Chimeric autoantibody receptor targeting acetylcholine receptor antibody, CAAR-T cell and application of chimeric autoantibody receptor in medicine for preventing and treating myasthenia gravis

Abstract

The invention provides a chimeric autoantibody receptor of a targeted acetylcholine receptor antibody, a CAAR-T cell and application of the chimeric autoantibody receptor in a medicine for preventing and treating myasthenia gravis, and belongs to the technical field of biological medicines. The invention provides a chimeric autoantibody receptor (CAAR) targeting an acetylcholine receptor antibody, which is characterized in that three extracellular domains of an AChR alpha subunit are connected in series through L1 and L2 connecting peptides or G4S connecting peptides to form an extracellular domain, and meanwhile, CD3 gamma only containing an ITAM motif is used as a signal transduction domain. The CAAR is expressed on the surface of a T cell, and the obtained CAAR-T not only has relatively strong binding capacity with an anti-AChR antibody, shows a relatively strong killing effect on pathogenic B cells expressing the anti-AChR antibody, can effectively relieve the symptom of myasthenia gravis, but also has relatively low risk of inducing cytokine storm, and has good drug safety.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological drugs, and particularly relates to a chimeric autoantibody receptor (CAAR) targeting acetylcholine receptor antibodies, CAAR-T cells and application of the CAAR-T cells in drugs for preventing and treating myasthenia gravis. BACKGROUND

[0002] Myasthenia gravis (MG) is an autoimmune disease of neuromuscular junction (NMJ) transmission disorder, and is clinically manifested as partial or general skeletal muscle weakness and fatigue. In serological detection of patients, 85% are positive for acetylcholine receptor (AChR) antibodies, and these antibodies are mainly directed against the alpha subunit of AChR, and the pathogenicity thereof is stronger than that of other subunits. The AChR antibodies can block the signal transmission of ACh at the NMJ by interfering with the recruitment of AChR, resulting in impaired ion transport across the muscle membrane and reduced muscle contraction. The conventional treatment means mainly includes non-specific immunotherapy of glucocorticoids and non-hormone immunosuppressants, and the adverse reactions and complications of the therapy increase the burden of disease treatment. SUMMARY

[0003] Therefore, the purpose of the application is to provide a chimeric autoantibody receptor (CAAR) targeting acetylcholine receptor antibodies, which is prepared by connecting the extracellular domains of AChR alpha subunits in series through L1 and L2 connecting peptides or G4S connecting peptides. The CAAR not only has a strong binding capacity to anti-AChR antibodies, but also is expressed on the surface of T cells, and shows a strong killing effect on pathogenic B cells expressing anti-AChR antibodies, and relieves the symptoms of myasthenia gravis.

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

[0005] ECD1, ECD2 and ECD3 in the extracellular domain of the AChR alpha subunit are connected in series through L1 and L2 connecting peptides or two G4S connecting peptides; the amino acid sequence of the L1 is shown as SEQ ID NO: 1; and the amino acid sequence of the L2 is shown as SEQ ID NO: 2.

[0006] Preferably, the intracellular domain of the costimulatory molecule comprises an intracellular costimulatory molecule CD28.

[0007] The transmembrane region domain comprises CD8a.

[0008] Preferably, the amino acid sequence of the chimeric autoantibody receptor targeting acetylcholine receptor antibody is shown as SEQ ID NO: 4 or SEQ ID NO: 5.

[0009] The present application provides a nucleic acid molecule encoding the chimeric autoantibody receptor targeting acetylcholine receptor antibody.

[0010] Preferably, the DNA sequence comprises a nucleotide sequence shown as SEQ ID NO: 6 or SEQ ID NO: 7.

[0011] The present application provides a recombinant vector comprising the nucleic acid molecule.

[0012] Preferably, the backbone vector of the recombinant vector comprises a lentiviral vector pLVX-EF1a-IRES-puro.

[0013] The present application provides a CAAR-T cell targeting acetylcholine receptor antibody, wherein the T cell surface expresses the chimeric autoantibody receptor targeting acetylcholine receptor antibody.

[0014] The present application provides a use of the CAAR-T cell targeting acetylcholine receptor antibody or the CAAR-T cell targeting acetylcholine receptor antibody prepared by the recombinant vector in the preparation of a drug for preventing and / or treating myasthenia gravis.

[0015] The present application provides a drug for treating myasthenia gravis, wherein the active ingredient comprises the CAAR-T cell targeting acetylcholine receptor antibody or the CAAR-T cell targeting acetylcholine receptor antibody prepared by the recombinant vector.

[0016] The application provides a chimeric autoantibody receptor targeting 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 signaling domain CD3 gamma; the amino acid sequence of the CD3 gamma is shown as SEQ ID NO: 3; ECD1, ECD2 and ECD3 in the AChR alpha subunit extracellular domain are connected in series by L1 connecting peptides and L2 connecting peptides or connected in series by two G4S connecting peptides; the amino acid sequence of the L1 is shown as SEQ ID NO: 1; and the amino acid sequence of the L2 is shown as SEQ ID NO: 2. The application evaluates the binding ability of the chimeric autoantibody receptor to AChR specific antibodies by surface plasmon resonance (SPR) technology and flow cytometry, and the two chimeric autoantibody receptors provided by the application both have good binding ability to AChR specific antibodies, and the affinity of the extracellular domain fECD-L1L2 connected in series by L1 and L2 is greater than that of the extracellular domain fECD-GS connected by G4S.

[0017] The application provides a CAAR-T cell targeting acetylcholine receptor antibody, and the T cell surface expresses the chimeric autoantibody receptor targeting acetylcholine receptor antibody. The CAAR-T cell can not only accurately recognize pathogenic cells with specific BCR and kill the pathogenic cells, but also effectively kill specific B cells derived from a clinical MG patient, so that the level of AChR specific antibody binding is significantly reduced. In a NSG mouse tumor cell transplantation model or an MG passive immunization model, the fECD-L1L2-A28gCAAR-T cell can completely remove pathogenic B 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

[0018] Figure 1 CAAR molecular structure and function detection results provided by the application, wherein A. schematic diagram of a natural AChR alpha subunit and schematic diagram of a CAAR molecular structure; B. affinity constant of a CAAR molecular extracellular segment (fECD-GS or fECD-L1L2) to Fab 637-Biotin; C. efficiency of lentivirus infection on T lymphocytes;

[0019] Figure 2Results of fECD-A28g-CAAR-T cell killing cells in vitro; A. Construction and identification results of target cell lines stably expressing Fab637; B. fECD-L1L2-A28g CAAR-T cell killing Raji cell results; C. fECD-L1L2-A28g CAAR-T cell specific killing of Fab637 + Results of fECD-A28g-CAAR-T cell killing cells in vitro; A. Construction and identification results of target cell lines stably expressing Fab637; B. fECD-L1L2-A28g CAAR-T cell killing Raji cell results; C. fECD-L1L2-A28g CAAR-T cell specific killing of Fab637

[0020] Figure 3 Results of cytokine release level detection after co-culturing 1928z CAR-T cells, a210-GS-BBz CAAR-T cells or fECD-L1L2-A28g CAAR-T cells with Fab637 + Results of cytokine release level detection after co-culturing 1928z CAR-T cells, a210-GS-BBz CAAR-T cells or fECD-L1L2-A28g CAAR-T cells with Fab637

[0021] Figure 4 Results of AChR specific antibody release level detection after a210-GS-BBz CAAR-T cells or fECD-L1L2-A28g CAAR-T cells killing MG patient peripheral blood B cells

[0022] Figure 5 Results of in vivo experiment verification of fECD-A28g-CAAR-T cells; Wherein A. Flow chart of NSG mouse in vivo experiment; B. Small animal live imaging monitoring of mouse disease progression; C. fECD-L1L2-A28g-CAAR-T cells have more significant killing effect on anti-AChR antibody positive target cells in mice; D. fECD-L1L2-A28g CAAR-T cells show more stable body weight change trend

[0023] Figure 6 Results of fECD-A28g-CAAR-T cells treating myasthenia gravis symptoms in NSG mice, wherein A. Flow chart of NSG mouse in vivo experiment; B. Results of fECD-L1L2-A28g CAAR-T cells affecting mouse body weight; C. Results of mouse falling off the instrument from the rotarod; D. Results of mouse footprint width detected by gait analysis instrument; E. Results of mouse footprint length detected by gait analysis instrument; F. Results of mouse footprint peak area detected by gait analysis instrument DETAILED DESCRIPTION

[0024] The 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 gamma; the amino acid sequence of the CD3 gamma is shown as SEQ ID NO: 3 (GQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN).

[0025] In the application, ECD1, ECD2 and ECD3 in the AChR alpha subunit extracellular domain are connected in series by L1 and L2 connection peptides to form a fECD-L1L2-A28g extracellular domain (see Figure 1 in the middle A). The L1 and L2 connection peptides are two end-advantage connection 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 and the anti-AChR antibody achieve the best affinity. 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. ECD1, ECD2 and ECD3 in the AChR alpha subunit extracellular domain also comprise two G4S connection peptides connected in series to form a fECD-GS-A28g extracellular domain (see Figure 1 in the middle A).

[0026] In the present application, the intracellular domain of the costimulatory molecule preferably comprises the intracellular costimulatory molecule CD28. The transmembrane domain preferably comprises CD8a. The CD3y is a modified signaling domain, containing only one ITAM motif. The amino acid sequence of the CAAR comprising fECD-L1L2 is preferably as set forth in SEQ ID NO: 4 (MEPWPLLLLFSLCSAGLVLGSEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRLKQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLLQYTGHITWTPPAIFKSYCEIIVTHFPFDEQNCSMKLGTWTYDGSVVAINPESDQPDLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLPLWFEENILKPVEEIDGPPPEELIEKILEEIPSTSSAVPLIGKKLLELEGKPPPPELPKEKLKEFLKRLIELNQQGFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSTSGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN).The amino acid sequence of the CAAR comprising fECD-GS is preferably as shown in SEQ ID NO: 5 (MEPWPLLLLFSLCSAGLVLGSEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRLKQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLLQYTGHITWTPPAIFKSYCEIIVTHFPFDEQNCSMKLGTWTYDGSVVAINPESDQPDLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLPLGGGGSGGGGSSTSSAVPLIGKGGGGSGGGGSRLIELNQQGFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSTSGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN).

[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 2:1, the fECD-L1L2-A28g CAAR-T cell has a significant killing intensity compared with 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-A28g CAAR-T cell is maintained at a lower level than that of the a210-GS-BBz CAAR-T cell. The analysis reason is that the signal domain CD3y of 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 CD3zeta, 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 a tumor cell transplantation model of immunodeficient animals.

[0038] In one embodiment of the application, the fECD-L1L2-A28g CAAR-T cell is administered to a passive immunization NSG mouse model, and results show that, compared with a model group, the CAAR-T cell of the targeting acetylcholine receptor antibody can reverse weight loss of the model mouse, prolong the time for the model mouse to fall off a rotating rod, and improve the gait of the model mouse, which indicates that the fECD-L1L2-A28g 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 more, can be 1x10 6 / mL to 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 of the targeting acetylcholine receptor antibody, the CAAR-T cell and the drug for preventing and treating myasthenia gravis will be described in detail in combination with examples below, but they should not be understood as limitations on the protection scope of the application.

[0042] Example 1

[0043] Construction and detection of fECD-L1L2-A28g

[0044] 1. Recombinant plasmid construction method

[0045] The three extracellular domains (ECD) of the natural AChR α subunit were connected in series by two superior linkers (L1, L2) designed by computational deep learning and de novo folding combined with homology modeling, as the extracellular domain (AChR α-ECD) of the CAAR molecule. The coding sequence of the CAAR molecule was composed of the extracellular domain AChR α-ECD, the transmembrane region CD8α, the intracellular costimulatory molecule CD28, and the signal transduction domain CD3γ, with pLVX-EF1ɑ-IRES-puro as the lentiviral vector backbone. The obtained recombinant plasmid was named fECD-L1L2-A28g Figure 1 A) in the middle.

[0046] The sequences of the extracellular domains in fECD-L1L2-A28g-CD3γ are as follows:

[0047] ECD1: SEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDE VNQIVTTNVRLKQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLLQYTGHITWTPPAIFKSYCEIIVTHFPFDEQNCSMKLGTWTYDGSVVAINPESDQPDLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQRLPL (SEQ ID NO: 8);

[0048] linker1 (L1): WFEENILKPVEEIDGPPPEELIEKILEEIP (SEQ ID NO: 1); ECD2: STSSAVPLIGK (SEQ ID NO: 9);

[0049] linker2 (L2): KLLELEGKPPPPELPKEKLKEFLK (SEQ ID NO: 2);

[0050] ECD3: RLIELNQQG (SEQ ID NO: 10).

[0051] Meanwhile, the three extracellular domains (ECD) of the natural AChR α subunit were connected in series by two G4S flexible linkers to obtain the extracellular domain of the CAAR molecule, and the obtained recombinant plasmid was named fECD-GS-A28g (see Figure 1A) of the middle.

[0052] The sequences of each of the extracellular domains in the ECD-GS-A28g 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 two recombinant plasmids described above were transformed into E. coli, cultured, and the bacterial liquid was obtained, and the bacterial liquid was subjected to PCR identification. The sequences of the PCR amplification primers are as follows:

[0059] F: 5'-TCAAGCCTCAGACAGTGGTTC-3' (SEQ ID NO: 12);

[0060] R: 5'-GCGTATCCACATAGCGTAAAAG-3' (SEQ ID NO: 13).

[0061] The reaction system (10 μL system) of PCR amplification was as follows: 5 μL of 2xT5 Super PCR Mix (Tiangen); 1 μL of bacterial liquid; 0.5 μL of each of the upstream and downstream primers (10 μM); and 3 μL of deionized water. The reaction program of PCR amplification was as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 sec; annealing at 56°C for 10 sec; and extension at 72°C for 10 sec. After 30 cycles of the above denaturation-annealing-extension program, the reaction was further extended at 72°C for 2 min to fully amplify the reaction.

[0062] The PCR amplification products were sequenced, and the sequencing results showed that they were consistent with the theoretical sequences.

[0063] 2. SPR technology for detecting the binding kinetics of recombinant proteins

[0064] 1) Using the mature mammalian cell protein expression platform in our 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;

[0065] 2) Antibody immobilization: the IgG heavy chain C-terminal biotin-modified AChR alpha antibody (Fab 637-Biotin) was coupled to the surface of the SA sensor chip (GE Healthcare Life Sciences), and the flow rate was set to 10 μL / min, and the injection time was 2 min;

[0066] 3) Test regeneration conditions: inject the protein to be tested, 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;

[0067] 4) Interaction: dilute the protein to be tested to 6 different concentration gradients, and sequentially inject them in order of concentration from low to high, with a flow rate of 30 μL / min, and a contact time of 100 s for each concentration. The regeneration condition is that the glycine pH is 2.5, the flow rate is 10 μL / min, the dissociation time is 30 s, and the stabilization period is 5 s;

[0068] 5) The whole experiment was carried out at 25°C using Biacore T200 instrument (GE Healthcare Life Sciences), and the data were analyzed using Biacore T200 Evaluation software, selecting 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's association rate constant (Association rate constant, Ka), dissociation rate constant (Dissociation rate constant, Kd) and equilibrium dissociation constant (Equilibrium dissociation constant, KD).

[0069] The binding rate constant and dissociation rate constant of fECD-L1L2 and fECD-GS recombinant proteins to AChR specific antibodies were analyzed by surface plasmon resonance (SPR) technology. 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 = 3.25 nM, and the dissociation equilibrium constant of fECD-GS was KD = 22.5 nM. It can be seen that the affinity size order is: fECD-L1L2 > fECD-GS.

[0070] 3. Monoclonal antibody detection of T cell surface lentivirus infection positive rate

[0071] 1) Lentivirus packaging: when the growth density of HEK293T cells reaches 80%, two kinds of recombinant plasmids with a concentration of 1 mg / ml are selected for transfection, and the virus supernatant is collected at 48 h and 72 h, respectively. After filtering through a 0.45 μm filter, it is stored in a -80℃ refrigerator; At the same time, controls are also set up, and the recombinant plasmid expressing α210-GS-BBz (CN114127287A) or the recombinant plasmid of 1928z (plasmid source Yescarta FMC63-28Z GenBank: HM852952.1) is transfected.

[0072] 2) Isolation and activation of human primary T lymphocytes: EDTA anticoagulated venous blood of healthy volunteers was collected, and PBMC was obtained by density gradient centrifugation. MojoSort TM Human CD3 T cell isolation kit (Biolegend, USA) was used to obtain primary T cells, and human T cell activation / amplification CD3 / CD28 magnetic beads (ACRO Biosystem company) were used to stimulate T cell activation.

[0073] 3) Lentivirus infection of human primary T lymphocytes: T cells were infected with virus stock solution by centrifugation, 800g centrifugation for 90 min. After 24 h of infection, the liquid was removed by centrifugation, and T cell culture solution was added at a cell density of 1×10 6 Normal culture;

[0074] 4) Flow cytometry to identify the binding efficiency of monoclonal antibody to CAAR: 100 μL of recombinant human anti-AChR monoclonal antibody Fab 637 was added to CAAR-T cells, respectively, and CD19-Biotin (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) was added to the prepared 1928z-CAR-T, 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, 1928z added Straptavidin-APC flow cytometry antibody (Biolegend, USA), mixed and vortexed the cells, incubated at 4°C for 30 min. After washing the cells, resuspend the cells with flow washing solution and sediment, and detect by flow cytometry.

[0075] Flow cytometry results showed that 1928z, a210-GS-BBz (CN 114127287 A), fECD-GS-A28g or fECD-L1L2-A28g could be normally expressed on the surface of T cells, and the binding efficiency of fECD-L1L2-A28g group to recombinant monoclonal antibody was the highest Figure 1 In the middle C). It is shown that the ECD full-length of L1 and L2 in series is used as the extracellular domain of CAAR, which can be more fully combined with anti-AChR antibody.

[0076] Based on the above structure design and result evaluation, fECD-L1L2 is used as the extracellular domain of CAAR molecule in the follow-up experiment, and the application of CAAR-T cells in the treatment of myasthenia gravis is explored.

[0077] Example 2

[0078] fECD-L1L2-A28g CAAR-T cell precise killing specific target cell experiment

[0079] 1. Fab 637 + Construction and identification method of Raji target cells

[0080] After it is determined that fECD-L1L2-A28g CAAR-T cells can be combined with natural anti-AChR antibody, in order to simulate the role of autoreactive B cells in MG patients, the killing function of CAAR-T cells is explored, and recombinant human anti-AChR antibody Fab 637 is stably expressed on Raji cells by lentivirus mediation Figure 2 In the middle A).

[0081] 1) The construction method of recombinant vector containing Fab 637 sequence is as follows: the Fab 637 gene fragment (heavy chain amino acid sequence:

[0082] MEFGLSWVFLVAIIKGVQCQVQLVESGGGLVKPGGSLRLSCAASGFKSTDYY

[0083] MSWVRQAPGRGLEWVSFISGRVFTNYTASVRGRFTVFREDDNTSVYLQMSR

[0084] LRVEDTAVYYCARLRGIFRGPLKPLEYYFDLWGRGTLVTVSSASTKGPSVFPL

[0085] APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS

[0086] LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL

[0087] LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN

[0088] AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK

[0089] AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY

[0090] KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; light chain amino acid sequence: MAWAPLLLTLLAHCTGSWANFMLTQPHSVSESPG KTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSNSGGRVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS), pLVX lentivirus vector was selected, and the fragment and the vector were double digested with restriction enzymes EcoRI and XbaI, respectively, and then connected and transformed. The double digestion reaction system and conditions were as follows: 10 μL of the Fab 637 gene fragment or the pLVX lentivirus vector; 3 μL of cutsmart (NEB, USA); 0.5 μL of each of restriction enzymes EcoRI and XbaI (NEB, USA); and 16 μL of deionized water. The reaction condition was 37 °C for 2 h. The connection reaction system and conditions were as follows: 5 μL of Ligation Mix connecting enzyme; 4 μL of the Fab 637 gene fragment digestion product; and 1 μL of the pLVX lentivirus vector digestion product. The reaction condition was 16 °C for 30 min. Transformation: the connection product was added to 40 μL of competent cells, mixed, and then sequentially subjected to ice incubation for 20 min, 42 °C heat shock for 90 s, and ice incubation for 2 min; 120 μL of liquid LB medium without antibiotics was added, and the mixture was cultured at 37 °C and 150 rpm for 30 min on a shaker; all the bacterial solution in the EP tube was inoculated on an LB solid culture dish containing Amp + (see prior art: Meng F, Stassen MH, Schillberg S, Fischer R, DeBaets 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.).

[0091] 2) The packaging method of the recombinant lentivirus containing the Fab 637 sequence is the same as in Example 1.

[0092] 3) Stable transfection: Raji cells were infected with the virus stock solution and cell culture solution at a volume ratio of 1:1. After 72 hours of infection, the solution was removed by centrifugation, and the cells were resuspended with cell culture solution and then normally cultured.

[0093] 4) Flow cytometry to identify infection efficiency: The cells were collected by centrifugation, resuspended with 100 μL of flow cytometry washing solution, and 5 μL of human mlgG-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 with flow cytometry washing solution and detected by flow cytometry.

[0094] 2. Gradient killing experiment of effector-target ratio

[0095] 1) Count the target cells stably expressing firefly luciferase that grow well, and collect the cells by centrifugation at a number of 2 x 10 4 per well for use; the target cells stably expressing firefly luciferase are obtained by directional cloning, in which the firefly luciferase (Firfly Luciferase, Fluc) coding gene is inserted into the pLVX-EF1α-IRES-puro lentivirus vector. 10 μg of lentivirus plasmid encoding Fluc molecules, 7.5 μg of pSPAX2, and 2.5 μg of pMD2G are co-transfected into HEK293T cells pre-seeded into culture dishes by 50 μL of Lipofectamine 2000. The culture supernatant containing virus particles is collected at 48 h and 72 h after transfection. The Raji cells are infected by centrifugation using the lentivirus stock solution. After 72 h of culture, 10 μg / ml of blastin is added to the culture solution as a final concentration for screening of the infected positive cell strain Fluc + Raji.

[0096] 2) CAAR-T cells are added to the corresponding wells at an effector-target ratio of 4:1, 2:1, 1:1, 0.5:1, and 0.25:1, wherein the number of CAAR-T cells is 8 x 10 4 , 4 x 10 4 , 2 x 10 4 , 1 x 10 4 , 0.5 x 10 4 , 1928z-CAR-T cells are added to the control group, and no T cells are added to the blank group, only the target cells are reserved, and three replicate wells are set for each group;

[0097] 3) After 48 h of co-culture, all suspended cells are collected by centrifugation and washed once with pre-cooled 1 x PBS;

[0098] 4) Add diluted 1x luciferase substrate (Shanghai Lijibiotech Co., Ltd.) to each well in batches of 100 μL / well, stand at room temperature for 2 min, then collect fluorescence values by Xenogen IVIS Lumina II live imaging instrument, and analyze data using Living Image software; calculate the specific killing rate according to formula I.

[0099] Specific killing rate = (blank group luciferase activity - experimental group luciferase activity) / blank group luciferase activity x 100% formula I.

[0100] The results show that fECD-L1L2-A28g CAAR-T cells only present effector-target ratio-dependent killing effect on pathogenic cells expressing specific anti-AChR antibodies, have more precise pathogenic cell lysis ability than 1928z-CAR-T cells, and the killing level is not different from that of α210-GS-BBz CAAR-T cells Figure 2 B, C).

[0101] Example 3

[0102] fECD-L1L2-A28g CAAR-T cells maintain cytokine release at a safe level

[0103] A large number of studies have shown that cytokines play an important role in the production of pathogenic autoantibodies in MG patients and the induction of neuromuscular junction inflammation. At the same time, the level of cytokine release is also a key indicator for evaluating the safety of CAR-T cells. Therefore, after CAAR-T cells were co-cultured with pathogenic cells expressing anti-AChR antibodies in vitro, the level of cytokine release in the supernatant was detected, and the specific steps were as follows:

[0104] 1. 1928z CAR-T cells, α210-GS-BBz CAAR-T cells and fECD-L1L2-A28g CAAR-T cells were respectively co-cultured with Fab637 + Raji cells at an effector-target ratio of 2:1 for 24 h without serum, and the cell culture supernatant was collected by centrifugation and stored at 4°C for use;

[0105] 2. Use human LEGENDplex TM Multi-Analys FlowAssay kit reagent kit (Biolegend, USA) to process the supernatant of each group;

[0106] 3. Detect the cytokine release level of each group by flow cytometry.

[0107] The results show that the cytokine release levels of the three groups of CAR-T cells are significantly different. Under the premise of no difference in killing level, the cytokine release of fECD-L1L2-A28g CAAR-T cells is maintained at a lower level than that of a210-GS-BBz CAAR-T cells. It is shown that the new CAAR-T cells containing only one ITAM motif of CD3y as the first intracellular activation signal produce a significantly reduced level of cytokines, and the risk of cytokine storm is greatly reduced. Compared with a210-GS-BBz CAAR-T containing three ITAM of natural motif CD3z, it is safer Figure 3 ).

[0108] Example 4

[0109] fECD-L1L2-A28g CAAR-T cells reduce pathogenic antibodies in peripheral blood of MG patients

[0110] Since the proportion of AChR-specific B cells in the peripheral blood of MG patients is very low, the research group developed a Feeder cell (3T3-CD40L-BAFF-IL2-IL4-IL21 cell) to culture B cells to differentiate into antibody-secreting plasma cells, and to evaluate the killing ability of CAAR-T cells on specific B cells from MG patients by detecting the level of specific antibodies. The steps of Feeder cell co-culture system and antibody level detection are as follows:

[0111] 1. B cell sorting kit (Mei Tianyin Biotechnology Co., Ltd., Germany) was used to obtain CD19 + B cells in the peripheral blood of MG patients, resuspended at 1x10 5 / ml for use;

[0112] 2. Add NT cells or CAAR-T cells to the B cell suspension at an effector-to-target ratio of 2:1, and kill for 48 h at 37℃ and 5% CO2;

[0113] 3. At 24 h of killing, count 3T3-CD40L-BAFF-IL2-IL4-IL21 cells, add an appropriate amount of Mitomycin (MMC) to inhibit cell proliferation at a cell density of 1x10 6 / ml, and after washing, seed the cells in a 12-well plate for use;

[0114] 4. After 48 h 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 the cell culture supernatant every 3-5 days, and transfer the co-culture system into a new well plate;

[0115] 5. The collected cell supernatant was concentrated and subjected to ELISA detection of anti-AChR antibody (RSR Limited, UK).

[0116] The results showed that the level of AChR-specific antibody produced by the B cells differentiated from the fECD-L1L2-A28g CAAR-T cell-killed B cells was significantly reduced, indicating that the fECD-L1L2-A28g CAAR-T cell could effectively kill the patient-derived specific B cells Figure 4 ), which also confirmed that the fECD-L1L2-A28g CAAR-T cell was more advantageous than the a210-GS-BBz CAAR-T cell for immunotherapy of MG patients.

[0117] Example 5

[0118] fECD-L1L2-A28g CAAR-T cells can completely eliminate pathogenic cells expressing specific BCR in NSG mice

[0119] Based on the above in vitro cell function experiment results, the Fab 637 + Raji cells were used to construct a tumor cell transplantation model in NSG mice. Figure 5 A).

[0120] 1. Establishment of a tumor cell transplantation model in NSG mice

[0121] 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;

[0122] 2) 5-7 days later, 2 x 10 5 Fab 637 + Raji cells were resuspended in 100 μL of 1 x PBS, and 100 μL of the cell suspension was drawn into a 1 mL syringe and injected into the mice via the tail vein;

[0123] 3) 7 days after injection of target cells, 100 μL of 1 x luciferase substrate was injected into the abdominal cavity of the mice, and after 5-10 min of anesthesia in a chamber containing isoflurane gas, the fluorescence signal was collected by the Xenogen IVIS Lumina II live imaging instrument, and the Living Image software was used to evaluate the distribution of anti-AChR antibody-positive target cells in the mice. The successfully modeled mice were randomly divided into 2 groups for use.

[0124] 2. Monitoring of disease progression by small animal live imaging technology

[0125] 1) Treatment was administered via tail vein injection of CAAR-T cells or NT cells (activated T cells not infected with any lentiviruses) according to the group assignments. The injection concentration of CAAR-T cells or NT cells was 2 × 10⁻⁶. 6 One cell suspension, with an injection volume of 200 μl;

[0126] 2) Imaging was performed weekly for a total of 4 weeks. Data was analyzed using Living Image software, and fluorescence intensity curves of pathogenic cells were plotted.

[0127] 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.

[0128] In vivo imaging results in small animals showed that, compared with the NT group, the fECD-L1L2-A28g 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-A28g 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-A28g group remained stable, gradually increasing after day 14. 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).

[0129] Example 6

[0130] fECD-L1L2-A28g CAAR-T cells can effectively alleviate myasthenia gravis symptoms in NSG mice.

[0131] Clinical studies have shown that the symptoms of myasthenia gravis are mainly caused by the overexpression of autoimmune antibodies in the body. Therefore, 6c7 cells with both membrane expression and antibody secretion types were selected to investigate the effect of CAAR-T cells on alleviating the symptoms of myasthenia gravis in mice.

[0132] 1. Establishment of NSG mouse MG passive immunization model: 2×10 5 6c7 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 a 10:1 effector-to-target ratio, using the same injection method as described in Example 5. The weight and vital signs of the mice were monitored and recorded every 3-4 days, and changes in behavioral function were also monitored (see [link to example]). Figure 6 (A)

[0133] 2. Rotarod test: To detect the motor ability of mice, and evaluate the symptoms of myasthenia gravis. One day before the experiment, mice were trained on the Rotarod Model LE8505 at 4 rpm to adapt to the environment of the Rotarod. On the day of the experiment, mice were placed on the Rotarod, and the speed was set in the range of 4-40 rpm. The speed and time at which the mice fell were recorded. Each mouse was trained for 3 times with an interval of 15 min.

[0134] 3. Gait analysis: Before the experiment, the mice were placed on the transparent runway, and the speed was set to 5 cm / s. The instrument was started, and the speed was gradually increased according to the adaptation of the mice. When the mice could walk steadily on the transparent runway, the experiment was started. The stable footstep action of the mice was captured for 5-10 s, and the recording was stopped. Each mouse was tested for 3 times with an interval of 15 min. After all the mice completed the test, the videos were analyzed using DigiGait software to evaluate and calculate the step length, step width, and maximum foot contact area.

[0135] The body weight monitoring curve showed that the body weight of the mice in the fECD-L1L2-A28g 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 Fig. 3B).

[0136] The Rotarod test results showed that the time for the mice in the fECD-L1L2-A28g group to fall off 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 symptoms of myasthenia gravis. Figure 6 Fig. 3C).

[0137] The gait analysis results 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-A28g group were significantly higher than those of the NT group, which could not walk normally. Figure 6 Fig. 3D, E, F).

[0138] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, which should be considered as the protection scope 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 gamma; 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 L1 is shown as SEQ ID NO: 1; the amino acid sequence of L2 is shown as SEQ ID NO: 2; The amino acid sequence of CD3 gamma is shown as SEQ ID NO:

3.

2. The chimeric autoantibody receptor targeting acetylcholine receptors of claim 1, wherein, The intracellular domain of the costimulatory molecule comprises intracellular costimulatory molecule CD28; The transmembrane region domain comprises CD8 alpha.

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 the 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 or 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 drug for treating myasthenia gravis, characterized by comprising the compound according to claim 1. The active ingredient comprises the CAAR-T cell of the targeting acetylcholine receptor antibody according to claim 8 or 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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