CN115976035A - Regulatable universal CAR, CAR-T cell and fusion leader polypeptide - Google Patents
Regulatable universal CAR, CAR-T cell and fusion leader polypeptide Download PDFInfo
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Abstract
The invention provides a regulatable universal CAR, CAR-T cell and variable fusion leader polypeptide for treating autoimmune nephropathy. The adjustable and controllable universal CAR-T cell has a Chimeric Antigen Receptor (CAR) which can specifically recognize and combine with a specific guide sequence, and can be activated, guided and regulated through variable fusion guide polypeptide, so that different autoreactive B cells can be accurately recognized and killed in a targeted manner, and the purpose of treating different autoimmune nephropathy is achieved.
Description
Technical Field
The invention relates to the technical field of immunotherapy, and particularly relates to a regulatable universal CAR and CAR-T cell for treating autoimmune nephropathy and a fusion guide polypeptide.
Background
Autoimmune nephropathy is an autoimmune-mediated kidney injury, a major cause of chronic kidney disease worldwide. Autoimmune nephropathy actually belongs to a class of autoimmune diseases, mainly including the manifestation of systemic autoimmune response diseases in the kidney and kidney-specific autoimmune diseases; the former is Lupus Nephritis (LN), and the latter is Idiopathic Membranous Nephropathy (IMN), igA nephropathy (IgAN), goodpasture syndrome, etc. Autoantibodies are a marker of many autoimmune renal diseases, where the detection of autoantibodies in the blood or kidney is critical to diagnosis. Currently, a number of key autoantibodies and their antigens in kidney disease have been identified, such as anti-PLA 2R, anti-Gd-IgA 1, anti-dsDNA, anti-ANCA and anti-GBM antibodies. These autoantibodies attack kidney tissue, form antigen-antibody complexes that deposit on the kidney and elicit antibody-mediated immune responses, leading to kidney damage. Each autoantibody is secreted by its corresponding autoreactive B cell clone. Thus, autoimmune nephropathy is essentially a B cell disease, as well as B cell malignancies.
Currently, the treatment regimen for autoimmune nephropathy mainly includes immunosuppressants and hormonal drugs. However, these drugs bring about a systemic immunosuppressive effect, thereby increasing the risk of serious complications such as infection and cancer. Antibody-based drugs or cell therapy protocols targeting B cells are one of the directions being explored. For example, anti-CD 20 monoclonal antibody (rituximab) can combine with CD20 molecules on the surface of B cells to mediate and kill B cells, and the clinical effect of the anti-CD 20 monoclonal antibody in treating systemic lupus erythematosus, particularly in severe lupus nephritis is very obvious. Rituximab can also significantly reduce the level of anti-PLA 2R antibodies in patients while treating membranous nephropathy. Rituximab may also be used in the treatment of ANCA-associated vasculitis. However, unlike B cell malignancies, pathogenic autoreactive B cell clones account for a very small percentage of human B cells and extensive B cell clearance is not the best choice.
CAR-T cell technology is a breakthrough innovation technology in the field of tumor therapy in recent years. The technology is characterized in that a Chimeric Antigen Receptor (CAR) is formed by coupling a single-chain antibody (scFv) capable of recognizing a tumor-associated antigen and an intracellular domain (comprising a co-stimulatory domain and a signal transduction domain), and a natural T cell is transfected in vitro by a gene transduction method to form a CAR-expressing T cell (CAR-T cell for short). CAR-T cells are able to bind tumor antigens in an antigen-dependent, non-MHC-restricted manner, initiating and activating 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 nephropathy by using a universal CAR-T cell fusion polypeptide guide, which can conveniently cope with the heterogeneity of autoreactive B cells in patients with autoimmune nephropathy. However, the sequence of the single-chain antibody of the universal CAR-T cell chimeric antigen receptor disclosed in the patent is short, and analysis shows that the CDR3 region of the heavy chain may be deleted, which will affect the recognition specificity and binding ability of the CAR-T cell, and further affect the application of the CAR-T cell in the treatment of autoimmune nephropathy.
Disclosure of Invention
Based on this, the invention provides a regulatable universal CAR, CAR-T cell and variable fusion leader polypeptide for treating autoimmune nephropathy.
The invention adopts the following technical scheme:
the invention provides a gene for coding an anti-leader polypeptide single-chain antibody (anti-GP scFv), the sequence of which is shown in SEQ ID NO. 11 or SEQ ID NO. 27. The sequence of the anti-leader polypeptide single-chain antibody is shown in SEQ ID NO. 12.
The invention also provides a coding gene of the controllable universal CAR, the sequence of the coding gene mainly comprises a CD3 zeta segment, a costimulation segment, a transmembrane segment, a hinge segment, an anti-leader polypeptide single-chain antibody segment and a signal peptide segment, and the gene sequence of the anti-leader polypeptide single-chain antibody segment is shown as SEQ ID NO. 11 or SEQ ID NO. 27. The gene sequence carried by the CD3 zeta segment is shown in SEQ ID NO 1. The gene sequence of the co-stimulation section is shown as SEQ ID NO. 3 or SEQ ID NO. 5. The gene sequence of the transmembrane segment is shown as SEQ ID NO. 7. The gene sequence of the hinge segment is shown as SEQ ID NO. 9. The gene sequence of the signal peptide segment is shown as SEQ ID NO. 13.
Preferably, the sequence of the gene encoding the regulatable universal CAR is shown as SEQ ID NO. 15 or SEQ ID NO. 17.
The invention also provides an adjustable universal CAR, and the sequence of the adjustable universal CAR is shown as SEQ ID NO. 16 or SEQ ID NO. 18.
The invention also provides a CAR-T cell comprising the above-described anti-homing polypeptide single-chain antibody or the above-described regulatable universal CAR.
The invention also provides a variable fusion leader polypeptide (vfGP) for matching the application of the regulatable universal CAR-T cell, which consists of a leader polypeptide, a linker and an autoantibody affinity peptide which are connected in sequence, wherein the sequence of the leader polypeptide is shown as SEQ ID NO. 19.
Preferably, the sequence of vfGP is shown in 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-leader polypeptide single-chain antibody (anti-GP scFv) expressed by the controllable universal CAR-T cell designed by the invention can recognize a specific leader polypeptide sequence and has extremely high affinity with the leader polypeptide sequence, so that the anti-leader polypeptide single-chain antibody can be matched with vfGP containing the leader polypeptide sequence to efficiently recognize and kill corresponding autoreactive B cells.
2) The adjustable and controllable universal CAR designed by the invention can be applied to different autoimmune nephropathy, is different from the traditional CAR-T cell which needs to design different CAR aiming at different diseases, and can shorten the CAR-T cell preparation time and reduce the preparation cost.
3) The regulatable universal CAR-T cells designed by the invention do not directly recognize target cells, so that abnormal autoreactive B cells cannot be attacked immediately after the cells are back-infused into a patient; only after subsequent infusion of vfGP will target cells be attacked under the guidance of the latter, and the efficacy of the attack is related to the dose of vfGP infused. Based on the characteristics, the immune effect strength of the CAR-T cells in vivo can be conveniently controlled, and side reactions such as cytokine storm and other risks in the general CAR-T cell treatment can be reduced.
4) The adjustable universal CAR-T cell has a Chimeric Antigen Receptor (CAR) which can specifically recognize and bind to a specific guide sequence, and can be activated, guided and adjusted through vfGP, so that different autoreactive B cells can be accurately recognized and killed in a targeted manner, and the purpose of treating different autoimmune nephropathy is achieved. The application of the adjustable and controllable universal CAR-T cells and the vfGP in the aspect of treating the autoimmune nephropathy can be expanded to the autoimmune nephropathy, and when a treatment system for a new disease species is developed, only the new vfGP needs 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 chart of the affinity and specificity test of the recombinant anti-GP scFv and vfGP in example 2.
Fig. 2 is a flow cytometry assay and a statistical chart of the vfGP activation-regulated universal CAR-T cells in example 5.
Fig. 3 is a statistical chart of the killing effect of vfGP mediated regulatable universal CAR-T cells on target cells detected by flow cytometry in example 5.
FIG. 4 is a graph of the competitive ELISA reaction in example 7.
FIG. 5 is a survival curve of the experimental mouse in example 8.
FIG. 6 shows the variation of the levels of 24h urine protein, serum urea nitrogen, serum creatinine and anti-ds-DNA antibody in the experimental mice of example 8.
Detailed Description
The present invention is further described in detail below with reference to specific examples so that those skilled in the art can more clearly understand the present invention.
The following examples are provided only for illustrating the present invention, and are not intended to limit the scope of the present invention. All other embodiments obtained by a person skilled in the art based on the specific embodiments of the present invention without any inventive step are within the scope of the present invention.
In the examples of the present invention, all the raw material components are commercially available products well known to those skilled in the art, unless otherwise specified; in the examples of the present invention, unless otherwise specified, all the technical means used are conventional means well known to those skilled in the art.
A gene encoding a regulatable universal Chimeric Antigen Receptor (CAR) comprises a CD3 zeta segment, a costimulatory segment, a transmembrane segment, a hinge segment, an anti-homing polypeptide single-chain antibody segment, and a signal peptide.
Wherein, the gene sequence carried by the CD3 zeta segment is as follows:
agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc(SEQ ID NO:1)。
the translated protein sequence of the gene carried by the CD3 ζ segment is as follows:
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:2)。
the gene sequence carried by the costimulatory segment is selected from one of the following 2 gene sequences:
gene sequence of CD28 costimulatory segment:
aggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctcc(SEQ ID NO:3)。
the protein sequence for translation of the CD28 costimulatory segment is as follows:
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO:4)。
gene sequence of CD137 costimulatory segment:
aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg(SEQ ID NO:5)。
the protein sequence for translation of the CD137 costimulatory segment is as follows:
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:6)。
the transmembrane segment carries the gene sequence:
atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaccacaggaac(SEQ ID NO:7)。
the sequence of the protein translated by 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 protein sequence translated by 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 for gene translation of the anti-leader polypeptide single chain antibody segment 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 translated protein sequence of the signal peptide segment gene is as follows:
MALPVTALLLPLALLLHAARP(SEQ ID NO:14)。
preferably, the complete gene sequence of the chimeric antigen receptor (svCAR) gene comprising the CD28 co-stimulatory segment is as follows:
atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggatgcggtggtgacccaggaaagcgcgctgaccaccagcccgggcgaaaccgtgaccctgacctgccgcagcagcaccggcgcggtgaccaccagcaactatgcgagctgggtgcaggaaaaaccggatcatctgtttaccggcctgattggcggcaccaacaaccgcgcgccgggcgtgccggcgcgctttagcggcagcctgattggcgataaagcggcgctgaccattaccggcgcgcagaccgaagatgaagcgatttatttttgcgcgctgtggtatagccatcattgggtgtttggcggcggcaccaaactgaccgtgctgggcggcagcaccagcggcagcggcaaaccgggcagcggcgaaggcagcaccaaaggcgatgtgcagctgcaggaaagcggcccgggcctggtggcgccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccgattatggcgtgaactgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggggcgatggcattaccgattataacagcgcgctgaaaagccgcctgagcgtgaccaaagataacagcaaaagccaggtgtttctgaaaatgaacagcctgcagagcggcgatagcgcgcgctattattgcgtgaccggcctgtttgattattggggccagggcaccaccctgaccgtgagcagcgagtccaagtatgggcctccatgccctccatgtcctatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaccacaggaacaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc(SEQ ID NO:15)。
the protein sequences translated by the chimeric antigen receptor (svCAR) gene comprising the CD28 costimulatory segment are as follows:
MALPVTALLLPLALLLHAARPDAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYASWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSHHWVFGGGTKLTVLGGSTSGSGKPGSGEGSTKGDVQLQESGPGLVAPSQSLSITCTVSGFSLTDYGVNWVRQSPGKGLEWLGVIWGDGITDYNSALKSRLSVTKDNSKSQVFLKMNSLQSGDSARYYCVTGLFDYWGQGTTLTVSSESKYGPPCPPCPIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:16)。
preferably, the sequence of the chimeric antigen receptor (svCAR) gene comprising the CD137 co-stimulatory segment is as follows:
atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggatgcggtggtgacccaggaaagcgcgctgaccaccagcccgggcgaaaccgtgaccctgacctgccgcagcagcaccggcgcggtgaccaccagcaactatgcgagctgggtgcaggaaaaaccggatcatctgtttaccggcctgattggcggcaccaacaaccgcgcgccgggcgtgccggcgcgctttagcggcagcctgattggcgataaagcggcgctgaccattaccggcgcgcagaccgaagatgaagcgatttatttttgcgcgctgtggtatagccatcattgggtgtttggcggcggcaccaaactgaccgtgctgggcggcagcaccagcggcagcggcaaaccgggcagcggcgaaggcagcaccaaaggcgatgtgcagctgcaggaaagcggcccgggcctggtggcgccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccgattatggcgtgaactgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggggcgatggcattaccgattataacagcgcgctgaaaagccgcctgagcgtgaccaaagataacagcaaaagccaggtgtttctgaaaatgaacagcctgcagagcggcgatagcgcgcgctattattgcgtgaccggcctgtttgattattggggccagggcaccaccctgaccgtgagcagcgagtccaagtatgggcctccatgccctccatgtcctatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaccacaggaacaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc(SEQ ID NO:17)。
the protein sequences translated by the chimeric antigen receptor (svCAR) gene comprising the CD137 co-stimulatory segment are as follows:
MALPVTALLLPLALLLHAARPDAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYASWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSHHWVFGGGTKLTVLGGSTSGSGKPGSGEGSTKGDVQLQESGPGLVAPSQSLSITCTVSGFSLTDYGVNWVRQSPGKGLEWLGVIWGDGITDYNSALKSRLSVTKDNSKSQVFLKMNSLQSGDSARYYCVTGLFDYWGQGTTLTVSSESKYGPPCPPCPIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ IDNO:18)。
a regulatable universal CAR-T cell, prepared by steps comprising: entrusting a biological company to synthesize a complete controllable universal CAR gene sequence with homologous arms at two ends, wherein the sequence is shown as SEQ ID NO. 15 or SEQ ID NO. 17; cloning the CAR gene sequence into a lentiviral vector by using a homologous recombinase in a homologous recombination mode, and transfecting 293T cells together with a lentiviral packaging plasmid to prepare CAR lentiviruses; transfecting the activated T cells with the CAR lentivirus to obtain regulatable universal CAR-T cells.
A variable fusion leader polypeptide (vfGP) matched with a controllable universal CAR-T cell consists of a leader polypeptide (GP), a linker and an 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 connecting the leader polypeptide and the autoantibody affinity peptide, and the amino acid sequence of the linker is as follows:
GGGGS(SEQ ID NO:20)。
autoantibody Affinity Peptides (AAPs) are polypeptide fragments that can specifically bind to the BCR molecule of autoreactive B cells, the amino acid sequence of which is variable and corresponds to autoreactive B cells as a target of attack.
In particular, vfGP may be a vfGP-IMN for the treatment of Idiopathic Membranous Nephropathy (IMN) in combination with regulatable universal CAR-T cells, the sequence being as follows:
HLENEVARLKKLVGERGGGGSWQDKGIFVIQSESLKKCIQAGKSVLTLENCK(SEQ ID NO:21)。
in particular, vfGP can be a vfGP-IgAN for the treatment of IgA nephropathy (IgAN) in combination with regulatable universal CAR-T cells, the sequence being as follows:
HLENEVARLKKLVGERGGGGSYCSKVCRPWNYRRPYYYGMDVW(SEQ ID NO:22)。
in particular, vfGP can be a vfGP-LN for 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, vfGP can be a vfGP-MA for treating MPO-ANCA-associated vasculitis (MPO-AAV) in combination with a regulatable universal CAR-T cell, the sequence being as follows:
HLENEVARLKKLVGERGGGGSRLDNRYQPMEPN(SEQ ID NO:24)。
in particular, vfGP may be a vfGP-PA for the treatment of PR 3-ANCA associated vasculitis (PR 3-AAV) in combination with regulatable universal CAR-T cells, the sequence being as follows:
HLENEVARLKKLVGERGGGGSVVLGAHNVRTQ(SEQ ID NO:25)。
in particular, vfGP may be vfGP-P14 for the treatment of Goodpasture syndrome in combination with regulatable universal CAR-T cells, the sequence being as follows:
HLENEVARLKKLVGERGGGGSTDIPPCPHGWISLWKGFSFIMF(SEQ ID NO:26)。
the sequences of the Autoantibody Affinity Peptides (AAPs) corresponding to the above-mentioned 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 examples illustrate:
example 1
The present embodiment provides a method for preparing an anti-leader polypeptide single-chain antibody (anti-GP scFv), comprising the following steps:
(1) Preparing an antigen: shanghai Qiaozhiya Bio was committed to synthesize the leader polypeptide (GP) as shown in SEQ ID NO:19 above, and the GP was conjugated to the KLH carrier protein using a KLH protein conjugation kit (Bioquest, USA).
(2) Immunizing a mouse: after emulsifying the antigen prepared in step 1 with Freund's adjuvant, balb/c mice were immunized by the conventional protocol.
(3) Preparing a hybridoma cell strain: after the three-immunization, spleen cells of the mice are extracted and fused with SP2/0 cells to prepare hybridoma cells. And culturing and subcloning hybridoma cells according to a conventional process, and screening a hybridoma cell strain capable of secreting the high-affinity anti-GP monoclonal antibody by enzyme-linked immunosorbent assay.
(4) Sequencing monoclonal antibody: entrusted Wu Hanpu Jian biological company to sequence the screened hybridoma cell strain 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 finished by Wu Hanpu Jian biological company, and the steps are as follows:
(1) connecting the anti-GP monoclonal antibody light chain and heavy chain genes determined in the step 4 by a linker gene sequence (see italic underlining in the sequence) to form a complete anti-GP scFv gene sequence:
(2) synthesizing a complete anti-GP scFv gene, and cloning the anti-GP scFv gene to a eukaryotic expression vector.
(3) Transient transfection of an anti-GP scFv expression vector into CHO cells to express recombinant anti-GP scFv, and collection of 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 the 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 Qianyao organism company was entrusted with the synthesis of polypeptides represented by SEQ ID NO 19, SEQ ID NO 21-26 and SEQ ID NO 28-33A peptide. (2) Taking a proper amount of the polypeptide, and dissolving the polypeptide into DMSO to obtain 2mg/mL stock solutions for later use. (3) coating an enzyme label plate: an appropriate amount of GP stock was diluted to 1. Mu.g/mL with coating buffer (0.05M, pH 9.6 carbonate buffer), and a 96-well microplate was added at 100. Mu.L per well and coated overnight at 4 ℃. (4) After the coating is finished, the antigen solution in the wells of the enzyme-labeled plate is discarded, and 200 μ L of PBST solution is added into each well for washing for 1 time. (5) sealing: mu.L of a 5% Bovine Serum Albumin (BSA) in PBS (0.01M, pH 7.4) was added to each well and blocked for 2 hours at room temperature. (6) An additional amount of the polypeptide stock solution from step 2 was diluted with 1% BSA in PBS to the following concentration gradient: 0.1, 5, 25, 125nM. (7) antigen pre-blocking: an appropriate amount of the recombinant anti-GP scFv obtained in the preparation of example 1 was taken and diluted to 0.1. Mu.g/mL with 1% BSA in PBS. And (4) respectively taking the polypeptides with different concentration gradients in the step (6) and the recombinant anti-GP scFv solution, mixing in an equal volume, and pre-incubating for 1 hour at room temperature. (8) After sealing is finished, removing sealing liquid in the hole of the enzyme label plate, adding 100 mu L of the mixed solution in the step 7, and designing 3 compound holes for each combination; separately, a blank well was placed, and 100. Mu.L of 1% BSA in PBS was added and incubated at room temperature for 2 hours. (9) After incubation, the wells were drained, washed 4 times with 250 μ L PBST solution and patted dry. (10) mu.L of HRP-labeled goat anti-mouse IgG Light-Chain Specific Antibody was added to the wells of the microplate and incubated at room temperature for 1 hour. (11) After incubation, the wells were drained, washed 5 times with 250 μ L PBST solution and patted dry. (12) Adding 100 mu L of TMB color development liquid into the wells of the ELISA plate, and reacting for 10-15 minutes at room temperature in a dark place. (13) Adding 100 mu L of stop solution into the wells of the ELISA plate, placing the ELISA plate in an ELISA reader to determine the absorbance value (OD) of 450nm 450nm )。
The results of the competitive ELISA assay are shown in FIG. 1. The results show that: GP (SEQ ID NO: 19) and vfGP (SEQ ID NO: 21-26) can compete to inhibit the combination of the recombinant anti-GP scFv and GP coated on the enzyme label 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 newly constructed recombinant anti-GP scFv can specifically recognize GP and vfGP containing GP sequences, and does not have cross reaction with autoantibody affinity peptide.
Furthermore, the results of the study also show: OD measured by pre-blocking anti-GP scFv with equivalent concentrations of either vfGP or GP 450nm There was no significant difference in the values, indicating that the anti-GP scFv has similar affinity to both vfGP and GP.
Since the anti-GP scFv is the antigen recognition domain of the regulatable universal CAR-T cell described in the application, the above experimental results also show that the CAR-T cell can specifically recognize and bind to vfGP.
Example 3
This example provides an experimental process for demonstrating that vfGP can bridge anti-GP scFv and kidney disease patient autoantibody by sandwich ELISA method, which comprises the following steps:
collecting serum from a patient with autoimmune kidney disease containing autoantibodies, comprising: the serum of IMN patients containing anti-PLA 2R antibody is confirmed by clinical examination; the serum of the IgAN patient containing the anti-GdIgA 1 antibody is confirmed by clinical examination; LN patient sera confirmed by clinical testing to contain anti-dsDNA antibodies; the serum of patients with MPO type-ANCA related vasculitis (MPO-AAV) containing anti-MPO antibody is confirmed by clinical examination; the serum of a PR3 type-ANCA associated vasculitis (PR 3-AAV) patient containing an anti-PR 3 antibody is confirmed by clinical examination; the serum of Goodpasture syndrome patients containing anti-GBM antibodies was confirmed by clinical examination.
Coating an enzyme label plate: the recombinant anti-GP scFv obtained in example 1 was diluted to 2ug/mL with coating buffer, added to a 96-well plate at 100. Mu.L per well and coated overnight at 4 ℃. After the coating is finished, the liquid in the wells of the enzyme label plate is discarded, and 200 mu L of PBST solution is added into each well for washing for 1 time. And (3) sealing: mu.L of PBS (0.01M, pH 7.4) containing 5% BSA was added to each well and blocked for 2 hours at room temperature. An appropriate amount of the stock solution of the variable fusion leader 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 gradients: 0.1, 10, 100, 1000pM. After the blocking is finished, the blocking solution in the hole of the enzyme label plate is discarded, the vfGP with different concentration gradients is added, each concentration of each polypeptide is provided with 3 multiple holes, each hole is 100 mu L, and the incubation is carried out for 1 hour at room temperature. After incubation, the wells were drained, washed 4 times with 250 μ L PBST solution and patted dry. 1/10 of the serum of the patients with autoimmune nephropathyDiluting, adding the diluted solution into the corresponding holes of an ELISA plate (for example, adding IMN patient serum into the holes of the ELISA plate incubated by the vfGP-IMN peptide), and arranging 3 multiple holes for each sample; after the sample addition was complete, incubation was carried out at room temperature for 1 hour. After incubation, the wells were discarded and 250. Mu.L of PBST solution was added and washed 4 times, patted dry. mu.L of HRP-labeled goat anti-human IgG was added to the wells of the plate and incubated at room temperature for 1 hour. After incubation, the wells were drained, washed 5 times with 250 μ L PBST solution and patted dry. Adding 100 mu L of TMB color development liquid into the wells of the ELISA plate, and reacting for 10-15 minutes at room temperature in a dark place. Adding 100 mu L of stop solution into the wells of the ELISA plate, placing the ELISA plate in an ELISA reader to measure the absorbance value (OD) of 450nm 450nm )。
The sandwich ELISA assay results are shown in table 1:
TABLE 1 Sandwich ELISA test results
The OD value measured without addition of vfGP was 2.1 times as the positive cut-off value, and the results showed that: the OD values measured by adding the vfGP with different concentrations are positive, the OD value is in positive correlation with the vfGP concentration, and the result shows that the vfGP combined with the anti-GP scFv can be continuously and specifically combined with the autoantibody of the nephropathy patient.
Since the autoantibody of the nephropathy patient has the same antigen binding sequence with the autoreactive B cell BCR, these experimental results prove that the vfGP can be combined with the single-chain antibody region (i.e. anti-GP scFv) of the regulatable universal CAR-T cell and the BCR of the autoreactive B cell at the same time, so as to mediate the targeting of the regulatable universal CAR-T cell to the autoreactive B cell.
Example 4
The implementation provides a method for preparing a controllable universal CAR-T cell, which comprises the following steps:
(1) General Biometrics was entrusted to synthesize a complete regulatable general CAR gene sequence with homology arms at both ends, as shown in SEQ ID NO 15 or SEQ ID NO 17 above.
(2) The controllable universal CAR gene sequence is cloned to a pCDH-CMV-MCS-EF1-copGFP vector (purchased from Youbao organisms with the code of VT 1479) by utilizing a homologous recombinase in a homologous recombination mode to obtain the pCDH-CMV-MCS-EF1-copGFP-CAR vector.
(3) pCDH-CMV-MCS-EF1-copGFP-CAR vector was ligated with lentivirus packaging plasmids pLP1-gag/pol, pLP2-Rev and pLP/VSVG (purchased from Invitrogen, trade name: viraPower) TM Lentiviral Packaging Mix, cat # K497500) co-transfected 293T cells to prepare CAR lentiviruses.
(4) Peripheral Blood Mononuclear Cells (PBMC) were isolated from fresh human whole blood by 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 mabs.
(6) Transfecting the activated T cells with the CAR lentivirus to obtain regulatable universal CAR-T cells.
(7) Expanding the regulatable universal CAR-T cells using a T cell expansion medium.
Example 5
This example provides an experimental method for verifying the killing of target cells by the regulatable universal CAR-T cells (CAR gene sequence shown in SEQ ID NO: 15) of example 4 via vfGP in vitro, taking the autoreactive B cells of Idiopathic Membranous Nephropathy (IMN) patients as an example, comprising the following steps:
(1) Construction of target cells: PBMC is separated from peripheral blood of an IMN patient, igG secretory B cells are separated by CD19 magnetic beads and anti-human IgG magnetic beads step by step, EB virus is converted to immortalize the cells, AAP-IMN (shown as SEQ ID NO: 28) positive reaction clone is selected by a limiting dilution method, and the EBV-IMN-B cells are obtained by subculture.
(2) EBV-IMN-B cells were seeded in 6-well plates with cell concentration adjusted to 2X 10 5 1mL per well; 1mL of cells at 2X 10 concentration was added to each well 6 a/mL of a regulatable universal CAR-T cell.
(3) An appropriate amount of the vfGP-IMN stock 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 plates of step 2, 20 μ L per well, i.e. the final vfGP-IMN concentrations were: 0. 0.1, 1, 10, 100pM; 3 multiple holes are arranged for each concentration, and the mixture is evenly mixed and then placed in a carbon dioxide incubator to be incubated for 24 hours.
(5) Lactate Dehydrogenase (LDH) release assay: the LDH content in the culture supernatant was assayed using an LDH cytotoxicity assay kit (purchased from Invitrogen, cat # C20300) to analyze the killing activity of the regulatable universal CAR-T cells (CAR gene sequence shown in SEQ ID NO: 15) against target cells. The statistical results are shown in table 2:
TABLE 2 LDH Release test results
The results of the above table show: after the vfGP-IMN is added, the LDH enzyme activity in the culture supernatant is obviously increased, and the correlation exists with 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) Detecting the activation condition of the regulatable universal CAR-T cells by adopting flow cytometry: the cells were labeled with antibodies against CD3-percp, CD25-PE, and CD69-APC, and then detected by flow cytometry. Wherein the activated CAR-T cells are CD3+/CD25+/CD69+.
The flow test results are shown in FIG. 2. The results show that: after the addition of the vfGP-IMN, the proportion of activated controllable universal CAR-T cells is remarkably increased, and the correlation exists with the concentration of the vfGP-IMN.
(7) Flow cytometry detection of target cell killing rate: labeling cells with CD3-percp and CD19-APC, detecting by using a flow cytometer, and analyzing the killing rate of target cells; wherein the target cell is CD3-/CD19+ and the regulatable universal CAR-T cell is CD3+/CD19-. The calculation method is as follows:
target cell killing rate (%) = [ (ratio of target cells in the case without addition of vfGP-IMN-ratio of target cells in the case with addition of vfGP-IMN)/(ratio of target cells in the case without addition of vfGP-IMN) ] × 100%.
The target cell killing rate statistics are shown in FIG. 3. The results show that: after the vfGP-IMN is added, the proportion of target cells is obviously reduced, and the correlation exists with the concentration of the vfGP-IMN; wherein, when the final concentration of the vfGP-IMN reaches 10pM, the target cells can not be basically detected, and the killing rate reaches 100 percent.
Example 6
The present example provides an experimental method for verifying that controllable universal CAR-T cells kill target cells through vfGP using an animal model, taking Goodpasture syndrome rat model as an example, and the experimental method comprises the following steps:
(1) Goodpasture's syndrome autoantibody-affinity peptide P14 (SEQ ID NO: 29) was synthesized by Shanghai Qiaozhizobium, dissolved in PBS at 0.4mg/mL, and sterilized by filtration.
(2) GBM rat model construction: healthy female WKY rats, 4 weeks old, 65-80g in weight, after 1 week of acclimatization, were treated with an equal volume of Freund's complete adjuvant to emulsify the P14 peptide from step 1, and single immunizations were performed by multi-point injection at the foot pad after rats at a dose of 200. Mu.g/kg.
(3) Controllable universal CAR-T cell preparation: PBMC cells were isolated from WKY rat whole blood; and after the T cell amplification culture medium is adopted for amplification, the T cells are obtained by sorting CD3 magnetic beads. Activating T cells with anti-CD 3 and anti-CD 28 mabs; transfecting the activated T cells with CAR lentivirus to obtain regulatable universal CAR-T cells; the regulatable universal CAR-T cells are expanded using a T cell expansion medium.
(4) Preparation of vfGP: entrusting Shanghai Qiaozhizog biological company to synthesize vfGP-P14, and dissolving with DMSO to be 2mg/mL storage solution; then, an appropriate amount of the stock solution was diluted to 1nM with sterile PBS for use.
(5) Grouping experiments: model rats after 4 weeks of P14 peptide immunization were randomly divided into a model group and a treatment group, 10 rats per group. Will be 1 × 10 7 The regulatable universal CAR-T cells obtained in step 3 (CAR gene sequence is shown in SEQ ID NO: 15) were infused into the treated rats from the tail vein, and after 6 hours 100. Mu.L of vfGP-P14 was infused via the tail vein at a concentration of 1 nM. The model group was infused with an equal volume of saline only via the tail vein.
(6) Monitoring indexes are as follows: and (3) detecting the change conditions of 24h urine protein, serum urea nitrogen and serum creatinine content of each group of rats by taking a week as a time observation unit.
The statistical results are shown in table 3 below:
TABLE 3 variation of 24h urine protein, serum urea nitrogen and serum creatinine content in rats
The results of the above table show: compared with a control group, the 24h urine protein, serum urea nitrogen and serum creatinine content of the rats in the treatment group are all obviously improved, and the application of the controllable universal CAR-T cell combined vfGP-P14 treatment in the application can obviously improve the renal function of the rats in the Goodpasture syndrome model.
Example 7
This example provides a comparison of the affinity of the anti-homing polypeptide single-chain antibody described above with that of the anti-GCN single-chain antibody disclosed in patent document CN 108795876. The affinity constants of the anti-leader polypeptide single-chain antibody and the anti-GCN single-chain antibody disclosed in patent document CN108795876 were determined by non-competitive ELISA. The method comprises the following specific steps:
(1) According to the anti-GCN single-chain antibody sequence disclosed in patent document CN108795876, a complete anti-GCN scFv gene is synthesized, and the anti-GCN scFv gene is cloned to a eukaryotic expression vector. Transient transfection of an anti-GCN scFv expression vector into CHO cells to express recombinant anti-GCN scFv, and collection of 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: the Shanghai Qianyao organism company was requested to synthesize a GCN peptide disclosed in patent document CN 108795876.
(3) The GCN peptide was dissolved in DMSO to a stock solution of 2 mg/mL.
(4) Coating an enzyme label plate: GP polypeptide stock prepared in example 2 was taken and diluted with carbonate buffer (0.05m, ph 9.6) to 3 concentrations: 2. Mu.g/mL, 1. Mu.g/mL, 0.5 μ g/mL, 100 μ L per well of 96-well plate, 4 ℃ coated overnight. GCN peptide-coated ELISA plates were prepared in the same manner. After the coating is finished, the antigen solution in the wells of the enzyme-labeled plate is discarded, and 200 μ L of PBST solution is added into each well for washing for 1 time. And (3) sealing: mu.L of a 5% Bovine Serum Albumin (BSA) in PBS (0.01M, pH 7.4) was added to each well and blocked for 2 hours at room temperature. An appropriate amount of the recombinant anti-GP scFv obtained in the preparation of example 1 was diluted to 0.5. Mu.g/mL (equivalent to 20 nM) in PBS containing 1% BSA (0.01M, pH 7.4) and then diluted to 1:2-128 times. The anti-GCN scFv with different concentration gradients is prepared by dilution in the same way. After the sealing is finished, removing the sealing liquid in the hole of the enzyme-labeled plate, adding 100 mu L of the gradient antibody solution in the step, and designing 3 compound holes for each combination; separately, a blank well was placed, and 100. Mu.L of a 1% BSA-containing PBS (0.01M, pH 7.4) was added thereto, followed by incubation at room temperature for 2 hours. After incubation, the wells were drained, washed 4 times with 250 μ L PBST solution and patted dry. mu.L of HRP-labeled goat anti-mouse IgG Light-Chain Specific Antibody was added to the wells of the microplate and incubated at room temperature for 1 hour. After incubation, the wells were drained, washed 5 times with 250 μ LPBST solution and patted dry. Adding 100 mu L of TMB color development liquid into the wells of the ELISA plate, and reacting for 10-15 minutes at room temperature in a dark place. Adding 100 mu L of stop solution into the wells of the ELISA plate, placing the ELISA plate in an ELISA reader to measure the absorbance value (OD) of 450nm 450 )。
The concentration of the monoclonal antibody is used as the abscissa and the corresponding OD 450nm The values are plotted on the ordinate and the response curve is shown in FIG. 4. Determining the binding reaction plateau of the competitive ELISA reaction curve by 5% subtraction, and calculating the antibody concentration at half of the maximum OD value 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 was calculated. Wherein n = [ Ag =] t /[Ag′] t ，[Ag] t And [ Ag'] t Concentration of different coating antigens; [ Ab ]] t 、[Ab′] t Is the maximum OD on the reaction curve corresponding to the concentration of different envelope antigens 450nm Half the value corresponds to the concentration of antibody. Due to the arrangement of 3 different kindsAntigen coating concentration, and combining two by two to obtain 3 Ks a The average value is the affinity constant of the antibody.
The statistical results of the affinity constants are given in table 4 below:
TABLE 4 affinity constants of anti-GP scFv and anti-GCN scFv
The results show that the OD of the anti-GP scFv at the same antibody concentration 450nm The value is much higher than that of anti-GCN scFv. According 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, the anti-GCN scFv did not detect positivity in the range of 10nM to 0.07813 nM. After repeated experiments are carried out by further increasing 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 anti-GP scFv for target is much higher than that of anti-GCN scFv disclosed in patent document CN108795876, suggesting that the target recognition specificity and binding ability of the regulatable universal CAR-T cell using anti-GP scFv as antibody recognition region in the present application are much higher than that of the universal CAR-T cell disclosed in patent document CN 108795876.
Example 8
The present example provides a comparative experiment of therapeutic effect of lupus nephritis model mice, and a comparative study of treating lupus nephritis by using the combination of universal CAR-T cells and PdN peptide disclosed in patent document CN 108795876.
Lupus nephritis mouse model: NZB and NZW cross F1 generation mice cultured to the 20 th week to construct spontaneous lupus nephritis mouse model.
CAR-T cell preparation of the present application: isolating PBMC cells from mouse whole blood; and after the T cell amplification culture medium is adopted for amplification, the T cells are obtained by sorting CD3 magnetic beads. The remaining steps the mouse CAR-T cells were constructed, with reference to the method of example 4.
CN108795876-CAR-T cells were prepared as described in example one of the CN108795876 patent.
The Shanghai Qiangyao biology company is entrusted to synthesize lupus nephritis vfGP-LN (SEQ ID NO: 23) and CN108795876 patent PdN peptides, which are dissolved in DMSO to be 2mg/mL stock solutions; an appropriate amount of stock solution was then diluted to 1nM in sterile PBS for use.
Grouping and processing: female NZB/W F mice at 20 weeks of age were randomly assigned to 3 groups of 15 mice each. The method comprises the following specific steps:
model group: 20 μ L of sterile PBS was injected via tail vein.
Group of the present application: will be 1 × 10 7 The present application can regulate universal CAR-T cell injection into mice from tail vein. After 6h, 20. Mu.L of vfGP-LN at a concentration of 1nM (final concentration of 10 pM) was injected intravenously into the tail of the mice.
Patent CN108795876 group: will be 1 × 10 7 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) was injected intravenously into the tail vein of the mice at a concentration of 1 nM.
After dosing was complete, the mortality of each group of mice was recorded until all of the model group mice died. Survival curves were generated at week 0 after completion of administration, as shown in fig. 5.
The results show that the survival time of mice in the patent CN108795876 group is improved to some extent compared with that of the model group, but the difference is not significant (p = 0.1894); the survival time of the mice in the group of the application is obviously prolonged compared with that in the CN108795876 group and the model group (the p value is less than 0.0001).
Mouse renal function monitoring: the change of the levels of urine protein, serum urea nitrogen, serum creatinine and anti-ds-DNA antibody in each group of experimental mice was measured for 24 hours every 2 weeks until the mortality of the model group mice exceeded 50% (23 weeks).
As shown in fig. 6, the results show that: the levels of 24h urine protein, serum urea nitrogen, serum creatinine and anti-ds-DNA antibody of mice in the CN108795876 patent group measured at multiple time points are all reduced compared with the levels in the model group, but the levels are still gradually increased along with time on the whole, which shows that the trend of the model mice for gradually deteriorating the renal function is not changed.
In contrast, the levels of 24h urine protein, serum urea nitrogen, serum creatinine and anti-ds-DNA antibodies of the model mouse are obviously reduced compared with those of the model mouse and the model mouse of patent CN108795876, and the whole model mouse shows a trend of gradual reduction along with time, which shows that the renal function of the lupus nephritis model mouse can be gradually recovered by applying the adjustable and universal CAR-T cell combined with vfGP-LN treatment.
In general, a method of treatment using the regulatable universal CAR-T cell and vfGP described above, comprising:
the first stage is as follows: controllable universal CAR-T cells are prepared and returned in vitro. Following the routine protocol, peripheral blood was drawn, T cells sorted, T cells activated (activated with anti-CD 3/CD28 or artificial APC etc.), CAR transfected (electrotransfer, lentivirus etc), CAR-T cells expanded and returned.
And a second stage: in vivo activation of infused vfGP. The different types and doses of vfGP were given depending on the disease type, disease activity (primary autoantibody titers, etc.) and could be subsequently adjusted according to clinical disease changes. After administration, the in vivo regulatable universal CAR-T cells will be mobilized and directed to attack autoreactive B cells, achieving the goal of precise treatment of disease.
It should be noted that the above examples are only for further illustration and description of the technical solution of the present invention, and are not intended to limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A gene for coding an anti-leader polypeptide single-chain antibody is characterized in that the sequence of the gene is shown as SEQ ID NO. 11 or SEQ ID NO. 27.
2. The single-chain antibody for resisting leader polypeptide is characterized in that the sequence is shown as SEQ ID NO. 12.
3. The encoding gene of the controllable universal CAR is characterized in that the sequence of the encoding gene mainly comprises a CD3 zeta segment, a costimulatory segment, a transmembrane segment, a hinge segment, an anti-leader polypeptide single-chain antibody segment and a signal peptide segment, and the gene sequence of the anti-leader polypeptide single-chain antibody segment is shown as SEQ ID NO. 11 or SEQ ID NO. 27.
4. The gene encoding the regulatable universal CAR of claim 3, wherein the CD3 ζ segment carries a gene sequence set forth in SEQ ID No. 1; and/or
The gene sequence of the co-stimulation section 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 as SEQ ID NO. 13.
5. The gene encoding the regulatable universal CAR of claim 4, wherein the sequence is set forth in SEQ ID No. 15 or SEQ ID No. 17.
6. An expression vector comprising a gene encoding the regulatable universal CAR of claim 5.
7. An adjustable and controllable universal CAR, which is characterized in that the amino acid sequence of the regulatable and controllable universal CAR is shown as SEQ ID NO. 16 or SEQ ID NO. 18.
8. A CAR-T cell comprising the anti-homing polypeptide single-chain antibody of claim 1 or the regulatable universal CAR of claim 7.
9. A fusion leader polypeptide for use with the regulatable universal CAR-T cell of claim 8, wherein the fusion leader polypeptide consists of a leader polypeptide, a linker and an autoantibody affinity peptide joined together in sequence, and the leader polypeptide has the sequence shown in SEQ ID NO. 19.
10. The fusion leader polypeptide of claim 9, wherein the sequence of the fusion leader polypeptide is set forth in 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.
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| WO2021014385A1 (en) * | 2019-07-24 | 2021-01-28 | Glaxosmithkline Biologicals Sa | Modified human cytomegalovirus proteins |
| CN113480663A (en) * | 2014-05-29 | 2021-10-08 | Ucb生物制药有限责任公司 | Novel bispecific formats suitable for high throughput screening |
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| CN110267982A (en) * | 2016-10-19 | 2019-09-20 | 斯克利普斯研究所 | With humanization targeting moiety and/or by optimization Chimeric antigen receptor interaction domain Chimeric antigen receptor effector cell switch with and application thereof |
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