WO2023044633A1

WO2023044633A1 - Application of bcma car-t in preparation of drug for treating autoimmune diseases

Info

Publication number: WO2023044633A1
Application number: PCT/CN2021/119720
Authority: WO
Inventors: 汪文; 胡广; 蔡松柏; 李大可; 操昌普; 谢白露; 吴明; 徐夏君; 姚正; 郭静静; 庞昊
Original assignee: 南京驯鹿医疗技术有限公司
Priority date: 2021-09-22
Filing date: 2021-09-22
Publication date: 2023-03-30
Also published as: WO2023045934A1

Abstract

Provided is an application of an immune effector cell in the preparation of a drug for preventing and/or treating autoimmune diseases. The immune effector cell comprises a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain targeting BCMA, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain, wherein the antigen binding domain is a fully human antigen binding domain.

Description

Application of BCMA CAR-T in the preparation of drugs for the treatment of autoimmune diseases

technical field

This application relates to the field of biomedicine, in particular to the application of CAR-T cells targeting BCMA in the preparation of drugs for the treatment of autoimmune diseases.

Background technique

Antibody-mediated idiopathic inflammatory disease of the nervous system (also known as autoimmunity) is an autoimmune disease in which autoimmune cells and molecules attack the nervous system as the main pathogenic mechanism. In the immune response, pathogens that act on the nervous system autoantigens are collectively called nervous system autoantibodies, and antibody-mediated nervous system idiopathic inflammatory diseases can occur in the central nervous system, peripheral nervous system, and nerve-muscle junctions.

Neuromyelitis optica (NMO) is an acute or subacute inflammatory demyelinating disease of the central nervous system and an antibody-mediated idiopathic inflammatory disease of the nervous system. NMO sectrum disorder (NMOSD) is marked by the presence of NMO-IgG antibodies in serum, covering NMO and NMO-related diseases. AQP4-Ab is the most important pathogenic antibody of NMOSD. A large number of basic research and clinical research It has been confirmed that the antibody can cause damage to the central nervous system of animals and humans, and its diagnostic specificity can be as high as more than 90%. The positive rate of AQP4-Ab in NMOSD patients is between 40% and 90%.

BCMA (B cell maturation antigen, B cell maturation antigen) is a cell surface receptor mainly expressed in long-lived plasma cells (effector B cells), germinal center B cells and human memory B cells. BCMA detaches from the cell surface to form free BCMA (sBCMA) in peripheral blood. Studies have shown that the level of sBCMA is correlated with the level of IgG. So far, the drugs marketed in China include hormones, immunosuppressants, etc., and patients frequently relapse after using them. There is no effective therapy for the treatment of neuromyelitis optica. Drugs used for off-label diseases are not effective. Relapse and refractory treatment are still a difficult problem for the majority of medical workers. Therefore, it is necessary and urgent to find new treatments.

Contents of the invention

The application provides the use of immune effector cells in the preparation of drugs for the prevention and/or treatment of autoimmune diseases, the immune effector cells include a chimeric antigen receptor (CAR) targeting BCMA, and the immune effector cells can effectively use For the prevention and/or treatment of autoimmune diseases such as neuromyelitis optica spectrum diseases.

In one aspect, the application provides the use of immune effector cells in the preparation of drugs for the prevention and/or treatment of autoimmune diseases, the immune effector cells comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a BCMA-targeted Antigen binding domain, transmembrane domain, co-stimulatory domain and intracellular signaling domain, wherein the antigen binding domain is a fully human antigen binding domain.

In certain embodiments, the autoimmune disease comprises an autoimmune inflammatory disease.

In certain embodiments, the inflammatory disease comprises neuritis.

In certain embodiments, the inflammatory disease comprises a demyelinating disease.

In certain embodiments, the inflammatory disease comprises an inflammatory disease of the central nervous system.

In certain embodiments, the inflammatory disease comprises an inflammatory demyelinating disease of the central nervous system.

In certain embodiments, the inflammatory disease comprises optic neuritis.

In certain embodiments, the inflammatory disease comprises myelitis.

In certain embodiments, the inflammatory disease comprises Neuromyelitis Optic Spectrum Disease (NMOSD).

In certain embodiments, the inflammatory disease comprises AQP4 (aquaporin 4)-Ab positive neuromyelitis optica spectrum disorder.

In certain embodiments, the autoimmune disease comprises an AQP4-Ab-mediated autoimmune disease.

In certain embodiments, the autoimmune disease comprises an AQP4-Ab positive disease and/or condition.

In certain embodiments, the AQP4-Ab positive disease and/or condition comprises central nervous system demyelinating disease.

In certain embodiments, the AQP4-Ab positive disease and/or condition comprises an AQP4-Ab positive neuromyelitis optica spectrum disorder.

In certain embodiments, the autoimmune disease comprises Neuromyelitis Optic Spectrum Disease (NMOSD).

In certain embodiments, the neuromyelitis optica spectrum disorder (NMOSD) is relapsed refractory NMOSD.

In some embodiments, the antigen binding domain comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, Said HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11.

In some embodiments, the antigen binding domain comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 17, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 18 , and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 19.

In certain embodiments, the antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:7.

In certain embodiments, the antigen binding domain comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 15.

In certain embodiments, the antigen binding domain comprises the amino acid sequence shown in SEQ ID NO:43.

In certain embodiments, the antigen binding domain comprises an antibody or fragment thereof.

In certain embodiments, the antigen binding domain comprises a scFv.

In certain embodiments, the antigen binding domain is capable of binding BCMA.

In certain embodiments, the transmembrane domain comprises a transmembrane domain derived from a protein selected from the group consisting of the alpha, beta or zeta chain of a T cell receptor, CD28, CD3e, CD45, CD4, CD5, CD8a , CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

In certain embodiments, the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:27.

In certain embodiments, the co-stimulatory domain comprises a polypeptide selected from the group consisting of CD28, 4-1BB, OX-40, and ICOS.

In certain embodiments, the co-stimulatory domain comprises the amino acid sequence set forth in SEQ ID NO:29 or SEQ ID NO:31.

In certain embodiments, the intracellular signaling domain comprises a signaling domain from CD3ζ.

In certain embodiments, the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:33.

In certain embodiments, the CAR further comprises a hinge region linking the antigen binding domain and the transmembrane domain.

In certain embodiments, the hinge region comprises the amino acid sequence shown in SEQ ID NO:25.

In some embodiments, the CAR is also linked to a signal peptide.

In some embodiments, the signal peptide comprises the amino acid sequence shown in SEQ ID NO:3.

In certain embodiments, the CAR is also linked to a cleavage peptide.

In certain embodiments, the cleaved peptide comprises an amino acid sequence from a T2A peptide.

In some embodiments, the cleaved peptide comprises the amino acid sequence shown in SEQ ID NO:35.

In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:49 or SEQ ID NO:51.

In certain embodiments, the immune effector cells comprise T cells and/or natural killer (NK) cells.

In certain embodiments, the medicament comprises optionally a pharmaceutically acceptable carrier.

On the other hand, the present application also provides an administration method, which comprises administering the immune effector cells described in the present application to a subject in need. In the present application, the administration method can be used for the prevention and/or treatment of autoimmune diseases.

In certain embodiments, the method of administering comprises administering the immune effector cells to the subject at a dose of about 0.25×10 ⁶ to 1×10 ⁶ cells/kg.

In certain embodiments, the method of administering comprises administering the immune effector cells to the subject at a dose of about 0.25×10 ⁶ to 0.5×10 ⁶ cells/kg.

In certain embodiments, the administration method comprises administering the immune effector cells to the subject at a dose of about 0.5×10 ⁶ to 1×10 ⁶ cells/kg

In certain embodiments, the method of administering comprises administering the immune effector cells to the subject at a dose of about 0.25 x ¹⁰⁶ cells/kg.

In certain embodiments, the method of administering comprises administering the immune effector cells to the subject at a dose of about 0.5 x ¹⁰⁶ cells/kg.

In certain embodiments, the method of administering comprises administering the immune effector cells to the subject at a dose of about 1 x ¹⁰⁶ cells/kg.

In certain embodiments, the method of administration comprises intravenous injection.

In certain embodiments, the immune effector cells are administered once.

In certain embodiments, the method of administration comprises treating the subject prior to infusion of the immune effector cells.

In certain embodiments, the treating of the subject comprises lymphoid depleting treatment.

In certain embodiments, said lymphoid depletion treatment is initiated about 4 days prior to infusion of said immune effector cells.

In certain embodiments, the period of the lymphoid depletion treatment is about 3 days.

In certain embodiments, the method of administration comprises a 3-day lymphodepletion treatment beginning 4 days prior to infusion of the immune effector cells.

In certain embodiments, the lymphoid depletion treatment comprises infusing the subject with cyclophosphamide prior to infusion of the immune effector cells.

In certain embodiments, the infusion dose of cyclophosphamide is about 500 mg/m ² /day.

In certain embodiments, said lymphoid depletion treatment comprises infusion of said cyclophosphamide 4 days, 3 days, or 2 days prior to infusion of said immune effector cells.

In certain embodiments, the method of administration comprises administering fludarabine to the subject following infusion of the cyclophosphamide.

In certain embodiments, the fludarabine is administered at a dose of about 30 mg/m ² .

In certain embodiments, the fludarabine is administered for more than about 30 minutes.

In certain embodiments, the lymphoid depletion treatment comprises receiving an infusion of cyclophosphamide at about 500 mg/m ² /day 4 days, 3 days, and 2 days prior to the infusion of the immune effector cells, and infusion of cyclophosphamide Administration of about 30 mg/ ^m2 of fludarabine over about 30 minutes immediately following the amide.

In another aspect, the present application provides a pharmaceutical kit, which includes: 1) immune effector cells, the immune effector cells comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain targeting BCMA, transmembrane domain, co-stimulatory domain and intracellular signal transduction domain, wherein the antigen binding domain is a fully human antigen binding domain; and 2) a depleting reagent.

In some embodiments, the depleting agent includes cyclophosphamide, and/or fludarabine.

In some embodiments, the immune effector cells in the pharmaceutical kit comprise CAR, the antigen binding domain of the CAR comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises amino acids shown in SEQ ID NO:9 Sequence, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11.

In some embodiments, the immune effector cells in the pharmaceutical kit comprise a CAR, and the antigen binding domain of the CAR comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises SEQ ID NO: 17 Amino acid sequence, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 18, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 19.

In certain embodiments, the immune effector cells in the pharmaceutical kit comprise a CAR, the antigen binding domain of the CAR comprises a heavy chain variable region, and the heavy chain variable region comprises SEQ ID NO: 7 Amino acid sequence shown.

In certain embodiments, the immune effector cells in the pharmaceutical kit comprise a CAR, and the antigen binding domain of the CAR comprises a light chain variable region comprising SEQ ID NO: 15 Amino acid sequence shown.

In certain embodiments, the immune effector cells in the pharmaceutical kit comprise CAR, and the antigen binding domain of the CAR comprises the amino acid sequence shown in SEQ ID NO:43.

Those skilled in the art can easily perceive other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will appreciate, the content of the present application enables those skilled in the art to make changes to the specific embodiments which are disclosed without departing from the spirit and scope of the invention to which this application relates. Correspondingly, the drawings and descriptions in the specification of the present application are only exemplary rather than restrictive.

Description of drawings

The particular features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates can be better understood with reference to the exemplary embodiments described in detail hereinafter and the accompanying drawings. A brief description of the accompanying drawings is as follows:

Figures 1A-1C show the results of serum AQP4-IgG concentration changes in NMOSD patients after reinfusion of BCMA CAR-T cells described in this application.

Figure 2 shows the results of serum sBCMA detection of NMOSD patients after reinfusion of BCMA CAR-T cells described in this application.

Figures 3A-3B show the changes in the BCMA CAR-T cell concentration-time curve and VCN-time curve described in this application.

Detailed ways

The implementation of the invention of the present application will be described by specific specific examples below, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

Definition of Terms

In this application, the term "chimeric antigen receptor" (Chimeric Antigen Receptor, CAR) is a fusion protein of the variable region of a single-chain antibody and a T cell signaling molecule. It allows T cells to recognize specific antigens in a non-MHC-restricted manner and play a killing role. CAR is the core component of chimeric antigen receptor T cells (CAR-T), which includes antigen (such as tumor-associated antigen, tumor-associated antigen, TAA) binding region, transmembrane domain, co-stimulatory domain and intracellular signal domain. In this application, the CAR can be a genetically engineered chimeric protein capable of redirecting the cytotoxicity of immune effector cells to B cells, which combines antibody-based antigen (such as BCMA) specificity with T cell receptor activation The intracellular domains are grouped together. T cells genetically modified to express CAR can specifically recognize and eliminate malignant cells expressing target antigens.

In this application, the terms "BCMA" and "B-cell maturation antigen" are used interchangeably, a member of the tumor necrosis factor receptor family. In the present application, the BCMA may be human BCMA, whose GenBank accession number is BAB60895.1. BCMA is a type III transmembrane protein with a cysteine-rich domain (CRD) characteristic of TNFR family members in the extracellular domain (ECD), which forms a ligand-binding motif. As a B cell biomarker, BCMA is expressed in tumor cells (eg, multiple myeloma cells) or located on the surface of tumor cells (eg, present in multiple myeloma malignant plasma cells). BCMA proteins may also include fragments of BCMA, such as the extracellular domain and fragments thereof.

In the present application, the term "BCMA binding domain" generally refers to a domain that can specifically bind to a BCMA protein or a fragment thereof. For example, the BCMA extracellular binding domain may comprise a chimeric antigen receptor capable of specifically binding to a human BCMA polypeptide expressed on a B cell, an anti-BCMA antibody or an antigen-binding fragment thereof. The terms "binding domain", "extracellular domain", "extracellular binding domain", "antigen-specific binding domain" and "extracellular antigen-specific binding domain" are used interchangeably in this application used, and a CAR having the ability to specifically bind a target antigen of interest (eg, BCMA) is provided. BCMA binding domains may be of natural, synthetic, semi-synthetic or recombinant origin.

In this application, the term "antibody" generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a specific antigen. For example, an antibody may comprise an immunoglobulin composed of at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, and includes any molecule comprising an antigen-binding portion thereof. The term "antibody" includes monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies (e.g., dAb), single chain antibodies (e.g., scFv), As well as antibody fragments (eg, Fab, Fab' and (Fab)2 fragments) that bind to the antigen. The term "antibody" also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof. Each heavy chain can be composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain can be composed of a light chain variable region (VL) and a light chain constant region. The VH and VL regions can be further distinguished into hypervariable regions called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs). Each VH and VL may consist of three CDR and four FR regions, which may be arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

In this application, the term "single-chain antibody" may be an antibody formed by linking the heavy chain variable region and the light chain variable region through a linker peptide.

In this application, the term "transmembrane domain" (Transmembrane Domain) generally refers to the domain in CAR that passes through the cell membrane, which is connected to the intracellular signal transduction domain and plays a role in transmitting signals.

In this application, the term "co-stimulatory domain" generally refers to an intracellular domain that can provide immune co-stimulatory molecules, which are cell surface molecules required for an effective response of lymphocytes to antigens. The costimulatory domain may include the costimulatory domain of CD28, and may also include the costimulatory domain of the TNF receptor family, such as the costimulatory domain of OX40 and 4-1BB.

In this application, the term "hinge region" generally refers to the structure used to connect the antigen recognition region and the transmembrane region.

In this application, the term "HA-tag" generally refers to a protein tag based on human influenza virus hemagglutinin (Human influenza hemagglutinin) antigen. short amino acid sequence. After the HA-tag sequence is joined to one end of the target protein by means of molecular biology, the specific antibody against the HA-tag can be used to bind the recombinant protein, which is beneficial to the carrying out of experiments such as immunohistochemistry (IHC) and Western Blotting (see Schembri, Laura et al. The HA tag is cleaved and loses immunoreactivity during apoptosis. Nature Methods. February 2007, 4(2):107-108).

In this application, the term "intracellular signaling domain" generally refers to the component of CAR located in intracellular signal transduction, which includes a signaling domain and a domain that specifically binds to the receptor component, for example: its It may be selected from CD3ζ intracellular domain, CD28 intracellular domain, CD28 intracellular domain, 4-1BB intracellular domain and OX40 intracellular domain.

In this application, the term "signal peptide" generally refers to a short (5-30 amino acids in length) peptide chain that directs the transfer of newly synthesized proteins to the secretory pathway.

In this application, the term "cleaved peptide" refers to self-cleaved 2A peptide, which can realize the function of cleaved protein through ribosome jumping instead of proteolytic hydrolysis, which may include T2A, F2A and P2A, etc.

In this application, the term "marker detection signal" generally refers to a gene, protein or other molecule with known function or sequence that can function as a specific marker and emit a detectable signal. The label detection signal can be a fluorescent protein, such as: GFP, RFP, YFP and the like. The marker detection signal may be EGFRt. The term "EGFRt" refers to the gene encoding a truncated human epidermal growth factor receptor polypeptide that lacks the distal membrane EGF-binding domain and cytoplasmic signaling tail, but retains extracellular epitopes recognized by anti-EGFR antibodies. EGFRt can be used as a non-immunogenic selection tool as well as a tracking marker for the function of genetically modified cells. In this application, it can be used as a marker molecule for CAR-T cells, and it can be used to clear CAR-T cells in vivo if necessary through the cetuximab mediated ADCC pathway (see US8802374B2).

The term "Kozak sequence" generally refers to the (gcc)gccRccAUGG sequence shared in eukaryotic mRNA, which plays an important role in the initiation of the translation process and is recognized by the ribosome as a translation initiation site (see De Angioletti M et al. A novel silent beta-thalassaemia mutation, the first in the Kozak sequence. Br J Haematol. 2004, 124(2):224–31.).

In this application, "sequence identity" generally refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino-acid-by-amino acid basis over a comparison window. "Percent sequence identity" can be calculated by comparing two optimally aligned sequences over a comparison window and determining the presence of identical nucleic acid bases (e.g., A, T, C, G, I) in the two sequences ) or the same amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) Number of positions To obtain the number of matching positions, the number of matching positions was divided by the total number of positions in the comparison window (ie, window size) and the result multiplied by 100 to yield the percent sequence identity. Optimal alignment for purposes of determining percent sequence identity can be achieved in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared or over a region of sequence of interest.

The term "variant" in relation to an antibody is used in this application to refer to an antibody that has been substituted by at least 1, such as 1-30, or 1-20 or 1-10, such as 1 or 2 or 3 or 4 or 5 amino acids, Antibodies with deletions and/or insertions of amino acid alterations to regions of the antibody of interest (e.g., heavy chain variable region or light chain variable region or heavy chain CDR region or light chain CDR region), wherein the variant substantially retains the antibody prior to the alteration Biological properties of molecules. In one aspect, this application encompasses variants of any of the antibodies described in this application. In certain embodiments, the antibody variant retains at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding ability) of the pre-altered antibody. In certain embodiments, the alterations do not result in the antibody variant losing binding to the antigen, but optionally may confer properties such as increased affinity for the antigen and different effector functions. It is understood that the antibody heavy chain variable region or light chain variable region, or each CDR region can be altered individually or in combination. In certain embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 amino acid changes in one or more or all three heavy chain CDRs or 10. In some embodiments, the amino acid changes may be amino acid substitutions, such as conservative substitutions. In certain embodiments, the antibody variant has at least 80%, 85%, 90% or 95% or 99% or more amino acid identity to the parent antibody over the antibody sequence region of interest.

In this application, the term "immune effector cells" generally refers to immune cells involved in clearing foreign antigens and performing effector functions in an immune response. For example, plasma cells, cytotoxic T cells, NK cells, APSC pluripotent cells, mast cells, etc.

In this application, the term "pharmaceutically acceptable carrier" generally refers to a pharmaceutically acceptable formulation carrier, solution or additive that enhances the properties of the formulation. Such additives are well known to those skilled in the art.

The term "and/or" should be understood as meaning any one of the alternatives or both of the alternatives.

As used in this application, the term "comprising" or "comprising" means including stated elements, integers or steps, but not excluding any other elements, integers or steps. In the present application, when the term "comprising" or "comprises" is used, unless otherwise specified, the situation consisting of the mentioned elements, integers or steps is also covered. For example, when referring to an antibody variable region that "comprises" a particular sequence, it is also intended to encompass an antibody variable region that consists of that particular sequence.

In this application, the term "about" generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.

In this application, the term "autoimmune disease" is used interchangeably with "autoimmune disease" and generally refers to any disease and/or condition in which the body mounts an immune system response to some component of its own tissues. For example, the immune system loses its ability to recognize a tissue or system in the body as itself, targeting and attacking it as if it were foreign. In the present application, the autoimmune disease may include autoimmune inflammatory disease.

In this application, the term "inflammatory disease" generally includes immune-mediated inflammatory processes, eg, components of the immune system in the body that cause, mediate or otherwise contribute to an inflammatory response, contributing to a disease or disorder.

In this application, the term "AQP4-Ab positive" can be used interchangeably with "AQP4 antibody positive" and "AQP4-IgG positive", and generally refers to the detection of AQP4-Ab by one or more detection means. For example, in this application, an in vitro diagnostic kit (enzyme-linked immunoassay) can be used for qualitative detection of AQP4-Ab, and the positive judgment value can be determined independently through experiments according to each kit. In the present application, the AQP4-Ab positive may include normal or pathologically significant references established in the clinic. For example, it can be determined whether it is AQP4-Ab positive by testing according to the positive determination part in the instruction manual of the ElisaRSRTM AQP4 Ab Version kit.

In the present application, the term "AQP4-Ab-mediated diseases and/or disorders" generally refers to diseases and/or disorders associated with AQP4-Ab expression. For example, the disease and/or condition can be caused by AQP4-Ab binding to AQP4, which binding can induce complement-dependent cytotoxicity and/or antibody-dependent cytotoxicity. For example, the binding can result in inflammation, oligodendrocyte damage demyelination, and/or neuronal loss.

In this application, the term "neuromyelitis optica spectrum disorder" can be used interchangeably with "NMOSD", and generally refers to an autoimmune disease of the central nervous system, which can include two groups of symptoms of the optic nerve and spinal cord. In the present application, the term can cover AQP4-Ab positive NMOSD as well as AQP4-Ab negative NMOSD. In this application, the terms "AQP4-Ab" and "AQP4-IgG" generally refer to anti-AQP4 antibodies.

Detailed description of the invention

In one aspect, the present application provides the use of immune effector cells comprising chimeric antigen receptors in the preparation of medicaments for preventing and/or treating autoimmune diseases. In certain embodiments, the immune effector cells comprise an antigen binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain.

In another aspect, the present application provides immune effector cells for preventing and/or treating autoimmune diseases.

In another aspect, the present application provides a method for preventing and/or treating an autoimmune disease, which comprises administering the immune effector cells described in the present application to a subject in need.

In the present application, the autoimmune disease may include autoimmune inflammatory disease. In the present application, the inflammatory disease may include neuritis.

In the present application, the inflammatory diseases may include demyelinating diseases.

In the present application, the inflammatory diseases may include central nervous system inflammatory diseases. In the present application, the inflammatory disease may include central nervous system inflammatory demyelinating disease.

In the present application, the inflammatory disease may include optic neuritis.

In the present application, the inflammatory disease may include myelitis.

In the present application, the inflammatory disease may comprise neuromyelitis optica (NMO). In the present application, the inflammatory disease may comprise Neuromyelitis Optic Spectrum Disease (NMOSD).

In the present application, the inflammatory disease may comprise AQP4 (aquaporin 4)-Ab positive neuromyelitis optica spectrum disorder.

In the present application, the autoimmune disease may include AQP4-Ab-mediated autoimmune disease. In the present application, the autoimmune disease may include AQP4-Ab positive diseases and/or disorders. In the present application, the AQP4-Ab-positive diseases and/or conditions may include central nervous system demyelinating diseases. In the present application, the AQP4-Ab-positive diseases and/or disorders may comprise AQP4-Ab-positive neuromyelitis optica spectrum disorders.

In the present application, the autoimmune disease may comprise neuromyelitis optica spectrum disease (NMOSD).

In the present application, a method of reducing the titer of AQP4-Ab in AQP4-Ab positive subjects in need of treatment for NMOSD is provided. In the present application, a method of reducing the occurrence and/or progression of NMOSD-related diseases and/or disorders in a subject in need of treatment for NMOSD is provided.

Neuromyelitis optica (NMO), also known as Devic's disease or Devic's syndrome, is an autoimmune inflammatory disease in which a person's own immune system attacks the optic nerve and spinal cord. The disease causes inflammation of the optic nerve (optic neuritis) and spinal cord (myelitis). Neuromyelitis optica is an acute or subacute inflammatory demyelinating disease of the central nervous system, usually an antibody-mediated idiopathic inflammatory disease of the nervous system.

NMO sectrum disorder (NMOSD) is marked by the presence of NMO-IgG antibodies in serum, covering NMO and NMO-related diseases. AQP4-Ab is the most important pathogenic antibody of NMOSD. A large number of basic and clinical studies have confirmed that this antibody can cause damage to the central nervous system of animals and humans, and its diagnostic specificity can be as high as 90%. NMOSD patients The positive rate of AQP4-Ab was between 40% and 90%.

AQP4-Ab is the pathogenic antibody of NMOSD. AQP4-IgG in serum and plasma cells producing AQP4-IgG infiltrate into the central nervous system, causing AQP4-IgG to bind to aquaporin 4 (AQP4) channels on astrocytes. Induction of complement-dependent cytotoxicity and antibody-dependent cytotoxicity leads to inflammation, oligodendrocyte injury demyelination and neuronal loss.

In the present application, the immune effector cells may comprise a chimeric antigen receptor, and the chimeric antigen receptor may comprise an antigen binding domain, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain.

In the present application, the antigen-binding domain of the CAR may include a fully human antigen-binding domain.

In the present application, the extracellular domain of the CAR may comprise the single-chain scFv antibody of the present invention. For example, the single chain antibody may be linked to the transmembrane domain via a hinge region, such as the CD8 hinge. In this application, the CAR can be used to transduce immune effector cells (such as T cells) and expressed on the cell surface.

In the present application, the chimeric antigen receptor (CAR) may comprise a BCMA binding domain, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain. In the present application, the BCMA-binding domain may comprise an antibody or fragment thereof that specifically binds BCMA. In the present application, the antigen binding domain may comprise heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3), the amino acids of the HCDR1-3 The sequence is shown in SEQ ID NO: 9-11, the antibody may comprise light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2) and light chain complementarity determining region 3 (LCDR3), the LCDR1 The amino acid sequence of -3 is shown in SEQ ID No:17-19. In the present application, the antigen-binding domain may comprise a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7. In the present application, the antibody may comprise a light chain variable region, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 15.

In the present application, the antigen-binding domain may comprise a single-chain antibody. In certain embodiments, the antigen binding domain may comprise the amino acid sequence shown in SEQ ID NO: 43 or a functional variant thereof. For example, the single chain antibody may comprise the amino acid sequence shown in SEQ ID NO:43.

The CAR described in the present application may include a transmembrane domain, which may comprise a polypeptide from a protein selected from the group consisting of α, β or ζ chains of T cell receptors, CD28, CD3e, CD45, CD4, CD5 , CD8a, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In the present application, the transmembrane domain may comprise the amino acid sequence shown by xx or a functional variant thereof. For example, the transmembrane domain of the present application may include CD8a, the sequence of which is shown in SEQ ID NO:27.

In the present application, the co-stimulatory domain may comprise a polypeptide from a protein selected from CD28, 4-1BB, OX40 and ICOS. In the present application, the co-stimulatory domain may comprise the amino acid sequence shown in SEQ ID NO: 29 or SEQ ID NO: 31 or a functional variant thereof.

The CAR described herein can include an intracellular signaling domain, which can comprise a signaling domain from CD3ζ. In the present application, the intracellular signaling domain may comprise the amino acid sequence shown in SEQ ID NO: 33 or a functional variant thereof.

The CAR described in the present application may include a hinge region, and the hinge region may connect the antibody and the transmembrane domain. In the present application, the hinge region may comprise the amino acid sequence shown in SEQ ID NO: 25 or a functional variant thereof.

The CAR described in the present application may also include an HA-tag, and the HA-tag may be located at the N-terminal of the CAR. In the present application, the HA-tag may comprise the amino acid sequence shown in SEQ ID NO: 5 or a functional variant thereof. In this application, the expression of the CAR described in this application can be detected by using an anti-HA antibody to specifically bind it and used to enrich CAR-T cells for functional research.

The CAR described in the present application may include a signal peptide, and the signal peptide may include the amino acid sequence shown in SEQ ID NO: 3 or a functional variant thereof. For example, the signal peptide can be a CD8a signal peptide, the sequence of which is shown in SEQ ID NO:3. For example, the CAR may include CAR0037, CAR0085, CAR0087.

In this application, the CAR can also be linked to a cleavage peptide. In the present application, the cleaved peptide may comprise an amino acid sequence derived from a T2A peptide. In the present application, the cleaved peptide may comprise the amino acid sequence shown in SEQ ID NO: 35 or a functional variant thereof. For example, the cleaved peptide can be T2A, the sequence of which is shown in SEQ ID NO:35. For example, the CAR may include CAR0037, CAR0087.

In the present application, the CAR can also be connected with a marker detection signal, and the marker detection signal can be located at the C-terminal of the CAR. In the present application, the label detection signal can be a fluorescent protein, which can be selected from the following group: GFP, RFP and YFP. In this application, the expression of CAR molecules can be evaluated indirectly by detecting the signal of GFP. For example, the CAR may include CAR0037, the marker detection signal sequence of which is shown in SEQ ID NO:37. In the present application, the marker detection signal may be EGFRt. For example, the CAR may include CAR0087, the marker detection signal sequence of which is shown in SEQ ID NO:39.

In the present application, the vector expressing the CAR may include the coding sequence of Kozak, the nucleotide sequence of which is shown in SEQ ID NO:2. In the present application, the vector expressing the CAR may include the coding sequence of Kozak. For example, the CAR may include CAR0037, CAR0085, CAR0087.

In the present application, the CAR may comprise the amino acid sequence shown in SEQ ID NO: 49 or SEQ ID NO: 51 or a functional variant thereof. For example, the CAR can be selected from CAR0037, the sequence of which is shown in SEQ ID NO:49. For another example, the CAR can be selected from CAR0085, whose sequence is shown in SEQ ID NO:51; the CAR can be selected from CAR0087, whose sequence is shown in SEQ ID NO:51.

In this application, the structures of CAR0037, CAR0085 and CAR0087 are shown in the following table:

In certain embodiments, the CAR described in the present application may sequentially include a BCMA-binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain from the N-terminus. Wherein, the CAR may include a BCMA binding domain, and the sequence of the BCMA binding domain is shown in SEQ ID NO:43. Wherein, the BCMA binding domain may include HCDR1-3, its sequence is shown in sequence as SEQ ID NO:9-11; and, the BCMA binding domain may include LCDR1-3, its sequence is as shown in SEQ ID NO:17- 19. For example, the CAR may include CAR0037 or the CAR described in this application having the same LCDR1-3 and HCDR1-3. The BCMA-binding domain can include a heavy chain variable region, whose sequence is shown in SEQ ID NO:7; and, the BCMA-binding domain can also include a light chain variable region, whose sequence is as shown in SEQ ID NO:15 shown. For example, the CAR may include CAR0037 or the CAR described in the present application having the same light chain variable region and heavy chain variable region as CAR0037. A connecting peptide may also be included between the light chain variable region and the heavy chain variable region, and its sequence is as shown in SEQ ID NO:23. For example, the CAR may include CAR0037 or the CAR described in the present application having the same linking peptide as CAR0037. The transmembrane domain may comprise a transmembrane domain from CD8a, and its sequence may be as shown in SEQ ID NO:27. For example, the CAR may include CAR0037 or the CAR described in the present application having the same transmembrane domain as CAR0037. The co-stimulatory domain may comprise a co-stimulatory structure from CD28, and its sequence may be as shown in SEQ ID NO:29. For example, the CAR may include CAR0037 or the CAR described in the present application having the same costimulatory domain as CAR0037. The intracellular signaling domain may comprise a signaling domain from CD3ζ, the sequence of which is shown in SEQ ID NO:33. For example, the CAR may include CAR0037 or the CAR described in the present application having the same intracellular signal transduction domain as CAR0037.

The CAR may also include a hinge region, which may be located at the C-terminus of the BCMA-binding domain and at the N-terminus of the transmembrane domain, the sequence of which is shown in SEQ ID NO: 25. For example, the CAR may include CAR0037 or the CAR described in this application having the same hinge region as CAR0037.

The CAR can also include an HA-tag, and the HA-tag can be located at the N-terminus of the BCMA-binding domain, and its sequence is shown in SEQ ID NO:5. For example, the CAR may include CAR0037 or the CAR described in this application having the same HA-tag as it.

The CAR can also be connected with a signal peptide, which can be located at the N-terminus of the CAR, and its sequence can be as shown in SEQ ID NO:3.

The CAR can also be linked to a cleavage peptide, such as T2A. The cleavage peptide may be located at the C-terminus of the intracellular signal transduction domain, and its sequence may be as shown in SEQ ID NO:35. The CAR can also be linked with a label detection signal, which can be located at the C-terminus of the CAR (or, the cleaved peptide). The marker detection signal can be selected from the following groups: GFP, RFP and YFP, the sequence of which is shown in SEQ ID NO:37.

For example, the CAR described in this application can be CAR0037, the amino acid sequences of its LCDR1-3 are respectively shown in SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19; the amino acid sequence of VL is shown in SEQ ID NO: 15; the amino acid sequence of HCDR1-3 is shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 respectively; the amino acid sequence of VH is shown in SEQ ID NO: 7; between VH and VL The sequence of the connecting peptide is shown in SEQ ID NO: 23; its hinge region is shown in SEQ ID NO: 25; its transmembrane domain is shown in SEQ ID NO: 27; its costimulatory domain is a CD28 costimulatory structure domain, as shown in SEQ ID NO:29; its CD3ζ intracellular signaling domain as shown in SEQ ID NO:33; the CAR0037 can also comprise a cleavage peptide as shown in SEQ ID NO:35, and as shown in SEQ ID NO:35 GFP marker detection signal shown in ID NO:37; the CAR0037 can also comprise the KOZAK sequence shown in SEQ ID NO:1, the CD8a signal peptide shown in SEQ ID NO:3, as shown in SEQ ID NO:5 HA-label shown.

For example, the CAR described in this application can be CAR0085, the amino acid sequences of its LCDR1-3 are respectively shown in SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19; the amino acid sequence of VL is shown in SEQ ID NO: 15; the amino acid sequence of HCDR1-3 is shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 respectively; the amino acid sequence of VH is shown in SEQ ID NO: 7; between VH and VL The sequence of the connecting peptide is shown in SEQ ID NO: 23; its hinge region is shown in SEQ ID NO: 25; its transmembrane domain is shown in SEQ ID NO: 27; its co-stimulatory domain is 4-1BB co- Stimulatory domain, as shown in SEQ ID NO: 31; Its CD3ζ intracellular signaling domain is shown in SEQ ID NO: 33; The vector expressing CAR0085 can also include the KOZAK sequence shown in SEQ ID NO: 1 ; The CAR0085 such as the CD8a signal peptide shown in SEQ ID NO: 3; the CAR0085 can also include a cleavage peptide as shown in SEQ ID NO: 35, and an EGFRt labeling detection signal as shown in SEQ ID NO: 39 .

For example, the CAR described in this application can be CAR0087, the amino acid sequences of its LCDR1-3 are respectively shown in SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19; the amino acid sequence of VL is shown in SEQ ID NO: 15; the amino acid sequence of HCDR1-3 is shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 respectively; the amino acid sequence of VH is shown in SEQ ID NO: 7; between VH and VL The sequence of the connecting peptide is shown in SEQ ID NO: 23; its hinge region is shown in SEQ ID NO: 25; its transmembrane domain is shown in SEQ ID NO: 27; its co-stimulatory domain is 4-1BB co- The stimulating domain is shown in SEQ ID NO:31; its CD3ζ intracellular signaling domain is shown in SEQ ID NO:33; the vector expressing CAR0087 can also include the KOZAK sequence shown in SEQ ID NO:2 ; The CAR0087 can also comprise a CD8a signal peptide as shown in SEQ ID NO:3.

In this application, NMOSD can be evaluated with reference to the EDSS neurological scale score, the number of active lesions on MRI, the annual recurrence rate and the number of inactive lesions, the annual recurrence rate, and the patient-free ratio.

In this application, the measured values of serum AQP4-Ab titers of NMOSD patients before and after treatment can be analyzed by descriptive statistics method, and the changes from the baseline can be used to evaluate the treatment effect.

In this application, the AQP4-Ab titer in the subject's serum can be detected by ELISA method to measure the change of AQP4-Ab in the subject's serum.

In the present application, the medicament for preventing and/or treating autoimmune diseases may comprise the immune effector cells described in the present application and optionally a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes and/or non-ionic surface Active agents, etc. In this application, the drug can be formulated for intravenous injection.

In another aspect, the present application provides an administration method, the method comprising administering the immune effector cells described in the present application to a subject in need. In the present application, the subject in need includes a subject suffering from an autoimmune disease.

In certain embodiments, the method of administering comprises administering the immune effector cells to the subject at a dose of about 0.5 x ¹⁰⁶ cells/kg. In certain embodiments, the method of administering comprises administering the immune effector cells to the subject at a dose of about 0.395 x ¹⁰⁶ cells/kg. In certain embodiments, the method of administering comprises administering the immune effector cells to the subject at a dose of about 0.425 x ¹⁰⁶ cells/kg. In certain embodiments, the method of administering comprises administering the immune effector cells to the subject at a dose of about 0.495 x ¹⁰⁶ cells/kg.

In the present application, the administration method may comprise intravenous injection. In the present application, the number of administration of the immune effector cells may be one time.

In certain embodiments, the lymphoid depletion treatment comprises receiving an infusion of cyclophosphamide at about 500 mg/m ² /day 4 days, 3 days, or 2 days prior to infusion of the immune effector cells, and Immediately thereafter, administer about 30 mg/ ^m2 of fludarabine over about 30 minutes.

On the other hand, the present application also provides a pharmaceutical kit, which includes: 1) immune effector cells, the immune effector cells comprise a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding antigen targeting BCMA domain, transmembrane domain, co-stimulatory domain and intracellular signaling domain, wherein the antigen-binding domain is a fully human antigen-binding domain; and a depleting reagent.

In the present application, the rinsing agent may include cyclophosphamide and/or fludarabine.

In the present application, the immune effector cells in the pharmaceutical kit may comprise CAR, and the antigen binding domain of the CAR may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1 may comprise SEQ ID NO:9 Amino acid sequence, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 10, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 11.

In the present application, the immune effector cells in the pharmaceutical kit may comprise CAR, and the antigen binding domain of the CAR may comprise LCDR1, LCDR2 and LCDR3, wherein the LCDR1 may comprise SEQ ID NO: 17 The amino acid sequence of the LCDR2 can include the amino acid sequence shown in SEQ ID NO: 18, and the LCDR3 can include the amino acid sequence shown in SEQ ID NO: 19.

In the present application, the immune effector cells in the pharmaceutical kit may comprise CAR, and the antigen binding domain of the CAR may comprise a heavy chain variable region, and the heavy chain variable region may comprise SEQ ID NO: The amino acid sequence shown in 7.

In the present application, the immune effector cells in the pharmaceutical kit may comprise a CAR, and the antigen binding domain of the CAR may comprise a light chain variable region, and the light chain variable region may comprise SEQ ID NO: The amino acid sequence shown in 15.

In the present application, the immune effector cells in the pharmaceutical kit may comprise CAR, and the antigen-binding domain of the CAR may comprise the amino acid sequence shown in SEQ ID NO:43.

Not intending to be limited by any theory, the following examples are only for explaining various technical solutions of the invention of the present application, and are not intended to limit the scope of the invention of the present application.

Example

Example 1 Preparation of BCMA CAR-T cells

The lentiviral vector PLVX-EF1alpha-IRES-Puro was double digested with NotI and MluI, and the vector fragment was recovered. The candidate scFv plasmid PXL0026 (its nucleotide sequence is SEQ ID NO: 44) was amplified by PCR, and the NotI restriction site (including protective bases), CD8a signal peptide, HA -label; hinge region, transmembrane region, CD28 co-stimulatory factor, CD3ζ intracellular signaling domain gene synthesis, PCR amplification; T2A cleavage peptide and eGFP PCR amplified from plasmid pMy-BirA-T2A-eGFP, 3 Carry MluI restriction site and protective base at the ' end; then use overlap PCR to obtain a PCR fragment with a NotI restriction site at the 5' end and a MluI site at the 3' end, and perform NotI and MluI double digestion on this fragment , and recycle. T4 connection was constructed to obtain the CAR plasmid numbered PXL0037 (the nucleotide sequence of CAR0037 is shown in SEQ ID NO: 50).

The CAR plasmid numbered PXL0085 was obtained in a similar way, the lentiviral vector PLVX-EF1alpha-IRES-Puro was double-digested with NotI and MluI, and the vector fragment was recovered. The candidate scFv plasmid PXL0026 was amplified by PCR, and the NotI restriction site (including protective bases), CD8a signal peptide, hinge region, transmembrane region, and 4-1BB co-stimulatory factor ( Its nucleotide sequence is SEQ ID NO: 32), CD3ζ intracellular signaling domain gene synthesis, PCR amplification; then use overlap PCR to obtain the 5' end band NotI restriction site 3' end band MluI site For the PCR fragment, NotI and MluI double enzyme digestion was performed on the fragment, and the fragment was recovered. T4 connection was constructed to obtain the CAR plasmid numbered PXL0085 (the nucleotide sequence of the CAR molecular part of CAR0085 is shown in SEQ ID NO: 52).

The CAR plasmid numbered PXL0087 was obtained in a similar way, the lentiviral vector PLVX-EF1alpha-IRES-Puro was double digested with NotI and MluI, and the vector fragment was recovered. The candidate scFv plasmid PXL0026 was amplified by PCR, and the NotI restriction site (including protective bases), CD8a signal peptide, hinge region, transmembrane region, and 4-1BB co-stimulatory factor ( Its nucleotide sequence is SEQ ID NO:32), CD3ζ intracellular signaling domain, T2A cleavage peptide, EGFRt (its nucleotide sequence is SEQ ID NO:40) gene synthesis, PCR amplification; Then use overlap A PCR fragment with a NotI restriction site at the 5' end and a MluI site at the 3' end was obtained by PCR, and the fragment was double digested with NotI and MluI and recovered. T4 connection was constructed to obtain the CAR plasmid numbered PXL0087 (the nucleotide sequence of the CAR molecular part of CAR0087 is shown in SEQ ID NO: 52).

After constructing CAR plasmids numbered PXL0037, PXL0085, and PXL0087 by lentiviral vector PLVX-EF1alpha-IRES-Puro (wherein the amino acid sequence of CAR0037 is: SEQ ID NO: 49, wherein the amino acid sequence of CAR0085 is: SEQ ID NO: 51 , wherein the amino acid sequence of CAR0087 is: SEQ ID NO: 51), the cells transfected with the CAR plasmid were subjected to lentivirus packaging to obtain LV0007, LV0020, LV0021 (corresponding to the CAR plasmids of PXL0037, PXL0085, and PXL0087) lentivirus. PBMCs were isolated from peripheral blood collected from healthy donors, and T cells were further sorted using CD3 MicroBeads. The sorted T cells were activated using CD3/CD28 Dynabeads, and then transduced with LV0007, LV0020, and LV0021 lentiviruses. The density of T cells during transduction was about 1.5×106 cells/ml. On day 3, a medium change was performed on the transduced T cells. According to the above process, LV0007-CAR-T, LV0020-CAR-T and LV0021-CAR-T cells were obtained. The preparation process of CAR-T cells is shown in the table below. The product must pass the quality inspection before it can be released. Before the release of each batch of finished products, the quality control department must collect and evaluate all the test records related to the batch of finished products. In the form of frozen suspension, filled in specific infusion bags for storage and transportation. The preparation process of CAR-T cells is shown in Table 1.

Table 1. Preparation process of CAR-T cells

Embodiment 2 Clearing lymph (lymphatic depletion) pretreatment

All examinations were completed within 1 week before the start of pretreatment for stranguria as a baseline, and pretreatment for stranguria clearing was performed 4 days to 2 days before the start of treatment. The pretreatment process for clearing stranguria is as follows: intravenous infusion of 500 mg/m ² cyclophosphamide, once a day for 3 consecutive days, each time over 30 minutes, and immediately after the completion of cyclophosphamide intravenous infusion, intravenous infusion of 30 mg/m ² Fludarabine, once a day for 3 consecutive days, each time more than 30 minutes. Taking subject Neu-003 as an example, the process of clearing stranguria is described in detail, and the pretreatment process of clearing stranguria is shown in Table 2. The degreasing treatment of other subjects was carried out in a similar manner.

Table 2 cleaning pretreatment situation

药物名称drug name	给药日期Dosing date	开始时间Starting time	结束时间End Time	剂量dose	单位unit
环磷酰胺cyclophosphamide	2020.12.312020.12.31	16:0516:05	16:3816:38	10201020	mgmg
氟达拉滨Fludarabine	2020.12.312020.12.31	16:4216:42	17:2017:20	6161	mgmg
环磷酰胺cyclophosphamide	2021.01.012021.01.01	11:4011:40	12:2612:26	10201020	mgmg
氟达拉滨Fludarabine	2021.01.012021.01.01	12:3012:30	13:1813:18	6161	mgmg
环磷酰胺cyclophosphamide	2021.01.022021.01.02	10:1610:16	10:5010:50	10201020	mgmg
氟达拉滨Fludarabine	2021.01.022021.01.02	10:5510:55	11:3211:32	6161	mgmg

The ELISA detection method and result of embodiment 3 AQP4-IgG

(1) Administration process

The subjects were AQP4-IgG positive relapsed refractory NMOSD patients. Three subjects (Neu-003, Neu-004, Neu-008) were given BCMA CAR-T cells at a dose of 0.5×10 ⁶ CAR+ cells/kg, and the actual infusion dose was 0.395×10 ⁶ CAR+ cells /kg, 0.425 ^{×10 6} CAR+ cells/kg and 0.495×10 ⁶ CAR+ cells/kg of BCMA CAR-T cells (containing the sequence of CAR0087). The BCMA CAR-T cells were infused intravenously two days after the end of the depletion preconditioning, and the number of infusions was once.

The timing of venous blood collection for changes in serum AQP4-IgG titer before and after treatment is as follows: screening period (D42-D20), twice a week from the screening period to before clearing the leprosy, 12 to 5 days before cell reinfusion (time window: no later than 1 minute before delineation), 1 day before cell reinfusion (time window: before reinfusion), 3 days (time window 8 hours), 7 days (time window 12 hours), 10 days (time window ± 12 hours ), 14 days (time window ± 1 day), 21 days (time window ± 1 day), 28 days (time window ± 3 days), 56 days (time window ± 3 days), 84 days (time window ± 7 days ), every 3 months (time window ± 14 days) within 2 years after 84 days.

CSF AQP4-IgG collection time: D12 to D5, 14 days (time window ± 1 day), 28 days (time window ± 3 days), 84 days (time window ± 7 days), 84 days later Every 3 months (time window ± 14 days) within 2 years and at the time of group visit.

(2) Detection method

The second-generation aquaporin 4 (AQP4) autoantibody ELISA detection kit (source: RSR company, batch number: 2KAQE66) was used for detection, and the absorbance value (OD value) was read at 450nm and 405nm on a microplate reader, and the OD value was obtained by fitting Values were compared with a standard curve to calculate the concentration levels of AQP4-IgG.

Descriptive statistics were used to analyze the measured values of AQP4-IgG concentrations in serum and cerebrospinal fluid of NMOSD patients before and after treatment. The serum AQP4-IgG concentration-time curves of NMOSD patients were drawn at each time point.

(3) Results

The concentration levels of AQP4-IgG in the serum of three patients were detected, and Figure 1A-1C shows the changing trend of AQP4-IgG in the serum of patients Neu-003, Neu-004, and Neu-008. The concentration of AQP4-IgG was high before cell reinfusion, and the concentration of AQP4-IgG decreased from the 14th day after reinfusion, suggesting that BCMA CAR-T cells can reduce AQP4-IgG in patients and have a significant therapeutic effect. Neu-003 and Neu-004 patient data cut off within 90 days after cell reinfusion. The data of Neu-008 patients were cut off within 14 days after cell reinfusion.

The detection results of AQP4-IgG in cerebrospinal fluid are shown in Table 3. The AQP4-IgG in Neu-003 patients was 1.279u/mL before reinfusion, and it could be reduced to 0.9628u/mL after BCMA CAR-T cell reinfusion. In patients with Neu-004, the concentration of AQP4-IgG in the cerebrospinal fluid reached 26.59u/mL 9 days before the cell reinfusion, and decreased significantly to 3.14u/mL on the 14th day after the cell reinfusion. down to 0.65u/mL. The AQP4-IgG in Neu-008 patient was 5.17u/mL before reinfusion, and it could be reduced to 3.88u/mL after cell reinfusion. The decrease of AQP4-IgG level after cell reinfusion indicates that CAR-T cell therapy is effective. Neu-003 and Neu-004 patient data cut off within 90 days after cell reinfusion. The data of Neu-008 patients were cut off within 28 days after cell reinfusion.

Table 3. Detection results of AQP4-IgG concentration levels in cerebrospinal fluid of NMOSD patients after BCMA CAR-T cell reinfusion

The ELISA detection method and result of embodiment 4 sBCMA

(1) Administration process

The collection time of peripheral blood free BCMA (sBCMA) before and after treatment is as follows: D12 to D5, 7 days (time window 12 hours), 14 days (time window 1 day), 21 days (time window 1 day) day), 28 days (time window 3 days), 56 days (time window ± 7 days), 84 days (time window 7 days), 84 days to 2 years every 3 months (time window 14 days) once and out of the group When visiting.

(2) Detection method

The Human BCMA/TNFRSF17 DuoSet ELISA detection kit (source: R&R company, article number: CAT#DY193) was used for detection, and the absorbance value (OD value) was read at 450nm and 570nm on the SpectraMax i3 microplate reader, and 570nm was used as the corrected wavelength. Fit the OD value to the standard curve to calculate the concentration level of sBCMA.

Descriptive statistics were used to analyze the measured values of sBCMA concentration in NMOSD patients before and after treatment. Concentration-time curves of sBCMA in NMOSD patients were drawn at each time point.

(3) Results

The sBCMA results and changing trends of the three patients are shown in Figure 2. The sBCMA concentration level was high before cell reinfusion, and the sBCMA level decreased significantly after reinfusion. Among them, Neu-004 and Neu-008 had sBCMA levels within 28 days after reinfusion. The concentration level dropped below the detection limit, suggesting that BCMA CAR-T cells can significantly reduce sBCMA in patients and indirectly reflect the reduction of plasma cells. Neu-003 and Neu-004 patient data cut off within 90 days after cell reinfusion. The data of Neu-008 patients were cut off within 14 days after cell reinfusion.

Embodiment 5 Pharmacokinetic (PK) experiment and result

(1) Experimental method

The concentration of BCMA CAR-T cells was detected by flow cytometry, and the main reagents and instruments involved were Anti-CD45 (PercP-labeled) and Anti-CD3, FITC-labeled BCMA protein from Acro BIOSYSTEMS, and Agilent’s Novocyte flow cytometer. The concentration of BCMA CAR-T cells is presented as the percentage (%) of CAR-T in lymphocytes.

The vector copy number (Vector copy number, VCN) of BCMA CAR-T cells was detected by droplet digital PCR (ddPCR). The main reagents and instruments involved are: Genomic DNA extraction kit from Qiagen, Thermo Fisher's NanoDrop Nucleic Acid Quantitative Instrument, BIO-RAD's droplet generator oil, droplet generator card and pad, ddPCR Supermix for Probes (no dUTP), droplet read oil and QX200 droplet digital PCR system. Theoretically, each BCMA CAR-T cell (CD3 ⁺ CAR ⁺ cell) contains 2 CAR VCNs. The increase in the number of VCN can reflect the expansion of CAR-T cells.

Sampling time: 12 to 5 days before the reinfusion of BCMA CAR-T cells (time window: before clearing lymph pretreatment), 1 day after reinfusion (time window ± 8 hours), 3 days (time window ± 8 hours), 5 days Days (time window ±12 hours), 7 days (time window ±12 hours), 10 days (time window ±12 hours), 14 days (time window ±1 day), 21 days (time window ±1 day), 28 days Blood samples were collected every 3 months (time window ± 14 days) after 84 days (time window ± 14 days) until CAR -2 years after the peak value was detected, no VCN was detected by droplet digital PCR (ddPCR), the disease worsened, and the group was discharged or reinfused. Among them: 1 day after reinfusion means 24 hours after reinfusion time, 3 days after reinfusion means 72 hours after reinfusion time, and so on for subsequent visits. Try to conduct blood routine sampling at the same time as PK sampling, and the time between blood routine sampling and PK blood collection should not exceed 24 hours at most.

Calculate the PK parameters of patients who have been reinfused with cells.

Plasma drug concentration-time data analysis: draw the concentration-time curves of BCMA CAR-T cells in peripheral blood and VCN-time curves at each time point; T concentrations and VCN values.

PK parameter analysis: Calculate the arithmetic mean, standard deviation coefficient of variation, median, and maximum value of PK parameters (C _max , T _max , AUC _0~ _28d , and AUC _0~last , etc.) through VCN and BCMA CAR-T cell concentrations , Minimum, Geometric Mean, and Geometric Mean Coefficient of Variation.

(2) Results

Figure 3A-3B shows the results of changes in BCMA CAR-T cell concentration and VCN. After administration, the concentration of CAR-T cells and VCN levels rise rapidly at first, and then decline slowly after reaching the peak. After administration, CAR-T cells expand in the patient's body. The increase was good, and VCN could still be detected 52 days after reinfusion. Neu-003 and Neu-004 patient data cut off within 90 days after cell reinfusion. The data of Neu-008 patients were cut off within 28 days after cell reinfusion.

The PK parameters are shown in Table 4. The median T _max of BCMA CAR-T cell concentration and VCN was 10 days, and the median C _max of BCMA CAR-T cell concentration and VCN were 77.78% and 55300 Copies/μg DNA, respectively. The median AUC _0-28 of VCN is 329184.4 Days×Copies/μg DNA (range 275819.600～1500890.100 Days×Copies/μg DNA), the median AUC _0-last of VCN is 329184.4 Days×Copies/μg DNA (range 275819.600～17060986.7 Days×Copies/μg DNA), it can be seen that the peak time of BCMA CAR-T cells in NMOSD patients is fast, and the cell expansion is good.

Table 4. PK parameters of BCMA CAR-T cells after reinfusion

Example 6 Inflammation factor detection method and result

(1) Detection method

The concentration of ferritin was detected by particle-enhanced immunoturbidimetry, the concentration of IL-6 was detected by electrochemiluminescence three-antigen sandwich turbidimetry, the concentration of C-reactive protein was detected by immune projection turbidimetry, and the concentration of C-reactive protein was detected by electrochemiluminescence three-antigen sandwich turbidimetry. Measure procalcitonin concentration.

(2) Detection results of inflammatory factors

The results of inflammatory factors in patients with Neu-003 are shown in Table 5. The highest concentration of ferritin after cell reinfusion was 654.6ug/mL, the highest concentration of IL-6 after cell reinfusion was 31.11pg/mL, and the concentration of C-reactive protein in cells The highest concentration after reinfusion was 16.7mg/L, and the concentration of procalcitonin after cell reinfusion was lower than that before reinfusion, and the concentration of various inflammatory factors did not increase after administration, which usually caused side effects. The results showed that BCMA CAR - Less risk of cytokine release syndrome from T cell therapy.

Table 5. Detection results of inflammatory factors

The foregoing detailed description has been offered by way of explanation and example, not to limit the scope of the appended claims. Variations on the presently recited embodiments of the present application will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and their equivalents.

Claims

The use of immune effector cells in the preparation of drugs for the prevention and/or treatment of autoimmune diseases, the immune effector cells comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain targeting BCMA, a transmembrane domain, co-stimulatory domain and intracellular signaling domain, wherein the antigen-binding domain is a fully human antigen-binding domain.
The use according to claim 1, wherein the autoimmune disease comprises autoimmune inflammatory disease.
The use according to claim 2, wherein the inflammatory disease comprises neuritis.
Use according to any one of claims 2-3, wherein the inflammatory disease comprises a demyelinating disease.
The use according to any one of claims 2-4, wherein the inflammatory disease comprises an inflammatory disease of the central nervous system.
The use according to any one of claims 2-5, wherein the inflammatory disease comprises central nervous system inflammatory demyelinating disease.
Use according to any one of claims 2-6, wherein the inflammatory disease comprises optic neuritis.
The use according to any one of claims 2-7, wherein the inflammatory disease comprises myelitis.
The use of any one of claims 2-8, wherein the inflammatory disease comprises Neuromyelitis Optic Spectrum Disease (NMOSD).
The use according to any one of claims 2-9, wherein the inflammatory disease comprises AQP4 (aquaporin 4)-Ab positive neuromyelitis optica spectrum disorder.
The use according to claim 1, wherein the autoimmune disease comprises AQP4-Ab-mediated autoimmune disease.
The use according to any one of claims 1-11, wherein the autoimmune disease comprises AQP4-Ab positive diseases and/or disorders.
The use according to claim 12, wherein the AQP4-Ab-positive diseases and/or conditions comprise central nervous system demyelinating diseases.
The use according to any one of claims 12-13, wherein the AQP4-Ab-positive diseases and/or disorders comprise AQP4-Ab-positive neuromyelitis optica spectrum disorders.
Use according to any one of claims 1-14, wherein the autoimmune disease comprises Neuromyelitis Optic Spectrum Disease (NMOSD).
The use according to any one of claims 1-15, wherein the antigen binding domain comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, and the HCDR2 comprises SEQ ID The amino acid sequence shown in NO: 10, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11.
Use according to any one of claims 1-16, wherein said antigen binding domain comprises LCDR1, LCDR2 and LCDR3, wherein said LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 17, said LCDR2 comprises SEQ ID NO: The amino acid sequence shown in ID NO: 18, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 19.
The use according to any one of claims 1-17, wherein the antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:7.
The use according to any one of claims 1-18, wherein the antigen binding domain comprises a light chain variable region, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:15.
The use according to any one of claims 1-19, wherein the antigen binding domain comprises the amino acid sequence shown in SEQ ID NO:43.
Use according to any one of claims 1-20, wherein the antigen binding domain comprises an antibody or fragment thereof.
The use according to any one of claims 1-21, wherein the antigen binding domain comprises a scFv.
The use according to any one of claims 1-22, wherein the antigen binding domain is capable of binding BCMA.
Use according to any one of claims 1-23, wherein said transmembrane domain comprises a transmembrane domain derived from a protein selected from the group consisting of α, β or ζ chains of T cell receptors, CD28, CD3e, CD45, CD4, CD5, CD8a, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.
Use according to any one of claims 1-24, wherein the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:27.
The use according to any one of claims 1-25, wherein the co-stimulatory domain comprises a polypeptide selected from the following proteins: CD28, 4-1BB, OX-40 and ICOS.
Use according to any one of claims 1-26, wherein the co-stimulatory domain comprises the amino acid sequence described in SEQ ID NO:29 or SEQ ID NO:31.
The use according to any one of claims 1-27, wherein the intracellular signaling domain comprises a signaling domain from CD3ζ.
The use according to any one of claims 1-28, wherein the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:33.
The use according to any one of claims 1-29, wherein the CAR further comprises a hinge region linking the antigen-binding domain and the transmembrane domain.
The use according to any one of claims 30, wherein the hinge region comprises the amino acid sequence shown in SEQ ID NO:25.
The use according to any one of claims 1-31, wherein the CAR is further linked to a signal peptide.
purposes according to claim 32, wherein said signal peptide comprises the aminoacid sequence shown in SEQ ID NO:3.
The use according to any one of claims 1-33, wherein the CAR is further linked to a cleavage peptide.
The use according to claim 34, wherein the cleaved peptide comprises an amino acid sequence from a T2A peptide.
Use according to any one of claims 34-35, wherein the cleaved peptide comprises the amino acid sequence shown in SEQ ID NO:35.
The use according to any one of claims 1-36, wherein the CAR comprises the amino acid sequence shown in SEQ ID NO:49 or SEQ ID NO:51.
The use according to any one of claims 1-37, wherein the immune effector cells comprise T cells and/or natural killer (NK) cells.
The use according to any one of claims 1-38, wherein the medicament comprises optionally a pharmaceutically acceptable carrier.
A method of administration comprising administering the immune effector cells of any one of claims 1-39 to a subject in need thereof.
The administration method according to claim 40, comprising administering the immune effector cells to the subject at a dose of about 0.25×10 6 to 1×10 6 cells/kg.
The method of administration according to any one of claims 40-41, comprising administering the immune effector cells to the subject at a dose of about 0.25 x 106 cells/kg.
The method of administration according to any one of claims 40-41, comprising administering the immune effector cells to the subject at a dose of about 0.5 x 106 cells/kg.
The method of administration according to any one of claims 40-41, comprising administering the immune effector cells to the subject at a dose of about 1 x 106 cells/kg.
The method of administration according to any one of claims 40-44 comprising intravenous injection.
The administration method according to any one of claims 40-45, wherein the number of administration of the immune effector cells is one.
The method of administration according to any one of claims 40-46, comprising treating the subject prior to infusion of the immune effector cells.
The method of administration according to claim 47, wherein said treatment comprises lymphoid depletion treatment.
The method of administration according to claim 48, wherein said lymphoid depletion treatment is started about 4 days before infusion of said immune effector cells.
The method of administration according to any one of claims 48-49, wherein the period of the lymphoid depletion treatment is about 3 days.
The method of administration according to any one of claims 48-50, comprising receiving a 3-day lymphoid depletion treatment starting 4 days before the infusion of the immune effector cells.
The method of administration according to any one of claims 48-51, wherein the lymphoid depletion treatment comprises infusing the subject with cyclophosphamide prior to infusion of the immune effector cells.
The administration method according to claim 52, wherein the infusion dose of cyclophosphamide is about 500 mg/m 2 /day.
The method of administration according to any one of claims 52-53, wherein the lymphoid depletion treatment comprises infusion of the cyclophosphamide 4 days, 3 days, and 2 days before the infusion of the immune effector cells.
The method of administration according to any one of claims 52-54, which administers fludarabine to the subject after infusion of the cyclophosphamide.
The administration method according to claim 55, wherein the dose of fludarabine administered is about 30 mg/m 2 .
The method of administration according to any one of claims 55-56, wherein the fludarabine is administered for more than about 30 minutes.
The administration method according to any one of claims 55-57, wherein the lymphoid depletion treatment comprises receiving about 500 mg/m 2 / Infusion of about 30 mg/ m2 of fludarabine over about 30 minutes immediately after cyclophosphamide.
Drug kit, including:

1) Immune effector cells comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain targeting BCMA, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling structure domain, wherein the antigen binding domain is a fully human antigen binding domain; and

2) Cleaning reagents.
According to the pharmaceutical kit according to claim 59, said cleaning agent comprises cyclophosphamide and/or fludarabine.
The pharmaceutical kit according to any one of claims 59-60, wherein the antigen binding domain comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9, and the HCDR2 comprises The amino acid sequence shown in SEQ ID NO: 10, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 11.
The pharmaceutical kit according to any one of claims 59-61, wherein the antigen binding domain comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 17, and the LCDR2 Comprising the amino acid sequence shown in SEQ ID NO: 18, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 19.
The pharmaceutical kit according to any one of claims 59-62, wherein the antigen binding domain comprises a heavy chain variable region, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:7.
The pharmaceutical kit according to any one of claims 59-63, wherein the antigen binding domain comprises a light chain variable region, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15.
The pharmaceutical kit according to any one of claims 59-64, wherein the antigen binding domain comprises the amino acid sequence shown in SEQ ID NO:43.