TABLE 2 construction of NK-92 cells overexpressing alpha FR-CAR

1.2 overexpression of alpha FR-CAR Virus infects NK-92:

1.3 NK-92 cells were seeded in 12-well plates at 2X 10 per well⁵One cell (1 ml of medium per well, interleukin-2100U/ml).

1.4 mu.l of the lentivirus concentrate prepared in example two and 10. mu.g/ml polybrene were added per well.

1.5 at 37 ℃ 5% CO₂The culture was carried out overnight and the medium was replaced with fresh medium (containing 100U/ml of interleukin-2).

1.6NK-92 cells five days after infection can be performed alpha FR-CAR-NK-92 screening:

to NK-92 cell culture medium infected with α FR-CAR lentivirus was added puromycin at 5. mu.g/ml for 6 days. Wherein, 5. mu.g/ml puromycin is added to the culture medium at each liquid change or passage.

1.7 after the screening of alpha FR-CAR-NK-92 is completed, the expression condition can be detected by flow cytometry, and the specific steps are as follows: the cells were collected and washed once with PBS; after incubation for 30 minutes at room temperature using antibody staining in table 2, the cells were washed once with PBS and collected by centrifugation; 100 μ l PBS resuspended cells were tested on the machine.

The flow cytometry results are shown in fig. 2, and it can be seen from the figure that the positive rate of the chimeric antigen receptor-modified NK-92 cell (α FR-CAR-NK-92) targeting folate receptor α is high.

The killing effect of the alpha FR-CAR-NK-92 cells obtained above was examined.

1. The desired materials are shown in table 3 below:

TABLE 3 detection of alpha FR-CAR-NK-92 cell killing effect materials

2. Detecting the killing of the colorectal cancer cells by the alpha FR-CAR-NK-92 obtained by the method by adopting a method for measuring LDH release:

liquid addition settings for each well of the LDH release detection experiment: respectively adding 50ul of effector cell suspension into ER and ESR wells, respectively adding 50ul of target cell suspension into ER, TMR and TSR wells, respectively adding 1640 culture medium, NK culture medium and NK culture medium into ESR, TMR and TSR wells, respectively adding 50ul of culture medium into CMB and VCC wells, respectively. (2 multiple holes per hole)

ER refers to LDH released by effector cells plus target cells; ESR refers to LDH spontaneously released by effector cells; TSR refers to LDH released spontaneously by target cells; TMR refers to the maximum release of target cells (added lysate); CMB refers to blank medium; VCC refers to volume calibration wells (blank medium plus lysate)

2.1Day 0: target cell plating: collecting HCT-116 cells by digestion and centrifugation, resuspending in serum-free 1640 medium, and counting cellsCell density to 5X 10⁴Perml, 50ul of cell suspension was seeded into each well of a 96-well plate and placed at 37 ℃ in 5% CO₂Culturing in an incubator.

2.2Day 1: plating effector cells: preparing NK-92, NK-92-EV and alpha FR-CAR-NK-92 cell suspensions with different effect-target ratios (1: 1; 5: 1; 10: 1; 20: 1) according to the NK-92 normal passage method, inoculating 50ul of the cell suspensions in corresponding wells, placing at 37 ℃ and 5% CO₂Culturing in an incubator for 18 h. Wherein, the alpha FR-CAR-NK-92 comprises two types respectively: one is alpha FR-CAR-NK-92(T cell element) is CAR-NK-92 cell constructed by the existing CAR structure, and the other is alpha FR-CAR-NK-92(NK cell element) is CAR-NK-92 cell constructed by the CAR structure of the invention. The α FR-CAR-NK-92(T cell element) structure is: a human CD8 a signal peptide, a folate receptor alpha binding polypeptide, a human immunoglobulin G1 Fc fragment, a human CD8 a hinge region, a human CD28 transmembrane fragment, a human CD28 intracellular fragment, a human CD3 ζ intracellular fragment.

2.3Day 2: and (3) detection: adding 10 mul lysate into TMR and VCC holes, cracking cell, and culturing for 30 min. ② adding 100 mul of working solution into each hole, keeping out of the sun for 5 minutes at room temperature. And adding 50 mul of stop solution into each hole, and detecting the absorbance at 490nm on an enzyme-linked immunosorbent assay.

And (4) calculating a result:

the killing efficiency was (ER-ESR-TSR + CMB)/(TMR-VCC-TSR + CMB) × 100%.

The results of the LDH release experiments are shown in FIG. 3, which shows that alpha FR-CAR-NK-92 (i.e. alpha FR-CAR-NK-92(NK cell element)) has a strong killing effect on HCT-116 cells, while the killing effect of alpha FR-CAR-NK-92 (i.e. alpha FR-CAR-NK-92(T cell element)) using the existing CAR structure designed for T cells is less than that of the invention for NK cells, indicating that the CAR structure designed by the present method is superior to the existing CAR structure in NK cells.

Enzyme-linked immunosorbent assay (ELISA) detects the release of TNF-alpha and IFN-gamma of alpha FR-CAR-NK-92:

day 0: target cell plating: target cells HCT116 were plated at 2X 10 per well⁴One was inoculated in a 24-well plate and cultured overnight for its adherence.

Day1: adding effectCell co-culture: the experimental group was added with 4X 10⁵Individual effector cells (NK-92, EV-NK-92,. alpha.FR-CAR-NK-92 (T cell element),. alpha.FR-CAR-NK-92 (NK cell element)) whereas control group was added with 4X 10 alone⁵And (4) effector cells. Each group was plated with 3 multiple wells for 24h of co-culture.

Day 2: collection of supernatants for TNF- α and IFN- γ detection by ELISA: the co-culture supernatant was collected, centrifuged at 1500rpm for 10min to remove cells and debris, and the supernatant was collected.

ELISA detection step: diluting a standard substance, preparing a biotin-labeled anti-human TNF-alpha/IFN-gamma antibody working solution and an avidin-peroxidase complex (ABC) working solution according to an instruction, and balancing the diluted ABC and TMB developing solution at 37 ℃ for more than 30 minutes in advance. Adding 100 mul of 7 standard products with different dilution concentrations into a row of enzyme label plate holes, and adding sample dilution solution into the other 1 hole to serve as a zero hole. The collected cell culture supernatant (100. mu.l) was added to the remaining wells, and the microplate was sealed with a sealing membrane and incubated at 37 ℃ for 90 minutes. And thirdly, throwing off liquid in the ELISA plate, beating the ELISA plate for several times against absorbent paper, adding a biotin anti-human TNF-alpha or IFN-gamma antibody working solution into each hole, sealing a plate sealing membrane without adding a TMB blank color developing hole, and incubating for 60 minutes at 37 ℃. And washing with washing buffer solution for 3 times, soaking for 1 minute each time, and washing with 300 mul of washing solution per hole. Fifthly, 100 mul of ABC working solution is added into each hole, and no TMB blank color developing hole is added. The microplate was sealed with a sealing membrane and incubated at 37 ℃ for 30 minutes. Sixthly, washing by the washing buffer solution for 5 times, soaking for 2 minutes each time, and washing by 300 mu l of washing liquid in each hole. Seventhly, adding 90 mu l of TMB color development liquid into each hole, and carrying out light-shielding reaction for 20 minutes at 37 ℃. And adding 100 mul of TMB stop solution into each hole, and measuring the OD value at the wavelength of 450nm by using a microplate reader.

The ELISA result graph is shown in FIG. 4, and the graph shows that when the alpha FR-CAR-NK-92(NK cell element) is co-cultured with HCT-116 cells, more TNF-alpha and IFN-gamma can be secreted, the effect of the alpha FR-CAR-NK-92(T cell element) is obviously stronger than that of the alpha FR-CAR-NK-92, and the CAR structure designed by the method is superior to the existing CAR structure in the NK cells.

From the above experimental results, it can be seen that: after the prepared chimeric antigen receptor targeting the folate receptor alpha is used for transfecting lentivirus and infecting NK-92 cells, the chimeric antigen receptor targeting the folate receptor alpha can be expressed in the NK-92 cells, namely the method can be used for constructing the chimeric antigen receptor modified NK-92 cells (alpha FR-CAR-NK-92) targeting the folate receptor alpha. In vitro experiments show that when the alpha FR-CAR-NK-92 and HCT-116 cells are co-cultured, more TNF-alpha and IFN-gamma can be secreted compared with the prior art, and the cell killing agent has strong killing effect on the HCT-116 cells.

The present invention is not limited to the above embodiments, and any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention are included in the scope of the claims of the present invention which are filed as the application.

SEQUENCE LISTING

<110> China people liberation army, military and medical university

<120> targeted folate receptor alpha chimeric antigen receptor, preparation method and application thereof

<130> 211-120

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 2403

<212> DNA

<213> Artificial Synthesis

<220>

<221> human CD8 alpha signal peptide

<222> (1)..(63)

<220>

<221> light chain variable region (VL)

<222> (64)..(393)

<220>

<221> Single chain variable region (scFv) against human folate receptor alpha (. alpha.FR)

<222> (64)..(798)

<220>

<221> variable region of heavy chain (VH)

<222> (439)..(798)

<220>

<221> human immunoglobulin G1 Fc fragment

<222> (799)..(1494)

<220>

<221> human CD8 alpha hinge region

<222> (1495)..(1638)

<220>

<221> human NKG2D transmembrane segment

<222> (1639)..(1701)

<220>

<221> human 2B4 intracellular segment

<222> (1639)..(2061)

<220>

<221> intracellular fragment of human CD3 ζ

<222> (2063)..(2403)

<400> 1

atggccctcc cagttaccgc ccttctcctg cccctggccc tgctgctgca cgccgcccgc 60

ccccagtctg ccctgactca gcctgcctcc gtgtctgggt ctcctggaca gagcatcacc 120

atctcctgca ctggaaccag cagtgatgtt gggagttata acctggtctc ctggtaccag 180

cagcacccag gcaaagcccc caaactcatg atttatgagg gcagtaagcg gccctcaggg 240

gtttctaatc gcttctctgg ctccaagtct ggcaacgccg cctccctgac aatctctggg 300

ctccaggctg aggacgaggc tgattattac tgccagtcct atgacagcag cctgagtgtg 360

gtgttcggcg gagggaccaa gctgaccgtc ctcggaggag gcggatcagg cggaggaggc 420

tctggcggag gcggaagcca ggtgcagctg gtggagtctg ggggaggcct ggtgcagcca 480

gggcggtccc tgagactctc ctgcacaact tctggattca cttttggcga ttatgctatg 540

atctgggccc gccaggctcc agggaagggg ctggagtggg tctcatccat tagtagtagt 600

agtagttaca tctactacgc agactcagtg aagggcagat tcaccatctc cagagacaac 660

gccaagaact cactgtatct gcagatgaac agcctgagag ccgaggacac cgctgtgtat 720

tactgtgcca gagaacggta cgatttttgg agtggaatgg acgtctgggg caaagggacc 780

accgtcaccg tgagcagtga gcccaaatct agcgacaaaa ctcacacatg cccaccttgc 840

ccagcacctg agctcctggg gggaccttca gtcttcctct tccccccaaa acccaaggac 900

accctcatga tctcccggac ccctggggtc acatgcgtgg tggtggacgt gagccacgaa 960

gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 1020

aagcctcggg aggagcagta caacagcacc tacagagtgg tcagcgtcct caccgtcctg 1080

caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 1140

gcccccatcg agaaaaccat ctccaaagcc aaagggcagc ccagagaacc acaggtgtac 1200

accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc 1260

aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gcctgagaac 1320

aactacaaga ccacccctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag 1380

ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 1440

gaggctctgc acaaccacta cacccagaag agcctctccc tgtctcctgg caaagccaag 1500

cccaccacta cacccgcccc acgccccccc acccccgccc ccaccatcgc cagccagccc 1560

ctgagcctgc gccccgaggc ctgccgcccc gccgccggcg gcgccgtgca cacccgcggc 1620

ctggacttcg cctgcgaccc attttttttc tgctgcttca tcgctgtagc catgggaatc 1680

cgtttcatta ttatggtaac atggaggaga aagaggaagg agaagcagtc agagaccagt 1740

cccaaggaat ttttgacaat ttacgaagat gtcaaggatc tgaaaaccag gagaaatcac 1800

gagcaggagc agacttttcc tggagggggg agcaccatct actctatgat ccagtcccag 1860

tcttctgctc ccacgtcaca agaacctgca tatacattat attcattaat tcagccttcc 1920

aggaagtctg gatccaggaa gaggaaccac agcccttcct tcaatagcac tatctatgaa 1980

gtgattggaa agagtcaacc taaagcccag aaccctgctc gattgagccg caaagagctg 2040

gagaactttg atgtttattc cagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 2100

cagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 2160

gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgca gagaaggaag 2220

aaccctcagg aaggcctgta caatgaactg cagaaagata agatggcgga ggcctacagt 2280

gagattggga tgaaaggcga gcgccggagg ggcaaggggc acgatggcct ttaccagggt 2340

ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct gccccctcgc 2400

taa 2403