Application of chimeric autoantibody receptor T cell of anti-AChR antibody in preparation of medicine for preventing and / or treating myasthenia gravis

Abstract

The invention provides application of chimeric autoantibody receptor T cells of an anti-AChR antibody in preparation of drugs for preventing and / or treating myasthenia gravis, and belongs to the technical field of biological drugs. The invention relates to a chimeric autoantibody receptor of a targeted acetylcholine receptor antibody. The chimeric autoantibody receptor comprises an AChR alpha subunit extracellular structural domain and a signal transduction structural domain CD3 epsilon, the CD3 epsilon only containing one ITAM motif is used as a signal transduction structural domain, and the safety level of cell factors can be effectively maintained. The CAAR-T cell prepared by the invention can accurately recognize and kill pathogenic cells with specific BCR, and can effectively relieve the symptom of mouse myasthenia. Therefore, the invention provides a more accurate and effective strategy for MG immune targeted therapy.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological drugs, and particularly relates to an application of a chimeric autoantibody receptor T cell of an anti-AChR antibody in preparation of a drug for preventing and / or treating myasthenia gravis. BACKGROUND

[0002] Myasthenia gravis (MG) mainly involves postsynaptic membrane acetylcholine receptors (AChR) at neuromuscular junctions, and is an autoimmune disease mediated by acetylcholine receptor antibodies (AChR-Ab). The conventional treatment methods are prone to adverse reactions and are difficult to meet the treatment needs. SUMMARY

[0003] Therefore, the purpose of the present application is to provide a chimeric autoantibody receptor of an anti-AChR antibody, which is prepared by using a CD3 epsilon with only one ITAM motif as a signal transduction domain and an extracellular domain of an AChR alpha subunit as an extracellular domain, and has the effects of binding to AChR antibodies and reducing the risk of cytokine storm in the body, and has good drug safety.

[0004] The present application provides a chimeric autoantibody receptor targeting acetylcholine receptor antibodies, which comprises the following domains in series: an AChR alpha subunit extracellular domain, a transmembrane region domain, an intracellular domain of a costimulatory molecule, and a signal transduction domain CD3 epsilon; the amino acid sequence of the CD3 epsilon is shown in SEQ ID NO: 3.

[0005] Preferably, the ECD1, ECD2 and ECD3 in the AChR alpha subunit extracellular domain are connected in series by L1 and L2 connection peptides or connected in series by two G4S connection peptides.

[0006] The amino acid sequence of the L1 is shown in SEQ ID NO: 1; and the amino acid sequence of the L2 is shown in SEQ ID NO: 2.

[0007] Preferably, the amino acid sequence is shown in SEQ ID NO: 4 or SEQ ID NO: 5.

[0008] The present application provides a nucleic acid molecule encoding the chimeric autoantibody receptor targeting acetylcholine receptor antibodies.

[0009] Preferably, the nucleic acid molecule comprises a DNA sequence with a nucleotide sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7.

[0010] The present application provides a recombinant vector comprising the nucleic acid molecule.

[0011] Preferably, the backbone vector of the recombinant vector comprises a lentiviral vector pLVX-EF1 alpha-IRES-puro.

[0012] The application provides a CAAR-T cell of an acetylcholine receptor antibody, and the T cell surface expresses a chimeric self-antibody receptor of the acetylcholine receptor antibody.

[0013] The application provides a CAAR-T cell of the acetylcholine receptor antibody, and a CAAR-T cell of the acetylcholine receptor antibody prepared from the recombinant vector, and application of the CAAR-T cell of the acetylcholine receptor antibody in preparation of a drug for preventing and / or treating myasthenia gravis.

[0014] The application provides a myasthenia gravis treatment drug, and an active ingredient comprises a CAAR-T cell of the acetylcholine receptor antibody, and a CAAR-T cell of the acetylcholine receptor antibody prepared from the recombinant vector.

[0015] The application provides a chimeric self-antibody receptor of an acetylcholine receptor antibody, comprising the following domains in series: an AChR alpha subunit extracellular domain, a transmembrane region domain, an intracellular domain of a costimulatory molecule and a signal transduction domain CD3 epsilon; and the amino acid sequence of the CD3 epsilon is shown as SEQ ID NO: 3. In the application, the CD3 epsilon containing only one ITAM motif is used as a signal transduction domain, so that the cytokine level of the prepared CAAR-T cell is significantly reduced, the risk of inducing cytokine storm is greatly reduced, and the safety is higher than that of the conventional CAAR-T cell containing three ITAM of the natural motif CD3 zeta. Meanwhile, the AChR alpha subunit extracellular domain is used as the extracellular domain in the CAAR molecule, so that the prepared CAAR-T cell can bind to the pathogenic anti-AChR antibody, accurately kill Nalm6 cells stably expressing the anti-AChR antibody (Fab 637), and the killing level is not different from that of the alpha210-GS-BBz CAAR-T cell. It can be seen that the chimeric self-antibody receptor of the acetylcholine receptor antibody provided in the application can provide an effective tool for treating myasthenia gravis.

[0016] The application also provides a CAAR-T cell of an antibody targeting an acetylcholine receptor, the T cell surface expressing a chimeric self-antibody receptor of the antibody targeting the acetylcholine receptor. The application proves that the CAAR-T cell can not only accurately recognize pathogenic cells with specific BCR and kill them, but also effectively kill specific B cells derived from a clinical MG patient, and the level of AChR-specific antibody binding is significantly reduced. In a tumor cell transplantation model of an NSG mouse or a MG passive immunization model, the fECD-L1L2-A28e CAAR-T cell can only completely eliminate pathogenic cells expressing anti-AChR antibodies, and effectively relieve the muscle weakness of the mouse. Therefore, the application provides a more accurate and effective strategy for immunotargeting treatment of MG. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Figure 5 is a performance detection result of fECD-L1L2-A28e-CAAR-T cell binding anti-AChR antibody, wherein A. schematic diagram of native AChR alpha subunit and schematic diagram of CAAR molecular structure; B. affinity constant detection result of CAAR extracellular segment (fECD-GS or fECD-L1L2) to Fab637-Biotin; C. efficiency of lentivirus infection of T lymphocytes;

[0018] Figure 2 Figure 6 is a killing effect determination result of fECD-L1L2-A28e CAAR-T cell to pathogenic cells expressing specific anti-AChR antibody; A. Fab 637 + Construction and identification of target cell lines; B. killing result of fECD-L1L2-A28e CAAR-T cell to non-specific target cells; C. killing result of fECD-L1L2-A28e CAAR-T cell to pathogenic cells expressing Fab 637;

[0019] Figure 3 Figure 7 is an effect result of fECD-L1L2-A28e CAAR-T cell on the level of cytokine release;

[0020] Figure 4 Figure 8 is a detection result of specific antibody of fECD-L1L2-A28e CAAR-T cell after killing pathogenic B cells in peripheral blood of a clinical MG patient;

[0021] Figure 5Figure 6 is a result of the clearance effect of fECD-L1L2-A28e CAAR-T cells on pathogenic cells expressing specific BCR in NSG mice in vivo, wherein A. a flow chart of the experiment in vivo in NSG mice; B. a graph of the disease progression of mice monitored by small animal live imaging; C. a result of the killing of anti-AChR antibody positive target cells in mice by fECD-L1L2-A28e CAAR-T cells in vivo; D. an effect of fECD-L1L2-A28e CAAR-T cells on the body weight of mice;

[0022] Figure 6 Figure 7 is a result of the effect of fECD-L1L2-A28e CAAR-T cells on the symptoms of myasthenia gravis in NSG mice, wherein A. a flow chart of the experiment in vivo in NSG mice; B. a result of the effect of fECD-L1L2-A28e CAAR-T cell treatment on the body weight of model mice; C. a result of the time for mice to fall off the apparatus detected by a rotarod; D. a result of the width of the footprints of mice detected by a gait analysis apparatus; E. a result of the length of the footprints of mice detected by a gait analysis apparatus; F. a result of the peak area of the footprints of mice detected by a gait analysis apparatus. DETAILED DESCRIPTION

[0023] The present application provides a chimeric autoantibody receptor targeting acetylcholine receptor antibody, comprising the following domains in series: AChR alpha subunit extracellular domain, transmembrane region domain, intracellular domain of a costimulatory molecule and signaling domain CD3 epsilon; the amino acid sequence of the CD3 epsilon is shown in SEQ ID NO: 3 (KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI).

[0024] In the present application, the CD3 epsilon is a modified domain, only containing one ITAM motif, which is conducive to reducing the release level of cytokines and greatly reducing the risk of inducing cytokine storm, and is safer compared with traditional CAAR-T cells (alpha210-GS-BBz CAAR-T) containing three ITAM of the natural motif CD3 zeta.

[0025] In the present application, the AChR alpha subunit extracellular domain is formed by the L1 connecting peptide and the L2 connecting peptide in series of ECD1, ECD2 and ECD3 in the AChR alpha subunit extracellular domain, forming a fECD-L1L2-A28e extracellular domain (see Figure 1The L1 connecting peptide and the L2 connecting peptide are two end-advantage connecting peptides designed by a method of combining deep learning and de novo folding and homology modeling, which are advantageous for the three extracellular domains ECD1, ECD2 and ECD3 to maintain a proper spatial conformation, thereby ensuring that the AChR alpha subunit extracellular domain reaches the best affinity with the anti-AChR antibody. The amino acid sequence of the L1 is shown as SEQ ID NO: 1; the amino acid sequence of the L2 is shown as SEQ ID NO: 2. The ECD1, ECD2 and ECD3 in the AChR alpha subunit extracellular domain further include a fECD-GS-A28e extracellular domain formed by two G4S connecting peptides in series (see Figure 1 In the middle A).

[0026] In the present application, the intracellular domain of the co-stimulatory molecule preferably comprises the intracellular co-stimulatory molecule CD28. The transmembrane domain preferably comprises CD8a. The amino acid sequence of the CAAR comprising fECD-L1L2 is preferably as set forth in SEQ ID NO: 4 (MEPWPLLLLFSLCSAGLVLGSEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRLKQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLLQYTGHITWTPPAIFKSYCEIIVTHFPFDEQNCSMKLGTWTYDGSVVAINPESDQPDLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLPLWFEENIIKPLLEIDGPPPKELLEEILKAVPSTSSAVPLIGKKLLEMMGIPPPPELPPEKLKELLKRLIELNQQGFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSTSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI).The amino acid sequence of the CAAR comprising fECD-GS is preferably as shown in SEQ ID NO: 5 (MEPWPLLLLFSLCSAGLVLGSEHETRLVAKLFKDYSSVVRPVEDHRQ VVEVTVGLQLIQLINVDEVNQIVTTNVRLKQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLLQYTGHITWTPPAIFKSYCEIIVTHFPFDEQNCSMKLGTWTYDGSVVAINPESDQPDLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLPLGGGGSGGGGSSTSSAVPLIGKGGGGSGGGGSRLIELNQQGFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSTSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI).

[0027] In one embodiment of the application, the binding properties of two extracellular domains in the CAAR of the application to anti-AChR antibodies were evaluated, and the association rate constant and dissociation rate constant of the fECD-L1L2 and fECD-GS recombinant proteins to anti-AChR antibodies were evaluated by SPR technology. The results showed that the association 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 the affinity of fECD-L1L2 to anti-AChR antibodies is greater than that of fECD-GS. In addition, at the cellular level, the binding efficiency of CAAR-T prepared from fECD-L1L2 to anti-AChR antibodies is superior to that of CAAR-T prepared from fECD-GS, and is superior to that of the control group (1928z CAR-T, a210-GS-BBzCAAR-T).

[0028] The application provides a nucleic acid molecule encoding the chimeric autoantibody receptor targeting the acetylcholine receptor antibody.

[0029]

[0030] The present application provides a recombinant vector comprising the nucleic acid molecule.

[0031] In the present application, the backbone vector of the recombinant vector preferably comprises a lentiviral vector pLVX-EF1a-IRES-puro. The present application does not have special restrictions on the construction method of the recombinant vector, and the construction method well known in the art can be used, such as homologous recombination, directional cloning. After construction, sequence verification is also included for the constructed product, including bacterial liquid PCR identification and sequencing. The recombinant vector is used for preparing CAAR-T cells.

[0032] The present application provides a CAAR-T cell of a targeted acetylcholine receptor antibody, and the T cell surface expresses a chimeric self-antibody receptor of the targeted acetylcholine receptor antibody.

[0033] In the present application, the preparation method of the CAAR-T cell of the targeted acetylcholine receptor antibody, the nucleic acid molecule encoding the targeted acetylcholine receptor antibody is inserted into the T cell genome by using lentivirus mediation, so as to obtain T cells stably expressing CAAR, preferably comprising the following steps: cloning the nucleic acid molecule into a lentiviral vector to obtain a recombinant lentiviral vector; using the recombinant lentiviral vector for recombinant lentivirus packaging; infecting the T cells with the recombinant lentivirus to obtain the CAAR-T cell of the targeted acetylcholine receptor antibody.

[0034] In the present application, the CAAR-T cell of the targeted acetylcholine receptor antibody can specifically kill pathogenic cells expressing Fab637, and the killing ability gradually increases with the increase of the effector-target ratio, and when the effector-target ratio is 1:1, the fECD-L1L2-A28e CAAR-T cell has a significantly higher killing intensity than the a210-GS-BBz CAAR-T cell.

[0035] In the present application, the CAAR-T cell of the targeted acetylcholine receptor antibody can control the cytokine release at a relatively safe level, and has good drug safety. The cell co-culture test shows that the cytokine release of the fECD-L1L2-A28e CAAR-T cell is maintained at a lower level than that of the a210-GS-BBz CAAR-T cell. The reason for analysis is that the signal domain CD3ε in the CAAR-T cell prepared in the present application only contains one ITAM motif, so that the level of produced cytokine is significantly reduced, and the risk of inducing cytokine storm is greatly reduced. Compared with the conventional CAAR-T cell containing three ITAM of the natural motif CD3ζ, the safety is higher.

[0036] The application provides application of the CAAR-T cell of the targeting acetylcholine receptor antibody or the CAAR-T cell of the targeting acetylcholine receptor antibody prepared from the recombinant vector in preparation of a drug for preventing and / or treating myasthenia gravis.

[0037] In the application, the prevention and / or treatment of myasthenia gravis preferably comprises killing of specific B cells from MG patients, and clearance of pathogenic B cells of anti-AChR antibodies in an immune-deficient animal tumor cell transplantation model.

[0038] In one embodiment of the application, the fECD-L1L2-A28e CAAR-T cell is administered to a passive immunization NSG mouse model, and the results show that, compared with the model group, the CAAR-T cell of the targeting acetylcholine receptor antibody can reverse the weight loss of the model mouse, prolong the time for the model mouse to fall off from a rotating rod, and improve the gait of the model mouse, which indicates that the fECD-L1L2-A28e CAAR-T cell can effectively relieve the myasthenia gravis symptoms of the NSG mouse.

[0039] The application provides a drug for treating myasthenia gravis, and an active ingredient comprises the CAAR-T cell of the targeting acetylcholine receptor antibody or the CAAR-T cell of the targeting acetylcholine receptor antibody prepared from the recombinant vector.

[0040] In the application, the dosage form of the drug comprises an injection. The concentration of the CAAR-T cell of the targeting acetylcholine receptor antibody in the injection is preferably 5x10 5 / mL or above, and can be 1x10 6 ~1x10 7 / mL. The application does not have special limitations on the preparation method of the injection, and a cell type injection known in the art can be used.

[0041] The application of the chimeric self-antibody receptor T cell of the anti-AChR antibody in preparation of a drug for preventing and / or treating myasthenia gravis is described in detail below in combination with examples, but they should not be understood as limitations on the protection scope of the application.

[0042] Example 1

[0043] Construction and identification of two CAAR-T cells

[0044] 1. Construction of a recombinant plasmid

[0045] The correlation between the actual rigid transformation parameters (such as contact, distance, omega, theta, phi, etc.) of the experimental structure and the predicted distribution pattern was analyzed by structure de novo folding modeling of trRosetta software, and the two connecting peptides L1 and L2 were optimized by template-modeling score (TM-score) algorithm.

[0046] The three extracellular domains (ECD) of the natural AChR alpha subunit were connected in series by two G4S linkers or L1 and L2 to form modified extracellular domains, respectively denoted as fECD-GS and fECD-L1L2.

[0047] The sequences in fECD-L1L2 are as follows:

[0048] ECD1: SEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRLKQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLLQYTGHITWTPPAIFKSYCEIIVTHFPFDEQNCSMKLGTWTYDGSVVAINPESDQPDLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLPL (SEQ ID NO: 8);

[0049] linker1 (L1): WFEENIIKPLLEIDGPPPKELLEEILKAVP (SEQ ID NO: 1); ECD2: STSSAVPLIGK (SEQ ID NO: 9);

[0050] linker2 (L2): KLLEMMGIPPPPELPPEKLKELLK (SEQ ID NO: 2);

[0051] ECD3: RLIELNQQG (SEQ ID NO: 10).

[0052] The sequences in fECD-GS are as follows:

[0053] ECD1: SEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDE VNQIVTTNVRLKQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLLQYTGHITWTPPAIFKSYCEIIVTHFPFDEQNCSMKLGTWTYDGSVVAINPESDQPDLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLPL (SEQ ID NO: 8);

[0054] G4S: GGGGSGGGGS (SEQ ID NO: 11);

[0055] ECD2: STSSAVPLIGK (SEQ ID NO: 9);

[0056] G4S: GGGGSGGGGS (SEQ ID NO: 11);

[0057] ECD3: RLIELNQQG (SEQ ID NO: 10).

[0058] The CAAR molecule is composed of an extracellular domain, a transmembrane region CD8a, an intracellular costimulatory molecule CD28 and a signal transduction domain CD3s. Using molecular cloning technology, a signal peptide sequence is added to the 5' end of the gene sequence of the modified extracellular domain configuration, and then connected in turn with the coding sequence of the intracellular domain of the CD28 costimulatory molecule and the intracellular domain of CD3s to construct the CAAR molecule (see Figure 1 Fig. A).

[0059] The CAAR molecule coding sequence was inserted into pLVX-EF1 alpha-IRES-puro lentivirus vector by directional cloning method, and fECD-L1L2-A28e / fECD-GS-A28e was obtained, respectively. The PCR amplification primers were F: 5'-TCAAGCCTCAGACAGTGGTTC-3'(SEQ ID NO: 12); R: 5'-GCGTATCCACATAGCGTAAAAG-3'(SEQ ID NO: 13). The PCR amplification reaction system (10 μL system) was 5 μL of 2xT5 SuperPCR Mix (Tiangen); 1 μL of bacterial solution; 0.5 μL of each of the upstream and downstream primers (10 μM); and 3 μL of deionized water. The PCR amplification reaction program was 98°C for 3 min for pre-denaturation; 98°C for 10 sec for denaturation; 56°C for 10 sec for annealing; and 72°C for 10 sec for extension. After 30 cycles of the above denaturation-annealing-extension program, the reaction was fully amplified by 72°C for 2 min for re-extension. The PCR product was sequenced, and the sequencing result showed that it was consistent with the theoretical sequence.

[0060] 2. SPR technology for detecting the binding kinetics of recombinant proteins

[0061] 1) Using the mature mammalian cell protein expression platform in the laboratory, the gene fragment encoding the extracellular segment of CAAR (fECD-GS or fECD-L1L2) was directionally cloned into a special expression vector, 293F cells were transiently transfected, recombinant proteins were expressed, and purified protein samples were obtained by affinity chromatography column;

[0062] 2) Antibody immobilization: the IgG heavy chain C-terminal biotin-modified AChR alpha antibody (Fab 637-Biotin) was coupled to the SA sensor chip (GE Healthcare Life Sciences) surface, the flow rate (Flow Rate) was set to 10 μL / min, and the injection time was 2 min;

[0063] 3) Test regeneration conditions: inject the protein to be detected, make it flow through the chip surface, and then add glycine solution with different pH values, and observe the response value after adding the regeneration solution to judge the removal effect of the analyte;

[0064] 4) Interaction: dilute the protein to be detected to 6 different concentration gradients, and inject them in order from low to high concentration, with a flow rate of 30 μL / min, a contact time of 100 s for each concentration, and a dissociation time of 200 s. The glycine pH in the regeneration condition was 2.5, the flow rate was 10 μL / min, the dissociation time was 30 s, and the stabilization period was 5 s;

[0065] 5) The experiment was performed at 25°C using a Biacore T200 instrument (GE Healthcare Life Sciences) throughout, and data analysis was performed using Biacore T200 Evaluation software, selecting the Kinetics / Affinity mode, subtracting the blank control value from the sample value to obtain the corrected value, and then fitting according to the 1:1 binding mode to obtain the antibody binding rate constant (Association rate constant, Ka), dissociation rate constant (Dissociation rate constant, Kd) and equilibrium dissociation constant (Equilibrium dissociation constant, KD).

[0066] The binding rate constant and dissociation rate constant of the AChR-specific antibody for fECD-L1L2 and fECD-GS recombinant proteins were analyzed by surface plasmon resonance (SPR) technology, and it was found 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 Figure 1 The dissociation equilibrium constant of fECD-L1L2 was KD = 2.57 nM, and the dissociation equilibrium constant of fECD-GS was KD = 16.0 nM, and the affinity fECD-L1L2 > fECD-GS.

[0067] 3. Monoclonal antibody detection of T cell surface lentivirus infection positive rate

[0068] 1) Lentivirus packaging: when the growth density of HEK293T cells reaches 80%, the above prepared recombinant plasmid is used for transfection, and the virus supernatant is collected at 48h and 72h, filtered through a 0.45μm filter, and stored in a -80℃ refrigerator; at the same time, the recombinant plasmid expressing 1928z (plasmid source Yescarta FMC63-28Z GenBank: HM852952.1) or α210-GS-BBz (CN 114127287A) is used as a control to construct a recombinant lentivirus;

[0069] 2) Isolation and activation of human primary T lymphocytes: collect EDTA anticoagulated venous blood from healthy volunteers, obtain PBMC by density gradient centrifugation, and use MojoSort TM Human CD3 T cell isolation kit (Biolegend, USA) to obtain primary T cells, and human T cell activation / amplification CD3 / CD28 magnetic beads (ACRO Biosystem company) to stimulate T cell activation;

[0070] 3) Lentivirus infection of human primary T lymphocytes: T cells were infected with virus stock solution by centrifugation at 800 g for 90 min. After 24 h of infection, the supernatant was removed by centrifugation, and the T cells were cultured at a cell density of 1 x 10 6

[0071] 4) Flow cytometry identification 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, and CD19-Biotin was added to the control group 1928z-CAR-T cells. The cells were resuspended and vortexed, and incubated at 4°C for 30 min. After washing the cells, 100 μL of flow washing solution was added to resuspend the cells, 5 μL of anti-human mlgG-APC flow cytometry antibody (Biolegend, USA) was added to 1928z, and Straptavidin-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 cells were resuspended in flow washing solution and detected by flow cytometry.

[0072] The flow cytometry results showed that 1928z, a210-GS-BBz, fECD-GS-A28e, or fECD-L1L2-A28e could be normally expressed on the surface of T cells, and the binding efficiency of the fECD-L1L2-A28e group to the recombinant monoclonal antibody was the highest (Fig. 2C). This indicates that the use of the ECD full-length of L1 and L2 in series as the extracellular segment of CAAR can bind more fully to anti-AChR antibodies. Figure 1

[0073] Based on the above structure design and result evaluation, fECD-L1L2 was used as the extracellular domain of CAAR molecules in subsequent experiments to explore the application of CAAR-T cells in the treatment of myasthenia gravis.

[0074] Example 2

[0075] fECD-L1L2-A28e CAAR-T cells precisely kill specific target cells

[0076] 1. After determining that fECD-L1L2-A28e CAAR-T cells can bind to natural anti-AChR antibodies, in order to simulate the role of autoreactive B cells in MG patients and explore the killing function of CAAR-T cells, recombinant human anti-AChR antibody Fab 637 was stably expressed on Nalm6 cells by lentivirus mediation. The specific steps are as follows:

[0077] 1) The recombinant vector containing the Fab 637 sequence was constructed as follows:

[0078] ​​The full gene synthesis Fab 637 gene fragment, select pLVX lentivirus vector, using restriction enzyme EcoRI and XbaI respectively on the fragment and the vector double enzyme digestion, ligation and transformation. Enzyme digestion reaction system and conditions: Fab 637 gene fragment or pLVX lentivirus vector sample amount 10 μL; cutsmart (American NEB company) 3 μL; restriction enzyme EcoRI and XbaI (American NEB company) each 0.5 μL; deionized water 16 μL. Reaction conditions: 37℃, 2h. Ligation reaction system and conditions: LigationMix ligase sample amount 5 μL; Fab 637 gene fragment enzyme digestion product 4 μL; pLVX lentivirus vector enzyme digestion product 1 μL. Reaction conditions: 16℃, 30min. Transformation: the ligation product is added to 40 μL competent cells, mixed, and then sequentially operated according to the order of ice incubation for 20min, 42℃ heat shock for 90s, and ice incubation for 2min; add 120 μL of liquid LB medium without antibiotics, 37℃, 150rpm, 30min shaking culture; inoculate all the bacterial liquid in the EP tube into LB solid culture dish containing Amp + (see prior art: Meng F, Stassen MH, Schillberg S, Fischer R, De Baets MH. Construction and characterization of a single-chain antibody fragment derived from thymus of a patient with myasthenia gravis. Autoimmunity. 2002 Mar; 35(2): 125-33.).

[0079] 2) Lentivirus packaging process same as example 1;

[0080] 3) Stable transfection: use lentivirus stock solution and cell culture fluid to infect Nalm6 cells at a volume ratio of 1:1, centrifuge and remove the liquid 72h after infection, resuspend with cell culture fluid and normal culture;

[0081] 4) Flow cytometry to identify infection efficiency: centrifugal collection of cells, add 100 μL flow cytometry washing fluid to resuspend cells, add 5 μL human mIgG-APC flow cytometry antibody (American Biolegend company), mix and vortex the cells, 4℃ incubate for 30min. Wash the cells and resuspend the cell pellet with flow cytometry washing fluid, and detect on the machine.

[0082] 2. Gradient killing experiment of effector to target

[0083] 1) Count the Nalm6 cells that grow well and stably express firefly luciferase, and add 2x10 4 Centrifuge the cells at a volume of 2x10

[0084] 2) Add CAAR-T cells to the corresponding wells at an effector-to-target ratio of 4:1, 2:1, 1:1, 0.5:1, 0.25:1, add 1928z-CAR-T cells to the control group, and add no T cells to the blank group, only target cells, with 3 replicates per group;

[0085] 3) After co-culturing for 48h, centrifuge all the suspended cells and wash them once with pre-cooled 1xPBS;

[0086] 4) Add 100μL / well of diluted 1x luciferase substrate (Shanghai Liji Biological Technology Co., Ltd.) to each well, and after 2min at room temperature, collect the fluorescence values by Xenogen IVIS Lumina II live imaging instrument, and analyze the data using Living Image software; calculate the specific killing rate according to formula I.

[0087] Specific killing rate = (blank group luciferase activity - experimental group luciferase activity) / blank group luciferase activity x 100% formula I.

[0088] To confirm the precise targeting of fECD-L1L2-A28e CAAR-T cells, CD19-targeting CAR-T cells were selected as the control group. The results showed that fECD-L1L2-A28e CAAR-T cells only exhibited effector-to-target ratio-dependent killing of pathogenic cells expressing specific anti-AChR antibodies, and had more precise pathogenic cell lysis ability than 1928z CAR-T cells, and the killing level was not different from that of α210-GS-BBz CAAR-T cells Figure 2 B, C).

[0089] Example 3

[0090] fECD-L1L2-A28e CAAR-T cells maintain cytokine release at a safe level

[0091] Cytokine release level is a key indicator for evaluating the safety of CAAR-T cell therapy. After co-culturing CAAR-T cells with pathogenic cells expressing AChR antibodies in vitro, the supernatant was collected to detect the cytokine release level. The method for detecting human cytokines is as follows:

[0092] 1. 1928z CAR-T cells, α210-GS-BBz CAAR-T cells and fECD-L1L2-A28e CAAR-T cells were co-cultured with Fab637+ Nalm6 cells were cultured in serum-free medium for 24 h at an effector-to-target ratio of 2:1. The cell culture supernatant was collected by centrifugation and stored at 4°C until use;

[0093] 2. Human LEGENDplex Multi-Analy FlowAssay kit (Biolegend, USA) was used to detect the cytokine release level of each group. TM Multi-Analy FlowAssay kit (Biolegend, USA) was used to detect the cytokine release level of each group.

[0094] 3. The cytokine release level of each group was detected by flow cytometry.

[0095] The results showed that there were significant differences in the cytokine release levels of the three groups of CAR-T cells. Under the premise of no difference in killing level, the cytokine release of fECD-L1L2-A28e CAAR-T cells was maintained at a lower level than that of a210-GS-BBz CAAR-T cells. This indicates that the new CAAR-T cell containing only one ITAM motif of CD3e as the first intracellular activation signal has a significantly reduced cytokine production level, a greatly reduced risk of inducing cytokine storm, and higher safety compared to the traditional CAAR-T cell containing three ITAM motifs of CD3zeta. Figure 3

[0096] Example 4

[0097] fECD-L1L2-A28e CAAR-T cells reduce pathogenic antibodies in peripheral blood of MG patients

[0098] Since the proportion of AChR-specific B cells in the peripheral blood of MG patients is extremely low, the research group developed a Feeder cell (3T3-CD40L-BAFF-IL2-IL4-IL21 cell) to culture B cells to differentiate into plasma cells capable of secreting antibodies, and to evaluate the killing ability of CAAR-T cells on specific B cells from MG patients by detecting the level of specific antibodies. Feeder cell co-culture system and antibody level detection:

[0099] 1. B cell sorting kit (Mitenyi, Germany) was used to obtain CD19 + B cells in the peripheral blood of MG patients, resuspended at 1×10 5 / ml for use;

[0100] 2. NT cells or CAAR-T cells were added to the B cell suspension at an effector-to-target ratio of 2:1, and incubated at 37°C in a 5% CO2 environment for 48 h;

[0101] ​3. At 24h, count 3T3-CD40L-BAFF-IL2-IL4-IL21 cells, add appropriate amount of Mitomycin (MMC) to inhibit cell proliferation at a cell density of 1 x 10 6 / ml, and after washing, seed the cells in 12-well plates for use;

[0102] 4. After 48h of killing, transfer NT cells or CAAR-T cells and B cells into the already adherent Feeder cells for continuous culture for three weeks, collect cell culture supernatant every 3-5 days, and transfer the co-culture system into a new well plate;

[0103] 5. Concentrate the collected cell supernatant and perform ELISA detection of anti-AChR antibodies (RSR Limited, UK).

[0104] The results show that the level of AChR-specific antibody binding produced by the B cells differentiated after being killed by fECD-L1L2-A28e CAAR-T cells is significantly reduced, indicating that fECD-L1L2-A28e CAAR-T cells can effectively kill patient-derived specific B cells Figure 4 , and the results also confirm that fECD-L1L2-A28e CAAR-T cells are more advantageous than a210-GS-BBz CAAR-T cells for immune therapy of MG patients.

[0105] Example 5

[0106] fECD-L1L2-A28e CAAR-T cells can completely eliminate pathogenic cells expressing specific BCR in NSG mice

[0107] 1. Based on the results of the above in vitro cell function experiments, this embodiment carries out in vivo experiments, and Fab 637 + Nalm6 cells are used to construct a tumor cell transplantation model in NSG mice, and the specific steps are as follows:

[0108] 1) 4-5 week old female NSG mice were purchased from Beijing Bao'esi Company and were raised in a standard animal house by professional personnel;

[0109] 2) 5-7 days later, 2 x 10 5 Fab 637 + Nalm6 cells were resuspended in 100 μL of 1 x PBS, and the cell suspension was drawn up with a 1 mL syringe and inoculated into the mice via the tail vein;

[0110] 3) 7 days after the injection of target cells, 100 μL of 1x luciferase substrate was injected into the abdominal cavity of the mice, and the mice were anesthetized in a chamber containing isoflurane gas for 5-10 min. The fluorescence signal was collected by the Xenogen IVIS Lumina II live imaging instrument, and the body weight of the mice was monitored and recorded, and the changes in the physical signs of the mice were observed. Figure 5 The distribution of anti-AChR antibody-positive target cells in the mice was evaluated using Living Image software. The mice that successfully modeled were randomly divided into two groups for use.

[0111] 2. Monitoring of disease progression by small animal live imaging technology

[0112] 1) The mice were treated by injecting CAAR-T cells or NT cells through the tail vein according to the grouping;

[0113] 2) The mice were imaged once a week, and the data were analyzed by Living Image software and the fluorescence intensity curve of the pathogenic cells was plotted.

[0114] 3) Monitoring of changes in the body weight of the mice: After each imaging, the body weight of the mice was measured using a precision electronic balance, and the body weight data were recorded and plotted as a body weight change curve.

[0115] The results of the small animal live imaging showed that the fECD-L1L2-A28e group exhibited obvious killing of pathogenic cells after the infusion of CAAR-T cells, and the anti-AChR antibody-expressing pathogenic B cells in the mice were completely eliminated on day 35 (Fig. 6B). Through the analysis of the imaging data of the mice, it was found that the fECD-L1L2-A28e group could effectively control the disease progression of the mice, and there was a significant statistical difference compared with the NT group (Fig. 6C). The body weight monitoring curve showed that the body weight of the mice in the fECD-L1L2-A28e group changed smoothly, and gradually increased after day 14. However, the mice in the NT group had a significant decrease in body weight starting from day 7 due to the load of pathogenic cells (Fig. 6D). Figure 5 Figure 5 Figure 5

[0116] Example 6

[0117] fECD-L1L2-A28e CAAR-T cells can effectively alleviate the symptoms of myasthenia gravis in NSG mice

[0118] 1. Hybridoma cells 6c7 that express both secreted and membrane-bound AChR antibodies were injected into NSG mice to establish a passive immunization model, and the alleviation of the symptoms of myasthenia gravis in the mice by CAAR-T cells was investigated. 2x10 5 ​​​Target cells were inoculated into mice via tail vein, and 7 days later, CAAR-T cells or control NT cells were injected into mice via tail vein at an effector-to-target ratio of 10:1. The body weight and physical signs of mice were monitored and recorded every 3-4 days, and the behavioral function changes of mice were monitored Figure 6 A).

[0119] 2. Rotarod test: used to detect the motor ability of mice to evaluate the symptoms of myasthenia gravis. One day before the experiment, mice were placed on a Rotarod Model LE8505 at 4 rpm for training to adapt to the environment of the rotarod. On the day of the experiment, mice were placed on the rotarod at a speed of 4-40 rpm, and the speed and time of the mice falling were recorded. Each mouse was trained for 3 times with an interval of 15 min.

[0120] 3. Body weight monitoring curve showed that the body weight of mice in the fECD-L1L2-A28e group changed smoothly and showed a gradual upward trend. The mice in the NT group could not eat independently due to the symptoms of myasthenia gravis, resulting in a progressive decrease in body weight Figure 6 B).

[0121] 4. Gait analysis of mice: before the experiment, the mice were placed on a transparent runway, and the instrument was started after setting the speed to 5 cm / s. According to the adaptation of the mice, the speed of the instrument was gradually increased, and when the mice could walk stably on the transparent runway, the experiment was started. The stable footstep action of the mice was captured for 5-10 s, the recording was stopped, and the mice were returned to the cage. Each mouse was tested three times with an interval of 15 min. After all the mice completed the test, the video was analyzed using DigiGait software to evaluate and calculate the step length, step width, and maximum foot contact area.

[0122] The results of the rotarod test showed that the duration of the mice in the fECD-L1L2-A28e group falling from the rotarod was significantly higher than that of the control group. The mice in the NT group could not stay on the rotarod for a long time due to the obvious symptoms of myasthenia gravis Figure 6 C). The results of gait analysis reflected the quantitative information of the footprints when the mouse's limbs were fully open. The step length, step width, and contact area of the mice in the fECD-L1L2-A28e group were significantly higher than those of the NT group which could not walk normally Figure 6 D, E, F).

[0123] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, which should also be considered within the scope of protection of the present application.

Claims

1. A chimeric autoantibody receptor targeting an acetylcholine receptor antibody, characterized in that, The structure comprises the following domains in series: AChR alpha subunit extracellular domain, transmembrane region domain, intracellular domain of a costimulatory molecule and signaling domain CD3 epsilon; the amino acid sequence of the CD3 epsilon is shown as SEQ ID NO:

3.

2. The chimeric autoantibody receptor targeting acetylcholine receptors of claim 1, wherein, ECD1, ECD2 and ECD3 in the AChR alpha subunit extracellular domain are connected in series by L1 connecting peptide and L2 connecting peptide or connected in series by two G4S connecting peptides; The amino acid sequence of the L1 is shown as SEQ ID NO: 1; the amino acid sequence of the L2 is shown as SEQ ID NO:

2.

3. The chimeric autoantibody receptor targeting acetylcholine receptors of claim 1 or 2, wherein, The amino acid sequence is shown as SEQ ID NO: 4 or SEQ ID NO:

5.

4. A nucleic acid molecule encoding the chimeric autoantibody receptor of the targeting acetylcholine receptor antibody according to any one of claims 1 to 3.

5. The nucleic acid molecule of claim 4, wherein, The DNA sequence comprises a nucleotide sequence shown as SEQ ID NO: 6 or SEQ ID NO:

7.

6. A recombinant vector, characterized in that, The nucleic acid molecule is according to claim 4 or 5.

7. The recombinant vector of claim 6, wherein, The backbone vector of the recombinant vector comprises lentiviral vector pLVX-EF1 alpha-IRES-puro.

8. A CAAR-T cell targeting an acetylcholine receptor antibody, characterized in that, The T cell surface expresses the chimeric autoantibody receptor of the targeting acetylcholine receptor antibody according to any one of claims 1 to 3.

9. Use of the CAAR-T cell of the targeting acetylcholine receptor antibody according to claim 8, the CAAR-T cell of the targeting acetylcholine receptor antibody prepared by the recombinant vector according to claim 6 or 7 in the preparation of a drug for preventing and / or treating myasthenia gravis.

10. A therapeutic agent for myasthenia gravis, characterized by comprising the compound or salt according to claim 1. The active ingredient comprises the CAAR-T cell of the targeting acetylcholine receptor antibody according to claim 8, the CAAR-T cell of the targeting acetylcholine receptor antibody prepared by the recombinant vector according to claim 6 or 7.

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