CAAR-T cell for treating myasthenia gravis and medicine thereof

Abstract

The invention provides a CAAR-T cell for treating myasthenia gravis and a medicine thereof, and belongs to the technical field of biological medicines. The chimeric autoantibody receptor of the targeted acetylcholine receptor antibody comprises the following structural domains which are connected in series: an AChR alpha subunit extracellular structural domain, a transmembrane region structural domain, a co-stimulatory molecule intracellular structural domain and a signal transduction structural domain. ECD1, ECD2 and ECD3 in the extracellular structural domain of the AChR alpha subunit are connected in series through an L1 connecting peptide and an L2 connecting peptide or two G4S connecting peptides, and the combining capacity with an anti-AChR antibody can be improved, so that a relatively strong killing effect on pathogenic B cells expressing the anti-AChR antibody is shown, the symptom of myasthenia gravis is effectively relieved, and good drug safety is achieved.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to a CAAR-T cell and its drug for treating myasthenia gravis. Background Technology

[0002] Myasthenia gravis (MG) is an acquired autoimmune disease characterized by impaired neuromuscular junction (NMJ) transmission, clinically manifested as partial or generalized skeletal muscle weakness and fatigue. Serological testing reveals that 85% of patients are positive for acetylcholine receptor (AChR) antibodies. Conventional treatment primarily involves nonspecific immunotherapy, mainly with glucocorticoids and non-hormonal immunosuppressants, but the treatment efficacy is poor. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a chimeric autoantibody receptor that targets acetylcholine receptor antibodies. CAAR is prepared by tandemly linking the extracellular domain of the AChRα subunit with L1 and L2 linker peptides or G4S linker peptide. It not only has a strong binding ability to anti-AChR antibodies, but also expresses CAAR on the surface of T cells, which shows a strong killing effect on pathogenic B cells expressing anti-AChR antibodies and alleviates the symptoms of myasthenia gravis.

[0004] This invention provides a chimeric autoantibody receptor that targets acetylcholine receptor antibodies, comprising the following tandem domains: an extracellular domain of the AChRα subunit, a transmembrane domain, an intracellular domain of the co-stimulatory molecule, and a signal transduction domain.

[0005] In the extracellular domain of the AChRα subunit, ECD1, ECD2, and ECD3 are linked in series via L1 and L2 linker peptides or via two G4S linker peptides; the amino acid sequence of L1 is shown in SEQ ID NO:1; the amino acid sequence of L2 is shown in SEQ ID NO:2.

[0006] Preferably, the signal transduction domain is the modified CD3δ; the amino acid sequence of the modified CD3δ is shown in SEQ ID NO:3;

[0007] The intracellular domain of the co-stimulatory molecule includes the intracellular co-stimulatory molecule CD28;

[0008] The transmembrane structural domain includes CD8α.

[0009] Preferably, the amino acid sequence of the chimeric autoantibody receptor targeting the acetylcholine receptor is shown in SEQ ID NO:4 or SEQ ID NO:5.

[0010] The present invention provides a nucleic acid molecule encoding a chimeric autoantibody receptor that targets the acetylcholine receptor.

[0011] Preferably, it includes DNA sequences with nucleotide sequences such as those shown in SEQ ID NO:6 or SEQ ID NO:7.

[0012] The present invention provides a recombinant vector comprising the nucleic acid molecule.

[0013] Preferably, the backbone vector of the recombinant vector includes the lentiviral vector pLVX-EF1α-IRES-puro.

[0014] This invention provides a CAAR-T cell that targets an acetylcholine receptor antibody, wherein the T cell surface expresses the chimeric autoantibody receptor that targets the acetylcholine receptor antibody.

[0015] This invention provides the use of CAAR-T cells targeting acetylcholine receptor antibodies or CAAR-T cells targeting acetylcholine receptor antibodies prepared from the recombinant vector in the preparation of drugs for the prevention and / or treatment of myasthenia gravis.

[0016] The present invention provides a therapeutic drug for myasthenia gravis, the active ingredient of which includes CAAR-T cells targeting acetylcholine receptor antibodies or CAAR-T cells targeting acetylcholine receptor antibodies prepared by the recombinant vector.

[0017] This invention provides a chimeric autoantibody receptor targeting acetylcholine receptor antibodies, comprising the following tandem domains: an extracellular domain of the AChRα subunit, a transmembrane domain, an intracellular domain of a co-stimulatory molecule, and a signal transduction domain; in the extracellular domain of the AChRα subunit, ECD1, ECD2, and ECD3 are tandemly linked by L1 and L2 linker peptides or by two G4S linker peptides; the amino acid sequence of L1 is shown in SEQ ID NO:1; the amino acid sequence of L2 is shown in SEQ ID NO:2. This invention evaluated the binding affinity of the chimeric autoantibody receptor to AChR-specific antibodies using surface plasmon resonance (SPR) technology and flow cytometry. Both chimeric autoantibody receptors provided by this invention exhibit good binding affinity to AChR-specific antibodies, and the affinity of the extracellular domain fECD-L1L2 linked by L1 and L2 is greater than that of the extracellular domain fECD-GS linked by G4S.

[0018] This invention provides CAAR-T cells targeting acetylcholine receptor antibodies, with the T cells expressing a chimeric autoantibody receptor on the surface of the acetylcholine receptor-targeting antibody. These CAAR-T cells can not only precisely recognize and kill pathogenic cells with specific BCRs, but also effectively kill specific B cells derived from clinical MG patients, significantly reducing the level of AChR-binding specific antibodies produced. In NSG mouse tumor cell transplantation models or MG passive immunization models, fECD-L1L2-A28dCAAR-T cells completely eliminated only pathogenic B cells expressing anti-AChR antibodies and effectively alleviated myasthenia gravis symptoms in mice. Therefore, this invention provides a more precise and effective strategy for MG immunotherapy. Attached Figure Description

[0019] Figure 1 The results show the performance of fECD-L1L2-A28d CAAR-T cells in binding to anti-AChR antibodies, including: A. Schematic diagram of the natural AChRα subunit and CAAR molecular structure; B. Detection results of the affinity constant of the extracellular domain of the CAAR molecule (fECD-GS or fECD-L1L2) for Fab637-Biotin; C. Efficiency of lentivirus infection of T lymphocytes.

[0020] Figure 2 The results show the killing effect of fECD-L1L2-A28d CAAR-T cells on pathogenic cells expressing specific anti-AChR antibodies; A.Fab 637 + Construction and identification of target cell lines; B. Results of killing non-specific target cells by fECD-L1L2-A28d CAAR-T cells; C. Results of specific killing of pathogenic cells expressing Fab 637 by fECD-L1L2-A28d CAAR-T cells;

[0021] Figure 3 Results of the effect of fECD-L1L2-A28d CAAR-T cells on cytokine release levels;

[0022] Figure 4 The results of specific antibody detection after fECD-L1L2-A28d CAAR-T cells killed pathogenic B cells in the peripheral blood of clinical MG patients;

[0023] Figure 5The results show the effect of fECD-L1L2-A28d CAAR-T cells on the clearance of pathogenic cells expressing specific BCR in NSG mice, including: A. Flowchart of the in vivo experiment in NSG mice; B. Diagram of mouse disease progression monitored by in vivo imaging in small animals; C. Killing effect of fECD-L1L2-A28d CAAR-T cells on anti-AChR antibody-positive target cells in mice; D. Effect of fECD-L1L2-A28d CAAR-T cells on mouse body weight.

[0024] Figure 6 The results of fECD-L1L2-A28d CAAR-T cell treatment on myasthenia gravis symptoms in NSG mice are shown below: A. Flowchart of the in vivo experiment in NSG mice; B. Changes in body weight of model mice after treatment with fECD-L1L2-A28d CAAR-T cells; C. Time taken for mice to fall from the instrument using a rotarod analyzer; D. Width of mouse footprints measured by a gait analyzer; E. Length of mouse footprints measured by a gait analyzer; F. Peak area of ​​mouse footprints measured by a gait analyzer. Detailed Implementation

[0025] This invention provides a chimeric autoantibody receptor targeting acetylcholine receptor antibodies, comprising the following tandem domains: an extracellular domain of the AChRα subunit, a transmembrane domain, an intracellular domain of a co-stimulatory molecule, and a signal transduction domain; wherein in the extracellular domain of the AChRα subunit, ECD1, ECD2, and ECD3 are tandemly linked by L1 and L2 linker peptides to form the fECD-L1L2-A28d extracellular domain (see...). Figure 1 (A)

[0026] In this invention, the L1 and L2 linker peptides are designed using computational deep learning and de novo folding combined with homology modeling. These end-capped linker peptides are advantageous in maintaining the proper spatial conformation of the three extracellular domains ECD1, ECD2, and ECD3, thereby ensuring optimal affinity between the AChRα subunit extracellular domain and the anti-AChR antibody. The amino acid sequence of L1 is shown in SEQ ID NO:1; the amino acid sequence of L2 is shown in SEQ ID NO:2. The AChRα subunit extracellular domains ECD1, ECD2, and ECD3 also include an fECD-GS-A28d extracellular domain formed by two G4S linker peptides linked in series (see...). Figure 1 (A)

[0027] In this invention, the signal transduction domain is a modified CD3δ. The amino acid sequence of the modified CD3δ is shown in SEQ ID NO:3 (GHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK). As a signal transduction domain, the modified CD3δ contains only one ITAM motif, which helps reduce cytokine release levels and significantly lowers the risk of inducing a cytokine storm. Compared to traditional CAAR-T cells (α210-GS-BBz CAAR-T) containing the natural CD3ζ motif with three ITAMs, it exhibits higher safety.

[0028] In this invention, the intracellular domain of the co-stimulatory molecule preferably includes the intracellular co-stimulatory molecule CD28. The transmembrane domain preferably includes CD8α. The amino acid sequence of the CAAR containing fECD-L1L2 is preferably as shown in SEQ ID NO:4 (MEPWPLLLLFSLCSAGLVLGSEHETRLVAKLFKDYSSVVRPVEDHRQVVEV TVGLQLIQLINVDEVNQIVTTNVRLKQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLLQYTGHITWTPPAIFKSYCEIIVTHFPFDEQNCSMKLGTWTYDGSVVAINPESDQPDLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLPLVFEKEILEPIKEIDGPPPKELLEKILEEVPSTS) SAVPLIGKELLKLSGIPPPPPLPEEELKKLLERLIELNQQGFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTC GVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSTSGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK).The preferred amino acid sequence of CAAR containing fECD-GS is as shown in SEQ ID NO:5 (MEPWPLLLLFSLCSAGLVLGSEHETRLVAKLFKDYSSVVRPVEDHRQVVEV) TVGLQLIQLINVDEVNQIVTTNVRLKQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLLQYTGHITWTPPAIFKSYCEIIVTHFPFDEQNCSMKLGTWTYDGSVVAINPESDQPDLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLPLGGGGSGGGGSSTSSAVP LIGKGGGGSGGGGSRLIELNQQGFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSTSGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK).

[0029] In one embodiment of the present invention, the binding characteristics of two extracellular domains in the CAAR of the present invention with anti-AChR antibodies were evaluated. The binding rate constants and dissociation rate constants of the recombinant proteins fECD-L1L2 and fECD-GS with anti-AChR antibodies were assessed using SPR technology. The results showed that the binding rate of fECD-L1L2 was greater than that of fECD-GS, while the dissociation rate was less than that of fECD-GS; the dissociation equilibrium constant of fECD-L1L2 was less than that of fECD-GS. This indicates that fECD-L1L2 has a greater affinity for anti-AChR antibodies than fECD-GS. Furthermore, at the cellular level, the binding efficiency of CAAR-T prepared from fECD-L1L2 with anti-AChR antibodies was superior to that prepared from fECD-GS, and also superior to the control groups (1928z CAR-T, α210-GS-BBzCAAR-T).

[0030] The present invention provides a nucleic acid molecule encoding a chimeric autoantibody receptor that targets the acetylcholine receptor.

[0031]

[0032] The present invention provides a recombinant vector comprising the nucleic acid molecule.

[0033] In this invention, the backbone vector of the recombinant vector preferably includes the lentiviral vector pLVX-EF1α-IRES-puro. This invention does not impose any particular limitation on the construction method of the recombinant vector; any construction method well-known in the art can be used, such as homologous recombination or directed cloning. After construction, the product is further validated through sequence verification, including bacterial PCR identification and sequencing. The recombinant vector is used to prepare CAAR-T cells.

[0034] This invention provides a CAAR-T cell that targets an acetylcholine receptor antibody, wherein the T cell surface expresses the chimeric autoantibody receptor that targets the acetylcholine receptor antibody.

[0035] In this invention, the method for preparing CAAR-T cells targeting acetylcholine receptor antibodies utilizes lentivirus-mediated insertion of nucleic acid molecules encoding acetylcholine receptor antibodies into the genome of T cells to obtain T cells stably expressing CAAR. Preferably, the method includes the following steps: cloning the nucleic acid molecule into a lentiviral vector to obtain a recombinant lentiviral vector; packaging the recombinant lentiviral vector with recombinant lentivirus; and infecting T cells with the recombinant lentivirus to obtain CAAR-T cells targeting acetylcholine receptor antibodies.

[0036] In this invention, the CAAR-T cells targeting acetylcholine receptor antibodies can specifically kill pathogenic cells expressing Fab637, and the killing ability gradually increases with the increase of the effector-to-target ratio.

[0037] In this invention, the CAAR-T cells targeting acetylcholine receptor antibodies can control cytokine release at a relatively safe level, exhibiting good drug administration safety. Cell co-culture experiments showed that fECD-L1L2-A28d CAAR-T cells maintained a lower level of cytokine release compared to α210-GS-BBz CAAR-T cells. The reason for this is that the signal domain CD3δ in the CAAR-T cells prepared in this invention contains only one ITAM motif, significantly reducing cytokine levels and greatly decreasing the risk of inducing a cytokine storm. Compared to traditional CAAR-T cells containing the natural CD3ζ motif with three ITAMs, this method offers higher safety.

[0038] This invention provides the use of CAAR-T cells targeting acetylcholine receptor antibodies or CAAR-T cells targeting acetylcholine receptor antibodies prepared from the recombinant vector in the preparation of drugs for the prevention and / or treatment of myasthenia gravis.

[0039] In this invention, the prevention and / or treatment of myasthenia gravis preferably includes the killing effect on specific B cells derived from MG patients and the ability to clear pathogenic B cells with anti-AChR antibodies in immunodeficient animal tumor cell transplantation models.

[0040] In one embodiment of the present invention, using an NSG mouse passive immunization model, fECD-L1L2-A28dCAAR-T cells were administered. The results showed that, compared with the model group, CAAR-T cells targeting acetylcholine receptor antibodies could reverse the weight loss in the model mice, prolong the time it took for them to fall off the rotarod, and improve their gait. This indicates that fECD-L1L2-A28d CAAR-T cells can effectively alleviate the symptoms of myasthenia gravis in NSG mice.

[0041] The present invention provides a therapeutic drug for myasthenia gravis, the active ingredient of which includes CAAR-T cells targeting acetylcholine receptor antibodies or CAAR-T cells targeting acetylcholine receptor antibodies prepared by the recombinant vector.

[0042] In this invention, the dosage form of the drug includes an injection. The concentration of CAAR-T cells targeting acetylcholine receptor antibodies in the injection is preferably 5 × 10⁻⁶. 5 For cells / mL or higher, it can be 1×10 6 ~1×10 7 Cells / mL. This invention does not impose any particular limitations on the preparation method of the injection; any cell type injection well-known in the art can be used.

[0043] The following detailed description, in conjunction with embodiments, illustrates a CAAR-T cell and its drug for treating myasthenia gravis provided by the present invention, but these should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] Construction and identification of two types of CAAR-T cells

[0046] 1. Construction of recombinant plasmids:

[0047] The CAAR molecule consists of the extracellular domain AChRα-ECD, the transmembrane region CD8α, the intracellular co-stimulatory molecule CD28, and the signal transduction domain CD3δ. The extracellular domain AChRα-ECD is formed by tandemly linking the three extracellular domains (ECDs) of the natural AChRα subunit through two G4S linkers, creating the tandem fragment fECD-GS, which serves as the extracellular domain of the CAAR molecule. Using pLVX-EF1α-IRES-puro as the lentiviral vector backbone, the full-length CAAR coding sequence was synthesized and inserted into the pLVX-EF1α-IRES-puro vector using the restriction endonucleases EcoRI and MluI. The resulting recombinant plasmid was named fECD-GS-A28d. Figure 1 (A)

[0048] The sequences of the extracellular domains in fECD-GS-A28d are as follows:

[0049] ECD1:SEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDE VNQIVTTNVRLKQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLLQYTGHITWTPPAIFKSYCEIIVTHFPFDEQNCSMKLGTWTYDGSVVAINPESDQPDLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLPL(SEQ ID NO:8);

[0050] G4S: GGGGSGGGGS (SEQ ID NO: 11);

[0051] ECD2:STSSAVPLIGK(SEQ ID NO:9);

[0052] G4S: GGGGSGGGGS (SEQ ID NO: 11);

[0053] ECD3: RLIELNQQG (SEQ ID NO: 10).

[0054] Furthermore, by employing computational deep learning and de novo folding combined with homology modeling, two dominant linkers (L1 and L2) were designed in tandem to form fECD-L1L2, the extracellular domain of the natural AChRα subunit. Using pLVX-EF1α-IRES-puro as the lentiviral vector backbone, the coding sequence of the CAAR molecule consists of the extracellular segment fECD-L1L2, the transmembrane region CD8α, the intracellular co-stimulatory molecule CD28, and the signal transduction domain CD3δ. The resulting recombinant plasmid was named fECD-L1L2-A28d( Figure 1 (A)

[0055] The sequences of the extracellular domains in fECD-L1L2-A28d are as follows:

[0056] ECD1:SEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDE VNQIVTTNVRLKQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLLQYTGHITWTPPAIFKSYCEIIVTHFPFDEQNCSMKLGTWTYDGSVVAINPESDQPDLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLPL(SEQ ID NO:8);

[0057] linker1(L1):

[0058] VFEKEILEPIKEIDGPPPKELLEKILEEVP(SEQ ID NO:1);

[0059] ECD2:STSSAVPLIGK(SEQ ID NO:9);

[0060] linker2(L2):

[0061] ELLKLSGIPPPPLPEEELKKLLE(SEQ ID NO:2);

[0062] ECD3: RLIELNQQG (SEQ ID NO: 10).

[0063] Two recombinant plasmids were identified by bacterial culture PCR. The PCR primers were F: 5'-TCAAGCCTCA GACAGTGGTTC-3' (SEQ ID NO: 12); R: 5'-GCGTATCCACATAGCGTAAAAG-3' (SEQ ID NO: 13). The PCR amplification reaction system (10 μL system) consisted of: 5 μL of 2×T5 SuperPCRMix (Tiangen); 1 μL of bacterial culture; 0.5 μL each of the upstream and downstream primers (10 μM); and 3 μL of deionized water. The PCR amplification reaction program was: pre-denaturation at 98℃ for 3 min; denaturation at 98℃ for 10 sec; annealing at 56℃ for 10 sec; and extension at 72℃ for 10 sec. After 30 cycles of this denaturation-annealing-extension program, a second extension at 72℃ for 2 min was performed to ensure sufficient amplification of the reaction products. Sequencing of the PCR products showed that they were consistent with the theoretical sequences.

[0064] 2. SPR technology for detecting the binding kinetics of recombinant proteins

[0065] 1) Using the mature mammalian cell protein expression platform of our laboratory, the gene fragment encoding the CAAR extracellular segment (fECD-GS or fECD-L1L2) was directionally cloned into a dedicated expression vector, transiently transfected into 293F cells, expressed recombinant protein, and purified protein samples were obtained by affinity chromatography.

[0066] 2) Antibody fixation: Biotin-modified AChRα antibody (Fab 637-Biotin) at the C-terminus of the IgG heavy chain was coupled to the surface of the SA sensor chip (GE Healthcare Life Sciences), with a flow rate of 10 μL / min and an injection time of 2 min.

[0067] 3) Test regeneration conditions: Inject the protein to be detected, let it flow through the chip surface, and then add glycine solutions with different pH values. Observe the response value after adding the regeneration solution to determine the effect of analyte removal.

[0068] 4) Interactions: Dilute the protein to be tested to six different concentration gradients, and load the protein sequentially in ascending order of concentration at a flow rate of 30 μL / min. The contact time for each concentration is 100 s, and the dissociation time is 200 s. Regeneration conditions include glycine at pH 2.5, a flow rate of 10 μL / min, a dissociation time of 30 s, and a stationary period of 5 s.

[0069] 5) The entire experiment was conducted at 25°C using a Biacore T200 instrument (GE Healthcare LifeSciences). Data analysis was performed using Biacore T200 Evaluation software. Kinetics / Affinity mode was selected, and the blank control value was subtracted from the sample values ​​to obtain the corrected values. Then, the data was fitted in a 1:1 binding mode to obtain the antibody binding rate constant (Ka), dissociation rate constant (Kd), and equilibrium dissociation constant (KD).

[0070] Analysis of the binding and dissociation rate constants of recombinant proteins fECD-L1L2 and fECD-GS against AChR-specific antibodies using surface plasmon resonance (SPR) technology revealed that the binding rate of fECD-L1L2 was greater than that of fECD-GS, while its dissociation rate was smaller. Figure 1 (B) The dissociation equilibrium constant of fECD-L1L2 is KD = 4.35 nM, and the dissociation equilibrium constant of fECD-GS is KD = 30.7 nM. Affinity comparison: fECD-L1L2 > fECD-GS.

[0071] 3. Construction of CAAR-T cells targeting anti-AChR antibodies and detection of lentiviral infection positivity rate on T cell surface using monoclonal antibodies.

[0072] 1) Lentiviral packaging: When HEK293T cells reached a growth density of 80%, the two recombinant plasmids prepared above were transfected under liposome Lipo conditions. Viral supernatant was collected at 48h and 72h, filtered through a 0.45μm filter, and the collected viral fluid was stored at -80℃. At the same time, two control plasmids were constructed: a recombinant plasmid expressing 1928z (plasmid source: Yescarta FMC63-28Z GenBank: HM852952.1) or a recombinant plasmid expressing α210-GS-BBz (CN 114127287A).

[0073] 2) Isolation and activation of human primary T lymphocytes: EDTA-anticoagulated venous blood was collected from healthy volunteers, and PBMCs were obtained by density gradient centrifugation. MojoSort was then used to further analyze the PBMCs. TMPrimary T cells were obtained using a human CD3 T cell isolation kit (Biolegend, USA), and human T cell activation / expansion CD3 / CD28 magnetic beads (ACRO Biosystems) were used to stimulate T cell activation.

[0074] 3) Lentiviral infection of human primary T lymphocytes: T cells were infected using the viral stock solution by centrifugation at 800g for 90 min. The virus stock solution was removed by centrifugation 24 h after infection, and the cells were then treated with 1×10⁻⁶ viral loads. 6 Add cells at a density of cells / mL to T cell culture medium and culture normally;

[0075] 4) Flow cytometry assessment of the binding efficiency of monoclonal antibodies to CAAR: 100 μL of recombinant human anti-AChR monoclonal antibody Fab 637 was added to CAAR-T cells, followed by CD19-Biotin at 1928 rpm. The cells were resuspended and vortexed, and incubated at 4°C for 30 min. After washing the cells, 100 μL of flow cytometry wash buffer was added to resuspend the cells. 5 μL of anti-human mIgG-APC flow cytometry antibody (Biolegend, USA) was added, followed by Straptavidin-APC flow cytometry antibody (Biolegend, USA) at 1928 rpm. The cells were mixed, vortexed, and incubated at 4°C for 30 min. After washing the cells, the cell pellet was resuspended in flow cytometry wash buffer and analyzed by flow cytometry.

[0076] Flow cytometry results showed that 1928z, α210-GS-BBz (CN 114127287 A), fECD-GS-A28d, or fECD-L1L2-A28d were all normally expressed on the surface of T cells, with the fECD-L1L2-A28d group exhibiting the highest binding efficiency to the recombinant monoclonal antibody. Figure 1 (C). This indicates that using the full-length ECD of L1 and L2 in tandem as the extracellular segment of CAAR can more fully bind to anti-AChR antibodies.

[0077] Based on the above structural design and results evaluation, subsequent experiments will use fECD-L1L2 as the extracellular domain of the CAAR molecule to explore the application of CAAR-T cells in the treatment of myasthenia gravis.

[0078] Example 2

[0079] fECD-A28d-CAAR-T cells precisely kill specific target cells.

[0080] 1. The method for constructing recombinant vectors containing the Fab 637 sequence is as follows:

[0081] The Fab 637 gene fragment was synthesized from its entirety. The pLVX lentiviral vector was selected, and both the fragment and the vector were double-digested, ligated, and transformed using restriction endonucleases EcoRI and XbaI, respectively. The double digestion reaction system and conditions were as follows: 10 μL of Fab 637 gene fragment or pLVX lentiviral vector; 3 μL of Cutsmart (NEB, USA); 0.5 μL each of EcoRI and XbaI (NEB, USA); and 16 μL of deionized water. The reaction conditions were 37℃ for 2 h. The ligation reaction system and conditions were as follows: 5 μL of LigationMix ligase; 4 μL of Fab 637 gene fragment digestion product; and 1 μL of pLVX lentiviral vector digestion product. The reaction conditions were 16℃ for 30 min. Transformation: Add the ligation product to 40 μL of competent cells, mix well, and then perform the following steps in sequence: ice incubation for 20 min, heat shock at 42℃ for 90 s, and ice incubation for 2 min; add 120 μL of antibiotic-free liquid LB medium and incubate at 37℃, 150 rpm for 30 min on a shaker; inoculate all the bacterial culture in the EP tube into a medium containing Amp... + Spread evenly on LB solid culture dishes, invert and air dry, then incubate overnight in a 37°C bacterial incubator (see existing technology: Meng F, Stassen MH, Schillberg S, Fischer R, De Baets MH. Construction and characterization of asingle-chain antibody fragment derived from thymus of a patient with myasthenia gravis. Autoimmunity. 2002 Mar; 35(2):125-33.).

[0082] 2. The lentivirus packaging process is the same as in Example 1;

[0083] 3. Stable transfection: CD19 cells were infected using lentivirus stock solution and cell culture medium at a 1:1 volume ratio. + K562 cells were centrifuged and removed from the culture medium 72 hours after infection, then resuspended in cell culture medium and cultured normally.

[0084] 4. Flow cytometry assessment of infection efficiency: Cells were collected by centrifugation, resuspended in 100 μL of flow cytometry wash buffer, and 5 μL of human mIgG-APC flow cytometry antibody (Biolegend, USA) was added. After mixing, the cells were vortexed and incubated at 4°C for 30 min. After washing the cells, the cell pellet was resuspended in flow cytometry wash buffer and analyzed by flow cytometry.

[0085] 5. Effectiveness-to-target ratio gradient kill experiment

[0086] 1) Select Fab 637, a strain that grows well and stably expresses firefly luciferase. + CD19 + K562 cells were counted at a rate of 2 × 10⁶ cells per well. 4 The cells were collected by centrifugation for later use.

[0087] 2) CAAR-T cells were added to the corresponding wells at effector-to-target ratio gradients of 4:1, 2:1, 1:1, 0.5:1, and 0.25:1, with each well containing 8 × 10⁶ CAAR-T cells. 4 4×10 4 2×10 4 1×10 4 0.5×10 4 The control group was given 1928z-CAR-T cells, while the blank group was given no T cells and only target cells were retained. Each group was set up with 3 replicates.

[0088] 3) After co-culturing for 48 hours, centrifuge to collect all suspended cells and wash once with pre-cooled 1×PBS;

[0089] 4) Add the diluted 1×luciferase substrate (Shanghai Liji Biotechnology Co., Ltd.) to each well in batches of 100 μL / well. After standing at room temperature for 2 min, collect the fluorescence value using the Xenogen IVIS Lumina II in vivo imaging system and analyze the data using Living Image software. Calculate the specific killing rate according to Formula I.

[0090] Specific killing rate = (luciferase activity in blank group - luciferase activity in experimental group) / luciferase activity in blank group × 100% Formula I.

[0091] After confirming that fECD-L1L2-A28d CAAR-T cells can bind to natural anti-AChR antibodies, in order to mimic the role of autoreactive B cells in MG patients and explore the cytotoxic function of CAAR-T cells, recombinant human anti-AChR antibody Fab 637 was stably expressed in CD19 cells via lentivirus-mediated expression. + On K562 cells ( Figure 2 (A). To confirm the precise targeting of fECD-L1L2-A28d CAAR-T cells, CD19-targeted CAR-T cells were selected as the control group. The results showed that fECD-L1L2-A28d CAAR-T cells exhibited effector-to-target dependent killing only against pathogenic cells expressing specific anti-AChR antibodies, demonstrating more precise pathogenic cell lysis ability than 1928z CAR-T cells, and the killing level was not significantly different from α210-GS-BBz CAAR-T cells. Figure 2 (B, C)

[0092] Example 3

[0093] fECD-L1L2-A28d-CAAR-T cells maintain cytokine release at safe levels.

[0094] Numerous studies have shown that cytokines play a crucial role in the production of pathogenic autoantibodies and the induction of neuromuscular junction inflammation in MG patients. Furthermore, cytokine release levels are a key indicator for evaluating the safety of CAR-T cells. After co-culturing CAAR-T cells with pathogenic cells expressing anti-AChR antibodies in vitro, the supernatant was collected to detect cytokine release levels. The method for detecting human cytokines is as follows:

[0095] 1.1928z CAR-T cells, α210-GS-BBz CAAR-T cells, or fECD-L1L2-A28dCAAR-T cells were used with Fab637, respectively. + CD19 + K562 cells were cultured in serum-free medium at an effector-to-target ratio of 2:1 for 24 h. The cell culture supernatant was collected by centrifugation and stored at 4°C for later use.

[0096] 2. Utilize human LEGENDplex TM Process the supernatant of each group using the Multi-Analys FlowAssay kit (Biolegend, USA);

[0097] 3. Cytokine release levels in each group were detected by flow cytometry.

[0098] The results showed significant differences in cytokine release levels among the three CAR-T cell groups. Under the premise of no difference in killing levels, fECD-L1L2-A28d CAAR-T cells maintained a lower cytokine release level than α210-GS-BBz CAAR-T cells. This indicates that using this novel CAAR-T cell line containing only one ITAM motif (CD3δ) as the first intracellular activation signal significantly reduces cytokine production and substantially lowers the risk of inducing a cytokine storm. Compared to α210-GS-BBz CAAR-T cells containing the natural CD3ζ motif (containing three ITAMs), it demonstrates higher safety. Figure 3 ).

[0099] Example 4

[0100] fECD-L1L2-A28d CAAR-T cells reduce pathogenic antibodies derived from peripheral blood in MG patients.

[0101] Because the proportion of AChR-specific B cells in the peripheral blood of MG patients is extremely low, our research group developed a feeder cell line (3T3-CD40L-BAFF-IL2-IL4-IL21 cells) to culture B cells and differentiate them into antibody-secreting plasma cells. We assessed the cytotoxic ability of CAAR-T cells against MG patient-derived specific B cells by detecting specific antibody levels. Feeder cell co-culture system and antibody level detection are as follows:

[0102] 1. CD19 levels in peripheral blood of MG patients were obtained using a B-cell sorting kit (Mitten Biotech GmbH, Germany). + B cells, at 1×10 5 / ml resuspended for later use;

[0103] 2. Add NT cells or CAAR-T cells to B cell suspension at an effector-target ratio of 2:1 and kill them at 37°C and 5% CO2 for 48 hours.

[0104] 3. At 24 hours after infection, count 3T3-CD40L-BAFF-IL2-IL4-IL21 cells at a concentration of 1×10⁻⁶. 6 Add an appropriate amount of mitomycin (MMC) to a cell density of / ml to inhibit cell proliferation. After washing, seed the cells into 12-well plates for later use.

[0105] 4. 48 hours after killing, NT cells or CAAR-T cells and B cells were co-transferred into the already adherent Feeder cells and cultured for three weeks. Cell culture supernatant was collected every 3-5 days and the co-culture system was transferred to a new well plate.

[0106] 5. The collected cell supernatant was concentrated and then subjected to ELISA detection of anti-AChR antibody (RSR Ltd, UK).

[0107] The results showed that after B cells were cytotoxic by fECD-L1L2-A28d CAAR-T cells and subsequently cultured and differentiated, the level of AChR-binding specific antibodies produced was significantly reduced, indicating that fECD-L1L2-A28d CAAR-T cells can effectively kill specific B cells derived from patients. Figure 4 This result also confirms that fECD-L1L2-A28d CAAR-T cells are more advantageous than α210-GS-BBz CAAR-T cells for immunotherapy in MG patients.

[0108] Example 5

[0109] fECD-L1L2-A28d CAAR-T cells can completely eliminate pathogenic cells expressing specific BCR in NSG mice.

[0110] Based on the results of the above in vitro cell function experiments, Fab 637 was selected for this in vivo experiment. + CD19 + A mouse tumor cell transplantation model was constructed using K562 cells.

[0111] 1. Establishment of NSG mouse tumor cell transplantation model

[0112] 1) Female NSG mice aged 4-5 weeks were purchased from Beijing Biocytogen Co., Ltd. and were raised by professionals in a standard animal facility;

[0113] 2) After 5-7 days, add 2×10 5 Fab 637 + CD19 + K562 cells were resuspended in 100 μL of 1×PBS, and 100 μL of the cell suspension was drawn up with a 1 mL syringe and injected into mice via the tail vein.

[0114] 3) Seven days after target cell injection, mice were intraperitoneally injected with 100 μL of 1× luciferase substrate. After anesthetizing the mice in a chamber containing isoflurane gas for 5-10 minutes, fluorescence signals were acquired using a Xenogen IVIS Lumina II in vivo imaging system. The distribution of anti-AChR antibody-positive target cells in the mice was assessed using Living Image software. Mouse body weight was monitored and recorded, and changes in vital signs were observed. Figure 5 (A) The mice that successfully modeled the mice were randomly divided into two groups for later use.

[0115] 2. Monitoring disease progression using small animal in vivo imaging technology

[0116] 1) Treatment was administered via tail vein injection of CAAR-T cells or NT cells (activated T cells that have not been infected with any lentiviruses) according to the group.

[0117] 2) Images are taken weekly, and the data is analyzed using Living Image software to plot the fluorescence intensity curves of pathogenic cells.

[0118] 3. Monitoring of mouse weight changes: After each imaging session, the weight of the mice was measured using a precision electronic balance, the weight data was recorded, and a weight change curve was plotted.

[0119] In vivo imaging results in small animals showed that, compared with the NT group, the fECD-L1L2-A28d group exhibited significant pathogenic cell killing after CAAR-T cell infusion, and pathogenic B cells expressing anti-AChR antibodies were completely eliminated in mice on day 35. Figure 5(B). Analysis of mouse imaging data showed that the fECD-L1L2-A28d group effectively controlled disease progression in mice, with a statistically significant difference compared to the NT group. Figure 5 (C). Weight monitoring curves showed that the weight of mice in the fECD-L1L2-A28d group remained stable, gradually increasing after day 7. In contrast, mice in the NT group experienced a significant weight loss starting from day 7 due to the burden of pathogenic cells. Figure 5 (D).

[0120] Example 6

[0121] fECD-L1L2-A28d CAAR-T cells can effectively alleviate myasthenia gravis symptoms in NSG mice.

[0122] Hybridoma cells 6c7, which simultaneously express secretory and membrane-bound AChR antibodies, were injected into NSG mice to establish a passive immunization model and investigate the effect of CAAR-T cells on alleviating the symptoms of myasthenia gravis in mice.

[0123] 1. Establishment of NSG mouse MG passive immunization model: 2×10 5 Target cells were injected into mice via the tail vein. Seven days later, CAAR-T cells or control NT cells were injected via the tail vein at an effector-target ratio of 10:1. Mouse weight and vital signs were monitored and recorded every 3-4 days, along with changes in behavioral function. Figure 6 (A)

[0124] 2. Rotarod Test: Used to assess the motor function of mice, thereby evaluating their myasthenia gravis symptoms. The day before the experiment, mice were trained on a Rotarod Model LE8505 rotarod at 4 rpm to acclimatize to the environment. On the day of the experiment, mice were placed on the rotarod at a speed between 4-40 rpm, and the speed and time of fall were recorded. Each mouse underwent three repetitions, with a 15-minute interval between each repetition.

[0125] 3. Mouse Gait Analysis: Before the experiment, mice were placed on a transparent running belt, and the instrument was started at a speed of 5 cm / s. The speed was gradually increased according to the mice's adaptation. Once the mice could walk stably on the transparent running belt, recording began. Recording was stopped after 5-10 seconds of stable walking motion, and the mice were returned to their cages. Each mouse underwent three repeated experiments with a 15-minute rest interval. After all mice had completed the test, the videos were analyzed using DigiGait software to evaluate and calculate stride length, stride width, and maximum foot contact area.

[0126] Weight monitoring curves showed that the weight of mice in the fECD-A28d group remained stable and showed a gradual upward trend. In contrast, mice in the NT group exhibited symptoms of myasthenia gravis, were unable to eat independently, and experienced progressive weight loss. Figure 6 (Middle B). The rotundus test results showed that the time it took for mice in the fECD-A28d group to fall off the rotundus was significantly longer than that in the control group. Mice in the NT group, exhibiting obvious symptoms of myasthenia gravis, were unable to remain on the rotundus for extended periods. Figure 6 (C). Gait analysis results reflect the quantitative information of footprints when the limbs of mice are fully extended. The stride length, stride width, and contact area of ​​the limbs of mice in the fECD-A28d group were significantly higher than those in the NT group, which was unable to walk normally and dragged on the transparent running belt. Figure 6 D in middle school, E in middle school, F in middle school).

[0127] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A chimeric autoantibody receptor targeting acetylcholine receptor antibodies, characterized in that, It includes the following tandem domains: the extracellular domain of the AChRα subunit, the transmembrane domain, the intracellular domain of the co-stimulatory molecule, and the signal transduction domain; In the extracellular domain of the AChRα subunit, ECD1, ECD2, and ECD3 are linked in series via L1 and L2 linker peptides or via two G4S linker peptides; the amino acid sequence of L1 is shown in SEQ ID NO:1; the amino acid sequence of L2 is shown in SEQ ID NO:

2.

2. The chimeric autoantibody receptor targeting acetylcholine receptors according to claim 1, characterized in that, The signal transduction domain is CD3δ; the amino acid sequence of CD3δ is shown in SEQ ID NO:

3.

3. The chimeric autoantibody receptor targeting acetylcholine receptors according to claim 1 or 2, characterized in that, The amino acid sequence is shown in SEQ ID NO:4 or SEQ ID NO:

5.

4. A nucleic acid molecule encoding a chimeric autoantibody receptor of an antibody targeting an acetylcholine receptor as described in any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that, This includes DNA sequences with nucleotide sequences such as those shown in SEQ ID NO:6 or SEQ ID NO:

7.

6. A recombinant vector, characterized in that, Includes the nucleic acid molecule described in claim 4 or 5.

7. The recombinant vector according to claim 6, characterized in that, The backbone vector of the recombinant vector includes the lentiviral vector pLVX-EF1α-IRES-puro.

8. A CAAR-T cell targeting an acetylcholine receptor antibody, characterized in that, T cells express a chimeric autoantibody receptor that targets the acetylcholine receptor as described in any one of claims 1 to 3 on their surface.

9. The use of the CAAR-T cells targeting acetylcholine receptor antibodies as described in claim 8 or the CAAR-T cells targeting acetylcholine receptor antibodies prepared from the recombinant vector as described in claim 6 or 7 in the preparation of medicaments for the prevention and / or treatment of myasthenia gravis.

10. A drug for treating myasthenia gravis, characterized in that, The active ingredient includes CAAR-T cells targeting the acetylcholine receptor antibody as described in claim 8 or CAAR-T cells targeting the acetylcholine receptor antibody prepared from the recombinant vector as described in claim 6 or 7.

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