CN115976035B

CN115976035B - Adjustable universal CAR, CAR-T cell and fusion guide polypeptide

Info

Publication number: CN115976035B
Application number: CN202211258438.0A
Authority: CN
Inventors: 王惠明; 王刚
Original assignee: Wuhan University WHU
Current assignee: Hefei Shenghuikang Medical Technology Co.,Ltd.
Priority date: 2022-10-14
Filing date: 2022-10-14
Publication date: 2024-01-09
Anticipated expiration: 2042-10-14
Also published as: CN115976035A

Abstract

The invention provides a controllable universal CAR, CAR-T cells and variable fusion guide polypeptides for treating autoimmune kidney disease. The adjustable universal type CAR-T cell has Chimeric Antigen Receptor (CAR) capable of specifically recognizing and combining a specific guide sequence, and can be activated, guided and adjusted through the variable fusion guide polypeptide, so that different autoreactive B cells can be precisely recognized and killed in a targeted manner, and the aim of treating different autoimmune nephrosis is fulfilled.

Description

Adjustable universal CAR, CAR-T cell and fusion guide polypeptide

Technical Field

The invention relates to the technical field of immunotherapy, in particular to a controllable universal CAR for treating autoimmune nephropathy, a CAR-T cell and fusion guide polypeptide.

Background

Autoimmune kidney disease is autoimmune mediated kidney injury, which is the leading cause of chronic kidney disease worldwide. Autoimmune kidney disease actually belongs to a class of autoimmune diseases, mainly including autoimmune diseases of systemic autoimmune reaction disease in the kidney manifestation and kidney specificity; the former such as Lupus Nephritis (LN), and the latter such as Idiopathic Membranous Nephropathy (IMN), igA nephropathy (IgAN), goodpasture syndrome, etc. Autoantibodies are a hallmark of many autoimmune kidney diseases, with detection of autoantibodies in the blood or kidneys being a key to diagnosis. Currently, key autoantibodies and their antigens in a variety of kidney diseases have been identified, such as anti-PLA 2R, anti-Gd-IgA 1, anti-dsDNA, anti-ANCA, and anti-GBM antibodies, among others. These autoantibodies attack kidney tissue, form antigen-antibody complexes that deposit in the kidneys and elicit antibody-mediated immune responses, resulting in kidney damage. Each autoantibody is secreted by its corresponding autoreactive B cell clone. Thus, it can be said that autoimmune kidney disease is a B-cell disease in essence, as is B-cell malignancy.

Currently, therapeutic regimens for autoimmune kidney disease mainly include immunosuppressants and hormonal drugs. However, these drugs bring about systemic immunosuppressive effects, thereby increasing the risk of serious complications such as infection and cancer. B cell-targeted antibody drugs or cell therapy regimens are one of the directions being explored. For example, anti-CD 20 mab (rituximab) can mediate killing B cells after binding to B cell surface CD20 molecules, which is a significant clinical effect in the treatment of systemic lupus erythematosus, especially severe lupus nephritis. Rituximab can also significantly reduce the level of anti-PLA 2R antibodies in patients while treating membranous nephropathy. Rituximab is also useful in the treatment of ANCA-related vasculitis. However, unlike B cell malignancies, pathogenic autoreactive B cell clones account for only a very small fraction of human B cells, and extensive B cell clearance is not the best choice.

CAR-T cell technology is a breakthrough innovative technology in the field of tumor therapy in recent years. This technology transfects natural T cells in vitro by gene transduction methods by coupling single chain antibodies (scFv) recognizing tumor associated antigens and intracellular domains (including co-stimulatory domains and signaling domains) to form Chimeric Antigen Receptors (CARs), forming CAR-expressing T cells (abbreviated as CAR-T cells). CAR-T cells are able to bind to tumor antigens in an antigen-dependent, non-MHC-restricted manner, initiate and activate specific killing of tumor cells bearing tumor-associated antigens.

Currently, CAR-T cells for targeted recognition and killing of autoreactive B cells have been studied. For example, patent document CN108795876 discloses a novel approach for treating autoimmune kidney disease using a universal CAR-T cell fusion polypeptide guide, which can cope with heterogeneity of autoreactive B cells in autoimmune kidney disease patients very conveniently. However, the sequence of the single-chain antibody sequence of the chimeric antigen receptor of the universal CAR-T cell disclosed in the patent is short, and analysis shows that the heavy chain CDR3 region of the chimeric antigen receptor may be deleted, which affects the recognition specificity and the binding capacity of the CAR-T cell, and further affects the application of the chimeric antigen receptor in the treatment of autoimmune nephropathy.

Disclosure of Invention

Based on this, the invention provides a regulatable universal CAR, CAR-T cells and variable fusion guide polypeptides for use in the treatment of autoimmune kidney disease.

The invention adopts the following technical scheme:

the invention provides a gene for encoding an anti-leader polypeptide single chain antibody (anti-GP scFv), the sequence of which is shown as SEQ ID NO. 11 or SEQ ID NO. 27. The sequence of the anti-leader polypeptide single chain antibody is shown as SEQ ID NO. 12.

The invention also provides a coding gene of the regulatable general CAR, the sequence of the coding gene mainly comprises a CD3 zeta section, a co-stimulatory section, a transmembrane section, a hinge section, an anti-leader polypeptide single-chain antibody section and a signal peptide section, and the gene sequence of the anti-leader polypeptide single-chain antibody section is shown as SEQ ID NO. 11 or SEQ ID NO. 27. The gene sequence carried by the CD3 zeta section is shown as SEQ ID NO. 1. The gene sequence of the costimulatory region is shown as SEQ ID NO. 3 or SEQ ID NO. 5. The gene sequence of the transmembrane segment is shown in SEQ ID NO. 7. The gene sequence of the hinge section is shown as SEQ ID NO. 9. The gene sequence of the signal peptide segment is shown in SEQ ID NO. 13.

Preferably, the sequence of the coding gene of the controllable general CAR is shown as SEQ ID NO. 15 or SEQ ID NO. 17.

The invention also provides a controllable general CAR, the sequence of which is shown as SEQ ID NO. 16 or SEQ ID NO. 18.

The invention also provides a CAR-T cell comprising the anti-leader polypeptide single chain antibody or the regulatable universal CAR.

The invention also provides a variable fusion guide polypeptide (variable fusion guide polypeptide, vfGP) for being matched with the application of the controllable universal CAR-T cell, which is formed by sequentially connecting a guide polypeptide, a connector and an autoantibody affinity peptide, wherein the sequence of the guide polypeptide is shown as SEQ ID NO. 19.

Preferably, the sequence of the vfGP is shown as SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, or SEQ ID NO. 26.

Compared with the prior art, the invention has the beneficial effects that:

1) The anti-guidance polypeptide single-chain antibody (anti-GP scFv) expressed by the adjustable and controllable general CAR-T cell can identify a specific guidance polypeptide sequence and has extremely high affinity with the specific guidance polypeptide sequence, so that the anti-guidance polypeptide single-chain antibody can be matched with the vfGP containing the guidance polypeptide sequence to efficiently identify and kill corresponding autoreactive B cells.

2) The adjustable universal CAR designed by the invention can be applied to different autoimmune kidney diseases, is different from the traditional CAR-T cells in that different CARs are designed aiming at different diseases, and can shorten the preparation time and reduce the preparation cost of the CAR-T cells.

3) The adjustable and controllable general CAR-T cells designed by the invention do not directly identify target cells, so that abnormal autoreactive B cells cannot be attacked immediately after the CAR-T cells are infused back into a patient; only after subsequent infusions of vfGP will target cells be challenged under the guidance of the latter, and the challenge efficacy is related to the dose of infused vfGP. Based on the characteristic, the immune effect intensity of the CAR-T cells in the body can be conveniently controlled, and the side effects such as cytokine storm and other risks in the common CAR-T cell treatment can be reduced.

4) The adjustable universal type CAR-T cell has Chimeric Antigen Receptor (CAR) capable of specifically recognizing and combining a specific guide sequence, and can be activated, guided and adjusted and controlled through vfGP, so that different autoreactive B cells can be precisely recognized and killed in a targeted manner, and the aim of treating different autoimmune nephrosis is fulfilled. The application of the adjustable general CAR-T cells and the vfGP in the aspect of treating autoimmune diseases can be expanded to autoimmune diseases, and when a treatment system aiming at new diseases is developed, only the new vfGP is required to be screened and designed, so that the research and development period is greatly shortened, and the research and development cost is saved.

Drawings

FIG. 1 is a statistical plot of affinity and specificity assays for recombinant anti-GP scFv and vfGP in example 2.

FIG. 2 is a flow cytometry detection results and statistical graphs of the vfGP activated regulatory universal CAR-T cells of example 5.

FIG. 3 is a statistical graph of the killing effect of the vfGP-mediated controllable universal CAR-T cells on target cells as detected by flow cytometry in example 5.

FIG. 4 is a competition ELISA response curve for example 7.

FIG. 5 is a survival curve of experimental mice in example 8.

FIG. 6 shows the variation of the levels of urine protein, serum urea nitrogen, serum creatinine and anti-ds-DNA antibodies in the experimental mice of example 8 for 24 h.

Detailed Description

The present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art.

The following examples are given for illustration of the invention only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art without creative efforts are within the protection scope of the present invention based on the specific embodiments of the present invention.

In the examples of the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise; in the embodiments of the present invention, unless specifically indicated, all technical means used are conventional means well known to those skilled in the art.

A coding gene of a regulatable universal Chimeric Antigen Receptor (CAR) consists of a cd3ζ segment, a costimulatory segment, a transmembrane segment, a hinge segment, an anti-leader polypeptide single chain antibody segment and a signal peptide.

Wherein the cd3ζ segment carries the following gene sequence:

agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc(SEQ ID NO:1)。

the translated protein sequence of the gene carried by the cd3ζ segment is as follows:

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:2)。

the gene sequence carried by the costimulatory region is selected from one of the following 2 gene sequences:

gene sequence of CD28 costimulatory segment:

aggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctcc(SEQ ID NO:3)。

the translated protein sequence of the CD28 costimulatory region is as follows:

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO:4)。

gene sequence of CD137 costimulatory segment:

aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg(SEQ ID NO:5)。

the translated protein sequence of the CD137 costimulatory region is as follows:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:6)。

the gene sequence carried by the transmembrane segment is:

atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaccacaggaac(SEQ ID NO:7)。

the translated protein sequence of the transmembrane segment is: IYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 8).

The gene sequence of the hinge segment is as follows: gagtccaagtatgggcctccatgccctccatgtcct (SEQ ID NO: 9).

The translated protein sequence of the hinge segment is: ESKYGPPCPPCP (SEQ ID NO: 10).

The gene sequence of the anti-leader polypeptide single chain antibody segment is as follows:

gatgcggtggtgacccaggaaagcgcgctgaccaccagcccgggcgaaaccgtgaccctgacctgccgcagcagcaccggcgcggtgaccaccagcaactatgcgagctgggtgcaggaaaaaccggatcatctgtttaccggcctgattggcggcaccaacaaccgcgcgccgggcgtgccggcgcgctttagcggcagcctgattggcgataaagcggcgctgaccattaccggcgcgcagaccgaagatgaagcgatttatttttgcgcgctgtggtatagccatcattgggtgtttggcggcggcaccaaactgaccgtgctgggcggcagcaccagcggcagcggcaaaccgggcagcggcgaaggcagcaccaaaggcgatgtgcagctgcaggaaagcggcccgggcctggtggcgccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccgattatggcgtgaactgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggggcgatggcattaccgattataacagcgcgctgaaaagccgcctgagcgtgaccaaagataacagcaaaagccaggtgtttctgaaaatgaacagcctgcagagcggcgatagcgcgcgctattattgcgtgaccggcctgtttgattattggggccagggcaccaccctgaccgtgagcagc(SEQ ID NO:11)。

the protein sequence of the anti-leader polypeptide single chain antibody segment gene translation is as follows:

DAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYASWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSHHWVFGGGTKLTVLGGSTSGSGKPGSGEGSTKGDVQLQESGPGLVAPSQSLSITCTVSGFSLTDYGVNWVRQSPGKGLEWLGVIWGDGITDYNSALKSRLSVTKDNSKSQVFLKMNSLQSGDSARYYCVTGLFDYWGQGTTLTVSS(SEQ ID NO:12)。

the gene sequence of the signal peptide segment is as follows:

atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg(SEQ ID NO:13)。

the protein sequence of the signal peptide segment gene translation is as follows:

MALPVTALLLPLALLLHAARP(SEQ ID NO:14)。

preferably, the complete gene sequence of the chimeric antigen receptor (svCAR) gene comprising the CD28 costimulatory region is as follows:

atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggatgcggtggtgacccaggaaagcgcgctgaccaccagcccgggcgaaaccgtgaccctgacctgccgcagcagcaccggcgcggtgaccaccagcaactatgcgagctgggtgcaggaaaaaccggatcatctgtttaccggcctgattggcggcaccaacaaccgcgcgccgggcgtgccggcgcgctttagcggcagcctgattggcgataaagcggcgctgaccattaccggcgcgcagaccgaagatgaagcgatttatttttgcgcgctgtggtatagccatcattgggtgtttggcggcggcaccaaactgaccgtgctgggcggcagcaccagcggcagcggcaaaccgggcagcggcgaaggcagcaccaaaggcgatgtgcagctgcaggaaagcggcccgggcctggtggcgccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccgattatggcgtgaactgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggggcgatggcattaccgattataacagcgcgctgaaaagccgcctgagcgtgaccaaagataacagcaaaagccaggtgtttctgaaaatgaacagcctgcagagcggcgatagcgcgcgctattattgcgtgaccggcctgtttgattattggggccagggcaccaccctgaccgtgagcagcgagtccaagtatgggcctccatgccctccatgtcctatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaccacaggaacaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc(SEQ ID NO:15)。

the translated protein sequence of the chimeric antigen receptor (svCAR) gene comprising the CD28 costimulatory region is as follows:

MALPVTALLLPLALLLHAARPDAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYASWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSHHWVFGGGTKLTVLGGSTSGSGKPGSGEGSTKGDVQLQESGPGLVAPSQSLSITCTVSGFSLTDYGVNWVRQSPGKGLEWLGVIWGDGITDYNSALKSRLSVTKDNSKSQVFLKMNSLQSGDSARYYCVTGLFDYWGQGTTLTVSSESKYGPPCPPCPIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:16)。

preferably, the sequence of the chimeric antigen receptor (svCAR) gene comprising the CD137 costimulatory region is as follows:

atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggatgcggtggtgacccaggaaagcgcgctgaccaccagcccgggcgaaaccgtgaccctgacctgccgcagcagcaccggcgcggtgaccaccagcaactatgcgagctgggtgcaggaaaaaccggatcatctgtttaccggcctgattggcggcaccaacaaccgcgcgccgggcgtgccggcgcgctttagcggcagcctgattggcgataaagcggcgctgaccattaccggcgcgcagaccgaagatgaagcgatttatttttgcgcgctgtggtatagccatcattgggtgtttggcggcggcaccaaactgaccgtgctgggcggcagcaccagcggcagcggcaaaccgggcagcggcgaaggcagcaccaaaggcgatgtgcagctgcaggaaagcggcccgggcctggtggcgccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccgattatggcgtgaactgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggggcgatggcattaccgattataacagcgcgctgaaaagccgcctgagcgtgaccaaagataacagcaaaagccaggtgtttctgaaaatgaacagcctgcagagcggcgatagcgcgcgctattattgcgtgaccggcctgtttgattattggggccagggcaccaccctgaccgtgagcagcgagtccaagtatgggcctccatgccctccatgtcctatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaccacaggaacaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc(SEQ ID NO:17)。

the translated protein sequence of the chimeric antigen receptor (svCAR) gene comprising the CD137 costimulatory region is as follows:

MALPVTALLLPLALLLHAARPDAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYASWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSHHWVFGGGTKLTVLGGSTSGSGKPGSGEGSTKGDVQLQESGPGLVAPSQSLSITCTVSGFSLTDYGVNWVRQSPGKGLEWLGVIWGDGITDYNSALKSRLSVTKDNSKSQVFLKMNSLQSGDSARYYCVTGLFDYWGQGTTLTVSSESKYGPPCPPCPIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:18)。

a regulatable universal CAR-T cell, the preparation steps comprising: the entrusted biological company synthesizes a complete adjustable general CAR gene sequence with homology arms at two ends, and the sequence is shown as SEQ ID NO. 15 or SEQ ID NO. 17; cloning the CAR gene sequence into a lentiviral vector by utilizing homologous recombination enzyme, and transfecting 293T cells together with a lentiviral packaging plasmid to prepare CAR lentivirus; the activated T cells are transfected by the CAR lentivirus, and the regulatable universal CAR-T cells are obtained.

A variable fusion guide polypeptide (vfGP) of a matched regulatable universal CAR-T cell, which consists of a Guide Polypeptide (GP), a linker and an autoantibody affinity peptide (Autoantibody affinity peptide, AAP).

Wherein the amino acid sequence of the guide polypeptide is as follows:

HLENEVARLKKLVGER(SEQ ID NO:19)。

the linker is used for linking the guide polypeptide and the autoantibody affinity peptide, and has the amino acid sequence as follows:

GGGGS(SEQ ID NO:20)。

autoantibody Affinity Peptides (AAPs) are polypeptide fragments capable of specifically binding to BCR molecules of autoreactive B cells, whose amino acid sequences are variable, corresponding to autoreactive B cells as targets of attack.

Specifically, the vfGP may be a vfGP-IMN for use in combination with a regulatable universal CAR-T cell to treat Idiopathic Membranous Nephropathy (IMN), the sequence being as follows:

HLENEVARLKKLVGERGGGGSWQDKGIFVIQSESLKKCIQAGKSVLTLENCK(SEQ ID NO:21)。

specifically, the vfGP may be a vfGP-IgAN for the treatment of IgA nephropathy (IgAN) in combination with a regulatable universal CAR-T cell, the sequence being as follows:

HLENEVARLKKLVGERGGGGSYCSKVCRPWNYRRPYYYGMDVW(SEQ ID NO:22)。

specifically, the vfGP may be a vfGP-LN for the treatment of Lupus Nephritis (LN) in combination with a regulatable universal CAR-T cell, the sequence being as follows:

HLENEVARLKKLVGERGGGGSDWEYSVWLSN(SEQ ID NO:23)。

specifically, the vfGP may be a vfGP-MA for the combined treatment of MPO-ANCA-related vasculitis (MPO-AAV) with a regulatable universal CAR-T cell, the sequence being as follows:

HLENEVARLKKLVGERGGGGSRLDNRYQPMEPN(SEQ ID NO:24)。

specifically, the vfGP may be a vfGP-PA for use in combination with a regulatable universal CAR-T cell to treat PR 3-ANCA-associated vasculitis (PR 3-AAV), the sequence being as follows:

HLENEVARLKKLVGERGGGGSVVLGAHNVRTQ(SEQ ID NO:25)。

specifically, the vfGP may be vfGP-P14 for the treatment of Goodpasture syndrome in combination with a regulatable universal CAR-T cell, the sequence being as follows:

HLENEVARLKKLVGERGGGGSTDIPPCPHGWISLWKGFSFIMF(SEQ ID NO:26)。

the sequences of the Autoantibody Affinity Peptides (AAPs) corresponding to the above vfGP were:

AAP-IMN：WQDKGIFVIQSESLKKCIQAGKSVLTLENCK(SEQ ID NO:28)。

AAP-IgAN：YCSKVCRPWNYRRPYYYGMDVW(SEQ ID NO:29)。

AAP-LN：DWEYSVWLSN(SEQ ID NO:30)。

AAP-MA：RLDNRYQPMEPN(SEQ ID NO:31)。

AAP-PA：VVLGAHNVRTQ(SEQ ID NO:32)。

AAP-P14：TDIPPCPHGWISLWKGFSFIMF(SEQ ID NO:33)。

the following is illustrative:

example 1

The embodiment provides a preparation method of an anti-guidance polypeptide single-chain antibody (anti-GP scFv), which comprises the following steps:

(1) Preparing an antigen: the Shanghai blaze Biocompany was commissioned to synthesize the Guide Polypeptide (GP) as shown in SEQ ID NO. 19 described above, and the GP was coupled to the KLH carrier protein using the KLH protein coupling kit (Bioquest Co. U.S.A.).

(2) Immunized mice: after emulsifying the antigen prepared in step 1 with Freund's adjuvant, bal b/c mice were immunized in a conventional procedure.

(3) Preparation of hybridoma cell lines: after the three-phase, the spleen cells of the mice are extracted and fused with SP2/0 cells to prepare hybridoma cells. Culturing and subcloning hybridoma cells according to a conventional process, and screening out hybridoma cell strains capable of secreting high-affinity anti-GP monoclonal antibodies by enzyme-linked immunosorbent assay.

(4) Monoclonal antibody sequencing: sequencing the selected hybridoma cell strain by the Wuhan Pujian biological company to obtain the light chain and heavy chain gene sequences of the anti-GP monoclonal antibody.

(5) The eukaryotic expression process of the recombinant anti-GP scFv is entrusted to be completed by the Wuhan Pujian biological company, and the steps are as follows:

(1) the anti-GP mab light and heavy chain genes measured in step 4 were joined in a linker gene sequence (see italic underlined in the sequence) to form the complete anti-GP scFv gene sequence:

gatgcggtggtgacccaggaaagcgcgctgaccaccagcccgggcgaaaccgtgaccctgacctgccgcagcagcaccggcgcggtgaccaccagcaactatgcgagctgggtgcaggaaaaaccggatcatctgtttaccggcctgattggcggcaccaacaaccgcgcgccgggcgtgccggcgcgctttagcggcagcctgattggcgataaagcggcgctgaccattaccggcgcgcagaccgaagatgaagcgatttatttttgcgcgctgtggtatagccatcattgggtgtttggcggcggcaccaaactgaccgtgctgggcggcagcaccagcggcagcggcaaaccgggcagcggcgaaggc agcaccaaaggcgatgtgcagctgcaggaaagcggcccgggcctggtggcgccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccgattatggcgtgaactgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggggcgatggcattaccgattataacagcgcgctgaaaagccgcctgagcgtgaccaaagataacagcaaaagccaggtgtttctgaaaatgaacagcctgcagagcggcgatagcgcgcgctattattgcgtgaccggcctgtttgattattggggccagggcaccaccctgaccgtgagcagc(SEQ ID NO:27)。

(2) and synthesizing an integral anti-GP scFv gene, and cloning the anti-GP scFv gene to a eukaryotic expression vector.

(3) Transiently transfecting the anti-GP scFv expression vector into CHO cells to express recombinant anti-GP scFv, and collecting culture supernatant. Coupling the guide polypeptide to N-hydroxysuccinimide activated agarose to prepare an antigen affinity purification column, carrying out affinity purification on the culture supernatant to collect recombinant anti-GP scFv, and determining the protein concentration by a Coomassie brilliant blue method.

Example 2

The embodiment provides a method for detecting affinity and specificity of recombinant anti-GP scFv and vfGP by a competition ELISA method, which comprises the following specific steps: (1) polypeptide synthesis: the Shanghai blaze biological company is entrusted to synthesize the polypeptides shown as SEQ ID NO 19, SEQ ID NO 21-26 and SEQ ID NO 28-33. (2) And taking a proper amount of the polypeptide, and dissolving the polypeptide into 2mg/mL stock solution respectively by using DMSO for standby. (3) coating an ELISA plate: a suitable amount of GP stock solution was diluted to 1. Mu.g/mL with coating buffer (0.05M, pH 9.6 carbonate buffer), and 96-well ELISA plates were added at a rate of 100. Mu.L per well, and coated overnight at 4 ℃. (4) After the coating, the antigen solution in the wells of the enzyme-labeled plate was discarded, and 200. Mu.L of PBST solution was added to each well and washed 1 time. (5) closing: 200. Mu.L of PBS (0.01M, pH 7.4) solution containing 5% Bovine Serum Albumin (BSA) was added to each well, and the wells were blocked at room temperature for 2 hours. (6) And (2) diluting the polypeptide stock solutions in the step (2) with PBS (phosphate buffered saline) solution containing 1% BSA to obtain the following concentration gradients: 0.1, 5, 25, 125nM. (7) Anti-cancer agentOriginal pre-blocking: an appropriate amount of the recombinant anti-GP scFv obtained in the preparation of example 1 was diluted to 0.1. Mu.g/mL with PBS containing 1% BSA. And (3) respectively mixing the polypeptides with different concentration gradients in the step (6) with the recombinant anti-GP scFv solution in equal volumes, and pre-incubating for 1 hour at room temperature. (8) After the sealing is finished, removing sealing liquid in the enzyme-labeled plate holes, adding 100 mu L of the mixed solution in the step 7, and designing 3 compound holes for each combination; a blank well was additionally provided, 100. Mu.L of 1% BSA in PBS was added and incubated at room temperature for 2 hours. (9) After the incubation, the wells were discarded, washed 4 times with 250. Mu.L of PBST solution, and patted dry. (10) 100. Mu.L of HRP-labeled goat anti-mouse IgG Light-Chain Specific Antibody was added to the wells of the enzyme-labeled wells and incubated for 1 hour at room temperature. (11) After the incubation, the wells were discarded, washed 5 times with 250. Mu.L of PBST solution, and patted dry. (12) 100 mu L of TMB color development solution is added into the wells of the enzyme-labeled plate, and the reaction is carried out at room temperature for 10-15 minutes in a dark place. (13) 100 mu L of stop solution is added into the plate hole of the enzyme label plate, and the enzyme label plate is placed in an enzyme label instrument to measure the absorbance value (OD) of 450nm _450nm )。

The competition ELISA assay results are shown in figure 1. The results show that: GP (SEQ ID NO: 19) and vfGP (SEQ ID NO: 21-26) can compete to inhibit the combination of recombinant anti-GP scFv and GP coated on an ELISA plate, and the inhibition effect is positively correlated with the concentration; in contrast, none of the autoantibody affinity peptides (SEQ ID NOS: 28-33) showed blocking effect. The recombinant anti-GP scFv constructed newly in the application can specifically recognize GP and vfGP containing GP sequence, and does not cross react with autoantibody affinity peptide.

Furthermore, the study results also show that: OD measured by pre-blocking anti-GP scFv with equal concentration of vfGP or GP _450nm The values were not significantly different, indicating that the affinity of anti-GP scFv was similar to vfGP and GP.

Since the anti-GP scFv is the antigen recognition domain of the regulatable general CAR-T cell, the experimental result also shows that the CAR-T cell can specifically recognize and bind to the vfGP.

Example 3

The embodiment provides an experimental process for proving that vfGP can bridge anti-GP scFv and autoantibodies of renal patients by a sandwich ELISA method, which comprises the following steps:

collecting serum from an autoimmune kidney disease patient containing autoantibodies, comprising: clinical tests prove that the serum of the IMN patient containing the anti-PLA 2R antibody; clinical tests prove that the serum of IgAN patients containing the anti-GdIgA 1 antibody; clinical tests prove that LN patient serum containing anti-dsDNA antibodies; clinical tests prove that the serum of the MPO type-ANCA related vasculitis (MPO-AAV) patient containing the anti-MPO antibody; clinical tests prove that the serum of PR3 type-ANCA related vasculitis (PR 3-AAV) patients containing anti-PR 3 antibodies; the serum of the Goodpasture syndrome patient containing the anti-GBM antibody is confirmed through clinical examination.

And (3) coating an ELISA plate: the recombinant anti-GP scFv obtained in example 1 was diluted to 2ug/mL with coating buffer, 96-well ELISA plates were added in an amount of 100. Mu.L per well, and coated overnight at 4 ℃. After the coating was completed, the wells of the enzyme-labeled plate were discarded, and 200. Mu.L of PBST solution was added to each well and washed 1 time. Closing: 200. Mu.L of a PBS (0.01M, pH 7.4) solution containing 5% BSA was added to each well, and the wells were blocked at room temperature for 2 hours. A suitable amount of stock solution of the variable fusion guide polypeptide (vfGP, SEQ ID NO: 21-26) prepared in step 2 of example 2 was diluted with 1% BSA in PBS to the following concentration gradient: 0.1, 10, 100, 1000pM. After the end of the blocking, the blocking solution in the wells of the enzyme-labeled plate was discarded, the above-mentioned vfGP with different concentration gradients was added, 3 multiplex wells were set per concentration of each polypeptide, 100. Mu.L per well, and incubated for 1 hour at room temperature. After the incubation, the wells were discarded, washed 4 times with 250. Mu.L of PBST solution, and patted dry. 1/10 of the serum of the autoimmune nephrosis patient is diluted and then added into the corresponding enzyme-labeled plate hole (for example, the serum of the IMN patient is added into the enzyme-labeled plate hole incubated by adding the vfGP-IMN peptide), and 3 compound holes are arranged for each sample; after the completion of the addition, the sample was incubated at room temperature for 1 hour. After the incubation, the wells were discarded, washed 4 times with 250. Mu.L of PBST solution, and patted dry. 100. Mu.L of HRP-labeled goat anti-human IgG was added to the wells of the enzyme-labeled plate and incubated for 1 hour at room temperature. After the incubation, the wells were discarded, washed 5 times with 250. Mu.L of PBST solution, and patted dry. 100 mu L of TMB color development solution is added into the wells of the enzyme-labeled plate, and the reaction is carried out at room temperature for 10-15 minutes in a dark place. 100 mu L of stop solution is added into the plate hole of the enzyme label plate, and the enzyme label plate is placed in an enzyme label instrument for measuring 450nmAbsorbance value (OD) _450nm )。

The sandwich ELISA assay results are shown in table 1:

TABLE 1 Sandwich ELISA detection results

The result shows that the OD value measured without adding the vfGP is 2.1 times as positive cut-off value: OD values measured by adding different concentrations of the vfGP are positive, and the OD values are positively correlated with the concentration of the vfGP, so that the vfGP combined with the anti-GP scFv can be continuously and specifically combined with autoantibodies of renal patients.

Because the autoantibodies of kidney disease patients are identical to the antigen binding sequences of autoreactive B cell BCRs, the experimental results prove that the vfGP can be combined with the single-chain antibody region (namely anti-GP scFv) of the regulatable universal CAR-T cell and the BCRs of the autoreactive B cell at the same time, thereby mediating the regulatable universal CAR-T cell to target the autoreactive B cell.

Example 4

The implementation provides a preparation method of a controllable general CAR-T cell, which comprises the following steps:

(1) The general biological company is entrusted to synthesize the complete controllable general CAR gene sequence with homology arms at two ends, and the sequence is shown as SEQ ID NO. 15 or SEQ ID NO. 17.

(2) The adjustable general CAR gene sequence is cloned to a pCDH-CMV-MCS-EF 1-copGGFP vector (purchased from Youbao organism, cat# VT 1479) by utilizing homologous recombination enzyme through a homologous recombination mode, so as to obtain the pCDH-CMV-MCS-EF 1-copGGFP-CAR vector.

(3) The pCDH-CMV-MCS-EF 1-copGGFP-CAR vector was combined with lentiviral packaging plasmids pLP1-gag/pol, pLP2-Rev and pLP/VSVG (available from Invitrogen under the trade name ViraPower) ^TM Lentiviral Packaging Mix, cat# K497500) co-transfected 293T cells, CAR lentiviruses were prepared.

(4) Peripheral Blood Mononuclear Cells (PBMCs) were isolated from fresh human whole blood using density gradient centrifugation and T cells were obtained by CD3 magnetic bead sorting.

(5) T cells were activated with anti-CD 3 and anti-CD 28 monoclonal antibodies.

(6) The activated T cells are transfected by the CAR lentivirus, and the regulatable universal CAR-T cells are obtained.

(7) The adjustable and controllable general CAR-T cells are amplified by adopting a T cell amplification culture medium.

Example 5

Taking Idiopathic Membranous Nephropathy (IMN) patient autoreactive B cells as an example, the embodiment provides an experimental method for in vitro verification of target cells killing by the adjustable universal CAR-T cells (CAR gene sequence is shown as SEQ ID NO: 15) of the embodiment 4 through vfGP, comprising the following steps:

(1) Target cell construction: PBMC are separated from peripheral blood of an IMN patient, igG secretion B cells are separated by CD19 magnetic beads and anti-human IgG magnetic beads in a step-by-step manner, the EB virus is converted to immortalize the B cells, AAP-IMN (with the sequence shown as SEQ ID NO: 28) positive reaction cloning is selected by a limiting dilution method, and subculture is carried out to obtain the EBV-IMN-B cells.

(2) EBV-IMN-B cells were seeded in 6-well plates to adjust the cell concentration to 2X 10 ⁵ 1 mL/well; 1mL of cells were added to each well at a concentration of 2X 10 ⁶ /mL of regulatable universal CAR-T cells.

(3) A suitable amount of the vfGP-IMN stock solution prepared in step 2 of example 2 was diluted with sterile PBS solution to the following concentration gradient: 0. 0.01, 0.1, 1, 10nM.

(4) Different concentrations of vfGP-IMN were added to the 6-well plate of step 2, 20 μl per well, i.e. the final vfGP-IMN concentrations were respectively: 0. 0.1, 1, 10, 100pM; 3 compound holes are arranged on each concentration, and the mixture is evenly mixed and then placed in a carbon dioxide incubator for incubation for 24 hours.

(5) Lactate Dehydrogenase (LDH) release assay: LDH content in the culture supernatant was detected using an LDH cytotoxicity detection kit (available from Invitrogen, cat# C20300), and the killing activity of the controllable universal CAR-T cells (CAR gene sequence shown in SEQ ID NO: 15) on target cells was analyzed. The statistical results are shown in Table 2:

TABLE 2 detection results of LDH Release test

The results of the table above show: after the vfGP-IMN is added, the LDH enzyme activity in the culture supernatant is obviously increased, and the concentration of the LDH enzyme is related to the concentration of the vfGP-IMN; wherein, when the final concentration of the vfGP-IMN reaches 10pM, the LDH enzyme activity reaches the peak value.

(6) Detection of regulatable universal CAR-T cell activation using flow cytometry: after labeling the cells with CD3-percp, CD25-PE, CD69-APC antibodies, they were detected by flow cytometry. Wherein the activated CAR-T cells are cd3+/cd25+/cd69+.

The results of the flow test are shown in figure 2. The results show that: after addition of vfGP-IMN, the proportion of activated regulatable universal CAR-T cells was significantly increased and correlated with the vfGP-IMN concentration.

(7) Flow cytometry detects target cell killing rate: after the cells are marked by CD3-percp and CD19-APC, the detection is carried out by a flow cytometer, and the killing rate of the target cells is analyzed; wherein the target cell is CD3-/CD19+, and the controllable universal CAR-T cell is CD3+/CD19-. The calculation method comprises the following steps:

target cell killing (%) = [ (proportion of target cells in no-vfGP-IMN group-proportion of target cells in vfGP-IMN group)/(proportion of target cells in no-vfGP-IMN group) ]. Times.100%.

The statistics of the killing rate of the target cells are shown in FIG. 3. The results show that: after the addition of the vfGP-IMN, the proportion of target cells is obviously reduced, and the relationship with the concentration of the vfGP-IMN exists; wherein, when the final concentration of the vfGP-IMN reaches 10pM, the target cells are basically undetectable, and the killing rate reaches 100%.

Example 6

Taking a Goodpasture syndrome rat model as an example, the embodiment provides an experimental method for verifying that regulatable general CAR-T cells kill target cells through vfGP by using an animal model, comprising the following steps:

(1) The Goodpasture syndrome autoantibody affinity peptide P14 (SEQ ID NO: 33) was synthesized by Shanghai blaze biological company, dissolved in PBS at 0.4mg/mL, and filtered for sterilization.

(2) Construction of GBM rat model: healthy female WKY rats, 4 weeks old, weighing 65-80g, were subjected to adaptive feeding for 1 week, and after emulsification of the P14 peptide in step 1 with an equal volume of Freund's complete adjuvant, were subjected to single immunization by multi-point injection into the hind foot pad of the rat at a dose of 200. Mu.g/kg.

(3) Preparation of the regulatable general CAR-T cells: PBMC cells were isolated from WKY rat whole blood; and (3) after the T cell expansion culture medium is adopted for expansion, the CD3 magnetic beads are used for sorting to obtain the T cells. Activating T cells with anti-CD 3 and anti-CD 28 monoclonal antibodies; transfecting the activated T cells with the CAR lentivirus to obtain regulatable universal CAR-T cells; the adjustable and controllable general CAR-T cells are amplified by adopting a T cell amplification culture medium.

(4) Preparing vfGP: the Shanghai blaze biological company is entrusted to synthesize the vfGP-P14, and the vfGP-P14 is dissolved into 2mg/mL stock solution by DMSO; an appropriate amount of stock solution was then diluted to 1nM in sterile PBS for use.

(5) Experimental grouping: model rats after 4 weeks of P14 peptide immunization were randomized into model and treatment groups of 10 animals each. Will be 1X 10 ⁷ The adjustable general CAR-T cells (CAR gene sequence is shown as SEQ ID NO: 15) obtained in the step 3 are infused into rats of the treatment group from the tail vein, and 100 mu L of vfGP-P14 with the concentration of 1nM is infused into the rats through the tail vein after 6 hours. The model group was fed with an equal volume of physiological saline only through the tail vein.

(6) Monitoring indexes: and (3) detecting the content change of 24h urine protein, serum urea nitrogen and serum creatinine of each group of rats by taking week as a time observation unit.

The statistical results are shown in Table 3 below:

TABLE 3 changes in 24h urine protein, serum Urea Nitrogen and serum creatinine levels in rats

The results of the table above show: compared with a control group, the 24h urine protein, serum urea nitrogen and serum creatinine content of rats in the treatment group are obviously improved, which indicates that the application of the adjustable universal CAR-T cell combined vfGP-P14 treatment obviously improves the kidney function of the rat model of Goodpasture syndrome.

Example 7

This example provides affinity comparison of the anti-leader polypeptide single chain antibody described above with the anti-GCN single chain antibody disclosed in patent document CN 108795876. The affinity constants of the anti-leader polypeptide single-chain antibody in the present application and the anti-GCN single-chain antibody disclosed in patent document CN108795876 were determined by a non-competitive ELISA method, respectively. The method comprises the following specific steps:

(1) The complete anti-GCN scFv gene was synthesized according to the anti-GCN single chain antibody sequence disclosed in patent document CN108795876, and the anti-GCN scFv gene was cloned into a eukaryotic expression vector. Transiently transfecting the anti-GCN scFv expression vector into CHO cells to express recombinant anti-GCN scFv, and collecting culture supernatant. Coupling the guide polypeptide to N-hydroxysuccinimide activated agarose to prepare an antigen affinity purification column, carrying out affinity purification on the culture supernatant to collect recombinant anti-GCN scFv, and determining the protein concentration by a Coomassie brilliant blue method.

(2) Polypeptide synthesis: GCN peptide disclosed in patent document CN108795876 was synthesized by Shanghai blaze biosystems.

(3) GCN peptide was dissolved in DMSO to 2mg/mL stock.

(4) And (3) coating an ELISA plate: the GP polypeptide stock solution prepared in example 2 was diluted with carbonate buffer (0.05 m, ph 9.6) to 3 concentrations: 2. Mu.g/mL, 1. Mu.g/mL, 0.5. Mu.g/mL, 96-well ELISA plates were added at 100. Mu.L per well and coated overnight at 4 ℃. GCN peptide coated ELISA plates were prepared in the same manner. After the coating, the antigen solution in the wells of the enzyme-labeled plate was discarded, and 200. Mu.L of PBST solution was added to each well and washed 1 time. Closing: 200. Mu.L of PBS (0.01M, pH 7.4) solution containing 5% Bovine Serum Albumin (BSA) was added to each well, and the wells were blocked at room temperature for 2 hours. An appropriate amount of the recombinant anti-GP scFv obtained in the preparation of example 1 was diluted to 0.5. Mu.g/mL (corresponding to 20 nM) with PBS (0.01M, pH 7.4) containing 1% BSA, and then subjected to a double dilution to 1:2-128 times. The anti-GCN scFv with different concentration gradients is diluted and prepared in the same way. After the end of the sealing, the enzyme label is discardedBlocking solution in the plate holes, adding 100 mu L of gradient antibody solution in the steps, and designing 3 compound holes for each combination; a blank well was additionally provided, 100. Mu.L of a 1% BSA in PBS (0.01M, pH 7.4) was added and incubated at room temperature for 2 hours. After the incubation, the wells were discarded, washed 4 times with 250. Mu.L of PBST solution, and patted dry. 100. Mu.L of HRP-labeled goat anti-mouse IgG Light-Chain Specific Antibody was added to the wells of the enzyme-labeled wells and incubated for 1 hour at room temperature. After the incubation, the wells were discarded, washed 5 times with 250. Mu.LPBST solution and patted dry. 100 mu L of TMB color development solution is added into the wells of the enzyme-labeled plate, and the reaction is carried out at room temperature for 10-15 minutes in a dark place. 100 mu L of stop solution is added into the plate hole of the enzyme label plate, and the enzyme label plate is placed in an enzyme label instrument to measure the absorbance value (OD) of 450nm ₄₅₀ )。

On the abscissa, the concentration of monoclonal antibody, and the corresponding OD _450nm The values are plotted on the ordinate and the response curve is shown in figure 4. Determining the binding reaction platform phase of the competitive ELISA reaction curve by a 5% progressive method, and taking the antibody concentration when the maximum OD value is half under different antigen coating concentrations by a mapping method to obtain [ Ab ]] _t 。

According to formula K _a ＝(n-1)/(2[Ab′] _t -[Ab] _t ) The affinity constant is calculated. Wherein n= [ Ag ]] _t /[Ag′] _t ，[Ag] _t And [ Ag ]'] _t For different coating antigen concentrations; [ Ab ]] _t 、[Ab′] _t Is the maximum OD on the reaction curve corresponding to the concentration of different coating antigens _450nm Half the value corresponds to the concentration of antibody. Because of setting 3 different antigen coating concentrations, the combination of two pairs can obtain 3K _a The average value is the affinity constant of the antibody.

The statistics of the affinity constants are shown in Table 4 below:

TABLE 4 affinity constants of anti-GP scFv and anti-GCN scFv

The results showed that at the same antibody concentration, the OD of anti-GP scFv _450nm The value is far higher than anti-GCN scFv. Root of Chinese characterAccording to the formula, the affinity constant of the anti-GP scFv is calculated as follows: (6.76.+ -. 1.79). Times.10 ⁹ L/mol. In contrast, no positive anti-GCN scFv was detected in the range of 10nM to 0.07813 nM. After repeated experiments are carried out to further increase the concentration of the anti-GCN scFv, the affinity constant of the anti-GCN scFv is measured as follows: (3.09.+ -. 0.19). Times.10 ⁶ L/mol。

These results indicate that the affinity of the anti-GP scFv for the target is much higher than the anti-GCN scFv disclosed in patent document CN108795876, suggesting that the target recognition specificity and binding capacity of the regulatable universal CAR-T cell using the anti-GP scFv as the antibody recognition region in the present application is much higher than the universal CAR-T cell disclosed in patent document CN 108795876.

Example 8

The embodiment provides a comparative test of treatment effect of a lupus nephritis model mouse, and is a comparative study of treating lupus nephritis by adopting a universal type CAR-T cell combined with vfGP and a patent document CN108795876 disclosed by the application.

Lupus nephritis mouse model: the NZB and NZW hybrid F1 generation mice were cultured until week 20 to construct a model of spontaneous lupus nephritis mice.

CAR-T cell preparation of the present application: isolating PBMC cells from mouse whole blood; and (3) after the T cell expansion culture medium is adopted for expansion, the CD3 magnetic beads are used for sorting to obtain the T cells. The remaining procedure was as described in example 4, with reference to the construction of mouse CAR-T cells.

CN108795876-CAR-T cells were prepared as described in example one of the CN108795876 patents.

The lupous nephritis vfGP-LN (SEQ ID NO: 23) and the CN108795876 patent PdN peptide are synthesized by Shanghai blaze biological company, and are dissolved into 2mg/mL stock solution by DMSO; an appropriate amount of stock solution was then diluted to 1nM in sterile PBS for use.

Grouping and processing: female NZB/W F1 mice of 20 weeks of age were randomly divided into 3 groups of 15 animals each. The method comprises the following steps:

model group: 20. Mu.L of sterile PBS was injected by tail vein.

The application group: will be 1X 10 ⁷ Individual regulatable universal CAR-T cells of the present application are injected into mice from tail veinsIn vivo. After 6h, 20. Mu.L of vfGP-LN (final concentration 10 pM) at a concentration of 1nM was injected intravenously into the tail of the mice.

Patent CN108795876 group: will be 1X 10 ⁷ The individual CN108795876-CAR-T cells were injected into mice from the tail vein. After 6h, 20. Mu.L of PdN peptide (final concentration 10 pM) at a concentration of 1nM was injected intravenously into the tail of the mice.

After the completion of the administration, the death of each group of mice was recorded until all mice in the model group died. Survival curves were generated at week 0 when dosing was completed, as shown in fig. 5.

The results show that the survival of the mice in the CN108795876 group is improved to a certain extent compared with the mice in the model group, but the difference is not obvious (p= 0.1894); the mice in the present application had significantly longer survival than the mice in the CN108795876 patent and model group (p values < 0.0001).

Renal function monitoring in mice: each group of mice was tested every 2 weeks for changes in the levels of urine protein, serum urea nitrogen, serum creatinine and anti-ds-DNA antibodies for 24h until the mortality of the model group mice exceeded 50% (week 23).

As shown in fig. 6, the results show: the levels of 24h urine protein, serum urea nitrogen, serum creatinine and anti-ds-DNA antibodies were reduced in the CN108795876 mice versus the model mice, but the overall was still gradually elevated over time, indicating that the trend of progressive deterioration of kidney function in the model mice was unchanged.

On the contrary, the levels of 24h urine protein, serum urea nitrogen, serum creatinine and anti-ds-DNA antibodies of the model mice in the application group are obviously reduced compared with those of the model mice in the patent CN108795876 group and the model group, and the whole model mice show a trend of gradually reducing along with time, which indicates that the kidney function of lupus nephritis model mice can be gradually recovered by applying the adjustable and controllable general CAR-T cell combined vfGP-LN treatment.

In general, a method of using the above-described regulatable universal CAR-T cell and vfGP therapy, comprising:

the first stage: and (3) in-vitro preparation and reinfusion of the adjustable general CAR-T cells. The procedures were performed according to conventional procedures, i.e., peripheral blood extraction, T cell sorting, T cell activation (activation with anti-CD 3/CD28 or artificial APC, etc.), CAR transfection (electrotransfection, lentivirus, etc.), CAR-T cell expansion and reinfusion.

And a second stage: infusion vfGP activated in vivo. Different kinds and dosages of vfGP are administered according to the kind of disease, the activity degree of the disease (main autoantibody titer, etc.), and can be adjusted later according to the clinical change of the disease. After administration, the in vivo regulatable general CAR-T cells will be mobilized and guided to attack self-reactive B cells, thus achieving the purpose of accurately treating diseases.

It is to be noted that the above examples are only limited to further explanation and illustration of the technical solution of the present invention, and are not to be construed as further limiting the technical solution of the present invention, but are only preferred embodiments of the method of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A gene for coding the single-chain antibody of anti-guide polypeptide features that its sequence is shown in SEQ ID NO. 11.

2. An anti-leader polypeptide single chain antibody, which is characterized in that the sequence is shown as SEQ ID NO. 12.

3. The coding gene of the regulatable general CAR is characterized in that the sequence of the coding gene mainly comprises a CD3 zeta section, a co-stimulatory section, a transmembrane section, a hinge section, an anti-leader polypeptide single-chain antibody section and a signal peptide section, and the gene sequence of the anti-leader polypeptide single-chain antibody section is shown as SEQ ID NO. 11.

4. The coding gene of the regulatable general CAR according to claim 3, wherein the gene sequence carried by the cd3ζ segment is shown in SEQ ID No. 1; and/or

The gene sequence of the costimulatory region is shown as SEQ ID NO. 3 or SEQ ID NO. 5; and/or

The gene sequence of the transmembrane segment is shown as SEQ ID NO. 7; and/or

The gene sequence of the hinge section is shown as SEQ ID NO. 9; and/or

The gene sequence of the signal peptide segment is shown in SEQ ID NO. 13.

5. The coding gene of the controllable universal CAR according to claim 4, wherein the sequence is shown in SEQ ID NO. 15 or SEQ ID NO. 17.

6. An expression vector comprising the gene encoding the regulatable universal CAR of claim 5.

7. The adjustable general CAR is characterized in that the amino acid sequence is shown as SEQ ID NO. 16 or SEQ ID NO. 18.

8. A CAR-T cell comprising the anti-leader polypeptide single chain antibody of claim 1 or the regulatable universal CAR of claim 7.

9. A fusion guide polypeptide for matching the application of the regulatable general CAR-T cell of claim 8, which is characterized in that the fusion guide polypeptide is formed by sequentially connecting a guide polypeptide, a connector and an autoantibody affinity peptide, the sequence of the guide polypeptide is shown as SEQ ID NO. 19, and the sequence of the fusion guide polypeptide is shown as SEQ ID NO. 21, SEQ ID NO. 23 and SEQ ID NO. 26.