TACI-FC fusion proteins for multifunctional inhibition of BAFF, APRIL, and neonatal FC receptors

Abstract

Methods and compositions are provided for TACI-Fc fusion proteins that bind to and inhibit the activity of BAFF and APRIL ligands, neutralizing their activity and blocking or antagonizing the activity of their corresponding B cell stimulatory receptors. The TACI-Fc fusion proteins contain mutant Fc domains designed to reduce one or more effector functions, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC), and inhibit IgG recycling by neonatal Fc receptor (FcRn) blockade. The TACI-Fc fusion proteins can be used to treat a variety of diseases, disorders, and conditions associated with dysregulated immune responses associated with inflammatory or autoimmune conditions.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 430,265, filed December 05, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] B cell proliferation, maturation, and survival depend on the signaling pathways of three receptors: TACI (transmembrane activator or calcium-regulated cyclophilin ligand interactor), BCMA (B cell maturation antigen), and BAFF-R (B cell-activating factor receptor). BCMA and BAFF-R are cell surface receptors of the TNF receptor superfamily and are encoded by the TNFRSF17 and TNFRSF13C genes, respectively.

[0003] TACI (encoded by the TNFRSF13B gene) is a transmembrane protein of the TNF receptor superfamily, which is found primarily on the surface of B cells. TACI recognizes three ligands: APRIL (a proliferation-inducing ligand encoded by the TNFSF1 gene), BAFF (a B cell-activating factor encoded by the TNFSF13B gene), and CAML (a calcium-modulating cyclophilin ligand). TACI is known to regulate T cell-independent B cell antibody responses, isotype switching, and B cell homeostasis. APRIL is a proliferation-inducing ligand that is a ligand for both TACI and BCMA. BAFF is a ligand for TACI, BCMA, and BAFF-R (Marsters et al. Curr Biol 2000;10(13):785-788, Thompson et al. Science 2001;293:2:108-111). BAFF is a ligand for TACI, BCMA, and BAFF-R, is expressed in B lineage cells, acts as a potent B cell activator, and plays an important role in the proliferation and differentiation of B cells.

[0004] BAFF and APRIL are potent stimulators of B cell maturation, proliferation, and survival (see, e.g., Gross et al., Nature 2000;404:995-999; Gross et al., Immunity 2001;15(2):289-302; and Groom et al., J Clin Invest 2002;109(1):59-68) and are known to independently form homodimers and BAFF / APRIL heterotrimers. BAFF and APRIL may be required for the maintenance of autoimmune diseases, particularly those involving B cells. Transgenic mice engineered to express high levels of BAFF exhibit immune cell dysfunction and display symptoms similar to those seen in patients with systemic lupus erythematosus (SLE) (see, e.g., Cheson et al. Blood 1996;87:4990-4997; and Cheema et al. Arthritis Rheum 2001;44(6):1313-1319). Similarly, elevated levels of BAFF and APRIL have been measured in serum samples taken from SLE patients and other patients with various autoimmune diseases such as rheumatoid arthritis (see, e.g., Roschke et al. J. Immunol 2002;169:4314-4321; Mariette et al. Ann Rheum Dis 2003;62(2):168-171; and Hahne et al. J Exp Med 1998;188(6):1185-1190), extending the association of BAFF and / or APRIL with B-cell mediated diseases from animal models to humans.

[0005] The current approach to treating autoimmune diseases is to suppress unwanted immune responses. For example, several immunosuppressants have proven beneficial in treating lupus nephritis (LN), a serious complication involving the kidneys of patients with systemic lupus nephritis (SLE). These drugs include cyclophosphamide (CYC), azathioprine (AZA), cyclosporine A (CSA), and mycophenolate mofetil (MMF) (e.g., Mok et al. (2003) Ann Rheum Dis 62, 799-804 and Iaccarino et al. (2007) Autoimmunity Reviews 6, 190-195). Although these drugs are beneficial, there remains a need in the art for improved therapeutic agents that improve response to immunosuppressive drugs, regulate immune responses, particularly B cell immune responses, and better treat inflammatory or autoimmune diseases, disorders, and conditions. Described herein are anti-FcRn BAFF / APRIL inhibitor compositions and methods for their use in the treatment of various diseases and disorders.

[0006] The present disclosure provides immunomodulatory TACI-Fc fusion proteins that exhibit neutralizing activity of BAFF and APRIL (or the BAFF / APRIL heterotrimer). The TACI-Fc fusion proteins are anti-FcRn BAFF / APRIL inhibitor compositions that contain variant extracellular DR1 and / or DR2 domains of TACI and variant Fc receptor binding domains. The present disclosure also provides nucleic acid molecules encoding the TACI-Fc fusion proteins, methods for making and using such proteins, and pharmaceutical compositions for treating various diseases, including immunological diseases, disorders, and conditions. Summary of the Invention

[0007] Provided herein are TACI-Fc fusion proteins that bind to and inhibit the activity of BAFF and APRIL ligands, neutralize their activity, and block or antagonize the activity of B cell stimulatory receptors, TACI, BCMA, and BAFF-R. The TACI-Fc fusion proteins further comprise a variant Fc domain containing mutations to block one or more effector functions, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC), and / or inhibit one or more activities, including FcRn activity. As described further below, the TACI-Fc fusion proteins can be used to treat various diseases, disorders, or conditions associated with a dysregulated immune response.

[0008] In one aspect, a TACI-Fc fusion protein comprises a TACI extracellular domain (ECD) portion and a hybrid immunoglobulin G (IgG) Fc domain, e.g., IgG1 / IgG4. In some embodiments, the TACI ECD portion comprises a TACI DR2 domain portion. In some embodiments, the TACI ECD portion comprises a contiguous ECD portion spanning at least a portion of the DR1 / DR2 region. In one embodiment, the TACI ECD portion comprises the amino acid sequence set forth in SEQ ID NO: 1 or 2.

[0009] In some embodiments, the TACI ECD portion comprises one or more mutations. In one embodiment, the mutation comprises L82H alone or in combination with a single histidine substitution mutation from Table 1, where amino acid identification and numbering is according to SEQ ID NO:40. In another embodiment, the mutation comprises F78H and D80H. In another embodiment, the mutation comprises F78H, D80H, and L82H. In another embodiment, the mutation comprises Y79H, D80H, and L82H. In another embodiment, the mutation comprises D80H, L82H, and L83H. In another embodiment, the mutation comprises Y79H, D80H, and L83H. In another embodiment, the mutation comprises D80H, L83H, and I87H. The SPR in Figures 2A, 2B, and 3 show weaker affinity compared to WT for some specific variants, e.g., those with one or more of the following mutations: L82H, F78H / D80H / L82H, Y79H / D80H / L82H, D80H / L82H / L83H, Y79H / D80H / L83H, D80 / L83H / I87H.

[0010] In one embodiment, the mutation is N434H. In another embodiment, the mutation is Y436L. In another embodiment, the mutation comprises L234F and L235E. In another embodiment, the mutation comprises M252Y, S254T, and T256E. In another embodiment, the mutation comprises L234F, L235E, and N434H. In another embodiment, the mutation comprises M252Y, S254T, T256E, and N434H. In another embodiment, the mutation comprises L234F, L235E, M252Y, S254T, and T256E. In another embodiment, the mutation comprises L234F, L235E, M252Y, S254T, T256E, and N434H. In another embodiment, the mutation comprises T307A, E380A, and N434A.

[0011] In some embodiments, the variant Ig Fc domain is derived from an IgG. In some embodiments, the variant Ig Fc domain is derived from an IgG1, IgG2, IgG3, IgG4, or a hybrid Fc domain thereof. In some embodiments, the variant Ig Fc domain comprises a hybrid IgG1-IgG4 Fc domain.

[0012] In some embodiments, the variant Ig Fc domain comprises an amino acid sequence at least 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 16-24 and 72. In one embodiment, the variant Ig Fc domain comprises an amino acid sequence identical to the variant Ig Fc domain set forth in any one of SEQ ID NOs: 16-24 and 72.

[0013] In some embodiments, the TACI ECD portion is linked to the mutant Ig Fc domain by a polypeptide linker. In one embodiment, the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-15.

[0014] In some embodiments, the TACI-Fc fusion protein comprises an amino acid sequence that is at least 96%, 97%, 98%, or 99% identical to the TACI-Fc amino acid sequence set forth in any one of SEQ ID NOs: 25-38, 46-48, 55, 56, and 67. In one embodiment, the fusion protein comprises an amino acid sequence that is identical to the TACI-Fc amino acid sequence set forth in any one of SEQ ID NOs: 25-38, 46-48, 55, 56, and 67. In some embodiments, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 25 or 67.

[0015] In some embodiments, the fusion protein has a binding affinity (K) for FcRn at pH 6.0 that is less than 50 nM, less than 20 nM, less than 12 nM, less than 6 nM, or less than 4 nM. D ) is shown.

[0016] In some embodiments, the fusion protein has a binding affinity (K) for FcRn that is 50-fold greater, 100-fold greater, or 200-fold greater at pH 6.0 compared to pH 7.4. D ) indicates an increase.

[0017] In another aspect, the present disclosure provides a pharmaceutical composition comprising a TACI-Fc fusion protein disclosed herein and a pharmaceutically acceptable carrier.

[0018] In another aspect, the disclosure provides a nucleic acid encoding a TACI-Fc fusion protein disclosed herein. In one embodiment, the nucleic acid encodes the fusion protein set forth in SEQ ID NO: 25 or 67. In another embodiment, the nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 69 or 70. In some embodiments, the nucleic acid encodes a codon-optimized TACI-Fc fusion protein.

[0019] In another aspect, the disclosure provides an expression vector encoding a TACI-Fc fusion protein disclosed herein, such as the fusion protein set forth in SEQ ID NO: 25 or 67 or a codon-optimized TACI-Fc fusion protein. In one embodiment, the expression vector is a viral vector. In another embodiment, the expression vector is an AAV vector.

[0020] In another aspect, the present disclosure provides a pharmaceutical composition comprising an expression vector encoding a TACI-Fc fusion protein disclosed herein and a pharmaceutically acceptable carrier.

[0021] In another aspect, the present disclosure provides a host cell comprising a nucleic acid or expression vector encoding a TACI-Fc fusion protein disclosed herein.

[0022] In another aspect, a method for treating a disease comprises administering to a patient an effective amount of a pharmaceutical composition comprising a TACI-Fc fusion protein or an expression vector encoding the TACI-Fc fusion protein in combination with a pharmaceutically acceptable carrier.

[0023] In some embodiments, the disease is an autoimmune disorder, ie, acute antibody-mediated rejection, chronic antibody-mediated rejection, primary Sjogren's syndrome, autoimmune hemolytic anemia, antiphospholipid syndrome (APS), catastrophic APS, refractory rheumatoid arthritis, autoimmune vasculitis, cryoglobulinemia, antineutrophil cytoplasmic autoantibodies (ANCA), IgA vasculitis, rheumatoid vasculitis, antisynthetase syndrome, primary membranous nephropathy, juvenile idiopathic arthritis (JIA), systemic sclerosis, or Guillain-Barré syndrome. , autoimmune pulmonary alveolar proteinosis, autoimmune hemolytic anemia (AIHA), hemolytic disease of the fetus and newborn (HDFN), acute hemolytic transfusion reaction, adeno-associated virus (AAV) prophylactic treatment, thyroid eye disease (TED), Huntington's disease, chronic inflammatory demyelinating polyneuropathy (CIDP), pemphigus vulgaris (PV), bullous pemphigoid (BP), immune thrombocytopenic purpura (ITP), myasthenia gravis (MG), and neuromyelitis optica (NMOSD).

[0024] In another aspect, a method for reducing an immune response comprises administering to a patient an effective amount of a pharmaceutical composition comprising a TACI-Fc fusion protein or an expression vector encoding a TACI-Fc fusion protein in combination with a pharmaceutically acceptable carrier.

[0025] In another aspect, a method for inhibiting the activity of one or more pro-inflammatory cytokines comprises administering to a patient an effective amount of a pharmaceutical composition comprising a TACI-Fc fusion protein or an expression vector encoding a TACI-Fc fusion protein in combination with a pharmaceutically acceptable carrier. In some embodiments, the pro-inflammatory cytokines comprise BAFF and / or APRIL.

[0026] In another aspect, a method for enhancing the clearance of IgG immunoglobulins comprises administering to a patient an effective amount of a pharmaceutical composition comprising a TACI-Fc fusion protein or an expression vector encoding a TACI-Fc fusion protein in combination with a pharmaceutically acceptable carrier.

[0027] In another aspect, a method for reducing levels of mature B cells, IgG, IgM, IgA, or a combination thereof comprises administering to a patient an effective amount of a pharmaceutical composition comprising a TACI-Fc fusion protein or an expression vector encoding a TACI-Fc fusion protein in combination with a pharmaceutically acceptable carrier. In some embodiments, administration of the pharmaceutical composition does not significantly reduce levels of IgD or IgE.

[0028] In another aspect, the disclosure provides a kit for treating a disease in a patient, the kit comprising at least one dose of a pharmaceutical composition comprising a TACI-Fc fusion protein or an expression vector encoding the TACI-Fc fusion protein, and instructions for use.

[0029] In another aspect, the disclosure provides a polypeptide linker comprising the amino acid sequence set forth in SEQ ID NO:4, 5, or 6. [Brief explanation of the drawings]

[0030] [Figure 1] Structural model based on 1XU1 and 1KD7 PBD entries showing the positions of sequentially and combinatorially mutated histidine residues (as black spheres) at / near the predicted interface between TACI and BAFF, using known contacts of TACI to BAFF and / or APRIL. [Figure 2A] The effect of a single histidine substitution in the TACI domain on binding to BAFF (FIG. 2A) and APRIL (FIG. 2B) by surface plasmon resonance (SPR) is shown. [Figure 2B] The effect of a single histidine substitution in the TACI domain on binding to BAFF (FIG. 2A) and APRIL (FIG. 2B) by surface plasmon resonance (SPR) is shown. [Figure 3] 1 shows the effect of multiple histidine substitutions in the TACI domain on binding to BAFF by SPR. [Figure 4]1 shows exemplary sensorgrams of TACI-Fc fusion protein against BAFF, APRIL, or FcRn by biolayer interferometry (BLI). [Figure 5] 1 is a sensorgram showing the binding of TPP-6269 to BAFF at pH 6.0 and pH 7.4 by BLI. [Figure 6] 1 shows the results of a functional assay demonstrating the inhibition of BAFF binding to a BCMA / nuclear factor kappa B (NF-κB)-luciferase reporter HEK293 cell line by different concentrations of TACI-Fc fusion protein or a control (TPP-4217). [Figure 7] Results of functional assays showing inhibition of BAFF binding and NF-κB-mediated activation in a BCMA / NF-κB-luciferase reporter HEK293 cell line by different concentrations of TACI-Fc variants, as indicated, are shown, including the associated corresponding IC50 values. [Figure 8] 1 shows the results of a functional assay demonstrating the inhibition of BAFF binding and NF-κB-mediated activation in a BCMA / NF-κB-luciferase reporter HEK293 cell line by different concentrations of the TACI-Fc fusion protein TPP-4286 and a control (TPP-4291), including the associated corresponding IC50 values. [Figure 9A] Biolayer interferometry sensorgrams showing binding of TACI-Fc variants to FcRn at pH 6.0 (left panel) and pH 7.4 (right panel) using the Octet Red 96 system. TACI-Fc variants include TPP-5260 (Figure 9A), TPP-5262 (Figure 9B), TPP-5317 (Figure 9C), TPP-5272 (Figure 9D), TPP-5273 (Figure 9E), TPP-5274 (Figure 9F), TPP-5275 (Figure 9G), and TPP-6269 (Figure 9H). [Figure 9B]Biolayer interferometry sensorgrams showing binding of TACI-Fc variants to FcRn at pH 6.0 (left panel) and pH 7.4 (right panel) using the Octet Red 96 system. TACI-Fc variants include TPP-5260 (Figure 9A), TPP-5262 (Figure 9B), TPP-5317 (Figure 9C), TPP-5272 (Figure 9D), TPP-5273 (Figure 9E), TPP-5274 (Figure 9F), TPP-5275 (Figure 9G), and TPP-6269 (Figure 9H). [Figure 9C] Biolayer interferometry sensorgrams showing binding of TACI-Fc variants to FcRn at pH 6.0 (left panel) and pH 7.4 (right panel) using the Octet Red 96 system. TACI-Fc variants include TPP-5260 (Figure 9A), TPP-5262 (Figure 9B), TPP-5317 (Figure 9C), TPP-5272 (Figure 9D), TPP-5273 (Figure 9E), TPP-5274 (Figure 9F), TPP-5275 (Figure 9G), and TPP-6269 (Figure 9H). [Figure 9D] Biolayer interferometry sensorgrams showing binding of TACI-Fc variants to FcRn at pH 6.0 (left panel) and pH 7.4 (right panel) using the Octet Red 96 system. TACI-Fc variants include TPP-5260 (Figure 9A), TPP-5262 (Figure 9B), TPP-5317 (Figure 9C), TPP-5272 (Figure 9D), TPP-5273 (Figure 9E), TPP-5274 (Figure 9F), TPP-5275 (Figure 9G), and TPP-6269 (Figure 9H). [Figure 9E] Biolayer interferometry sensorgrams showing binding of TACI-Fc variants to FcRn at pH 6.0 (left panel) and pH 7.4 (right panel) using the Octet Red 96 system. TACI-Fc variants include TPP-5260 (Figure 9A), TPP-5262 (Figure 9B), TPP-5317 (Figure 9C), TPP-5272 (Figure 9D), TPP-5273 (Figure 9E), TPP-5274 (Figure 9F), TPP-5275 (Figure 9G), and TPP-6269 (Figure 9H). [Figure 9F]Biolayer interferometry sensorgrams showing binding of TACI-Fc variants to FcRn at pH 6.0 (left panel) and pH 7.4 (right panel) using the Octet Red 96 system. TACI-Fc variants include TPP-5260 (Figure 9A), TPP-5262 (Figure 9B), TPP-5317 (Figure 9C), TPP-5272 (Figure 9D), TPP-5273 (Figure 9E), TPP-5274 (Figure 9F), TPP-5275 (Figure 9G), and TPP-6269 (Figure 9H). [Figure 9G] Biolayer interferometry sensorgrams showing binding of TACI-Fc variants to FcRn at pH 6.0 (left panel) and pH 7.4 (right panel) using the Octet Red 96 system. TACI-Fc variants include TPP-5260 (Figure 9A), TPP-5262 (Figure 9B), TPP-5317 (Figure 9C), TPP-5272 (Figure 9D), TPP-5273 (Figure 9E), TPP-5274 (Figure 9F), TPP-5275 (Figure 9G), and TPP-6269 (Figure 9H). [Figure 9H] Biolayer interferometry sensorgrams showing binding of TACI-Fc variants to FcRn at pH 6.0 (left panel) and pH 7.4 (right panel) using the Octet Red 96 system. TACI-Fc variants include TPP-5260 (Figure 9A), TPP-5262 (Figure 9B), TPP-5317 (Figure 9C), TPP-5272 (Figure 9D), TPP-5273 (Figure 9E), TPP-5274 (Figure 9F), TPP-5275 (Figure 9G), and TPP-6269 (Figure 9H). [Figure 10] 1 shows the crystal structure of the FcRn:Fc complex (PDB: 4N0U), showing the acidic residues E115, E116, D130, and E133 in processed human FcRn for targeting by the TACI-Fc fusion proteins of the present disclosure. [Figure 11A]Octet traces comparing FcRn binding of a single histidine-substituted TACI-Fc variant (Figure 11A) and both single and double histidine-substituted TACI-Fc variants (Figure 11B) compared to the wild-type ("WT" parent non-histidine-substituted TPP-5274) TACI-Fc fusion protein at pH 7.4 are shown. [Figure 11B] Octet traces comparing FcRn binding of a single histidine-substituted TACI-Fc variant (Figure 11A) and both single and double histidine-substituted TACI-Fc variants (Figure 11B) compared to the wild-type ("WT" parent non-histidine-substituted TPP-5274) TACI-Fc fusion protein at pH 7.4 are shown. [Figure 11C] Octet traces comparing FcRn binding of a single histidine-substituted TACI-Fc fusion protein variant (Figure 11C) and both single and double histidine-substituted TACI-Fc fusion protein variants (Figure 11D) compared to wild-type ("WT" TPP-5274) TACI-Fc fusion protein at pH 6.0 are shown. [Figure 11D] Octet traces comparing FcRn binding of a single histidine-substituted TACI-Fc fusion protein variant (Figure 11C) and both single and double histidine-substituted TACI-Fc fusion protein variants (Figure 11D) compared to wild-type ("WT" TPP-5274) TACI-Fc fusion protein at pH 6.0 are shown. [Figure 12A] Octet traces comparing FcRn binding by TPP-5958 (L234F, L235E, M252Y, S254T, T256E, N434H) and TPP-5957 (L234F, L235E, M252Y, S254T, T256E) at pH 6.0 are shown. [Figure 12B] Octet traces comparing FcRn binding by TPP-5958 (L234F, L235E, M252Y, S254T, T256E, N434H) and TPP-5957 (L234F, L235E, M252Y, S254T, T256E) at pH 7.4 are shown. [Figure 13]Figure 13A shows the binding kinetics of TPP-6269 to FcRn at various concentrations at pH 6.0 by surface plasmon resonance (SPR) / Biacore. Figure 13B shows the binding kinetics of TPP-6269 to FcRn at various concentrations at pH 7.4 by SPR / Biacore. Figure 13C shows the corresponding equilibrium dissociation constants (KD) determined by SPR / Biacore. [Figure 14] Figures 14A-14D show flow cytometry analysis demonstrating simultaneous binding of TACI-Fc variants TPP-5954 (Figures 14A, 14B), TPP-5957 (Figures 14A, 14C), and TPP-5958 (Figures 14A, 14D) to BAFF and human FcRn in human FcRn-expressing cell lines. [Figure 15] Figures 15A and 15B show flow cytometry analyses demonstrating the ability of TPP-6269 (Figure 15A) and other TACI-Fc variants (Figure 15B, as indicated) to block human IgG1 binding to FcRn in human FcRn-expressing cell lines. [Figure 16] Figure 16A is a graph showing the stability of selected TACI-Fc variants from an accelerated stability study using size exclusion chromatography to analyze the percentage of TACI-Fc monomer as a function of time, and Figure 16B is a table showing the percentage monomer numbers for each of the selected TACI-Fc variants at time zero (T0) and after 1, 2, and 3 weeks of incubation at 37°C. [Figure 17]Figure 17A shows the in vivo study design of an experiment to evaluate the ability of TPP-6269 to reduce levels of IgG, IgM, and mature B cells after in vivo treatment of mice with keyhole limpet hemocyanin (KLH). Figure 17B shows the absolute numbers of B220+ cells and CD4+ T cells in mouse splenocytes after co-administration of TPP-6269 and KLH compared to administration of a KLH control ("KLH+PBS") or no administration ("healthy control"), as determined by flow cytometry. Figure 17C shows the resulting serum levels of anti-KLH IgM and IgG antibodies after administration. Figure 17D shows the resulting serum levels of total IgM and total IgG antibodies after administration. [Figure 18] Figure 18A shows the experimental design to evaluate the clearance of human IgG (biotin-hIgG) antibodies after administration of TPP-6269 in muFcRn- / -, huFcRn Tg32 transgenic mice in vivo. Figure 18B shows the percent reduction of total human IgG antibodies as a function of time at dosages of 20 mg / kg and 60 mg / kg TPP-6269 compared to administration of PBS alone. [Figure 19] Figure 19A shows the design of the experiment to evaluate the pharmacokinetics of TPP-6269 over a 21 day period, and Figure 19B is a graph showing the results of this analysis. [Figure 20] Figure 20A shows the experimental design for evaluating the pharmacokinetics and pharmacodynamics of TPP-6269 over a 10-day period. Figure 20B shows graphs depicting the results of the pharmacokinetic analysis. Figure 20C shows graphs depicting the results of the pharmacodynamic analysis. DETAILED DESCRIPTION OF THE INVENTION

[0031] definition As used herein, the word "a" or "plurality" before a noun refers to one or more of that particular noun. For example, the phrase "mammalian cells" refers to "one or more mammalian cells."

[0032] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0033] Particularly with respect to a given amount or number, the term "about" is meant to encompass variations within plus or minus ten percent (±10%), five percent (e.g., ±5%), two percent (±2%), one percent (±1%), or one-half percent (±0.5%).

[0034] The terms "portion," "part of," and "portion of" refer to a portion or the whole of the subject component. Thus, it can refer to the whole component or a portion of the component.

[0035] The terms "polypeptide" and "protein" are used interchangeably herein and refer to a molecular chain of two or more amino acids linked via peptide bonds. The term does not refer to a specific length of the product. Thus, "peptide" and "oligopeptide" are included within the definition of polypeptide. The term includes post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like. The term also includes molecules containing one or more amino acid analogs, or non-standard or unnatural amino acids, such as may be recombinantly synthesized or expressed using known protein engineering techniques. Additionally, proteins can be derivatized as described herein by well-known organic chemistry techniques.

[0036] As used herein, the terms "domain" and "functional domain" refer to a functional polypeptide or nucleic acid domain that typically contains or encodes an amino acid sequence of three or more, generally five or seven or more, amino acids, e.g., 10-200 amino acid residues, that is structurally and / or functionally distinct and identifiable from other portions of the molecule. For example, a domain includes a portion of a polypeptide chain that can form an independently folded structure within a protein composed of one or more structural motifs and / or that is recognized by a functional activity (e.g., binding activity). Proteins, such as the fusion proteins described herein, can contain one or more distinct domains. Domains can be identified, defined, or distinguished by primary sequence or structural homology to related family members, such as homology to a motif. In another example, a domain can be distinguished by its function, e.g., its ability to interact with a biomolecule, such as a cognate binding partner. A domain can exhibit an independent biological function or activity, whereby a domain can perform an activity, e.g., binding, either independently or fused to another molecule. In some cases, adjacent N- and / or C-terminal amino acids of a given domain (e.g., a cysteine-rich domain CRD) may be directly linked by a single peptide bond or a polypeptide linker containing one or more amino acid residues. Generally, the two moieties and the polypeptide linker are in reading frame with each other. Alternatively, the two moieties may be covalently linked together using one or more cross-linking agents known in the art.

[0037] As used herein, the term "TACI protein" (transmembrane activator or calcium-modulating cyclophilin ligand interactor) refers to a transmembrane protein of the TNF receptor superfamily (encoded by the TNFRSF13B gene) that antagonizes or blocks the activity of the B cell stimulatory receptor B cell maturation antigen (BCMA), B cell activating factor receptor (BAFF-R), by binding to their ligands APRIL (proliferation-inducing ligand encoded by the TNFSF1 gene) and BAFF (B cell activating factor encoded by the TNFSF13B gene).

[0038] The terms "TACI extracellular domain (ECD)" and "TACI ECD portion" refer to the portion of the protein that contains the binding sites for APRIL and BAFF, and is composed of the cysteine-rich TNFR repeat region including the TACI DR1 / DR2 domain.

[0039] The terms "Fc region," "Fc domain," and "immunoglobulin (Ig) Fc" refer to the C-terminal region of an antibody heavy chain, which includes the Ig heavy chain constant region domains CH2 and CH3, and may include all or part of the Ig hinge domain. The Fc domain typically encodes various effector function(s), which may be modified or deleted. The Fc domain can form a monomer or dimer of two polypeptide chains linked via hinge region cysteine ​​residues that form interpolypeptide disulfide bonds.

[0040] The terms "mutant" and "variant" refer to a protein obtained after modification of a parent protein in a protein modification process. Specifically, a mutant or variant is a protein derived from a parent protein by one or more amino acid substitution(s), deletion(s), and / or addition(s) relative to the parent protein, which may add, retain, remove, or enhance some or all of the parent's functions.

[0041] As used herein, the terms "TACI mutant" and "TACI variant" are used interchangeably to refer to a TACI extracellular domain (ECD) portion that differs from a parent TACI ECD sequence by at least one amino acid modification. The amino acid modification may be a substitution mutation, an amino acid deletion, and / or an amino acid addition. The TACI mutant may bind to APRIL and / or BAFF and exhibit enhanced binding to its ligand at low pH (e.g., pH 6.0), for example, compared to the wild-type TACI ECD or the TACI reference protein from which the TACI mutant is derived. See, e.g., Tables 1 and 2.

[0042] As used herein, the terms "Fc mutant," "Fc variant," and "Ig Fc variant" are used interchangeably to refer to an Fc sequence that differs from a parent Fc sequence by virtue of at least one amino acid modification. An Fc variant may comprise only the Fc region, or may exist in the context of an antibody, an Fc fusion, an isolated Fc, an Fc region, or other polypeptide substantially encoded by an Fc. An Fc variant may refer to the Fc polypeptide itself, a composition comprising the Fc variant polypeptide, or the amino acid sequence encoding it.

[0043] The terms "TACI-Fc variant" and "TACI-Fc fusion protein" are used interchangeably to refer to hybrid proteins that combine the binding specificity of a TACI binding domain containing a TACI extracellular domain (ECD) portion containing a TACI DR2- or TACI DR1 / DR2 domain portion fused or chemically conjugated to a variant immunoglobulin (Ig) that exhibits reduced effector function due to the inclusion of one or more mutations therein. The TACI ECD contains a contiguous or noncontiguous amino acid sequence that provides one or more binding sites for APRIL and BAFF. The APRIL and BAFF binding portions are derived from the TACI ECD without the transmembrane or cytoplasmic sequences of the TACI receptor.

[0044] The terms "Fc region," "Fc domain," and "Ig Fc" refer to the C-terminal region of an antibody heavy chain, comprising Ig heavy chain constant region domains CH2 and CH3, and may include all or a portion of an Ig hinge domain. The Fc domain typically encodes various effector function(s), which may be modified or deleted. Fc domains can form monomers or dimers of two polypeptide chains linked by one or more disulfide bonds. The Fc domains of the present disclosure are variant or mutant forms of wild-type Fc domains that exhibit reduced effector function (e.g., greater than about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 5-fold, or more reduced).

[0045] As used herein, references to amino acid substitutions in the Fc region utilize the EU numbering system, unless otherwise specified with respect to a specific SEQ ID NO. EU numbering is known and follows the latest IMGT Scientific Chart (IMGT®, the international ImMunoGeneTics information System™, http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html (created May 17, 2001, last updated January 10, 2013) and Kabat, E. A. et al., Sequences of Proteins of Immunological Interest. 5th ed., U.S. Department of Health and Human Services, NIH publication No. 91-3242 (1991).

[0046] The term "polypeptide linker" refers to a polypeptide defined by a sequence of amino acids that functions to connect the TACI domain to the Ig Fc domain in-frame, resulting in a TACI-Fc fusion protein.

[0047] As used herein, the term "hybrid Ig Fc" refers to a variant Fc region that contains portions from at least two IgG1 subclasses.

[0048] "Conjugate" refers to any hybrid molecule that contains protein domains and / or amino acids or other molecules that contain both protein and non-protein portions. Conjugates may be synthesized by a variety of techniques known in the art, including, for example, recombinant DNA techniques, solid-phase synthesis, solution-phase synthesis, organic chemical synthesis techniques, or a combination of these techniques. The choice of synthesis depends on the molecule being produced. For example, hybrid molecules that are not entirely "proteinaceous" in nature may be synthesized by a combination of recombinant and solution-phase techniques.

[0049] The terms "bind," "bound," or grammatical variations thereof, are used in reference to the participation of a molecule in any attractive interaction with another molecule that results in a stable association in which the two molecules are in close proximity to one another. Binding includes, but is not limited to, non-covalent bonds, covalent bonds (such as reversible and irreversible covalent bonds), and includes interactions between molecules such as, but not limited to, proteins, nucleic acids, carbohydrates, lipids, and small molecules such as compounds including drugs.

[0050] With respect to the binding of TACI-Fc fusion proteins of the present disclosure, the terms "bind" and "bound" or grammatical variations thereof are used in reference to a TACI-Fc fusion protein or domain thereof that specifically binds to each of APRIL, BAFF, and neonatal Fc receptor (FcRn) in vitro and / or in vivo, but does not bind to other ligands or antigens. Typically, a TACI-Fc fusion protein or domain binds to each of APRIL, BAFF, and FcRn at approximately 10 -7 Less than M, e.g., approximately 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 The equilibrium dissociation constant (K D) but does not bind to another molecule with similar affinity, as determined, for example, by surface plasmon resonance (SPR) techniques on a BIACORE® 2000 surface plasmon resonance instrument using the TACI-Fc fusion protein as the analyte and APRIL, BAFF, or FcRn as the ligand. Thus, unless otherwise specified, the TACI-Fc fusion protein binds to soluble or cell-bound APRIL, BAFF, or FcRn at a concentration of 10 -7 M or less, e.g., about 10 -8 M, 10 -9 M, 10 -10 Less than M or less than K D Combine with.

[0051] The term "binding activity" refers to a characteristic of a molecule, e.g., a polypeptide or polypeptide domain, relating to whether or how it binds to one or more binding partners. Binding activity can include any measure of the binding of a molecule to a binding partner. Binding activity includes the ability to bind to a binding partner, the affinity (e.g., high affinity) of binding to a binding partner, the avidity of binding to a binding partner, the strength of binding to a binding partner, and / or the specificity or selectivity of binding to a binding partner.

[0052] The term "binding affinity" refers to the specific binding affinity of a protein for its binding partner (i.e., its counterstructure) under specific binding conditions. Binding affinity refers to the strength of the interaction between two or more molecules, such as binding partners, typically the strength of a noncovalent interaction between two binding partners. The increased or decreased binding affinity of an affinity-modified domain, or a TACI-Fc fusion protein comprising an affinity-modified domain, for a binding partner is determined relative to the binding affinity of the unmodified domain (e.g., native or wild-type TACI or Ig Fc domain). Methods for determining binding affinity or relative binding affinity are known in the art, including Biacore measurements or flow cytometry, solid-phase ELISA immunoassays, ForteBio Octet, etc. In some embodiments, binding affinity can be measured, for example, based on mean fluorescence intensity (MFI) in a flow binding assay.

[0053] As used herein, "k assoc " or "k a The term "k" is intended to refer to a particular protein-protein interaction, e.g., the association rate of a TACI-Fc fusion protein or domain thereof to a ligand such as APRIL, BAFF, or FcRn, while "k" as used herein is intended to refer to a specific protein-protein interaction, e.g., the association rate of a TACI-Fc fusion protein or domain thereof to a ligand such as APRIL, BAFF, or FcRn. dis " or "k d The term "K" is intended to refer to the dissociation rate of a particular protein-protein interaction. D The term "k" is intended to refer to the dissociation constant, which is k d k a (i.e., k d / k a ) and is expressed as molar concentration (M). D The K value can be determined using methods well established in the art. DMethods for determining include surface plasmon resonance, biolayer interferometry, flow cytometry, and Scatchard analysis, preferably using a biosensor system such as a Biacore system.

[0054] Such determinations are typically measured, for example, at 25° C. or 37° C. For example, the kinetics of fusion protein binding to APRIL, BAFF, or FcRn can be determined via surface plasmon resonance (“SPR”) on a BIAcore 3000 instrument at pH 8.0, 7.4, 7.0, 6.5, and 6.0 using an appropriate capture method to immobilize the fusion protein or its binding ligand.

[0055] As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows for the analysis of real-time biomolecular specific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using a BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, New Jersey). For further explanation, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., et al. (1991) Biotechniques 11:620-627; Johnson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnson, B., et al. (1991) Anal. Biochem 198:268-277.

[0056] The term "corresponding" with respect to a protein or polynucleotide position means that an amino acid or nucleotide position in a reference sequence "corresponds" to a nucleotide or amino acid position in a disclosed sequence as set forth in a sequence listing, such that corresponding amino acid or nucleotide positions are identified upon structural alignment of two or more sequences using standard alignment algorithms such as the GAP algorithm. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides.

[0057] As used herein, the term "sequence identity" refers to sequence identity between genes or proteins at the nucleotide or amino acid level, respectively. "Sequence identity" is a measure of identity between proteins at the amino acid level and between nucleic acids at the nucleotide level. Protein sequence identity may be determined by comparing the amino acid sequences at each position when the sequences are aligned. Similarly, nucleic acid sequence identity may be determined by comparing the nucleotide sequences at each position when the sequences are aligned. Methods for aligning sequences for comparison are well known in the art, and such methods include GAP, BESTFIT, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software, FASTA, and TFASTA.

[0058] The term "expression vector" refers to a nucleic acid molecule containing nucleic acid sequences necessary for expression in prokaryotes, including, for example, a promoter, optionally an operator sequence, a ribosome binding site, and possibly other sequences, or expression in eukaryotic cells, including, for example, a promoter, an enhancer, termination and polyadenylation signals, etc. A secretory signal peptide sequence is also optionally encoded by the recombinant expression vector and operably linked to the coding sequence so that the expressed protein can be secreted by the recombinant host cell, e.g., for its expression as a secreted protein, or, if desired, for easier isolation or purification of the TACI-Fc fusion protein from the cell. The term includes vectors as self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they are introduced.

[0059] An expression vector contains sufficient regulatory sequence(s), e.g., expression control sequences, to direct transcription of an operably linked nucleic acid under at least some conditions. Other elements necessary or helpful for expression can be supplied, for example, by the host cell or by an in vitro expression system. Such regulatory sequences typically include a promoter and may include enhancer sequences or upstream activator sequences. In some embodiments, vectors may also include sequences encoding 5' and / or 3' untranslated regions, which may include cleavage and / or polyadenylation signals. Generally, regulatory elements may be included in the vector prior to insertion of the nucleic acid desired to be expressed, may be included with the inserted nucleic acid, or may be inserted into the vector after insertion of the nucleic acid desired to be expressed. Expression vectors include non-viral vectors, such as plasmid vectors, and viral vectors, such as adeno-associated virus (AAV) vectors and lentiviral vectors.

[0060] As used herein, the term "disease" is used in reference to any disease, disorder, or condition that directly or indirectly causes or contributes to a state other than physiological homeostasis that exists in the absence of such disease, disorder, or condition.

[0061] As used herein, the term "treating" includes prophylactic and / or therapeutic treatment of one or more diseases. The term "prevention or treatment" is art-recognized and includes administration to a host of one or more TACIFc fusion protein compositions described herein. If administered before the clinical manifestation of disease, the treatment is prophylactic (i.e., it protects the host from developing the disease or reduces clinical manifestations that would otherwise result in the absence of treatment), while if administered after the manifestation of disease, the treatment is therapeutic (i.e., it is intended to reduce, ameliorate, or stabilize an existing disease or its side effects). Preferably, it is intended that the severity of the subject's condition be reduced or at least partially ameliorated or altered, and that some relief, alleviation, reversal, or reduction in at least one clinical symptom (e.g., reduction in autoimmune symptoms, weight loss in a subject compared to normal subjects) be achieved.

[0062] The term "pharmaceutical composition" refers to a preparation that is in a form that clearly allows the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are significantly toxic to the subject to which the formulation is administered.

[0063] As used herein, the term "subject" includes both human and non-human subjects (e.g., veterinary or wild animals). Preferably, a "subject" includes a human patient.

[0064] The term "effective amount" or "therapeutically effective amount" refers to the amount of a TACI-Fc fusion protein, alone or in combination with one or more other therapeutic agents, that provides a desired biological, therapeutic, and / or prophylactic result. The result can be reduction, amelioration, palliative, relief, delay, and / or alleviation of one or more of the signs, symptoms, or causes of a disease in a subject, or any other desired alteration of a biological system. An effective amount can be administered in one or more administrations.

[0065] As used herein, "effective treatment" refers to treatment that results in a beneficial effect, e.g., an improvement in at least one symptom of the subject's disease. The beneficial effect can take the form of an improvement over baseline, e.g., an improvement over a measurement or observation made before initiation of therapy according to the method.

[0066] As used herein, the term "biological half-life" refers to the amount of time it takes for a TACI-Fc fusion protein to lose half of its pharmacological or physiological activity or concentration. Biological half-life may be affected by elimination, excretion, degradation (e.g., enzymatic degradation / digestion) of the fusion protein, or absorption and concentration in specific organs or tissues of the body. In some embodiments, biological half-life can be assessed by determining the time it takes for the plasma concentration of a substance to reach half of its steady-state level ("plasma half-life").

[0067] I. TACI-Fc Fusion Protein Provided herein are TACI-Fc fusion proteins that bind to and inhibit the activity of BAFF and APRIL ligands, neutralize their activity, and block or antagonize the activity of B cell stimulatory receptors, TACI, BCMA, and BAFF-R. The TACI-Fc fusion proteins further comprise a variant Fc domain containing mutations to block one or more effector functions, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC), and / or inhibit one or more activities, including FcRn activity. As described further below, the TACI-Fc fusion proteins can be used to treat a variety of diseases associated with dysregulated immune responses associated with inflammatory or autoimmune conditions, including those associated with a wide range of inflammatory and autoimmune diseases and disorders.

[0068] The TACI-Fc fusion proteins of the present disclosure comprise a TACI extracellular domain (ECD) portion and a variant Ig Fc domain.

[0069] In some embodiments, the TACI ECD portion comprises a TACI DR2 domain. In some embodiments, the TACI ECD portion comprises a contiguous ECD portion spanning at least a portion of the CRD1 / CRD2 region. In some embodiments, the TACI ECD portion comprises the amino acid sequence set forth in SEQ ID NO: 1 or 2.

[0070] In some embodiments, the TACI ECD portion comprises one or more mutations such as those identified in Tables 1-3, where the amino acids are numbered and identified according to the amino acid sequence set forth in SEQ ID NO:40.

[0071] In another embodiment, the mutations include F78H and L83H.

[0072] In another embodiment, the mutations include F78H and D80H.

[0073] In another embodiment, the mutations include F78H, D80H, and L82H.

[0074] In another embodiment, the mutations include Y79H and L83H.

[0075] In another embodiment, the mutations include Y79H, D80H, and L83H.

[0076] In another embodiment, the mutations include Y79H, D80H, and L82H.

[0077] In one embodiment, the mutation comprises D80H.

[0078] In another embodiment, the mutations include D80H and L82H.

[0079] In another embodiment, the mutations include D80H and L83H.

[0080] In another embodiment, the mutations include D80H and I87H.

[0081] In another embodiment, the mutations include D80H, L82H, and L83H.

[0082] In another embodiment, the mutations include D80H, L83H, and I87H.

[0083] In one embodiment, the mutation comprises L82H.

[0084] In another embodiment, the mutations include L82H and I87H.

[0085] In another embodiment, the mutations include L82H and I92H.

[0086] In another embodiment, the mutations include L82H and Q95H.

[0087] In another embodiment, the mutation comprises L83H.

[0088] In another embodiment, the mutations include L83H and I87H.

[0089] In another embodiment, the mutations include I87H and I92H.

[0090] In some embodiments, the TACI-Fc fusion protein comprises a variant Ig Fc domain, which may contain various mutations that act to reduce Fc effector functions, including downregulation of inflammatory functions such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and Clq binding, downregulate B cell activation and B cell receptor expression, enhance binding to the neonatal Fc receptor (FcRn), increase protein stability, and / or render the variant Fc domain inactive with respect to one or more of these functions.

[0091] The FcRn-binding sequence in the Fc region plays an important role in regulating serum IgG levels. FcRn regulates serum IgG concentrations by binding to endocytosed monomeric IgG, protecting it from degradation in the lysosomal compartment, and transporting it to the cell surface for release at neutral extracellular pH. Through this mechanism, FcRn is responsible for the long serum half-life of IgG, as IgG not bound by FcRn enters the lysosomal pathway and is degraded.

[0092] High-dose administration of IgG in patients with autoimmune disease has a palliative effect that may be explained, at least in part, by saturation of FcRn-mediated protection of IgG, shortening the half-life of pathogenic IgG (Jin & Balthasar, 2005, Hum. Immunol. 66:403-410; Akilesh et al., 2004, J. Clin. Invest. 113:1328-1333; Li et al., 2005, J. Clin. Invest. 115:3440-3450). Therefore, specific blockade of FcRn-IgG interactions can be used to promote the degradation of pathogenic IgG antibodies, for example, to treat IgG-mediated autoimmune diseases. For example, in one embodiment, the TACI-Fc fusion protein of the present disclosure is used in a method for enhancing the clearance of IgG immunoglobulins in patients with an inflammatory or autoimmune disease, disorder, or condition.

[0093] The TACI-Fc fusion proteins of the present disclosure comprise an IgG Fc domain containing one or more mutations that enhance binding of the variant Fc domain to FcRn, thereby allowing the TACI-Fc fusion protein to outcompete IgG for binding to FcRn, thereby reducing IgG levels. The variant Fc domain may further comprise one or more mutations that confer improved functional activity, anti-inflammatory function, or pharmacokinetic properties associated with the TACI-Fc fusion proteins of the present disclosure. Improved functional activity or anti-inflammatory function relates to improvements related to Clq binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), B cell activation, and / or increased protein stability.

[0094] In some embodiments, the TACI-Fc fusion protein comprises an Ig Fc domain comprising an N434H mutation.

[0095] In another embodiment, the TACI-Fc fusion protein comprises an Ig Fc domain containing a Y436L mutation.

[0096] In another embodiment, the TACI-Fc fusion protein comprises an Ig Fc domain comprising the L234F and L235E mutations.

[0097] In another embodiment, the TACI-Fc fusion protein comprises an Ig Fc domain comprising M252Y, S254T, and T256E mutations.

[0098] In another embodiment, the TACI-Fc fusion protein comprises an Ig Fc domain comprising the L234F / L235E / N434H mutations.

[0099] In another embodiment, the TACI-Fc fusion protein comprises an Ig Fc domain comprising the following mutations: M252Y, S254T, T256E, N434H.

[0100] In another embodiment, the TACI-Fc fusion protein comprises an Ig Fc domain comprising the L234F, L235E, M252Y, S254T, and T256E mutations.

[0101] In another embodiment, the TACI-Fc fusion protein comprises an Ig Fc domain comprising the following mutations: L234F, L235E, M252Y, S254T, T256E, and N434H.

[0102] In another embodiment, the TACI-Fc fusion protein comprises an Ig Fc domain comprising the T307A, E380A, and N434A mutations.

[0103] Additional useful mutations or combinations of mutations for incorporation into the TACI-Fc fusion proteins of the present disclosure are disclosed in U.S. Patent Application Publication Nos. 2015 / 0017164, 2020 / 0095310, and 2021 / 0388054, the disclosures of which are incorporated herein by reference.

[0104] In some embodiments, the variant Ig Fc domain comprises an amino acid sequence at least 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NOs: 16-24 and 72. In other embodiments, the variant Ig Fc domain comprises an amino acid sequence identical to the variant Ig Fc domain set forth in any one of SEQ ID NOs: 16-24 and 72. In some embodiments, the variant Ig Fc domain comprises a variant Ig Fc domain according to any one of SEQ ID NOs: 16-24 and 72, and optionally a terminal lysine residue attached thereto. In one embodiment, the variant Ig Fc domain comprises the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the variant Ig Fc domain comprises the amino acid sequence set forth in SEQ ID NO: 72.

[0105] In some embodiments, a TACI-Fc fusion protein comprises a TACI ECD portion having any of the mutations or combinations of mutations described above, and any of the Ig Fc domain mutations or combinations of mutations described above.

[0106] Preferably, the variant Fc domain is derived from IgG. Various IgG isotypes may be used, including Fc domains derived from IgG1, IgG2, IgG3, IgG4, or a combination thereof. In a preferred embodiment, the Fc domain comprises a hybrid IgG1-IgG4 Fc domain. In one embodiment, the hybrid IgG1-IgG4 Fc domain is constructed from nucleic acids encoding the IgG1 gamma-1 chain C region (e.g., accession number P01857) and the IgG4 gamma-4 chain C region (e.g., accession number P01861).

[0107] The TACI ECD portion is linked (e.g., fused or conjugated) to the mutant Ig Fc domain. In some embodiments, the TACI ECD portion is fused to the mutant Ig Fc domain by a polypeptide linker. In some embodiments, the TACI ECD portion is chemically conjugated to the mutant Ig Fc domain.

[0108] In some embodiments, the polypeptide linker comprises an amino acid sequence having a length of at least 3, at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 amino acids, comprising one or more amino acids selected from G (glycine), alanine (A), serine (S), proline (P), or a combination thereof.

[0109] In one embodiment, the polypeptide linker comprises the amino acid sequence (AG x ) n ,(GA x ) n (A x G) n or (G x A) nwhere x=1, 2, 3, 4, 5, 6, 7, or 8 and n=1, 2, 3, 4, 5, 6, 7, or 8.

[0110] In another embodiment, the polypeptide linker comprises the amino acid sequence (SG x ) n ,(GS x ) n ,(S x G) n or (G x S) n where x=1, 2, 3, 4, 5, 6, 7, or 8 and n=1, 2, 3, 4, 5, 6, 7, or 8.

[0111] In another embodiment, the polypeptide linker comprises the amino acid sequence (EA x K) n where x=2, 3, 4, 5, 6, 7, or 8 and n=1, 2, 3, 4, 5, 6, 7, or 8.

[0112] In some embodiments, the TACI ECD portion is fused to the variant Ig Fc domain by a polypeptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-15.

[0113] In some embodiments, the present disclosure provides a polypeptide linker comprising an amino acid sequence selected from the group consisting of 4, 5, and 6.

[0114] In some embodiments, a TACI Fc fusion protein comprises a polypeptide linker according to any one of SEQ ID NOs: 3-15 fused to an Ig Fc domain portion according to any one of SEQ ID NOs: 16-24 and 72, and optionally comprising a C-terminal lysine attached thereto. For example, in one embodiment, the TACI Fc fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 61. In another embodiment, the TACI Fc fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 62. In another embodiment, the TACI Fc fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63. In another embodiment, the TACI Fc fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 64. In another embodiment, the TACI Fc fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 65. In another embodiment, the TACI Fc fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 66.

[0115] In some embodiments, the TACI Fc fusion protein comprises a TACI ECD portion with or without any of the above TACI mutations or combinations of TACI mutations, and the TACI ECD portion is fused to any of the polypeptide linkers set forth in any one of SEQ ID NOs: 3-15.

[0116] In some embodiments, a TACI Fc fusion protein comprises a TACI ECD portion according to SEQ ID NO: 1 or SEQ ID NO: 2 fused to a polypeptide linker according to any one of SEQ ID NOs: 3-15.

[0117] In some embodiments, the TACI-Fc fusion protein comprises a TACI ECD portion comprising any of the above-described TACI mutations or combinations of TACI mutations in combination with any of the above-described Ig Fc domain mutations or combinations of Ig Fc mutations, and any of the above-described polypeptide linkers.

[0118] In some embodiments, the TACI ECD portion is conjugated to an Fc domain. In one embodiment, the TACI ECD portion is crosslinked to the Fc domain using any of a number of known chemical crosslinkers. Exemplary crosslinkers are those that link two amino acid residues via a bond containing a "hindered" disulfide bond. In these bonds, the disulfide bond within the crosslinking unit is protected from reduction (by blocking groups on either side of the disulfide bond), for example, by the action of reduced glutathione or the enzyme disulfide reductase. One suitable reagent, 4-succinimidyloxycarbonyl-α-methyl-α(2-pyridyldithio)toluene (SMPT), utilizes a terminal lysine on one side of the protein and a terminal cysteine ​​on the other to form such a bond between two proteins. Heterobifunctional reagents that crosslink via different coupling moieties on each protein can also be used. Other useful cross-linking agents include, but are not limited to, reagents that link two amino groups (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide), reagents that link two sulfhydryl groups (e.g., 1,4-bis-maleimidobutane), reagents that link an amino group and a sulfhydryl group (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), reagents that link an amino group and a carboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and reagents that link an amino group and a guanidinium group present in the side chain of arginine (e.g., p-azidophenylglyoxal monohydrate).

[0119] In some embodiments, the TACI-Fc fusion protein is conjugated to polyethylene glycol (PEG). PEG can be attached to any amino acid side chain or terminal amino acid functional group, such as a free amino, imino, thiol, hydroxyl, or carboxyl group. Methods for attaching PEG to antibodies known in the art may be used.

[0120] In some embodiments, the fusion protein comprises an amino acid sequence that is at least 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 25-38, 46-48, 55, 56, and 67. In some embodiments, the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25-38, 46-48, 55, 56, and 67.

[0121] In one embodiment, the TACI-Fc fusion protein is TPP-6269, which comprises the amino acid sequence of SEQ ID NO:25.

[0122] In another embodiment, the TACI-Fc fusion protein is TPP-6270, which comprises the amino acid sequence of SEQ ID NO:26.

[0123] In another embodiment, the TACI-Fc fusion protein is TPP-6267, which comprises the amino acid sequence of SEQ ID NO:27.

[0124] In another embodiment, the TACI-Fc fusion protein is TPP-5776, which comprises the amino acid sequence of SEQ ID NO:28.

[0125] In another embodiment, the TACI-Fc fusion protein is TPP-5954, which comprises the amino acid sequence of SEQ ID NO:29.

[0126] In another embodiment, the TACI-Fc fusion protein is TPP-5957, which comprises the amino acid sequence of SEQ ID NO:30.

[0127] In another embodiment, the TACI-Fc fusion protein is TPP-5958, which comprises the amino acid sequence of SEQ ID NO:31.

[0128] In another embodiment, the TACI-Fc fusion protein is TPP-5975, which comprises the amino acid sequence of SEQ ID NO:32.

[0129] In another embodiment, the TACI-Fc fusion protein is TPP-5977, which comprises the amino acid sequence of SEQ ID NO:33.

[0130] In another embodiment, the TACI-Fc fusion protein is TPP-5791, which comprises the amino acid sequence of SEQ ID NO:34.

[0131] In another embodiment, the TACI-Fc fusion protein is TPP-5780, which comprises the amino acid sequence of SEQ ID NO:35.

[0132] In another embodiment, the TACI-Fc fusion protein is TPP-5779, which comprises the amino acid sequence of SEQ ID NO:36.

[0133] In another embodiment, the TACI-Fc fusion protein is TPP-5792, which comprises the amino acid sequence of SEQ ID NO:37.

[0134] In another embodiment, the TACI-Fc fusion protein is TPP-5298 comprising the amino acid sequence of SEQ ID NO:38.

[0135] In another embodiment, the TACI-Fc fusion protein is TPP-7165, which comprises the amino acid sequence of SEQ ID NO:46.

[0136] In another embodiment, the TACI-Fc fusion protein is TPP-7166, which comprises the amino acid sequence of SEQ ID NO:47.

[0137] In another embodiment, the TACI-Fc fusion protein is TPP-7167, which comprises the amino acid sequence of SEQ ID NO:48.

[0138] In another embodiment, the TACI-Fc fusion protein is TPP-6265, which comprises the amino acid sequence of SEQ ID NO:55.

[0139] In another embodiment, the TACI-Fc fusion protein is TPP-6458 comprising the amino acid sequence of SEQ ID NO:56.

[0140] In another embodiment, the TACI-Fc fusion protein is TPP-6265, which comprises the amino acid sequence of SEQ ID NO:67.

[0141] The TACI-Fc fusion proteins of the present disclosure are engineered to exhibit increased binding at pH 6.0 and low or negligible binding at pH 7.4. In one embodiment, the TACI-Fc fusion protein has a binding affinity (K) for FcRn at pH 6.0 that is less than 50 nM, less than 20 nM, less than 12 nM, less than 6 nM, or less than 4 nM. D TACI-Fc fusion proteins are characterized by a 50-fold, 100-fold, or 200-fold greater binding affinity (K) for FcRn at pH 6.0 compared to pH 7.4. D Furthermore, to the extent that the mutant Fc region in the TACI-Fc fusion protein exhibits increased binding to FcRn compared to the wild-type Fc region, circulating IgG levels are reduced.

[0142] In certain embodiments, a TACI-Fc fusion protein of the present disclosure exhibits an increase in in vivo half-life of about 1.2-fold, about 1.5-fold, about 2.0-fold, about 3.0-fold, about 4.0-fold, about 5.0-fold, or about 6.0-fold or more. In some embodiments, the biological half-life is extended by more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, more than 72 hours, more than 96 hours, or more than 1 week after in vivo administration compared to the corresponding TACI-Fc fusion protein comprising the wild-type or non-mutated parent Fc region. Biological half-life can be determined, for example, by using an ELISA assay or activity assay.

[0143] In another aspect, the present application provides a fusion protein comprising a polypeptide linker fused to a hybrid Ig Fc domain. The polypeptide linker may be fused to the N-terminus or C-terminus of the hybrid Ig Fc domain. The fusion protein may be further linked (or fused) to any TACI domain in accordance with the present application. In one embodiment, the polypeptide linker comprises the amino acid sequence set forth in any one of SEQ ID NOs: 3-15, and the hybrid Ig Fc domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 16-24 or 72, optionally containing a lysine residue added to the C-terminus. In some embodiments, the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 61-66, optionally containing a lysine residue added to the C-terminus.

[0144] II. Nucleic Acids, Expression Vectors, and Cells In another aspect, the disclosure provides nucleic acids encoding TACI-Fc fusion proteins, expression vectors for expressing TACI-Fc fusion proteins, as well as host cells, including stably transformed cells, that express the TACI-Fc fusion proteins. Cell lines that express the TACI-Fc fusion proteins can be used to produce TACI-Fc fusion proteins for use in pharmaceutical compositions for treatment.

[0145] In one aspect, the present disclosure provides an expression vector for expressing a TACI-Fc fusion protein in a suitable cell under conditions suitable for expressing the TACI-Fc fusion protein. The nucleic acid molecule or expression vector comprises a DNA molecule encoding a TACI-Fc fusion protein operatively linked to appropriate expression control sequences. Expression control sequences include promoters, activators, enhancers, operators, ribosome binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved in regulating transcription or translation. The selection and design of appropriate vectors and regulatory element(s) is within the ability and discretion of one of ordinary skill in the art. For example, one of ordinary skill in the art can select an appropriate promoter or other expression control sequence for expression in a desired species (e.g., a mammalian species) or cell type.

[0146] In some embodiments, the expression vector comprises a promoter capable of directing expression in mammalian cells, such as a suitable viral promoter derived from cytomegalovirus (CMV), retrovirus, simian virus (e.g., SV40), papillomavirus, herpesvirus, or other viruses that infect mammalian cells; or a mammalian promoter derived from genes such as EF1α, ubiquitin (e.g., ubiquitin B or C), globin, actin, phosphoglycerate kinase (PGK), or a composite promoter such as the CAG promoter-CMV early enhancer (combining elements with the chicken β-actin promoter). In some embodiments, a human promoter may be used. In some embodiments, a promoter that normally directs transcription by eukaryotic RNA polymerase II (a "pol II promoter") or a functional variant thereof is used. In some embodiments, a promoter that normally directs transcription by a eukaryotic RNA polymerase I promoter is used, such as a promoter for transcription of a ribosomal RNA (other than 5S rRNA) or a functional variant thereof. In some embodiments, a promoter that typically directs transcription by eukaryotic RNA polymerase III (pol III promoter), such as (U6, H1, 7SK, or tRNA promoter or functional variants thereof) can be used.

[0147] Exemplary promoters include the cytomegalovirus (CMV) immediate early promoter, RSV LTR, MoMLV LTR, phosphoglycerate kinase-1 (PGK) promoter, simian virus 40 (SV40) promoter, CK6 promoter, transthyretin promoter (TTR), TK promoter, tetracycline-responsive promoter (TRE), HBV promoter, hAAT promoter, neuron-selective promoters, such as the human synapsin promoter, muscle-specific promoters, such as the human creatine kinase (MCK) promoter, liver-specific promoters, such as the human phosphoenolpyruvate carboxykinase (PEPCK) promoter, and rhodopsin kinase promoter. Examples of promoters include, but are not limited to, a motor, an opsin promoter, a U6 promoter, an E2F promoter, a telomerase (hTERT) promoter, an H1 promoter, a cytomegalovirus enhancer / chicken β-actin / rabbit β-globin promoter (CAG) promoter, an elongation factor 1-α promoter (EF1-α) promoter, a human β-glucuronidase promoter, a chicken β-actin (CBA) promoter, a retroviral Rous sarcoma virus (RSV) LTR promoter, a dihydrofolate reductase promoter, and an 13-actin promoter.

[0148] In some embodiments, the expression vector comprises a codon-optimized TACI-Fc fusion protein. The terms "codon optimized" and "codon optimization" refer to a process for modifying a nucleic acid sequence according to one or more of the following: (1) matching codon frequencies in the host organism target, (2) promoting increased expression, (3) ensuring proper folding, (4) providing suitable GC content to increase mRNA stability or reduce secondary structure, (5) minimizing tandemly repeated codons or base runs that may impair gene assembly or expression, (6) customizing transcriptional and translational control regions, (7) inserting or removing protein trafficking sequences, (8) removing / adding post-translational modification sites (e.g., glycosylation sites) in the encoded protein, (9) adding, removing, or shuffling protein domains, (10) inserting or deleting restriction sites, (11) modifying ribosome binding sites and mRNA degradation sites, (12) adjusting translation rates so that various domains of the protein fold properly, and (13) reducing or eliminating problematic secondary structures within the polynucleotide. Codon optimization tools, algorithms, and services are known in the art, and non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Rowe Park, CA), and / or proprietary methods.

[0149] In one embodiment, the nucleic acid encodes the fusion protein set forth in SEQ ID NO: 25 or 67.

[0150] In another embodiment, the nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO:69 or 70.

[0151] In some embodiments, a nucleic acid encoding a TACI-Fc fusion protein is provided that is codon-optimized for human expression. A codon-optimized polynucleotide may be prepared by, for example, substituting codons in a polynucleotide encoding a TACI-Fc fusion protein with codons that appear more frequently in highly expressed human genes. Codon optimization methods are known in the art and can be used as provided herein.

[0152] In some embodiments, the codon-optimized polynucleotide sequence shares less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, or less than 50% sequence identity with the corresponding naturally occurring or wild-type sequence from which the TACI-Fc fusion protein is derived. In some embodiments, the codon-optimized polynucleotide sequence is 50% to 95%, 50% to 90%, 50% to 85%, 50% to 80%, 50% to 75%, 50% to 70%, 50% to 65%, 50% to 60%, 50% to 55%, 55% to 95%, 55% to 90%, 55% to 85%, 55% to 80%, 55% to 75%, 55% to 70%, 55% to 65%, 55% to 60%, 60% to 95%, 60% to 9 ... sharing 0%, 60% to 85%, 60% to 80%, 60% to 75%, 60% to 70%, 60% to 65%, 65% to 95%, 65% to 90%, 65% to 85%, 65% to 80%, 65% to 75%, 65% to 70%, 70% to 95%, 70% to 90%, 70% to 85%, 70% to 80%, 70% to 75%, 75% to 95%, 75% to 90%, 75% to 85%, 75% to 80%, 80% to 95%, 80% to 90%, 80% to 85%, 85% to 95%, 85% to 90%, or 90% to 95% sequence identity.

[0153] In some embodiments, a heterologous (non-native) signal peptide is added to or utilized in the appropriate expression of a nucleic acid encoding a TACI-Fc fusion protein. The signal peptide is operably linked to the nucleic acid encoding the TACI-Fc fusion protein such that the resulting soluble TACI-Fc fusion protein is recovered from the culture medium, the host cell, or the host cell periplasm. In some embodiments, the signal peptide is derived from an immunoglobulin (e.g., an IgG heavy chain or an IgG-kappa light chain), a cytokine (e.g., interleukin-2 (IL-2) or CD33), a serum albumin protein (e.g., HSA or albumin), a human azurocidin preprotein signal sequence, luciferase, trypsinogen (e.g., chymotrypsinogen or trypsinogen), or other signal peptide that enables efficient expression and / or secretion of the TACI-Fc fusion protein.

[0154] The expression vector may be in the form of a viral or non-viral vector, e.g., a plasmid. In some embodiments, the expression vector is a viral vector. Viral vectors for expression of antibodies can be derived, for example, from adenoviruses, adeno-associated viruses (AAV), retroviruses (including lentiviruses such as HIV-1 and HIV-2), vaccinia virus and other poxviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, polioviruses, Sindbis virus and other RNA viruses, alphaviruses, astroviruses, coronaviruses, orthomyxoviruses, papovaviruses, paramyxoviruses, parvoviruses, picornaviruses, togaviruses, and the like. Viral vectors contain sufficient viral genes for the production of infectious virus when introduced into a host cell. Viral vectors may or may not contain genetic information and / or structural components, i.e., they may be replication-competent or replication-defective. For example, in some embodiments lacking the structural components for the production of infectious virus, the necessary functional components may be supplied in trans by the host cell or by another vector introduced into the cell when production of recombinant virus is desired. In preferred embodiments, replication-defective recombinant viruses are administered for therapy. The nucleic acid for delivery may be integrated into a naturally occurring or modified viral genome (or portion thereof) or may be present within the viral capsid as a separate nucleic acid molecule.

[0155] In some cases, viral vectors may be engineered to target specific cells affected by a disease, disorder, or condition, either by using targeting properties inherent in the viral vector or by being engineered into the viral vector. Specific cells may be "targeted" for delivery and expression of a polynucleotide. Thus, "targeting" in this context may involve the use of endogenous or heterologous binding factors in the form of capsids, envelope proteins, antibodies for delivery to specific cells, the use of tissue-specific regulatory elements to restrict expression to specific subsets of cells, or both.

[0156] In some embodiments, the viral vector is an AAV vector. AAV vectors provide a preferred delivery system for the nucleic acid therapeutic agents of the present application because they can enable long-term, continuous expression of functional alleles and silencing of corresponding mutant alleles. AAV vectors can contain or be modified to control the expression of the first and second active agents for various regulatory elements, including various promoter and / or enhancer elements for constitutive or cell-type-specific expression.

[0157] In another aspect, a method for producing a TACI-Fc fusion protein includes culturing a host cell containing an expression vector encoding TACI-Fc, and recovering the TACI-Fc fusion protein from the cell or from the culture supernatant.

[0158] Many well-known and publicly available host cells can be used, for example, in preparing polypeptides from engineered cells or cell lines. The choice of cell or cell line depends on many factors recognized in the art. These include, for example, compatibility with the selected expression vector, toxicity of the peptide encoded by the DNA molecule, rate of transformation, ease of peptide recovery, expression characteristics, biosafety, and cost.

[0159] In some embodiments, the host cell is a mammalian cell. Examples of suitable mammalian host cells include African green monkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1; ATCC CCL61; CHO DG44), rat pituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548), SV40-transformed monkey kidney cells (COS-1; ATCC CRL 1650), and mouse embryonic cells (NIH-3T3; K. ATCC CRL 1658).

[0160] In some embodiments, the host cells are Chinese hamster ovary (CHO) cells or HEK293 cells. In some embodiments, the host cells are suspension cells, and the polypeptide is engineered or produced in suspension culture, such as suspension CHO cells, e.g., CHO-S cells. In some examples, the cell line is a DHFR-deficient (DHFR-) CHO cell line, e.g., DG44 and DUXB11. In some embodiments, the cells are glutamine synthase (GS)-deficient, e.g., CHO-S cells, CHOK1 SV cells, and CHOZN((R))GS- / - cells. In some embodiments, the CHO cells, e.g., suspension CHO cells, can be CHO-S-2H2 cells, CHO-S-clone 14 cells, or ExpiCHO-S cells. In other embodiments, the host cells are yeast cells.

[0161] In some embodiments, the host cell is a prokaryotic cell, such as E. coli. The transformed prokaryotic cells are cultured under conditions for polypeptide expression, followed by purification to obtain soluble protein. The host cells can be cultured under conventional fermentation conditions to express the desired polypeptide. The TACI-Fc fusion protein can be recovered and purified from the recombinant cell culture by methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, and affinity chromatography. Protein refolding steps can be used, if desired, to complete the construction of the mature protein. Finally, high-performance liquid chromatography (HPLC) can be used for final purification.

[0162] III. Pharmaceutical Compositions In another aspect, a pharmaceutical composition comprises a TACI-Fc fusion according to the present disclosure and a pharmaceutically acceptable carrier. The pharmaceutical composition can contain, for example, one or more excipients to modify, maintain, or preserve the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or penetration of the composition. Such compositions may include a buffer, such as neutral buffered saline, phosphate buffered saline, or the like; a carbohydrate, such as glucose, mannose, sucrose, or dextran, mannitol; a polypeptide or amino acid, such as glycine; an antioxidant; a chelating agent, such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); a preservative; or a combination thereof.

[0163] In some embodiments, the pharmaceutical composition is a solid, such as a powder, capsule, or tablet. For example, the components of the pharmaceutical composition may be lyophilized. In some embodiments, the solid pharmaceutical composition is reconstituted or dissolved in a liquid prior to administration.

[0164] In some embodiments, the pharmaceutical composition is a liquid, e.g., a TACI-Fc fusion protein dissolved in an aqueous solution (e.g., physiological saline or Ringer's solution). In some embodiments, the pH of the pharmaceutical composition is about 4.0 to about 8.5 (e.g., about 4.0 to about 5.0, about 4.5 to about 5.5, about 5.0 to about 6.0, about 5.5 to about 6.5, about 6.0 to about 7.0, about 6.5 to about 7.5, about 7.0 to about 8.0, or about 7.5 to about 8.5).

[0165] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient, such as a filler, binder, coating, preservative, lubricant, flavoring agent, sweetener, colorant, solvent, buffer, chelating agent, or stabilizer. Exemplary pharmaceutically acceptable fillers include cellulose, calcium hydrogen phosphate, calcium carbonate, microcrystalline cellulose, sucrose, lactose, glucose, mannitol, sorbitol, maltol, pregelatinized starch, corn starch, or potato starch. Exemplary pharmaceutically acceptable binders include polyvinylpyrrolidone, starch, lactose, xylitol, sorbitol, maltitol, gelatin, sucrose, polyethylene glycol, methylcellulose, or cellulose. Exemplary pharmaceutically acceptable coatings include hydroxypropylmethylcellulose (HPMC), shellac, corn protein zein, or gelatin. Exemplary pharmaceutically acceptable disintegrants include polyvinylpyrrolidone, carboxymethylcellulose, or sodium starch glycolate. Exemplary pharmaceutically acceptable lubricants include polyethylene glycol, magnesium stearate, or stearic acid. Exemplary pharmaceutically acceptable preservatives include methylparaben, ethylparaben, propylparaben, benzoic acid, or sorbic acid. Exemplary pharmaceutically acceptable sweeteners include sucrose, saccharin, aspartame, or sorbitol. Exemplary pharmaceutically acceptable buffers include carbonates, citrates, gluconates, acetates, phosphates, or tartrates.

[0166] In some embodiments, the pharmaceutical composition further comprises an agent for controlled or sustained release of the product, such as injectable microspheres, bioerodible particles, polymeric compounds (polylactic acid, polyglycolic acid), beads, or liposomes.

[0167] Preferably, the pharmaceutical composition is sterile. Sterilization may be achieved by filtration through sterile filtration membranes or by irradiation. If the composition is lyophilized, sterilization using this method may be performed either before or after lyophilization and reconstitution. Compositions for parenteral administration may be stored in lyophilized form or in a solution. Furthermore, parenteral compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0168] A pharmaceutically acceptable carrier may be a pharmaceutically acceptable material, composition, or vehicle. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or some combination thereof. Each component of the carrier must be "pharmaceutically acceptable" in that it must be compatible with the other components of the formulation. It must also be suitable for contact with any tissue, organ, or body part that it may encounter, which means that it must not pose the risk of toxicity, irritation, allergic reaction, immunogenicity, or any other complications that excessively outweigh its therapeutic benefits.

[0169] IV. Products and Kits Articles of manufacture comprising the pharmaceutical compositions described herein in suitable packaging. Suitable packaging for the compositions (such as ophthalmic compositions) described herein is known in the art and includes, for example, vials (such as sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles may be further sterilized and / or sealed.

[0170] Additionally provided are kits comprising the pharmaceutical compositions (or articles of manufacture) described herein, and optionally further including instruction(s) regarding how to use the composition, such as the uses described herein. The kits described herein may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts containing instructions for practicing any of the methods described herein.

[0171] V. Treatment Methods and Indications In another aspect, a method for treating a disease in a patient comprises administering to a patient in need thereof an effective amount of a pharmaceutical composition comprising a TACI-Fc fusion protein of the present disclosure. In a preferred embodiment, the disease is an autoimmune disease or disorder.

[0172] Pharmaceutical compositions according to the present disclosure can be used to treat a variety of inflammatory or autoimmune disorders, cancer, organ transplants, viral infections, and / or bacterial infections in mammals, particularly when a reduction in the immune response is desired. Thus, in certain embodiments, the pharmaceutical compositions may be useful in treating inflammatory or autoimmune disorders, or organ transplants.

[0173] The prophylactic or therapeutic methods may be used to treat the therapeutic indications disclosed herein. In a therapeutic setting, the mammalian subject is typically a subject with a disease, such as a disease or condition of the immune system, and administration is performed to prevent further progression of the disease or condition. Furthermore, suppression of the immune response may be beneficial in prophylactic and / or therapeutic methods to inhibit rejection of a tissue, cell, or organ transplant in a donor by the recipient.

[0174] TACI-Fc fusion proteins may be used to treat one or more diseases, including autoimmune diseases, B cell cancers, antibody-mediated conditions (e.g., ITCP, myasthenia gravis, etc.), renal disease, indirect T cell immune response, graft rejection, and graft-versus-host disease. Administration of TACI-Fc fusion proteins can specifically regulate B cell responses during an immune response. Additionally, administration of TACI-Fc fusion proteins can be used to regulate B cell development, other cell development, antibody production, and cytokine production. Administration or use of the provided TACI-Fc fusion proteins can also regulate B cell communication, for example, by neutralizing the proliferative effects of BAFF or APRIL.

[0175] In certain embodiments, the pharmaceutical composition is used to treat a disease, such as an autoimmune disease, disorder, or condition. In some embodiments, administration of the pharmaceutical composition to a subject suffering from a disease can result in the suppression or inhibition of such immune system attack or biological responses associated therewith. By suppressing this immune system attack against healthy body tissues, the resulting physical symptoms (e.g., pain, joint inflammation, joint swelling, or tenderness) resulting from or associated with such attack against healthy tissue can be reduced or alleviated, and the biological and physical damage resulting from or associated with the immune system attack can be reduced, delayed, or stopped. In a prophylactic context, the subject may be one who has, is susceptible to, or is thought to exhibit a disease (e.g., an immune system disease, disorder, or condition), and administration is typically carried out to prevent the progression of the disease, disorder, or condition, inhibit or alleviate symptoms, signs, or biological responses associated therewith, prevent physical damage that may result therefrom, and / or maintain or improve the subject's physical function.

[0176] In some embodiments, the disease that can be treated by the pharmaceutical compositions described herein is any disease mediated by immune complex deposition (e.g., lupus nephritis, vasculitis); direct interference with the pathway (e.g., fulminant antiphospholipid syndrome, myasthenia gravis crisis; anti-Jo-1 disease); opsonization or direct damage to cells (e.g., idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia; antibody-mediated rejection of allografts (e.g., highly sensitized kidney transplant patients); or anti-drug antibodies against biological replacement factors, vectors (e.g., anti-Factor VIII).

[0177] In some embodiments, the disease is systemic lupus erythematosus (SLE), including glucocorticoid-free flare prophylaxis, Sjogren's syndrome, primary biliary cirrhosis (PBC), systemic sclerosis, polymyositis, diabetes prophylaxis, IgA nephropathy, IgA vasculitis, B-cell cancers such as myeloma, multiple sclerosis, or optic neuritis.

[0178] In some embodiments, the pharmaceutical compositions may be used to treat pre-B cell or B cell leukemias, such as plasma cell leukemia, chronic or acute lymphocytic leukemia, myelomas, such as multiple myeloma, plasma cell myeloma, endothelial myeloma, and giant cell myeloma, and lymphomas, such as non-Hodgkin's lymphoma. In some of any of the embodiments, the type of myeloma includes multiple myeloma, plasmacytoma, multiple solitary plasmacytoma, and / or extramedullary myeloma. In some of any of the embodiments, the type of myeloma includes light chain myeloma, non-secretory myeloma, and / or IgD or IgE myeloma.

[0179] In some embodiments, the provided TACI-Fc fusion proteins can be used as immunosuppressants to selectively block the action of B lymphocytes for use in the treatment of diseases. For example, certain autoimmune diseases are characterized by the production of autoantibodies, which contribute to tissue destruction and disease exacerbation. Autoantibodies can also lead to the development of immune complex deposition complications, resulting in many symptoms of systemic lupus erythematosus, including renal failure, neuralgia symptoms, and death. Regulating antibody production independently of cellular responses is also beneficial in many disease states. B cells have also been shown to play a role in the secretion of arthritis-inducing immunoglobulins in rheumatoid arthritis. Methods and uses of the provided TACI-Fc fusion proteins to inhibit, block, or neutralize the action of B cells, thereby suppressing antibody production, would be beneficial in the treatment of autoimmune diseases such as myasthenia gravis, rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, and psoriatic arthritis.

[0180] In some embodiments, TACI-Fc fusion proteins may be used to block or neutralize the action of B cells associated with end-stage renal disease, which may or may not be associated with an autoimmune disease. Such methods may also be useful for treating immunological renal diseases. Furthermore, such methods may be useful for treating glomerulonephritis associated with diseases such as membranous nephropathy, IgA nephropathy or Berger's disease, IgM nephropathy, IgA vasculitis, Goodpasture's disease, post-infectious glomerulonephritis, mesangial proliferative disease, chronic lymphocytic leukemia, and minimal change nephrotic syndrome. Such methods also serve as therapeutic applications for treating secondary glomerulonephritis or vasculitis associated with diseases such as lupus, polyarteritis nodosa, Henoch-Schönlein, scleroderma, HIV-associated disease, amyloidosis, or hemolytic uremic syndrome. The provided methods are also useful as part of therapeutic applications for treating interstitial nephritis or pyelonephritis associated with chronic pyelonephritis, analgesic abuse, nephrocalcinosis, nephropathy caused by other drugs, nephrolithiasis, or chronic or acute interstitial nephritis. The methods provided herein also include the use of the provided TACI-Fc fusion proteins in the treatment of hypertension or macrovascular disease, including renal artery stenosis or occlusion and cholesterol embolism or renal embolism. The provided methods and uses can also be used in the treatment of renal or urinary neoplasms, multiple myeloma, lymphoma, light chain neuropathy, or amyloidosis.

[0181] In some embodiments, provided TACI-Fc fusion proteins may be used in the treatment of asthma and other chronic airway diseases, such as bronchitis and emphysema. In some embodiments, TACI-Fc fusion proteins may be used for immunosuppression in graft-versus-host disease and transplant rejection. In some embodiments, methods and uses of TACI-Fc fusion proteins include the treatment of autoimmune diseases, such as insulin-dependent diabetes mellitus (IDDM) and Crohn's disease. Additional applications include the treatment of chronic inflammatory diseases, e.g., to relieve joint pain, swelling, anemia, and other associated symptoms, as well as the treatment of septic shock.

[0182] In some embodiments, the inflammatory and autoimmune disorders include achalasia, Addison's disease; adult Still's disease; agammaglobulinemia; alopecia areata; amyloidosis; ankylosing spondylitis; anti-GBM / anti-TBM nephritis; antiphospholipid syndrome; autoimmune adrenalitis (Addison's disease); autoimmune angioedema; autoimmune autonomic neuropathy; autoimmune encephalomyelitis; autoimmune hepatitis; autoimmune inner ear disease (AIED); autoimmune myocarditis; autoimmune oophoritis; autoimmune orchitis; autoimmune pancreatitis; autoimmune polyglandular syndrome type II (APS). II); autoimmune retinopathy; autoimmune thyroid disease (AITD), i.e., Hashimoto's disease; autoimmune urticaria; axonal and neuronal neuropathy (AMAN); Barrow's disease; Behçet's disease; benign mucous membrane pemphigoid; bullous pemphigoid; Castleman's disease (CD); celiac disease; Chagas' disease; chronic inflammatory demyelinating polyneuropathy (CIDP); chronic relapsing multifocal osteomyelitis (CRMO); Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis (EGF) PA); Cicatricial pemphigoid; Cogan's syndrome; Cold agglutinin disease; Congenital heart block; Coxsackie myocarditis; CREST syndrome; Crohn's disease; Dermatitis herpetiformis; Dermatomyositis; Devic's disease (neuromyelitis optica); Discoid lupus; Dressler's syndrome; Endometriosis; Eosinophilic esophagitis (EoE); Eosinophilic fasciitis; Erythema nodosum; Essential mixed cryoglobulinemia; Evans syndrome; Fibromyalgia; Fibrosing alveolitis; Giant cell arteritis (temporal arteritis); Giant cell Myocarditis; Glomerulonephritis; Goodpasture's syndrome; Granulomatosis with polyangiitis; Graves' disease; Guillain-Barré syndrome; Hashimoto's thyroiditis; Hemolytic anemia; Henoch-Schönlein purpura (HSP); Herpes gestationis or pemphigoid of gestationis (PG); Hidradenitis suppurativa (HS) (acne inversa); Hypogammaglobulinemia; IgA nephropathy; IgA vasculitis; IgG4-related sclerosing disease; Immune thrombocytopenic purpura (ITP); Inclusion body myositis (IBM); Interstitial Cystitis (IC); Juvenile arthritis; Juvenile diabetes mellitus (type 1 diabetes); Juvenile myositis (JM); Kawasaki disease; Lambert-Eaton syndrome; Leukocytoclastic vasculitis; Lichen planus; Lichen sclerosus; Lignin conjunctivitis; Linear IgA disease (LAD); Lupus; Chronic Lyme disease; Meniere's disease; Microscopic polyangiitis (MPA); Mixed connective tissue disease (MCTD); Mooren's ulcer; Much-Habermann disease; Multifocal motor neuropathy (MMN) or MMNCB; Multiple sclerosis;Myasthenia gravis; Myositis; Narcolepsy; Neonatal lupus; Neuromyelitis optica; Neutropenia; Ocular cicatricial pemphigoid; Optic neuritis; Relapsing rheumatoid arthritis (PR); PANDAS; Paraneoplastic neurological syndromes (PCD); Paroxysmal nocturnal hemoglobinuria (PNH); Parry-Romberg syndrome; Parsplanitis (peripheral uveitis); Parsonage-Turner syndrome; Pemphigus, pemphigus vulgaris; Perivenous encephalomyelitis; Pernicious anemia (PA); POEMS syndrome; Polyarteritis nodosa; Polyglandular syndrome type I, type II , Type III; Polymyalgia rheumatica; Polymyositis; Post-myocardial infarction syndrome; Post-pericardiotomy syndrome; Primary biliary cirrhosis; Primary sclerosing cholangitis; Progestational dermatitis; Psoriasis; Psoriatic arthritis; Pure red cell aplasia (PRCA); Pyoderma gangrenosum; Raynaud's phenomenon; Reactive arthritis; Reflex sympathetic dystrophy; Relapsing polychondritis; Restless legs syndrome (RLS); Retroperitoneal fibrosis; Rheumatic fever; Rheumatoid arthritis; Sarcoidosis; Schmidt's syndrome; Scleritis; Scleroderma; Sjogren's syndrome; Sperm and sperm Focal autoimmunity; stiff-person syndrome (SPS); subacute infective endocarditis (SBE); Susac syndrome; sympathetic ophthalmia (SO); Takayasu's arteritis; temporal arteritis / giant cell arteritis; thrombocytopenic purpura (TTP); Tolosa-Hunt syndrome (THS); transverse myelitis; type 1 diabetes; ulcerative colitis (UC); undifferentiated connective tissue disease (UCTD); uveitis; vasculitis; vitiligo or Vogt-Koyanagi-Harada disease, antiphospholipid Ab syndrome; multiple sclerosis; IgG4-related disease; type 1 diabetes; glucocorticoids Rheumatoid arthritis, including rheumatoid arthritis (GC) or acute lupus nephritis, amyotrophic lateral sclerosis, neuromyelitis optica, transverse myelitis, CNS autoimmunity, Guillain-Barré syndrome, neurocysticercosis, sarcoidosis (T / seroneg), Churg-Strauss syndrome, Hashimoto's thyroiditis, Graves' disease, immune thrombocytopenia (ITP), Addison's disease, polymyositis, or dermatomyositis; IgA nephropathy; chronic inflammatory demyelinating polyneuropathy (CIDP); antisynthetic enzyme disorders, such as Jo-1 syndrome and ANCA vasculitis;

[0183] In some embodiments, provided TACI-Fc fusion proteins can be used to treat B-cell cancer. In some embodiments, the B-cell cancer is a cancer in which BAFF and APRIL are implicated or associated with providing an autocrine survival loop to B cells. In some embodiments, the cancer is B-cell chronic lymphocytic leukemia, non-Hodgkin's lymphoma, or myeloma. In some embodiments, the cancer is myeloma.

[0184] In preferred embodiments, the autoimmune disorder is selected from the group consisting of acute antibody-mediated rejection, primary Sjogren's syndrome, autoimmune hemolytic anemia, antiphospholipid syndrome (APS), catastrophic APS, refractory rheumatoid arthritis, autoimmune vasculitis, cryoglobulinemia, antineutrophil cytoplasmic autoantibodies (ANCA), IgA vasculitis, rheumatoid vasculitis, antisynthetase syndrome, primary membranous nephropathy, juvenile idiopathic arthritis, systemic sclerosis, Guillain-Barré syndrome, autoimmune pulmonary alveolar proteinosis, autoimmune hemolytic anemia, hemolytic disease of the fetus and newborn, acute antibody-mediated rejection, and acute hemolytic transfusion reaction.

[0185] In another aspect, a method for enhancing clearance of IgG immunoglobulin in a patient in need thereof comprises administering to the patient an effective amount of a pharmaceutical composition comprising a TACI-Fc fusion protein of the present disclosure.

[0186] In another aspect, a method for reducing levels of mature B cells, IgG, IgM, IgA (or a combination thereof) in a patient in need thereof comprises administering to the patient an effective amount of a pharmaceutical composition comprising a TACI-Fc fusion protein of the present disclosure. Further, in some embodiments, administration of the pharmaceutical composition does not significantly reduce levels of IgD or IgE.

[0187] In another aspect, a method for inhibiting the activity of one or more proinflammatory cytokines in a patient in need thereof comprises administering to the patient an effective amount of a pharmaceutical composition comprising a TACI-Fc fusion protein of the present disclosure. In one embodiment, the proinflammatory cytokines comprise BAFF and / or APRIL.

[0188] In general, the dosage and route of administration of a pharmaceutical composition are determined according to standard pharmaceutical practice, based on the size and condition of the subject. For example, a therapeutically effective dose can be initially estimated using either cell culture assays or animal models (e.g., mice, rats, rabbits, dogs, pigs, or monkeys). Animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes for administration in humans. The exact dosage can be determined taking into account factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active compound or to maintain the desired effect. Factors to consider for treatment include the severity of the disease state, the subject's overall health, the subject's age, weight, and sex, the time and frequency of administration, the drug combination(s), reaction sensitivities, and response to treatment.

[0189] Long-acting pharmaceutical compositions may be administered every 3-4 days, weekly, or biweekly, depending on the half-life and clearance rate of the formulation. The frequency of administration depends on the pharmacokinetic parameters of the molecule in the formulation used. Typically, the composition is administered until a dosage that achieves the desired effect is reached. Thus, the composition may be administered as a single dose, as multiple doses over time (at the same or different concentrations / dosages), or as a continuous infusion. Further refinement of the appropriate dosage is routine. The appropriate dosage may be confirmed using appropriate dose-response data.

[0190] In some embodiments, the pharmaceutical composition is administered to a subject via any route, including orally, transdermally, by inhalation, intravenously, intraarterially, intramuscularly, directly at a wound site, at a surgical site, intraperitoneally, by suppository, subcutaneously, intradermally, transdermally, by spray, intrapleurally, intraventricularly, intraarticularly, intraocularly, or intrathecally. In some embodiments, the pharmaceutical composition is administered in a form suitable for intravenous infusion.

[0191] In some embodiments, the administered dosage of the pharmaceutical composition is about 1 mg or more of protein per kg of subject body weight (e.g., about 2 mg or more of protein per kg of subject body weight, about 5 mg or more of protein per kg of subject body weight, about 10 mg or more of protein per kg of subject body weight, about 25 mg or more of protein per kg of subject body weight, about 50 mg or more of protein per kg of subject body weight, about 100 mg or more of protein per kg of subject body weight, about 250 mg or more of protein per kg of subject body weight, about 500 mg or more of protein per kg of subject body weight, about 1000 mg or more of protein per kg of subject body weight, about 2000 mg or more of protein per kg of subject body weight, or about 5000 mg or more of protein per kg of subject body weight).

[0192] The dosage of the pharmaceutical composition may be administered as a single dose or multiple doses. In some embodiments, doses are given to a subject once a day, twice a day, three times a day, or four or more times a day. In some embodiments, about one or more doses (e.g., about two or more, about three or more, about four or more, about five or more, about six or more, or about seven or more) are given per week. In some embodiments, multiple doses are given over the course of days, weeks, months, or years. In some embodiments, a treatment course is about one or more doses (such as about two or more doses, about three or more doses, about four or more doses, about five or more doses, about seven or more doses, about ten or more doses, about fifteen or more doses, about twenty-five or more doses, about forty or more doses, about fifty or more doses, or about one hundred or more doses).

[0193] VI. Evaluation of immune responses to TACI-Fc fusion protein compositions TACI-Fc fusion proteins modulate B cell activities, such as B cell proliferation, differentiation, or survival. Assessment of these activities can be performed in vitro or in vivo to monitor response to TACI-Fc therapy.

[0194] The functional activity of a TACI-Fc fusion protein can be tested using various approaches to assess the protein's ability to bind to its cognate binding partner. For example, a TACI-Fc fusion protein may be assessed for binding to APRIL or BAFF. Various assays are known for assessing binding affinity and / or determining whether a TACI-Fc fusion protein specifically binds to APRIL, BAFF, or FcRn. Several binding assays known in the art can be utilized, including, but not limited to, BIAcore®, OCTET®, and other surface plasmon resonance (SPR) devices and methodologies; flow cytometry, kinetic exclusion assay (KinExA), and enzyme-linked immunosorbent assay (ELISA). For example, in some embodiments, a BIAcore® instrument can be used to determine the binding kinetics and constants of the complex between a TACI-Fc fusion protein and APRIL, BAFF, or FcRn using surface plasmon resonance analysis. SPR measures the change in concentration of a molecule at a sensor surface as the molecule binds to or dissociates from the surface. The change in SPR signal is directly proportional to the change in mass concentration near the surface, thereby allowing for the measurement of binding kinetics between the two molecules. The dissociation constant of the complex can be determined by monitoring the change in refractive index over time as a buffer solution passes over the chip. Other suitable assays for measuring the binding of one protein to another include, for example, immunoassays (e.g., ELISA assays), radioimmunoassays (RIA), or determining binding by monitoring changes in the spectroscopic or optical properties of the proteins via fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR).

[0195] TACI-Fc fusion proteins can also be evaluated in any of a variety of assays well known in the art for assessing modulation of B cell activity. One such assay is a cell proliferation assay. Cells are cultured in the presence or absence of a test compound (e.g., a TACI-Fc fusion protein), and cell proliferation is detected, for example, by measuring the incorporation of tritiated thymidine or by a colorimetric assay based on the metabolic degradation of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Another assay format uses cells further engineered to express a reporter gene. The reporter gene is linked to a promoter element responsive to a receptor-associated pathway, and the assay detects transcriptional activation of the reporter gene. Numerous reporter genes that are easily assayed in cell extracts are known in the art, such as E. coli lacZ, chloramphenicol acetyltransferase (CAT), and serum response element (SRE). An exemplary reporter gene is the luciferase gene. Expression of the luciferase gene is detected by luminescence using methods and kits well known in the art.

[0196] TACI-Fc fusion proteins can be characterized by their ability to inhibit stimulation of human B cells by soluble APRIL or BAFF, as described by Gross et al., International Publication No. WO 00 / 40716. Briefly, human B cells are isolated from peripheral blood mononuclear cells, such as by using CD19 magnetic bead separation (e.g., Miltenyi Biotec, Auburn, CA). Purified B cells can be incubated under stimulatory conditions, for example, in the presence of soluble APRIL and titrated concentrations of TACI-Fc fusion protein. B cells can be labeled with a proliferation dye to measure proliferation, or 1 μCi of TACI-Fc fusion protein can be used. 3 They can be labeled with 3H-thymidine. The number of B cells can be determined over time.

[0197] Reporter cell lines expressing reporter genes under the operable control of transcription factors such as NF-κB, NFAT-1, and AP-1 can be engineered to express TACI or BCMA. For example, Jurkat and other B lymphoma cell lines can be used as suitable reporter cells. Incubation of these cells with soluble BAFF or APRIL ligand transduces signals through the reporter genes in these constructs. The effect of the provided TACI-Fc fusion protein on modulating this signaling can be assessed.

[0198] For example, in some embodiments, a mouse model of Sjögren's syndrome can be used. Sjögren's syndrome disease, as well as the accelerated onset of diabetes, can be induced in diabetes-prone non-obese diabetic (NOD) female mice using repeated administration of anti-mouse (m)PD-L1 antibodies based on a modified version of the protocol published by Zhou et al., 2016; Sci. Rep. 6, 39105. Starting at 6 weeks of age, mice are intraperitoneally (IP) injected with 100 μg of anti-PD-L1 antibody on study days 0, 2, 4, and 6, and then treated with the provided TACI-Fc fusion protein on various days. Naive mice are included as controls for endpoint analysis. All mice are typically terminated on study day 10, and the submandibular gland (SMG) and pancreas from each mouse are collected for histopathological evaluation to assess the signs and severity of sialadenitis and insulitis. Blood glucose levels can be measured on various days.

[0199] In some embodiments, a collagen-induced arthritis (CIA) model can be used in which mice develop chronic inflammatory arthritis that closely resembles human rheumatoid arthritis (RA). Because CIA shares similar immunological and pathological characteristics with RA, it is an ideal model for screening potential human anti-inflammatory compositions. Another advantage of using the CIA model is that the mechanism of pathogenesis is known. T cell and B cell epitopes on type II collagen have been identified, and various immunological parameters (delayed-type hypersensitivity and anti-collagen antibodies) and inflammatory parameters (cytokines, chemokines, and matrix-degrading enzymes) associated with immune-mediated arthritis have been determined and can be used to evaluate the efficacy of test compounds in models (Wooley, Curr. Opin. Rheum. 3:407-20, 1999; Williams et al., Immunol. 89:9784-788, 1992; Myers et al., Life Sci. 61:1861-78, 1997; and Wang et al., Immunol. 92:8955-959, 1995). Administration of TACI-Fc fusion proteins to ameliorate the symptoms and course of disease or disorders can be evaluated.

[0200] Pharmacokinetic studies can be used with radiolabeled TACI-Fc fusion proteins to determine the distribution and half-life of such polypeptides in vivo.

[0201] The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The sequence listing, figures, and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference. The sequence listings referenced throughout this application follow the examples and are set forth in Figures 20A-20K. [Example]

[0202] Example 1: Engineering the TACI extracellular domain to enhance pH-dependent binding of BAFF and APRIL cytokines The transmembrane activator or calcium-regulated cyclophilin ligand interactor (TACI) receptor has high affinity for the cytokines BAFF and APRIL. The extracellular domain of TACI has been demonstrated to block BAFF and APRIL as an Fc fusion, and blocking BAFF and APRIL prevents activation of receptors in this family (Merrill et al. (2018) Arthritis & Rheumatology 70, 2, 266-276).

[0203] Following the published crystal structure of the TACI cysteine-rich CRD2 domain in complex with mouse APRIL (PDB: 1XU1), single-addition histidine mutants (fused to the Fc domain with a C-terminal Flag tag) were generated to sequentially replace all non-cysteine ​​residues at or near the binding interface formed with APRIL (Figure 1). These histidine mutants were expressed from pCDNA3.1 (containing a single sequence) in Expi293 cells, and purified from cell supernatants using a HiTrap MabSelect SuRe 1 mL affinity column on an AKTApure 25 M2 purification system by protein elution with 100 mM citrate, pH 3.2.

[0204] The pH dependence of the binding of TACI histidine mutants to BAFF and APRIL was determined using surface plasmon resonance (SPR) (Biacore™ T200 instrument), as shown in Figures 2A-2B. After immobilizing biotinylated BAFF (Figure 2A) or biotinylated APRIL (Figure 2B) on a streptavidin sensor, BAFF-biotin and APRIL-biotin were flowed at 100 nM in PBS + 0.05% Tween® at pH 7.4. The dissociation rate was then measured at pH 7.4, followed by injection of PBS + 0.05% Tween® 20 at pH 6.0, and the new dissociation rate was measured as shown in Figure 2. By comparing the relative dissociation rates at pH 6.0 and pH 7.4 (Figure 2), the most promising single histidine-added TACI-DR1 / DR2 Fc variants (with relatively fast dissociation at pH 6.0) were identified, as summarized in Table 1. TACI variants are named based on their amino acid position in the native TACI amino acid sequence (SEQ ID NO: 39; NCBI reference sequence NP_036584).

[0205] [Table 1]

[0206] A second panel of double- and triple-tagged histidine TACI-Fc variants was then designed based on combining the most effective mutations identified in round 1. These new variants were similarly tested for BAFF binding by the same Biacore method as above to determine relative dissociation rates at pH 6.0 and pH 7.4, as well as overall binding stability (Figure 3).

[0207] [Table 2]

[0208] The entire panel of single-, double-, and triple-tagged histidine-tagged TACI-Fc variants was also tested in an ELISA assay to determine the pH dependence of BAFF binding. Biotinylated BAFF (1 μg / mL in 1x PBST) was immobilized on a pre-blocked 96-well plate coated with streptavidin. The wells were washed three times, and then serial dilutions of the TACI-Fc panel (0.003–10 μg / mL in 1x PBST at either pH 5.5 or 7.4) were applied for 45 minutes. The wells were washed three times, and HRP-conjugated anti-FLAG M2 mouse antibody (diluted 1:20,000 in 1x PBST) was applied. The wells were washed three times, and TMB substrate solution was added. The solution was then quenched with 2N sulfuric acid after 15 minutes, after which the absorbance at 450 nm was read on a Spectramax i3X plate reader.

[0209] Table 3 summarizes the observed relative shifts in BAFF binding affinity for the His variants compared to WT at pH 5.5 and 7.4, as determined by ELISA assay. A higher net pH-dependent shift indicates a greater difference in affinity from pH 7.4 to pH 5.5 (weaker affinity at pH 5.5). These results indicate a decreased affinity of the TACI-Ig Fc fusion protein for BAFF under low pH conditions (e.g., pH 5.5) and a higher affinity under neutral pH conditions (e.g., pH 7.4). The results of this analysis allowed the identification of several promising double- and triple-added histidine TACI-Fc variants for further characterization.

[0210] [Table 3]

[0211] Example 2: TACI-Fc fusion protein binds to BAFF and APRIL in a pH-dependent manner Fusion proteins consisting of TACI and Ig Fc regions were designed using Geneious software (Biomatters LLC), submitted to GeneArt for codon optimization, cloned into pCDNA3.1 (including the signal sequence) using HindIII and ApaI restriction enzymes, expressed using ExpiCHO-S or Expi293F (Thermo Fisher Scientific) cell expression systems, and purified on an AKTApure 25 M2 purification system (Cytiva) by MabSelect SuRe Protein A affinity chromatography with citrate pH 3.8 elution, followed by size-exclusion chromatography in PBS on a column packed with Superdex 200 pg resin (Cytiva).

[0212] Octet binding analysis Biolayer interferometry was performed using an Octet RED96e system (ForteBio) to examine the kinetics of TACI-Fc fusion protein binding to the TACI ligands BAFF and APRIL, as well as binding to the neonatal Fc receptor (FcRn), as described in Example 3 below. In this assay, the addition of protein mass to the biosensor is detected by biolayer interferometry (BLI) as a deflection or increase in the detectable signal observed in the sensorgram. In the absence of mass change, a flat line or baseline is observed, as shown in the buffer-only step. Furthermore, as the protein dissociates from the biosensor, a negative slope is observed in the sensorgram. In the exemplary assay shown in Figure 4, the sensorgram can show the following time phases: (1) baseline (buffer only), (2) biotinylated analyte loading, (3) baseline (buffer only), (4) TACI-Fc fusion protein association, and (5) TACI-Fc fusion protein dissociation.

[0213] To demonstrate binding of an exemplary TACI-Fc fusion protein (i.e., TPP-6269, SEQ ID NO: 22) to BAFF, TPP-6269 (20 μg / mL) was diluted in phosphate-buffered saline (PBS) pH 6.0 or 7.4 and analyzed for binding to human biotinylated BAFF (Biotechne catalog no. 7537-BF-025 / CF) loaded onto a streptavidin biosensor (Sartorius, catalog no. 18-5019) using an Octet RED96e system (ForteBio). Negative control assays were performed at pH 6.0 and pH 7.4 using PBS buffer alone. The results of this binding experiment are shown in the sensorgrams shown in FIG. 5.

[0214] The sensorgrams in Figure 5 show the latter three phases shown in Figure 4, including the buffer-only baseline, the TACI-Fc / BAFF association phase, and the TACI-Fc / BAFF dissociation phase. From these traces, the on-rate and off-rate binding profiles of the variant TACI-Fc fusion proteins to BAFF were determined.

[0215] BAFF inhibition by TPP-6269 was further tested in a functional assay performed in a BCMA / nuclear factor kappa B (NF-κB)-luciferase reporter HEK293 cell line. This cell line contains a stably integrated full-length human BCMA cDNA (Genbank #NM_001192) under the control of a CMV promoter (Amsbio LLC) and a reporter in which expression of the firefly luciferase gene (in relative light units (RLU)) is under the control of four copies of an NF-κB response element upstream of a minimal promoter. In this assay, binding of the BAFF ligand to BCMA activates the NF-κB signaling pathway, resulting in expression of the NF-κB-regulated luciferase reporter. Inhibition of BAFF binding by the TACI-Fc fusion protein inhibits NF-κB signaling, which is reflected in a decrease in luciferase signal (i.e., RLU).

[0216] TPP-6269 (SEQ ID NO: 25) contains a portion of the TACI extracellular domain consisting of the DR1-DR2 region, lacking the N-terminal portion of DR1 and the C-terminal portion of DR2. For comparison, TPP-6269 was tested against a TACI-Fc fusion protein (TPP-4217) containing the full-length TACI extracellular domain. The results of this assay are shown in Figure 6. In this assay, TPP-6269 was found to completely inhibit luciferase expression at a concentration of 5.3 nM, a higher level of inhibition compared to TPP-4217. As expected, a control Fc protein lacking the TACI domain showed no inhibition.

[0217] Additional TACI-Fc variants were similarly found to exhibit potent inhibition of BAFF binding to BCMA-HEK293 cells (Figure 7). Furthermore, in a similar functional assay using a BCMA / NF-κB-luciferase reporter HEK293 cell line with APRIL (Biotechne, Cat. No. 5860-AP / CF) as the BCMA ligand, TACI variants TPP-4286 (SEQ ID NO: 57) and TPP-4291 (SEQ ID NO: 58) were found to exhibit potent inhibition of APRIL-mediated BCMA activation, as shown in Figure 8.

[0218] Example 3: Crystallizable fragment (Fc) sequence variants of human IgG1, IgG2, IgG3, and IgG4 enhance binding to neonatal Fc receptors To investigate whether enhanced binding profiles and pH dependence could be engineered, a series of TACI-Fc fusion proteins containing chimeric Fc regions assembled from different IgG1 / IgG2 / IgG4 subclass moieties were designed and purified essentially as described in Example 2. The resulting TACI-Fc chimeric IgG subclass fusion proteins, containing the amino acid sequences set forth in SEQ ID NOs: 20 and 39-45, were diluted in phosphate-buffered saline (PBS) pH 6.0 or 7.4 and analyzed for binding to human biotinylated FcRn (Biotechne, catalog no. 8639-FC) loaded onto a streptavidin biosensor (Sartorius, catalog no. 18-5019), essentially as described in Example 2. Negative control assays were performed at pH 6.0 and pH 7.4 using PBS buffer alone.

[0219] These variant chimeric Fc fusion proteins were similarly evaluated for binding to neonatal FcRn and pH dependence by biolayer interferometry using the Octet Red 96 system (ForteBio), as described in Example 2. The results of these assays are shown in Figures 9A-9H. An additional assay employed surface plasmon resonance (SPR / Biacore). Similar to the assay in Figure 5, the sensorgrams in Figures 9A-9G show the buffer-only baseline phase, the Fc variant association phase, and the Fc dissociation phase. From this data, the on-rate and off-rate binding profiles of the variant TACI-Fc fusion proteins to FcRn can be determined.

[0220] As shown in Figures 9A-9H, TACI-Fc fusion variants exhibited varying levels of binding to FcRn with observable differences in on-rate and off-rate profiles at pH 6.0 and pH 7.4. For example, fusions of different IgG1 / IgG2 / IgG4 subclass regions with M252Y, S254T, and T256E mutations exhibited enhanced binding, as shown by TPP-5260 (SEQ ID NO: 50), compared to TPP-5272 (SEQ ID NO: 51), as shown in Figures 9A and 9D, respectively. An example demonstrating this observation for IgG1 / IgG4 Fc subclass variants is shown by TPP-5262 (SEQ ID NO: 49), which was found to exhibit increased on-rate and amplitude at pH 7.4 compared to TPP-5274 (SEQ ID NO: 45) (see Figures 9B and 9F, respectively).

[0221] The TACI-Fc variants in Figures 9A-9H show slower dissociation rates for human FcRn when the M252Y, S254T, and T256E mutations are present in the different hybrid subclass fusion proteins. TPP-6269 (SEQ ID NO: 25), which contains a hybrid IgG1 / IgG4 Fc domain in combination with the L234F, L235E, M252Y, S254T, T256E, and N434H mutations, showed exceptional pH dependence, with an observed decrease in on-rate and total amplitude of binding and a faster off-rate at pH 7.4 compared to pH 6.0, as confirmed by surface plasmon resonance (SPR / Biacore) in Figure 13 of Example 4 (Figure 9H).

[0222] Example 4: Rationally designed mutagenesis of human IgG1 / IgG4 Fc to enhance pH-dependent binding to FcRn Guided by the published crystal structure of the FcRn:Fc complex (PDB: 4N0U; Figure 10), a series of single- and double-substituted histidine IgG1 / 4 Fc mutants (as TACI-D1 / D2 Fc fusions with a C-terminal His tag) were designed to target the interface with FcRn, specifically the acidic residues E115, E116, D130, and E133 in processed human FcRn (E138, E139, D253, and E256 in UniProtKB / Swiss-Prot: P55899.1). Selected mutations were introduced into constructs containing the TACI D1 / D2 domain region, a chimeric IgG1 / IgG4 Fc region, and a 6xHis tag. These proteins were expressed from pCDNA3.1 in Expi293 cells and purified from cell supernatants using a HisTrap HP 1 mL nickel affinity column on an AKTApure 25 M2 purification system using an imidazole gradient to separate monomeric proteins from aggregates.

[0223] The relative effects of mutations on FcRn binding at pH 7.4 and 6.0 were assessed using the Octet system: Streptavidin chips were immersed in PBS for 15 min, a 60-second baseline was recorded, and biotinylated FcRn (10 μg / mL in PBS) was loaded for 200 seconds, followed by a 30-second immersion in PBS followed by 120 seconds of association with Fc variants (400 nM in PBS) and subsequent dissociation, all steps performed at either pH 6.0 or 7.4.

[0224] Figures 11A-11D show Octet traces demonstrating the binding kinetics of the single- and double-histidine-tagged TACI-Fc variants compared to TPP-5274 "WT" (SEQ ID NO: 45), which contains the parent IgG1 / IgG4 hybrid Fc with no mutations added to the variants. The Octet traces in Figures 11A-11B were performed at pH 7.4, and the Octet traces in Figures 11C-11D were performed at pH 6.0. Results from this study identified the novel N434H variant, TPP-5721 (SEQ ID NO: 60), as improving FcRn binding affinity in a pH-dependent manner (see Figures 11A, 11C).

[0225] To further investigate the improved pH-dependent binding properties conferred by the N434H mutation, additional binding studies were performed using TPP-5957 (SEQ ID NO: 30) and TPP-5958 (SEQ ID NO: 31) at pH 6.0 (FIG. 12A) and pH 7.4 (FIG. 12B) on the Octet RED96e system (ForteBio). Both TPP-5957 and TPP-5958 contain the amino acid substitutions L234F, L235E, M252Y, S254T, and T256E. However, TPP-5958 also contains the N434H mutation. The results of this comparison are consistent with the N434H mutation conferring improved pH-dependent binding properties.

[0226] The binding properties of the TACI-Fc variants were further evaluated by SPR / Biacore analysis. In these experiments, the TACI-Fc variants were directly immobilized onto the surface of a CM5 chip exposed to injections of FcRn (Biotechne, catalog no. 8639-FC) at various concentrations at pH 7.4 and pH 6.0 using a Biacore T200 system. The surface was regenerated with 50 mM NaOH, and binding kinetic parameters were determined. All Biacore analyses were performed according to WPD-PC-050-02D, "Kinetic analysis of protein-protein interactions with direct ligand immobilization using surface plasmon resonance." pH 7.4 and pH 6.0 data were processed according to the Biacore protocol using Biacore evaluation software with double referencing.

[0227] In an exemplary embodiment, TPP-6269 was evaluated by SPR / Biacore. TPP-6269 (SEQ ID NO: 25) was engineered with a hybrid IgG1 / IgG4 Fc region bearing the previously described mutations in TPP-5958 (L234F, L235E, M252Y, S254T, T256E, and N434H). From this analysis, TPP-6269 was found to exhibit exceptional pH-dependent binding to human FcRn by SPR / Biacore (Figures 13A and 13B). In phosphate-buffered saline (PBS) at pH 7.4, TPP-6269 exhibited a binding affinity (K) of 928 nM at pH 7.4. D ) and a binding affinity (K ) of 3.82 nM at pH 6.0 D ) bound to FcRn, a 244-fold difference (Fig. 13C).

[0228] Example 5: Simultaneous binding of TACI-Fc variants to human FcRn-expressing cell lines and BAFF by flow cytometry Flow cytometry analysis was performed to determine whether TACI-Fc variants could simultaneously bind to BAFF (Biotechne, catalog no. 7537-BF-025 / CF, biotinylated) and human FcRn using the CHO-K1 human FcRn stable cell line (Genscript, catalog no. M00603). CHO-K1 / FcRn cells were seeded at 100,000 cells per well in PBS (pH 6.0), combined with 1 μM TACI-Fc variants in 0.5% BSA (pH 6.0), and incubated at 4°C for 60 minutes. Cells were washed three times with 100 μL of 0.5% BSA (pH 6.0), and 10 μg / mL of BAFF-biotin in 0.5% BSA (pH 6.0) was added to the cells, followed by incubation at 4°C for 60 minutes. The cells were washed three times, and 5 μg / mL of 0.5% BSA (pH 6.0) was added to the cells, which were then incubated on ice for 30 minutes. The cells were washed, labeled with 5 μg / mL streptavidin-Alexa-Fluor™ 488 (Invitrogen, Cat. No. 532354) in 0.5% BSA (pH 6.0), and incubated on ice for 30 minutes. The cells were washed three times, resuspended in 100 μl of 0.5% BSA (pH 6.0), and analyzed using an LSRII Fortessa (BD, custom-built) flow cytometer.

[0229] In this assay, a positive median fluorescence intensity (MFI) greater than that of labeled BAFF indicates simultaneous binding of the TACI-Fc variant to BAFF and FcRn on cells. Figure 14A shows a graph summarizing the mean fluorescence intensity (MFI) ratios, consistent with simultaneous binding of the TACI domain to both labeled BAFF and FcRn in a human FcRn-expressing cell line. Figures 14B-14D are histograms showing simultaneous binding of TPP-5954 (Figure 14B), TPP-5957 (Figure 14C), and TPP-5958 (Figure 14D) to FcRn and BAFF binding compared to labeled BAFF binding only to the TACI-Fc variant (i.e., BAFF-biotin + streptavidin-Alexa-Fluor™ 488 (SA-488)).

[0230] Example 6: TACI-Fc variants block IgG binding to human FcRn-expressing cell lines Flow cytometry analysis was used to examine the ability of TACI-Fc variants to block IgG1 binding to FcRn in the CHO-K1 / hu FcRn cell line. Briefly, HO-K1 / hu FcRn cells were seeded at 100,000 cells per well in PBS (pH 6.0), combined with 1 μM FcRn Fc variants in 0.5% BSA (pH 6.0), and then incubated at 4°C for 60 minutes. Cells were washed three times with 100 μL of 0.5% BSA (pH 6.0). Various amounts of 100 μg / mL phycoerythrin-labeled human IgG1 (IgG1-PE, Southern Biotech, catalog number 0151K-09) in 0.5% BSA (pH 6.0) were added and incubated at 4°C for 30 minutes. Cells were washed twice, resuspended in 100 μl of 0.5% BSA (pH 6.0), and analyzed using an LSRII Fortessa (BD, custom-built) flow cytometer.

[0231] FcRn-expressing CHO-K1 cells treated with IgG exhibited a basal level of fluorescence (MFI) reflecting IgG1-PE binding to cellular FcRn. However, as shown in Figures 15A and 15B, when increasing concentrations of TACI-Fc fusion proteins were titrated into the cell culture, they reduced (i.e., outcompeted) the signal resulting from IgG1-PE binding to FcRn. The reduction in signal from IgG1-PE was plotted as the ratio of mean fluorescence intensity (MFI) to background signal (Figure 15A) or raw MFI (Figure 15B). The different binding profiles of the TACI-Fc variants to FcRn in Figure 15B indicate that weaker affinity for FcRn translates into lower potency in this cell-based human IgG1 competition assay.

[0232] The results in Figures 15A and 15B confirm that TACI-Fc fusion proteins can block IgG binding to human FcRn-expressing cell lines.

[0233] Example 7: The linker sequence between the TACI and FC domains improves protein stability To investigate whether the inclusion of certain amino acids or peptides can increase the isoelectric point of TACI-Fc fusion proteins, a high isoelectric point (pI) polypeptide linker was inserted between the TACI domain and the IgG Fc domain and compared with other variants that did not contain a linker. Among the tested TACI-Fc variants identified in Figures 16A-16B, TPP-5776 (SEQ ID NO: 28) and TPP-5780 (SEQ ID NO: 35) contain the high pI linker GGRSSKARSSSRGGGRGG ("PI linker," SEQ ID NO: 8). TPP-5776 further contains the mutations M252Y, S254T, and T256E. TPP-5777 (SEQ ID NO: 71) and TPP-5779 (SEQ ID NO: 36) do not contain a PI linker, while TPP-5791 (SEQ ID NO: 34) contains the linker (AGGGG)3 (SEQ ID NO: 7).

[0234] The TACI-Fc fusion proteins were subjected to accelerated stability testing by incubating them at 37°C for a total of 3 weeks. Size-exclusion chromatography (SEC) using 0.15 M sodium phosphate buffer, pH 7.0, was performed on 20 μg of on-column TACI-Fc fusion at a flow rate of 0.7 mL / min and a wavelength of 280 nm. As shown in Figures 16A-16B, at 37°C for a total of 3 weeks, the TPP-5776 (SEQ ID NO: 28) and TPP-5780 (SEQ ID NO: 35) fusion proteins containing a high isoelectric point (PI) linker exhibited a higher percent of monomer and reduced aggregation over time compared to TPP-5777 (SEQ ID NO: 71) and TPP-5779 (SEQ ID NO: 36), which did not contain a PI linker. It was further observed that the inclusion of the PI linker resulted in less aggregation during the purification process.

[0235] Example 8: Enhancement of FcRn binding by blocking BAFF and APRIL additively reduces immunoglobulin levels in vitro and in vivo TPP-6269 (SEQ ID NO: 25) was evaluated for its ability to reduce IgG, IgM, and mature B cells after in vivo treatment of mice with keyhole limpet hemocyanin (KLH). Figure 17A shows the in vivo study design for this experiment. On day 0, mice were subcutaneously immunized with 0.5 mg of KLH in 0.1 mL of PBS. Control mice received PBS only. On days 1–7, mice were intravenously administered TPP-6269 at a daily dose of 5 mg / kg. Serum was collected after the 7-day dose to measure anti-KLH IgM and IgG, as well as total IgM and IgG levels. On day 8, five mice per group were euthanized, and their spleens were collected and individually processed as single-cell suspensions. The absolute numbers of B220+ cells and CD4+ T cells from mouse splenocytes were counted by flow cytometry, and the results are shown in Figure 17B. The resulting serum levels of anti-KLH IgM and IgG antibodies are shown in Figure 17C. The resulting serum levels of total IgM and total IgG antibodies are shown in Figure 17D.

[0236] The results in Figures 17B-17D show that TPP-6269 exhibits similar results to the individual protein components tested in functional assays. Figure 17B shows that mouse B220+ cell levels were significantly reduced in response to administration of 5 mg / kg of TPP-6269. In contrast, CD4+ T cell levels were not significantly affected. Figures 17C and 17D show that TPP-6269-mediated blockade of FcRn activity resulted in a significant reduction in anti-KLH IgM and anti-KLH IgG levels (Figure 17C), as well as a significant reduction in total IgM and total IgG levels (Figure 17D).

[0237] Example 9: Effect of an enhanced Fc domain on in vivo human IgG clearance in human FcRn mice Using its enhanced Fc domain, TPP-6269 was further evaluated for its ability to promote the clearance of human IgG (biotin-hIgG) antibodies in muFcRn- / -, huFcRn Tg32 transgenic mice. Tg32 mice suffer from hypogammaglobulinemia due to the low affinity of mouse IgG for hFcRn, providing a useful model for directly tracking IgG degradation in vivo.

[0238] Figure 18A shows the experimental design for evaluating IgG reduction by TPP-6269 in vivo. On day 0, Tg32 transgenic mice were administered a large bolus dose of biotin-hIgG (245 mg / kg) by intravenous injection (iv). TPP-6269 (20 mg / kg and 60 mg / kg) or vehicle control was administered intravenously. Serum samples were collected 49 hours after IVIg administration, and serially at 48, 72, 96, and 120 hours after IVIg administration. Circulating levels of biotin-IgG remaining in serum at various collection time points were measured using a Gyrolab generic PK kit (Gyrolab, catalog number P0020499). By running serum over streptavidin-coated beads in an affinity capture column as a first step according to Gyrolab Bioaffy CD, circulating biotin-human IgG was directly captured, allowing IgG to be detected using the included detection reagents to determine the % of total human IgG in the mice.

[0239] As shown in Figure 18B, treatment of Tg32 mice with TPP-6269 (20 mg / kg and 60 mg / kg) dose-dependently significantly reduced plasma concentrations of biotin-hIVIg compared to the vehicle-treated control group.

[0240] Example 10: Enhanced Fc domain pharmacokinetics in human FcRn mice To determine whether an enhanced Fc domain provides improved pharmacokinetic (PK) properties, human FcRn mice (mFcRn- / -, hFcRn Tg32) were utilized and TPP-6269 was tested as an example. After injecting TPP-6269 into mice, Cmax was determined from blood drawn 10 minutes after drug injection. Continuous blood sampling was performed over a 21-day period, and circulating drug levels were directly measured using a Gyrolab generic PK kit (catalog number P0020499). Based on the results shown in Figure 19, the engineered Fc variant was found to exhibit a half-life of 53.7 hours, calculated using a two-phase decay nonlinear regression in GraphPad Prism version 9.3.1.

[0241] Array Overview

[0242] [Table 4]

[0243] [Table 5]

[0244] [Table 6]

[0245] [Table 7]

[0246] [Table 8]

[0247] [Table 9]

[0248] [Table 10]

[0249] Table 11

[0250] Table 12

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[0252] Table 14

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[0255] Table 17

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[0310] Table 72

[0311] [Table 73]

[0312] [Table 74]

[0313] [Table 75]

[0314] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A fusion protein comprising a TACI extracellular domain (ECD) portion and a hybrid IgG Fc domain, 10. A fusion protein wherein the Ig Fc domain comprises one or more mutations selected from the group consisting of N434H, Y436L, L234F / L235E, M252Y / S254T / T256E, L234F / L235E / N434H, L234F / L235E / N434H, M252Y / S254T / T256E / N434H, L234F / L235E / M252Y / S254T / T256E, L234F / L235E / M252Y / S254T / T256E / N434H, and T307A / E380A / N434, and combinations thereof.

2. The fusion protein of claim 1 , wherein the TACI ECD portion comprises a TACI DR2 portion.

3. The fusion protein of claim 2 , wherein the TACI ECD portion further comprises a TACI DR1 portion.

4. 2. The fusion protein of any one of the preceding claims, wherein the TACI ECD portion comprises the amino acid sequence set forth in SEQ ID NO: 1 or 2.

5. The fusion protein of claim 4 , wherein the TACI ECD portion consists of the amino acid sequence set forth in SEQ ID NO: 1 or 2.

6. 10. The fusion protein of any one of the preceding claims, wherein the TACI ECD portion comprises one or more mutations selected from the group consisting of L82H, F78H / D80H / L82H, Y79H / D80H / L82H, D80H / L82H / L83H, Y79H / D80H / L83H, D80 / L83H / I87H, and combinations thereof.

7. 10. The fusion protein of any one of the preceding claims, wherein the variant Ig Fc domain is derived from an IgG1, IgG2, IgG3, IgG4, or hybrid Fc domain thereof.

8. The fusion protein of claim 7, wherein the variant Ig Fc domain comprises a hybrid IgG1-IgG4 Fc domain.

9. 10. The fusion protein of any one of the preceding claims, wherein the variant Ig Fc domain comprises an amino acid sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 16-24 and 72.

10. 10. The fusion protein of claim 9, wherein the variant Ig Fc domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 16-24 and 72.

11. 10. The fusion protein of any one of the preceding claims, wherein the TACI ECD portion is linked to the mutant Ig Fc domain by a polypeptide linker.

12. The fusion protein of claim 11, wherein the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-15.

13. 10. The fusion protein of any one of the preceding claims, comprising an amino acid sequence that is at least 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 25-38, 46-48, 55, 56, and 67.

14. The fusion protein of claim 13, comprising an amino acid sequence set forth in any one of SEQ ID NOs: 25 to 38, 46 to 48, 55, 56, and 67.

15. The fusion protein of claim 14, comprising the amino acid sequence set forth in SEQ ID NO: 25 or 67.

16. The fusion protein has a binding affinity for FcRn (K) at pH 6.0 of less than 50 nM, less than 20 nM, less than 12 nM, less than 6 nM, or less than 4 nM. Ｄ 2. The fusion protein of any one of the preceding claims, wherein

17. The fusion protein has a binding affinity (K) for FcRn that is 50-fold greater, 100-fold greater, or 200-fold greater at pH 6.0 compared to pH 7.

4. Ｄ 2. The fusion protein of any one of the preceding claims, exhibiting an increase in IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL

18. A pharmaceutical composition comprising the fusion protein of any one of the preceding claims and a pharmaceutically acceptable carrier.

19. A nucleic acid encoding a fusion protein according to any one of the preceding claims.

20. 20. The nucleic acid of claim 19, encoding a fusion protein as set forth in SEQ ID NO: 25 or 67.

21. 21. The nucleic acid of claim 20, comprising the nucleotide sequence set forth in SEQ ID NO: 69 or 70.

22. 22. The nucleic acid of any one of claims 19 to 21, encoding a codon-optimized fusion protein.

23. An expression vector comprising the nucleic acid of any one of claims 19 to 22.

24. The expression vector of claim 23 , wherein the expression vector is a viral vector.

25. 25. The expression vector of claim 24, wherein the viral vector is an AAV vector.

26. A pharmaceutical composition comprising the expression vector of any one of claims 23 to 25 and a pharmaceutically acceptable carrier.

27. A host cell comprising the nucleic acid of any one of claims 19 to 22.

28. A host cell comprising the expression vector of any one of claims 23 to 25.

29. 20. A method of treating a disease in a patient, comprising administering to a patient in need of treatment an effective amount of the pharmaceutical composition of claim 18.

30. 27. A method of treating a disease in a patient, comprising administering to a patient in need of treatment an effective amount of the pharmaceutical composition of claim 26.

31. 31. The method of claim 29 or 30, wherein the disease is an autoimmune disorder.

32. The autoimmune disorder may be acute antibody-mediated rejection, chronic antibody-mediated rejection, primary Sjogren's syndrome, autoimmune hemolytic anemia, antiphospholipid syndrome (APS), catastrophic APS, refractory rheumatoid arthritis, autoimmune vasculitis, cryoglobulinemia, antineutrophil cytoplasmic autoantibody (ANCA), IgA vasculitis, rheumatoid vasculitis, antisynthetase syndrome, primary membranous nephropathy, juvenile idiopathic arthritis (JIA), systemic sclerosis, Guillain-Barré syndrome, autoimmune alveolar protein 32. The method of claim 31 , wherein the disease is selected from the group consisting of: autoimmune hemolytic anemia (AIHA), hemolytic disease of the fetus and newborn (HDFN), acute hemolytic transfusion reaction, prophylactic treatment with adeno-associated virus (AAV), thyroid eye disease (TED), Huntington's disease, chronic inflammatory demyelinating polyneuropathy (CIDP), pemphigus vulgaris (PV), bullous pemphigoid (BP), immune thrombocytopenic purpura (ITP), myasthenia gravis (MG), and neuromyelitis optica (NMOSD).

33. 30. A method of reducing an immune response in a patient in need thereof, comprising administering to said patient an effective amount of the pharmaceutical composition of claim 18 or 26.

34. 30. A method of inhibiting the activity of one or more pro-inflammatory cytokines in a patient in need thereof, comprising administering to said patient in need thereof an effective amount of the pharmaceutical composition of claim 18 or 26.

35. 35. The method of claim 34, wherein the one or more pro-inflammatory cytokines comprise BAFF and / or APRIL.

36. 27. A method of enhancing the clearance of IgG immunoglobulins in a patient in need thereof, comprising administering to said patient an effective amount of the pharmaceutical composition of claim 18 or 26.

37. 30. A method of reducing levels of mature B cells, IgG, IgM, IgA, or a combination thereof in a patient in need thereof, comprising administering to the patient an effective amount of the pharmaceutical composition of claim 18 or 26.

38. 38. The method of claim 37, wherein administration of the pharmaceutical composition does not significantly reduce IgD or IgE levels.

39. 30. A kit for treating one or more disorders in a patient, comprising at least one dose of the pharmaceutical composition of claim 18 or 26 and instructions for use.

40. A polypeptide linker comprising the amino acid sequence set forth in any one of SEQ ID NOs: 4, 5, and 6.

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