B cell-specific mAb-siRNA conjugate ameliorates myasthenia gravis

Abstract

The present invention relates to a combination of two antibody-RNA conjugates comprising an anti-BAFF receptor antibody or binding fragment thereof bound or conjugated to a small RNA targeting BAFF receptor mRNA, and an anti-BCMA specific antibody or binding fragment thereof bound or conjugated to a small RNA targeting BAFF receptor mRNA. A composition comprising the combination is to be used for the treatment of an autoimmune disorder, preferably myasthenia gravis.

Description

[Technical Field]

[0001] The present invention relates to a combination of two antibody-RNA complexes, to compositions comprising said combinations, and to compositions for use as pharmaceuticals, particularly for the treatment of autoimmune muscle disorders, in particular myasthenia gravis. [Background technology]

[0002] Myasthenia gravis (MG) is a chronic autoimmune neuromuscular disorder characterized by muscle fatigue and weakness caused by antibody- and complement-mediated damage to the neuromuscular junction.

[0003] Patients with MG who are seropositive for autoantibodies against the acetylcholine receptor (AChR), muscle-specific tyrosine (MuSK), or low-density lipoprotein receptor-related protein 4 (LRP4) exhibit voluntary muscle weakness due to dysfunction of the neuromuscular junction and impaired neuromuscular transmission. 70% of patients with MG are seropositive for AChR-specific autoantibodies and have significantly reduced muscle function. Clinical signs of muscle wasting include ptosis, diplopia, slurred speech, appendage tremor, and even respiratory failure due to disease exacerbation.

[0004] To date, approximately 700,000 cases of MG with either systemic or ocular involvement have been reported worldwide.

[0005] There is an increasing emphasis on developing new therapies for MG. In addition to conventional treatments such as thymectomy, intravenous immunoglobulin, plasma exchange, or corticosteroids, new treatments are needed for MG patients who do not achieve disease remission with conventional treatments. In recent years, new non-immunosuppressive therapies for MG have focused primarily on target-specific therapies, particularly monoclonal antibody (mAb)-based therapies, such as rituximab, belimumab, and other neonatal Fc receptor-targeting mAbs. Several mAbs have been developed against B cell-specific cluster of differentiation biomarker proteins to control B cell maturation, differentiation, survival, and pathogenic autoantibody production. The main goal of these treatments is to prevent pathogenic autoantibody production and complement activation. Reducing circulating levels of autoantibodies blocks AChR depletion and complement activation at the postsynaptic neuromuscular junction, thus preserving muscle function.

[0006] The first therapeutic mAb for targeted therapy in patients was muromonab, a murine anti-CD3 mAb used to prevent tissue rejection. Subsequently, mAbs were engineered to incorporate both human and murine sequences (humanization), followed by the production of fully human recombinant mAbs.

[0007] Rituximab or RTX (also known as Rituxan, Rixathon, or Truxima [Genentech, San Francisco, GA, USA]) is a chimeric mouse-human IgG1k mAb that targets CD20, a 33-kDa protein expressed on pro-B cells and all mature B cells, but not on long-lived plasma cells or plasmablasts. CD20 plays a key role in B cell proliferation and differentiation into plasma cells, and rituximab can efficiently deplete CD20-positive B cells in MG patients. However, this is not effective in reducing pathogenic AChR-Ab levels. Long-lived plasma cells, which are the major producers of autologous Abs, lack CD20. Therefore, rituximab likely targets only short-lived plasma cells and CD20+, IL10-producing B-reg or B10 cells. Reduction of autologous Abs is generally short-lived and insufficient, resulting in only transient clinical improvement. Thus, AChR-MG and MuSK-MG patients treated with rituximab often have relapses or recurrences of disease after an initial phase of disease peptides from IgG immune complexes.

[0008] Thus, there remains a need for ever more effective treatments that reduce pathogenic AChR-Ab levels. Summary of the Invention [Problem to be solved by the invention]

[0009] The technical problem underlying the present invention is to prevent pathogenic autoantibody production and complement activation. Reduction of circulating levels of autoantibodies blocks AChR depletion and complement activation at the postsynaptic neuromuscular junction, thus preserving muscle function.

[0010] The technical problem is solved by the embodiments provided herein below and characterized in the appended claims. [Means for solving the problem]

[0011] In a first aspect, the present invention provides a combination of two antibody-RNA complexes, an anti-BAFF receptor antibody or a binding fragment thereof bound or conjugated to a small RNA that targets the BAFF receptor mRNA; - an anti-BCMA specific receptor antibody or binding fragment thereof bound or conjugated to a small RNA that targets the BCMA receptor mRNA.

[0012] In another aspect, the present invention relates to a composition comprising a combination according to the present invention.

[0013] In a further aspect, the present invention relates to a composition according to the invention for use as a medicament.

[0014] In a particular embodiment, the present invention relates to a composition for use according to the present invention for the treatment of an autoimmune disorder.

[0015] In a more particular embodiment, the present invention relates to a composition for use according to the present invention for the treatment of an autoimmune myopathic disorder, preferably myasthenia gravis.

[0016] Applicants have been able to demonstrate that the duoconjugate has potential therapeutic value for the treatment of MG and other autoimmune diseases.

[0017] Applicants were also able to observe that PEGylation further improved the therapeutic efficacy of the duoconjugate in terms of higher levels of autoantibody reduction and clinical improvement in a mouse model of myasthenia gravis.

[0018] Further aspects and advantages of the present invention are described in the following description (with reference to FIGS. 1-6), which description should be considered illustrative and not limiting of the scope of the present application. [Brief explanation of the drawings]

[0019] FIG. 1. Preparation of mAb / siRNA conjugates. [Figure 1A] Purification of protaminated mAb. [Top left] Purification of protaminated mAb. Protamine alone (lane 1) and contaminating unreacted or free protamine removed (or not removed) from the protamine-linked mAb through gel filtration (lanes 2 and 3, respectively). [Top center] Verification of the presence of mAb and siRNA in the conjugate (representative gel image). The conjugate with appropriate controls was electrophoresed in a native polyacrylamide gel. Strong association of siRNA with the mAb alters the electrophoretic mobility of both the mAb and both siRNAs. Lanes 3 and 4 (top panel) show the siRNA component of the conjugate, detected via staining the gel with ethidium bromide (top panel). The mAb component was detected by restaining the gel with Coomassie brilliant blue (bottom panel). [Top right] The conjugate eluted from the 10% native polyacrylamide gel was then separated in a 4%-20% sodium dodecyl sulfate-polyacrylamide gel. Gel eluted conjugate band with reduced and non-reduced loading dye and bovine IgG control (lanes 1, 2, and 5, respectively). [Figure 1B] Zeta potential (surface charge) measurements of siRNA, protaminate mAb (mt), and duoconjugate (smt). [Figure 1C] Serum stability of the conjugate. Figure 2 Dose kinetics: Conjugate-mediated suppression of target receptor-expressing B cells. [Figure 2A] Flow analysis of isolated splenocytes (3 dpt) stained with fluorochrome-conjugated antibodies specific for B cells expressing CD268, CD269, and B220 on their surface (indicated by columns). [Figure 2B] The BCMA+ cell frequencies (3 dpt) in the spleen, lymph nodes, and PBMCs by three doses of mAb-siRNA conjugate are also shown in bar graphs (bottom). [Figure 2C]Relative expression of BR and BCMA normalized to beta-actin via qRT-PCR. Fig. 3 CD19+ cell frequency after IFNAR neutralization or siRNA GC content changes in the conjugates (6 wpt). [Figure 3A] Flow cytometry analysis of LN cells (6 wpt) showing the frequency of CD19+ B cells in EAMG mice pretreated (or not) with IFNAR antibody prior to conjugate treatment, or treated with anti-IFNAR antibody alone. [Figure 3B] Frequency of CD19+ cells after treatment of mice with duoconjugates containing siRNA and siRNA with high or low GC content (6 wpt). [Figure 3C] Effect of GC content of siRNA on the frequency of AChR+ B cells and CD95+ cells (6 wpt). Figure 4. Frequency of total CD27+ memory cells. [Figure 4A] Frequencies of total CD27+, B220+, and CD4+ cells after treatment with the conjugates (3 mpt). [Figure 4B] Frequencies of total CD27+, B220+, and CD4+ cells after treatment with the conjugates (3 mpt). [Figure 4C] Frequencies of total CD27+, B220+, and CD4+ cells after treatment with the conjugates (3 mpt). [Figure 4D] Gating used. Vertical bars represent mean ± SEM, calculated p-value indicates significance at p<0.001 (***). All experiments were repeated three times. Figure 5. Anti-AChR antibody affinity / levels in mice treated with conjugates. [Figure 5A] Measurement of serum levels of pathogenic anti-AChR lgG2b by ELISA and a novel serum antibody immunoprecipitation assay. [Figure 5B] Measurement of serum levels of pathogenic anti-AChR IgG2b by ELISA and a novel serum antibody immunoprecipitation assay. Figure 6. [Figure 6A] Grip strength of EAMG mice in the treatment and control groups. [Figure 6B]ELISA results for functional muscle AChRs at the experimental endpoint of 4 mpt. [Figure 7] A comparison of class-switched memory B cell (CD27+IgD-) frequencies between mice treated with the conjugate and untreated mice is shown. DETAILED DESCRIPTION OF THE INVENTION

[0020] As used herein, unless otherwise specified, when an interval is given, it is understood to include the upper and lower limits of that interval.

[0021] The present invention provides materials and methods useful for therapy, more particularly for the treatment of autoimmune diseases. More specifically, the present invention provides a combination of mAb and siRNA that can potentially inhibit the function of all pathogenic autoantibody-producing B cells and their precursor cells.

[0022] The advantages of the present invention are more particularly demonstrated with respect to the treatment of myasthenia gravis.

[0023] In a first aspect, the present invention relates to the combination of two antibody-RNA complexes.

[0024] The terms "antibody," "immunoglobulin," and "antibody / immunoglobulin-binding fragment," as used herein, refer to polypeptides, and more precisely to preparations including polyclonal and monoclonal antibody preparations, as well as hybrid antibodies, modified antibodies, F(ab')2 fragments, F(ab) fragments, Fv fragments, single domain antibodies, chimeric antibodies, humanized antibodies, and functional fragments or proteins thereof that exhibit the immunological binding properties of an antibody molecule for a specific antigen target.

[0025] The term "polypeptide," as used herein, refers to any peptide or protein comprising two or more amino acids joined together by peptide bonds or modified peptide bonds. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides, and oligomers, and to longer chains, commonly referred to as proteins. Polypeptides may contain amino acids other than those encoded by genes. "Polypeptides" include those modified either by natural processes, such as processing and other post-translational modifications.

[0026] As used herein, the term "antigen-binding site" or "binding portion" refers to the portion of an antibody or immunoglobulin molecule that is involved in specific antigen binding. The antigen-binding site is formed by amino acid residues from the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches within the V regions of the heavy and light chains are called "hypervariable regions" and are interposed between more conserved adjacent stretches known as "framework regions" or "FRs." Thus, the term "FR" refers to the amino acid sequences naturally found between and adjacent to the hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are positioned relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are called "complementarity-determining regions" or "CDRs."

[0027] The term "complex," as used herein, preferably refers to an association of two molecules held together by chemical forces.

[0028] In a preferred embodiment, the antibody is a monoclonal antibody (mAb). As used herein, the term "monoclonal antibody" refers to an antibody composition having a homogeneous antibody population. The term is not limited regarding the species or source of the antibody, nor is it intended to be limited by the manner in which it is produced. The term encompasses whole immunoglobulins as well as fragments such as Fab, F(ab')2, Fv, and other fragments that exhibit the immunological binding properties of the parent monoclonal antibody molecule.

[0029] The combination according to the present invention first comprises an anti-BAFF receptor antibody or a binding fragment thereof bound or conjugated to a small RNA that targets the BAFF receptor mRNA, such that the anti-BAFF receptor antibody or a binding fragment thereof bound or conjugated to the small RNA degrades the BAFF receptor mRNA.

[0030] As used herein, the term "BAFF receptor" refers to the BAFF receptor (B-cell activating factor receptor, BAFF-R, BR), also known as tumor necrosis factor receptor superfamily member 13C (TNFRSF13C), which is a membrane protein of the TNF receptor superfamily that recognizes BAFF. In humans, the BAFF receptor is encoded by the TNFRSF13C gene (UniProt Q96RJ3, GenBank AF373846.1), the relevant sequence of which is incorporated herein by reference.

[0031] The term "conjugated," as used herein, refers to a compound formed by the joining of two or more chemical / biological compounds.

[0032] The combination according to the invention secondly comprises an anti-BCMA specific receptor antibody or binding fragment thereof bound or conjugated to a small RNA that targets the BCMA receptor mRNA, such that the anti-BCMA specific receptor antibody or binding fragment thereof bound or conjugated to the small RNA degrades the BCMA receptor mRNA.

[0033] As used herein, "BCMA-specific receptor" refers to the BCMA receptor (B-cell maturation antigen receptor, BCMA-R), also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17), which is a membrane protein of the TNF receptor superfamily that recognizes BCMA. In humans, the BCMA receptor is encoded by the TNFRSF17 gene (UniProt Q02223, GenBank Z14954.1), the relevant sequence of which is incorporated herein by reference.

[0034] As used herein, the term "RNA" refers to ribonucleic acid, a polynucleotide molecule essential in a variety of biological roles in coding, decoding, regulating, and expressing genes. RNA is a nucleic acid.

[0035] The term "polynucleotide," as used herein, generally refers to a plurality, two or more, of "nucleotides," i.e., any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Polynucleotides include, but are not limited to, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single- and triple-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules containing DNA and RNA that may be single-stranded, or more typically double-stranded, or triple-stranded regions, or a mixture of single- and double-stranded regions. As used herein, the term "polynucleotide" also includes DNA or RNA, as described above, that contains one or more modified bases. Thus, DNA or RNA with backbones modified for stability or other reasons is a "polynucleotide" as the term is intended herein. Furthermore, DNA or RNA that contains unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, is a polynucleotide as the term is used herein. It will be understood that a wide variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term "polynucleotide," as used herein, encompasses chemically, enzymatically, or metabolically modified forms of such polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells (including, e.g., simple and complex cells). "Polynucleotide" also encompasses short polynucleotides often referred to as oligonucleotides.

[0036] "Small RNA" or "small RNA" refers to small, non-coding silencer RNAs (ribonucleic acids) with a maximum length of 150 nucleotides, including all types of small, single-stranded, and / or double-stranded non-messenger RNAs, which can inhibit target gene expression through post-transcriptional and chromatin-dependent gene silencing, or nuclease-mediated degradation in both the cytoplasm and the nucleus.

[0037] In the combination according to the present invention, the small RNA is preferably miRNA, shRNA, siRNA, antisense RNA, and / or a specific mRNA-targeting base / nucleotide sequence, more preferably siRNA.

[0038] As used herein, the term "miRNA" refers to microRNAs, which inhibit the expression of multiple mRNAs. In fact, miRNAs are partially complementary to their target mRNAs.

[0039] As used herein, the term " siRNA " refers to small interfering RNA. They inhibit the expression of one specific target mRNA. In fact, siRNA is usually completely complementary to the coding region of its target mRNA. Alternatively, short hairpin RNA (shRNA) can be used to achieve specific gene silencing effect through RNAi mechanism. shRNA is a stem-loop RNA, which is typically expressed in the nucleus through the delivery of viral vector. Once expressed, they are transported to the cytoplasm for further processing, and then loaded into RISC for specific gene silencing activity in the same manner as synthetic siRNA.

[0040] The small RNA used in the present invention is an antisense silencer (in vitro synthesized) RNA.Antisense RNA, also called antisense transcript, natural antisense transcript, or antisense oligonucleotide, is a single-stranded RNA that is complementary to the protein-encoded messenger RNA to which it hybridizes, thereby blocking its translation into protein.

[0041] In a preferred embodiment, the combination of two antibody-RNA complexes comprises: an anti-BAFF receptor antibody or binding fragment thereof bound or conjugated to an siRNA, shRNA, or both, that targets the BAFF receptor mRNA; an anti-BCMA specific receptor antibody or binding fragment thereof bound or conjugated to an siRNA, shRNA, or both, that targets the BCMA receptor mRNA.

[0042] In a more preferred embodiment, the combination of two antibody-RNA complexes comprises: an anti-BAFF receptor antibody or binding fragment thereof bound or conjugated to an siRNA targeting the BAFF receptor mRNA; an anti-BCMA specific receptor antibody or binding fragment thereof bound or conjugated to an siRNA targeting the BCMA receptor mRNA.

[0043] Preferably, the combination according to the invention comprises: -An anti-BAFF receptor antibody and siRNA, an anti-BCMA-specific receptor antibody and siRNA, The antibody and siRNA are electrostatically linked via a small protein that is chemically cross-linked to the antibody by a linker.

[0044] The linker is associated with a small protein that binds electrostatically to the siRNA.

[0045] Electrostatic interaction is the force between nearby atoms and molecules: like charges repel each other and opposite charges attract each other.

[0046] The term "small protein" or "low molecular weight protein" as used herein means a polypeptide having a maximum length of 100 amino acids.

[0047] In preferred embodiments, the linker is a conditional self-cleaving RNA sequence, a pH-sensitive linker, a hydrophobic-sensitive linker, a cleavable linker, a linker that provides a sorting signal, a linker that reduces steric hindrance, a linker that contributes to the condensation ability of the nucleic acid binding domain, a peptide or protein linker, a protamine linker, a polyK linker, or an HIV-TaT protein translocation (TPTV) linker.

[0048] In a more preferred embodiment, the linker is glutaraldehyde, bissulfosuccinimidyl suberate, carbodiimide, bis(succinimidyl)penta(ethylene glycol), bis(succinimidyl)nona(ethylene glycol), bis(sulfosuccinimidyl)suberate, dimethylsuberimidate, ethylene glycol, having the formula ICH,OH— (where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 9, 20, 21, 22, 23, 24, or 25, wherein one or both ends of the ethylene glycol are substituted with a succinimide or maleimide group).

[0049] In a particular embodiment, the combination according to the present invention comprises: -An anti-BAFF receptor antibody and siRNA, an anti-BCMA-specific receptor antibody and siRNA, The antibody components are each conjugated to a small basic protein, preferably protamine.

[0050] Synthetic techniques for the preparation of suitable peptide / protein-RNA linker molecules are conventional and any of the techniques listed below may be used in the context of the present invention, such as those described in WO2009083738, the relevant portions of which are incorporated herein by reference.U.S. Patent Nos. 5,138,045 and 5,218,105 for polyamine-conjugated oligonucleotides; U.S. Patent No. 5,212,295 for monomers for the preparation of oligonucleotides with chiral phosphorus linkages; U.S. Patent Nos. 5,378,825 and 5,541,307 for oligonucleotides with modified backbones; U.S. Patent No. 5,386,023 for backbone-modified oligonucleotides and their preparation by reductive coupling; and U.S. Patent No. 5,386,023 for modified nucleobases based on 3-deazapurine ring systems and methods for their synthesis. U.S. Patent No. 5,457,191 relating to modified nucleobases based on N-2 substituted purines; U.S. Patent No. 5,459,255 relating to modified nucleobases based on N-2 substituted purines; U.S. Patent No. 5,521,302 relating to processes for preparing oligonucleotides with chiral phosphorus linkages; U.S. Patent No. 5,539,082 relating to peptide nucleic acids; U.S. Patent No. 5,554,746 relating to oligonucleotides with lactam backbones; U.S. Patent No. 5,571,902 relating to methods and materials for the synthesis of oligonucleotides; alkylthio groups (such groups are not included in the nucleoside species) U.S. Patent No. 5,578,718 for nucleosides having phosphorothioate linkages (which may be used as linkers to other moieties attached at any of the positions); U.S. Patent Nos. 5,587,361 and 5,599,797 for oligonucleotides having phosphorothioate linkages of high chiral purity; U.S. Patent No. 5,506,351 for a process for the preparation of 2-O-alkylguanosines and related compounds (including 2,6-diaminopurine compounds); U.S. Patent No. 5,587,466 for oligonucleotides having N-disubstituted purines. No. 9; U.S. Pat. No. 5,587,470 relating to oligonucleotides having 3-deazapurines; U.S. Pat. Nos. 5,223,168 and 5,608,046, both relating to conjugated 4'-desmethyl nucleoside analogs; U.S. Pat. Nos. 5,602,240 and 5,610,289 relating to backbone-modified oligonucleotide analogs; and U.S. Pat. Nos. 6,262,241 and 5,459,255, particularly relating to methods for synthesizing 2-fluoro-oligonucleotides, are incorporated herein by reference.

[0051] Many different linkers can be used in the present invention. In the present invention, linkers have been used to associate protamine or small proteins with BAFF receptor-specific mAbs and BCMA receptor-specific mAbs. In one non-limiting example, a protamine linker (also referred to herein as a coupling reagent) can be used. Protamine linkers generally contain amino acids 8-29 of protamine, i.e., RSQSRSRYYRQRQRSRRRRRRS, SEQ ID NO: 1. Other protamine sequences (e.g., peptides containing at least amino acids 12-20, at least amino acids 10-24, or at least amino acids 10-26 of protamine) are also suitable. Linkers can be incorporated at the N- or C-terminus of the translocation component or within surface-exposed loop regions of the translocation component. In another example, a polylysine linker (also known as a polyK linker) is used. A polyK linker contains 5-30, 5-20, 5-15, or 7-10 lysine residues, and optionally contains one or more (but preferably no more than five) non-lysine residues. The linker can be incorporated at the N-terminus or C-terminus of the translocation component, or within a surface-exposed loop region of the translocation component. In another example, a TPTV linker (also known as an HIV-TaT protein translocation domain linker) can be used. One such TPTV linker contains residues 47-57 of HIV TAT. TPTV linkers typically contain 5-30, 5-20, 5-15, or 7-10 amino acid residues. The linker can be incorporated at the N-terminus or C-terminus of the translocation component, or within a surface-exposed loop region of the translocation component.

[0052] Preferably, the antibody-siRNA complex of the combination according to the present invention is PEGylated.

[0053] PEGylation is the biochemical modification process of biologically active molecules with polyethylene glycol (PEG) to impart several desirable properties to proteins / peptides, antibodies, and vesicles that may be used for cellular therapy or genetic modification. PEGylation is the process of both covalent and non-covalent attachment or amalgamation of polyethylene glycol polymer chains to molecules and macrostructures, such as drugs, therapeutic proteins, or vesicles, which are then described as PEGylated.

[0054] Even more preferably, the disulfide bonds in the mAb are first reduced and then the antibody-RNA complex of the combination according to the invention is PEGylated.

[0055] The present inventors observed that partial reduction of disulfide bonds in mAbs prior to PEG treatment of the mAbs significantly improved conjugate-mediated reduction of autoantibody levels, likely through reduced aggregate formation and enhanced cellular uptake of the PEGylated conjugates, resulting in greater suppression of key receptors. Thus, the present inventors demonstrated that PEGylation further improved the efficacy of duoconjugate treatment with respect to higher levels of autoantibody reduction. Again, the attachment of PEG polymers to antibody-siRNA conjugates (PEGylated conjugates) further enhanced the immunosuppressive effect.

[0056] The present invention also relates to a composition comprising a combination according to the invention.

[0057] Preferably, compositions according to the invention are adapted for intravenous, intramuscular, oral, parenteral, enteral, intraperitoneal, pulmonary, nasal, subcutaneous, rectal, or transdermal administration.

[0058] The present invention also relates to a composition according to the invention for use as a medicament, which also comprises a pharmaceutically acceptable vehicle.

[0059] Pharmaceutically acceptable media include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. suitable for administration to mammalian hosts. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the pharmaceutical preparations of the present invention.

[0060] The compositions according to the present invention are preferably used for the treatment of autoimmune disorders.

[0061] Autoimmunity, and more generally "immunity," refers to the body's ability to defend itself against foreign substances, such as pathogenic microorganisms or cancer cells, by eliciting an immune response. When a foreign substance enters the body, the immune system is triggered by the production of messengers such as interleukins and cytokines.

[0062] The term "autoimmune disorder," as used herein, refers to a condition in which the body's immune system mistakes its own healthy tissues for foreign bodies and attacks them. Most autoimmune diseases cause inflammation that can affect many parts of the body. The affected parts of the body depend on which autoimmune disease a person has. Common signs and symptoms include fatigue, fever, muscle aches, joint pain and swelling, skin problems, abdominal pain, digestive problems, and glandular swelling. Symptoms come and go frequently and can be mild or severe. There are many different types of autoimmune diseases.

[0063] The composition for use according to the present invention is preferably used for the treatment of an autoimmune myopathic disorder, more preferably myasthenia gravis.

[0064] As used herein, the term "myasthenia gravis" or MG refers to any chronic progressive muscle weakness. Myasthenia gravis is an autoimmune disease resulting from antibodies that block nicotinic acetylcholine receptors at the junction between nerves and muscles, preventing nerve impulses from triggering muscle contraction.

[0065] Patients with MG who are seropositive for autoantibodies against the acetylcholine receptor (AChR), muscle-specific tyrosine kinase (MuSK), or low-density lipoprotein receptor-related protein 4 (LRP4) exhibit voluntary muscle weakness due to dysfunction of the neuromuscular junction and impaired neuromuscular transmission. 70% of MG patients are seropositive for AChR-specific autoantibodies and have significantly reduced muscle function. Clinical signs of muscle wasting include ptosis, diplopia, slurred speech, appendicular tremors, and even respiratory failure due to exacerbation of the disease.

[0066] Preferably, in the composition for use according to the present invention, the combination of two antibody-RNA complexes is an immunosuppressant.

[0067] "Immunosuppressant" or "immunosuppressive agent" or "immunosuppressive drug," as used herein, refers to an agent that can suppress or prevent an immune response. Immunosuppressants are used to prevent rejection of transplanted organs and to treat autoimmune diseases. Immunosuppressants interfere with different stages and levels of the immune response.

[0068] As illustrated in the examples, the inventors were able to highlight that the combination of the two conjugates (anti-BAFF receptor antibody siRNA and anti-BCMA receptor antibody siRNA) results in a synergistic / additive immunosuppressive effect, which in fact reduces the production of pathogenic antibodies from B cells.

[0069] Preferably, the composition for use according to the invention is provided in a dose of 5 to 25 mg / kg of total mammalian body weight.

[0070] In a more preferred embodiment, the composition is provided at a dose of 5-22 mg / kg of total mammalian body weight.

[0071] In an even more preferred embodiment, the composition is provided in a dosage of 5-20 mg / kg of total mammalian body weight.

[0072] The present invention also relates to a composition for use according to the invention, the composition comprising: an anti-BAFF receptor antibody or a binding fragment thereof bound or conjugated to a small RNA that targets the BAFF receptor mRNA; - an anti-BCMA specific receptor antibody or binding fragment thereof linked or conjugated to a small RNA targeting BCMA receptor mRNA, as a combination of the two antibody-RNA complex products for simultaneous, separate or spaced administration.

[0073] Preferably, the composition for use according to the invention comprises: an anti-BAFF receptor antibody or binding fragment thereof electrostatically bound or conjugated to an siRNA, shRNA, or both, that targets the BAFF receptor mRNA; - an anti-BCMA specific receptor antibody or binding fragment thereof electrostatically bound or conjugated to siRNA, shRNA, or both targeting BCMA receptor mRNA, as a combination of two antibody-RNA conjugate products for simultaneous, separate, or spaced administration. [Example]

[0074] Each experiment was repeated at least three times. Data were compared and evaluated using one-way analysis of variance, followed by Holm-Sidak or Tukey's post hoc analysis, where applicable. Statistical significance was set at p<0.05. The UTMB Biostatistics Core further performed a power determination analysis for each experiment. All statistical analyses assumed a 95% level of confidence.

[0075] Example 1: Animals, EAMG induction, and clinical evaluation C57BL / 6 mice (Jackson Lab, Bar Harbor, ME) were housed and maintained in a barrier facility at the University of Texas Medical Branch (UTMB) in Galveston, Texas, in accordance with the guidelines of the National Institutes of Health and the UTMB Animal Care and Use Committee. The UTMB Animal Care and Use Committee reviewed and approved this animal study.

[0076] MG-like disease (EAMG) is induced in mice through immunization with Torpedo AChR emulsified in complete Freund's adjuvant (CFA). This model typically displays clinical signs and features similar to those characteristic of MG in humans, including high-affinity anti-AChR antibodies, immunoglobulin G (IgG), and complement deposition at the neuromuscular junction, and a reduced number of AChRs in postsynaptic membrane folds.

[0077] Affinity-purified Torpedo AChR was emulsified in CFA and used to immunize C57BL / 6 mice (8 weeks old, male) three times. MG disease in these mice was confirmed by elevated serum anti-AChR antibodies. Clinical grade of disease was determined through blinded observation (grade 0, normal muscle strength; grade 1, normal at rest but weak during exercise, hunched head posture, and reduced mobility; grade 2, grade 1+ weakness at rest; and grade 3, moribund, dehydrated, and paralyzed). Digital recordings (dynamometer) of forelimb grip strength were performed once before treatment and then at 2 and 6 weeks and 3 and 4 months after treatment.

[0078] Example 2: Preparation and validation of mAb-siRNA conjugates Both BR-specific and BCMA-specific siRNAs (Ambion In Vivo Pre-designed, HPLC-purified; Genbank accession numbers NM_028075.2 and NM_011608.1) and in vivo negative control siRNAs were purchased from ThermoFisher Scientific (Waltham, MA). BR siRNAs with GC content of 57.9% vs. 36.8% and BCMA siRNAs with GC content of 42% vs. 33% were compared. Internalizing antibodies, anti-mouse antibodies against BR (tumor necrosis factor receptor superfamily 13c) and BCMA (Tnfrsf17), were purchased from LS Bioscience (Seattle, WA) or Adipogen (San Diego, CA), or obtained from custom hybridoma clones (RayBiotech, Peachtree Corners, GA). A BCMA antibody (Vicky-1) (ThermoFisher) was also evaluated for cross-reactivity in mice and used in the experiments. All mAb and siRNA activities were verified through flow cytometry, quantitative reverse transcription-PCR (qRT-PCR), or Western blot using splenocytes and lymph node (LN) cells obtained from EAMG mice.

[0079] BR-specific and BCMA-specific smt conjugates were prepared using protamine (a small, <7 kDa protein (Sigma, St. Louis, MO)) and a heterobifunctional linker (Trilink Biotech, San Diego, CA). Briefly, buffer-exchanged mAb (3 mg / mL) and protamine (0.5 mg) were modified for 2 hours using Sulfo(S)-4FB and S-HyNic (Trilink Biotech). Both molecules were then desalted and contaminants were removed by using a Zeba column (Pierce Biotechnology, Rockford, IL). The modified mAb and protamine were then combined and incubated overnight at 4°C. Unbound protamine was removed by gel filtration. The protaminate-ylated mAb (mt) was then conjugated to siRNA (1:1 or 4:1) and separated using a concentration column (Abcam, Waltham, MA). The mt was quantified by BCA protein assay (Pierce) using purified mouse IgG as a standard and stored in phosphate buffered saline (PBS) at 4°C until further use. Conjugates (smt) with appropriate controls were electrophoresed using native 10%-20% polyacrylamide gels. The presence of siRNA and mAb components in each conjugate was verified by staining the gel first with ethidium bromide and then with Coomassie blue. Protaminate-ylated mAb:siRNA ratios of 4:1 or 1:1 resulted in equivalent yields of conjugates.

[0080] The zeta potential values (quantifying surface charge) of the conjugates were measured in triplicate at 25°C by laser Doppler velocimetry using a Zetasizer 2000 (Malvern Instruments, Malvern, UK) after diluting the samples to 10% with distilled water. The conjugates were also assessed for stability by incubating aliquots of the BR-BCMA conjugates with fresh mouse serum in a 1:1 ratio (10 μL each) at 4°C for various periods up to 24 hours. The complexes were then resolved via polyacrylamide gel electrophoresis (PAGE; 10% gel) to check for signs of degradation.

[0081] To target specific B cell subsets, each fusion mAb-siRNA conjugate was constructed using protamine covalently linked to the B cell-targeting mAb at its Fc region via a heterobifunctional crosslinker. The protaminate-containing mAb then formed strong electrostatic bonds with BR- or BCMA-specific siRNA. Data further revealed that gel filtration removed contaminating free protamine from the protaminate-containing mAb (BRmt and BCMAmt), and both the siRNA and mAb components of the conjugates appeared as the same band in a native 20% polyacrylamide gel stained with ethidium bromide and Coomassie blue, respectively (Figure 1A).

[0082] Unconjugated siRNA alone showed no protein bands in Coomassie-stained gels. The gel-eluted conjugate retained both Fab and Fc, as evidenced by gradient SDS-PAGE performed under denaturing / non-denaturing conditions (Figure 1A). The zeta potential of the conjugate particles (smt) was approximately -22 mV, indicating moderate stability of the complex (Figure 1B). Incubation of the conjugate with serum at a 1:1 ratio showed that the conjugate (mAb-siRNA) was fairly stable even after 24 h (Figure 1C), by which time the conjugate was expected to reach the target organ and exert its desired effect.

[0083] Example 3: In vivo imaging of EAMG mice and localization of conjugates Whole-body IVIS Spectrum fluorescence imaging was performed noninvasively to determine the distribution and persistence of the conjugates in vivo in EAMG mice. After the second booster immunization, EAMG mice were intraperitoneally administered either PBS or conjugates of anti-mouse BR mAb and Alexa Fluor 555-labeled nonspecific siRNA and anti-mouse BCMA mAb and Alexa Fluor 488-labeled nonspecific siRNA. The mAb and siRNA were used at 100 μg and 25 μg per mouse, respectively. One day before treatment, the abdomens of the mice were depilated using a commercially available depilator (Nair, Church & Dwight). All mice were sedated with isoflurane and placed on the imaging shuttle of an IVIS Spectrum In Vivo Imaging System (Perkin Elmer, MA). The shuttle was equipped with a gas anesthesia connection for live imaging. Three hours after conjugate administration, Alexa-555 conjugates were imaged using excitation / emission wavelengths of 535 nm / 580 nm. + Images were acquired and then analyzed using Alexa-488 at 465 nm / 520 nm. + Images were captured and recaptured 24 hours later using the CCD camera of the IVIS system. Mice were then euthanized, and inguinal LNs and kidneys were excised to directly compare lymphoid and non-lymphoid organs for Alexa Fluor 488 and 555 fluorescence intensity.

[0084] Initial studies have shown that mAb-siRNA conjugates can be internalized by transformed B cells in vitro and primary B cells isolated from EAMG mice. To further investigate the time-dependent biodistribution of B cell-targeting conjugates in vivo, EAMG mice were intraperitoneally injected with BR-specific and BCMA-specific mAbs conjugated to fluorescent siRNA labeled with Alexa-555 (red / orange) and Alexa-488 (green), respectively. Both conjugates exhibited fluorescence near the intraperitoneal injection site 3 hours after injection. After 24 hours, the Alexa-555-containing conjugate was not detected at the injection site but was observed in the upper left abdomen, suggesting its possible accumulation in the spleen. The Alexa488 conjugate was detectable 3 hours after injection but not at 24 hours, likely due to its weak fluorescence that photobleached over time; therefore, fluorescence was undetectable on the skin surface. No background fluorescence was detected in control mice. Inguinal LNs from PBS control-treated and fluorescent conjugate-treated mice were excised to observe fluorescence in the LNs, but not on the surface of the mouse skin. EAMG mice injected with Alexa-555 conjugates exhibited fluorescence relatively higher than autofluorescence in the LNs of PBS-treated mice, but no fluorescence was detected in the kidneys, a nonlymphoid organ.

[0085] The results show that B cell-targeting conjugates with specific affinity for B cells are preferentially directed to these cells, which reside predominantly in lymphoid organs.

[0086] Example 4: Duoconjugates reduce target receptor expression Dose Optimization and Treatment To optimize the dose of the duoconjugate, two booster immunizations were provided over a 2-week period. EAMG mice with grade 2 or 3 disease were randomized into various groups (n=7). Mice were treated intraperitoneally once with 100, 125, or 150 μg of the BR-specific and BCMA-specific conjugates (smt). Some groups of mice received protaminate- ed mAb (mt) or PBS to serve as controls. Treatment with the BCMA-specific conjugate was initially performed one day after the BR-specific conjugate to avoid potential dose overload. However, mice were able to tolerate subsequent co-administration of the duoconjugate without signs of stress or adverse effects.

[0087] Another set of EAMG mice (n = 5) was treated or pretreated once with anti-interferon (IFN)-α / β receptor (IFNAR) monoclonal antibody (MAR1-5A3) (Leinco Technologies, Fenton, MO) at a dose of 1–2.5 mg per mouse according to the manufacturer's recommendations. Conjugates containing siRNA with high or low GC content (Assay IDs: s90521 [57.9%] and s90522 [36.8%] for BR and s75270 [42%] and s75269 [33%] for BCMA; ThermoFisher Scientific) were also administered to EAMG mice. To improve serum stability and cellular uptake, the conjugates were PEGylated before treatment. Briefly, MS(PEG)12 (ThermoFisher Scientific) was dissolved in dimethylformamide and added to 2-mercaptoethylamine (ThermoFisher Scientific)-treated mAb in an amine-free buffer in the presence of the linker S-4FB. The mixture was incubated at room temperature for 30 minutes and then desalted. The PEGylated linker-modified mAb was then incubated with linker-modified protamine and subsequently reacted with siRNA to obtain PEGylated mAb-siRNA conjugates. To verify whether non-targeting siRNA nonspecifically reduces BR or BCMA transcripts, EAMG mice were treated with conjugates containing either BR or BCMA mt along with non-specific siRNA. All conjugates were quantified, diluted in PBS, and injected intraperitoneally into mice.

[0088] All mice were euthanized at 3 days post-treatment (dpt), 6 weeks post-treatment (wpt), and 3 and 4 months post-treatment (mpt) for different sets of experiments.

[0089] Cell preparation and flow cytometry analysis Mice were bled via the tail vein, peripheral blood mononuclear cells (PBMCs) were isolated, and serum was collected. Mice were euthanized at various time points (dpt / wpt / mpt), tissues were removed, and splenocytes and LN cells (inguinal and brachial) were isolated. PBMCs or splenocytes (4 s) were isolated using Lymphoprep on SepMate tubes (StemCell Technologies, Cambridge, MA). x Cells (>106 cells) were processed. Cells were treated with red blood cell (RBC) lysis solution (Sigma) to remove contaminating RBCs. Cells were then subjected to flow cytometry and qRT-PCR. Sampled tissues were snap-frozen in small pieces for qRT-PCR and other downstream analyses.

[0090] All single-cell preparations (without RBCs) were washed with PBS-1% heat-inactivated fetal bovine serum. Cells were subjected (or not) to Fc receptor blockade and stained with fluorescent dye (e.g., FITC, PE, Alexa, APC, and PE-Cy7)-conjugated mAbs and secondary antibodies (as needed). Cells were either analyzed immediately or fixed with 1% paraformaldehyde. All cells were analyzed on a BD LSR Fortessa flow cytometer using FACSDiva software (BD Biosciences, San Jose, CA) at the UTMB Flow Cytometry and Cell Sorting Core.

[0091] qRT-PCR Total RNA was isolated from PBMCs and small pieces of snap-frozen spleen and LN (inguinal) using TRIzol (Invitrogen, Carlsbad, CA). Genomic DNA-free complementary DNA was prepared from the RNA using Superscript II, random primers, a nucleotide mix, DNAse, and the appropriate buffer (Invitrogen). The relative expression of BR, BCMA, B220, and beta-actin (for normalization) was analyzed using FAM-conjugated gene-specific probe-primer mixes and AmpliTaq Gold TaqMan PGR Master Mix (Invitrogen) on a CFX 96 Real-Time system (Bio-Rad, Hercules, CA). The relative fold change of mRNA compared to the change in beta-actin mRNA (standard) was calculated using CFX software (Bio-Rad) (2 -ΔΔCt method).

[0092] result It has previously been reported that a paradoxical increase in autoantibody levels (despite a slight reduction in B cell frequency) is associated with high expression of type 1 IFN following treatment with a conjugate at high doses (>350 μg, approximately 20 μg / g body weight). Therefore, dose optimization of the conjugate was performed by evaluating a 50% or lower dose reduction to induce maximal inhibition of the target receptor without eliciting any interferonogenic or humoral responses. Furthermore, because BCMA is a highly specific receptor for plasma cells that produce chronic MG-specific autoantibodies, a duoconjugate (a BCMA-specific conjugate was used in addition to a BR-specific conjugate) was used. Therefore, the goal was to maximally suppress target mRNA and B cell receptor expression in both precursor and terminally differentiated plasma B cells, which cooperate to exert a stronger autoantibody-reducing effect. The study included four sets of experiments, as outlined in Table 1.

[0093] [Table 1]

[0094] To confirm the inhibitory effect of the conjugates, the frequency of BR- and BCMA-expressing B cells in the spleen 3 days after treatment was assessed as a measure of the reduction in the number of mature and plasmatic B cells. Flow cytometry showed that all three doses of the duoconjugate (100, 125, and 150 μg) significantly reduced BR and BCMA expression and total B220 expression compared to PBS, mAb alone, or monoconjugate treatment. + The results demonstrated a substantial reduction (20%-60%) in splenocytes (Figure 2A, a-c). Notably, due to plasma cell-specific surface expression, BCMA-specific antibody treatment significantly reduced BR + Similarly, BR-specific antibody treatment did not significantly reduce BCMA + Although it did not appear to affect B cell frequencies, a slight reduction in the proportion of both cell types was nevertheless found caused by both antibodies (Figure 2A, a and b). Higher levels of antibody resulted in higher frequencies of BCMA in separate experiments. + BCMA produced cells + The low frequency of cells was likely due to insufficient amounts of antibody (Fig. 2B).

[0095] CD3 + Because T cells also express low levels of BR, we investigated how CD3 cell frequency was affected by conjugate treatment. Only BR mt significantly reduced the frequency of these cells. In contrast, BR-specific conjugates significantly reduced the frequency of CD3 + significantly increased the number of T cells (Fig. 2A, d).

[0096] Flow cytometry revealed that BCMA in the spleen was more abundant than cells in PBMCs or LNs. +The duoconjugate demonstrated a higher potency in depleting B cells (Figure 2B, upper and lower panels). Consistent with the flow cytometry results, qRT-PCR results further demonstrated a reduction in BR and BCMA mRNA levels (up to a -6-fold reduction) in response to three doses of the duoconjugate treatment (Figure 2C).

[0097] In follow-up repeated experiments, further dose escalation of the mAb and siRNA was determined to be approximately 200 μg per gram of mouse body weight, 10 μg of protaminate mAb and 4 μg of siRNA, without overhauling the EAMG mice with severe or overdose-induced adverse effects. Higher doses appear to be ineffective in achieving greater inhibition of target receptors or reduction of target B cell subsets, and higher doses also exert the opposite effect.

[0098] In conclusion, the results show that a single treatment of EAMG mice with optimized doses of the conjugates, especially the duoconjugates and duomAbs, significantly suppresses the key receptors BR and BCMA on target B cells at both the mRNA and protein levels.

[0099] Example 5: Neutralization of Type 1 IFN and the effect of the GC content of the siRNA component of the conjugate and AChR-specific B cells Despite the use of modified siRNA to prevent the elicitation of innate immune responses, nonspecific induction of type 1 IFN by high doses of BR-specific monoconjugates has previously been reported. Type 1 IFN is known to promote antigen processing, which likely interfered with the autoantibody-reducing effect of the conjugates. To mitigate this plausible effect of IFN-α / β, we evaluated whether antibody-mediated pre-neutralization of the IFN-α / β receptor (IFNAR) would increase the efficacy of conjugate treatment by further reducing the frequency of target cells and, therefore, autoantibody levels. Pretreatment with 2 mg of anti-IFNAR mAb per mouse followed by duoconjugate treatment resulted in CD19 cytotoxicity at 6 wpt. +The cell frequency was slightly reduced (Figure 3A). However, treatment with IFNAR antibody alone (unconjugated) produced a paradoxical effect (increased surface expression of the target receptor, Figure 3A). The data also support the conclusion that Fc-blocked CD19 + B cell numbers (representing the same BR-positive transitional / mature B cell subset) were substantially lower in LNs at 6 wpt. Due to plasma cell-specific expression of BCMA, BCMA-only conjugates were significantly lower than CD19 + It did not reduce the number of transitional B cells (Fig. 3A).

[0100] Upon resumption, the inhibitory effect of BR and BCMA receptors resulted in a modest reduction in the number of B cells (CD19 and B220) for at least 6 wpt.

[0101] Next, we compared the effect of the GC content of the siRNA component of the conjugate on B cell receptor suppression. The GC content of siRNA reportedly affects its activity; for example, a high GC content can confer stability to siRNA through secondary structure. In contrast, a low GC content lacks this property but facilitates the binding of siRNA to target mRNA. To determine whether the differential GC content of siRNA further reduces surface receptor expression through increased mRNA degradation, we used 21-mer siRNAs containing approximately 20% more or less GC content. Higher GC content of siRNAs significantly reduced CD19 expression compared to lower GC content. + Although efficacy in reducing B cells was demonstrated, the differences were not statistically significant between treatment groups (Figure 3B).

[0102] Previously, a positive correlation between AChR-specific B cells and autoantibody levels, detectable using the fluorescent dye Alexa647 conjugated to Torpedo AChR, was shown. A correlation was detected between changes in GC content (high vs. low) and increased apoptosis of AChR antibody-producing B cells and Fas receptor (CD95)-coexpressing B cells. While the low GC content of the conjugate correlated with relatively low Fas receptor expression in LN cells, a significant difference in AChR expression between the high and low GC content of the siRNA-containing conjugate was observed.+ CD95 + The difference in cell frequency was not statistically significant (Fig. 3C).

[0103] Example 6: CD27 + Plasmablasts Splenic B cells from a separate set of 3 mpt mice were purified to assess the generation of total spleen-resident memory B or T cells. Total CD27, representing the splenic plasmablast population, was also assessed. + The frequency of cells co-expressing CD27 B220 or CD19 was determined. + There are conflicting reports about the cells. + Total spleen CD27, not the percentage of cells + The percentage of B220 cells was significantly reduced at 3 months after treatment with the duoconjugate (Figure 4). + The deficiency in subset proportions appeared to gradually recover within 3 mpt. + The cell number was also slightly reduced to 3 mpt due to the presence of BR on these cells (Fig. 4C).

[0104] In conclusion, the number of splenic CD27+ total memory B / T cells (also representing plasmablasts) was significantly reduced within 3 mpt, suggesting an association between reduced memory B cell frequencies and impaired autoantibody function.

[0105] Example 7: AChR-specific autoantibody levels Anti-AChR antibody levels were measured by ELISA using pre-titrated serum dilutions and affinity-purified mouse muscle AChR as the coating antigen, horseradish peroxidase (HRP)-conjugated rat anti-mouse IgG2b (Caltag, Burlingame, CA, USA and BD Biosciences) as the secondary antibody, and 2'azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) or 3,3',5,5'-tetramethylbenzidine (ThermoFisher) as the substrate.

[0106] We developed a highly specific, non-radioactive method to accurately measure relative serum levels of autoantibodies in samples using biotinylated α-bungarotoxin (BTX) and magnetic streptavidin beads. For antibody assays, serum anti-AChR IgG / IgG2b was immunoprecipitated using biotinylated BTX (ThermoFisher Scientific) and affinity-purified AChR in Tris-EDTA-Tween 20 buffer, followed by overnight incubation on a rotating platform with magnetic streptavidin (Cell Signaling Technology, Danvers, MA). The pulled-down complexes in the beads were washed, mixed with loading dye, and then resolved in sodium dodecyl sulfate (SDS)-PAGE (4%–20%; Bio-Rad). Proteins were transferred onto polyvinylidene difluoride membranes blocked with 5% nonfat dry milk and probed with HRP-conjugated goat anti-mouse IgG2b (Santa Cruz Biotechnology, Santa Cruz, CA and Caltag) diluted 2:5000 in Tris-buffered saline-Tween 20 (0.05%) for 1 hour at room temperature. Enhanced chemiluminescence (Pierce Biotechnology) assays were performed, and gel images were captured using a GE Amersham Imager 680. Images revealed bands representing anti-AChR IgG2b levels in each sample.

[0107] To assess the extent to which the BR-BCMA-specific conjugate reduced autoantibody levels and the duration of reduced autoantibodies, AChR-specific antibodies in EAMG mice in different sets of experiments were assessed throughout the study period. Pathogenic anti-AChR IgG / IgG2b levels were measured by ELISA and a custom-developed antibody precipitation assay (more specific than ELISA). High-affinity AChR-specific antibodies were detected in the serum of PBS-treated EAMG mice compared with the serum of mice receiving other treatments. At any post-treatment time point evaluated, duoconjugate and BR-specific monoconjugate treatment significantly reduced anti-AChR antibody levels (Figures 5A and 5B). PEGylation of the duoconjugate reduced autoantibody levels more effectively than the non-PEGylated duoconjugate. At 4 mpt, only duoconjugate and PEGylated duoconjugate treatment still significantly reduced autoantibody levels. Pretreatment of EAMG mice with anti-IFNAR mAb reduced autoantibody levels compared with EAMG mice alone. However, pretreatment, even at higher doses, did not demonstrate efficacy at any time point in further reducing the level of conjugate-mediated reduction of autoantibodies.

[0108] Treatment-mediated loss of B220+ B cells was substantially reversed within 3 mpt, whereas reductions in autoantibody levels persisted from 6 wpt to 3 mpt and beyond to the experimental endpoint of 4 mpt, as found in a different set of experiments.

[0109] Example 8: Clinical grades of EAMG mice At 6 mpt, 70% of EAMG mice treated with either the monoconjugate or the duoconjugate exhibited grade 0 disease with normal motility, and 30% had a clinical grade of 1-2. All EAMG mice treated with PBS developed grade 3 disease. Mice treated with the PEGylated conjugate or anti-IFNAR mAb developed grade 2 disease. The differences in body weight between the various mouse groups were not statistically significant.

[0110] Example 9: Grip strength of EAMG mice Digital recordings (dynamometer) of forelimb grip strength in EAMG mice revealed a significant improvement in muscle strength at 6 weeks and 4 months (but not earlier than 2 or 4 weeks) after treatment when compared with PBS-treated controls. Mouse weight did not fluctuate significantly at any of the experimental time points (Figure 6A).

[0111] Example 10: Functional AChR levels in muscle For functional AChR analysis, crude AChRs were obtained from membrane fractions derived from homogenized mouse forelimb muscle (proximal) tissue. ELISA was performed according to previously described methods.

[0112] Forelimb muscle samples from EAMG mice that were untreated, treated with PBS or BR-BCMA conjugates, or left untreated were quantified by ELISA as described. Muscle AChR levels were significantly preserved only with duo and PEGylated duo conjugate treatment (Figure 6B).

[0113] Clinical improvements, including disease grade, grip strength, and functional muscle AChR levels, resulting from treatment with the duoconjugate (BR and BCMA) and PEGylated duoconjugate were significantly and significantly better than those from monoconjugate treatment.

[0114] Example 11: Determination of class-switched memory B cell frequencies in EAMG mice after treatment with conjugates Because class-switched memory B cells tend to differentiate into pathogenic plasma cells after activation (Dogan I et al. Natl Immunol, 2009 10:1292-9), we wanted to investigate whether their frequency was altered in EAMG mice after treatment with the conjugate.

[0115] PBMCs were isolated from groups of EAMG mice treated with or without the conjugate (3 months after treatment) and subjected to flow cytometry analysis using fluorochrome-labeled antibodies specific for GD19, CD27, and IgD. + B cells were selected and further gated on subpopulations positive for CD27 and IgD. Results showed that conjugate treatment significantly reduced the number of class-switched memory B cells in EAMG mice, suggesting a reduced ability to generate pathogenic autoantibody-producing plasma cells.

[0116] In Figure 7, mice in group 1 were immunized with CFA and AChR (acetylcholine receptor) to induce autoantibodies that cause myasthenia gravis disease in mice.

[0117] Groups 3 and 5 were treated with conjugates still containing BAFF receptor (BR)- and BCMA-specific monoclonal antibodies in addition to nonspecific (scrambled) siRNA. These mAbs primarily or exclusively target mature and plasma B cells, destroying these cells to some extent, regardless of siRNA efficacy. As a result, we observed that class-switched memory B cells, which have the potential to produce antibodies that cause pathogenic MG, were much fewer in the BR-specific / BCMA-specific monoconjugate (Groups 4 and 6) and duoconjugate-treated MG mice (Groups 2 and 7) compared with conjugates containing not only scrambled siRNA but also cell-specific mAbs. Therefore, the addition of mRNA-targeting siRNA to these B cell-specific mAbs contributes to a more cell-reducing effect, reducing the formation of class-switched memory B cells. However, the difference in B cell percentages is significant between Groups 3 and 5 versus Groups 2, 4, 6, and 7.

[0118] We can conclude that class-switched memory B cells functionally contribute to autoimmune-associated pathological antibody levels.

Claims

1. A combination of two antibody-RNA complexes, an anti-BAFF receptor antibody or a binding fragment thereof bound or conjugated to a small RNA targeting the BAFF receptor mRNA; - an anti-BCMA-specific receptor antibody or binding fragment thereof bound or conjugated to a small RNA that targets the BCMA receptor mRNA.

2. The combination according to claim 1 , wherein the small RNA is a miRNA, shRNA, siRNA, antisense RNA, and / or a specific mRNA-targeting base / nucleotide sequence, preferably a siRNA.

3. the anti-BAFF receptor antibody and the siRNA, the anti-BCMA-specific receptor antibody and the siRNA; The combination of claim 2 , wherein the antibody and siRNA are electrostatically linked via a small protein that is chemically cross-linked to the antibody by a linker.

4. 4. The combination of claim 3, wherein the linker is a conditional self-cleaving RNA sequence, a pH-sensitive linker, a hydrophobic-sensitive linker, a cleavable linker, a linker that provides a sorting signal, a linker that reduces steric hindrance, a linker that contributes to the condensation ability of the nucleic acid binding domain, a peptide or protein linker, a protamine linker, a polyK linker, or an HIV-TaT protein translocation (TPTV) linker.

5. The linker is selected from the group consisting of glutaraldehyde, bissulfosuccinimidyl suberate, carbodiimide, bis(succinimidyl)penta(ethylene glycol), bis(succinimidyl)nona(ethylene glycol), bis(sulfosuccinimidyl)suberate, dimethylsuberimidate, ethylene glycol, and the formula ICH,OH-) (wherein n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, wherein one or both termini of the ethylene glycol are substituted with a succinimide or maleimide group.

6. the anti-BAFF receptor antibody and the siRNA, - the anti-BCMA-specific receptor antibody and the siRNA, each of the antibody components being conjugated to a small basic protein, preferably protamine.

7. The combination according to any one of claims 1 to 6, wherein the antibody-siRNA conjugate is PEGylated.

8. A composition comprising a combination according to any one of claims 1 to 7.

9. 9. The composition of claim 8, adapted for intravenous, intramuscular, oral, parenteral, enteral, intraperitoneal, pulmonary, nasal, subcutaneous, rectal, or transdermal administration.

10. 10. A composition according to claim 8 or 9 for use as a medicament.

11. 11. A composition for use according to claim 10 for the treatment of autoimmune disorders.

12. 12. A composition for use according to claim 11 for the treatment of autoimmune muscle disorders, preferably myasthenia gravis.

13. The composition for use according to claim 12, wherein said combination of said two antibody-RNA complexes is an immunosuppressant.

14. The composition for use according to any one of claims 10 to 13, wherein said composition is provided in a dose of 5 to 25 mg / kg of total mammalian body weight.

15. The composition comprises: an anti-BAFF receptor antibody or binding fragment thereof that is electrostatically bound or conjugated to a small RNA that targets the BAFF receptor mRNA; an anti-BCMA-specific receptor antibody or binding fragment thereof that is electrostatically bound or conjugated to a small RNA that targets the BCMA receptor mRNA; A composition for use according to any one of claims 10 to 14, comprising as a combination two antibody-RNA complex products for simultaneous, separate or spaced administration.

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