Antibody binding to april and use thereof

Abstract

Provided is an isolated monoclonal antibody capable of specifically binding to APRIL or an antigen-binding portion thereof. Further provided are a nucleic acid molecule encoding the antibody or the antigen-binding portion thereof, an expression vector, host cell and method for expressing the antibody or the antigen-binding portion thereof, and a treatment method using the antibody or the antigen-binding portion thereof, the nucleic acid molecule, the expression vector and / or the host cell.

Description

Antibodies that bind to APRIL and their uses Invention Field

[0001] This invention relates to an antibody or its antigen-binding moiety that can specifically bind to human APRIL, its preparation and use, and in particular its use in the treatment of human autoimmune diseases associated with APRIL, such as IgA nephropathy, inflammatory diseases and other immune diseases associated with B cell dysfunction. Background Technology

[0002] Immunoglobulin A nephropathy (IgAN) is the most common type of primary glomerulonephritis worldwide, with an annual global prevalence of approximately 3 per 100,000 adults, and an incidence rate as high as 40-50% in East Asia. IgAN is characterized by the deposition of immune complexes formed by galactose-deficient IgA1 (Gd-IgA1) and autoantibodies targeting Gd-IgA1 in the glomerular mesangial region. This usually leads to a decline in renal function, and up to 25%-50% of patients will gradually develop end-stage renal disease within 10-20 years after onset (Cheung CK, et al., (2024) The role of BAFF and APRIL in IgA nephropathy:pathogenic mechanisms and targeted therapies. Front Nephrol. 3:1346769).

[0003] In normal individuals, IgA1 typically has an O-glucose terminally attached to its hinge region. The absence of the terminal galactose in the IgA1 hinge region (i.e., the formation of Gd-IgA1) exposes the N-acetylgalactosamine (GalNAc) residues in the O-glucose, which are then recognized by anti-glucose autoantibodies in susceptible individuals, forming an immune complex that deposits on the glomerular mesangium, triggering a series of inflammatory responses. This is the "four-hit" hypothesis of IgAN pathogenesis (Suzuki H, et al., (2011) The pathophysiology of IgA nephropathy. J Am Soc Nephrol. 22(10):1795-803). Furthermore, the co-distribution of IgA and complement C3 has been observed in most IgAN patients, suggesting that the complement system may also be activated by Gd-IgA1. The generation of Gd-IgA1 and autoantibodies targeting Gd-IgA1 is mainly driven by mucosal plasma cells (Cheung CK, et al., (2024) ibid.).

[0004] Currently, international guidelines recommend only RAS blockers (ACE inhibitors / ARBs) as first-line treatments, but these only reduce proteinuria to a limited extent and often fail to achieve therapeutic targets. Powerful drugs for reducing proteinuria—hormones / immunosuppressants—are not very sensitive to IgA and have significant side effects. The world's first targeted therapy for IgA is Nefecon (budesonide delayed-release capsules) from Calliditas Therapeutics, which received FDA approval in December 2021 for treating proteinuria in adult patients with primary IgA nephropathy at risk of progression. Currently, more than 20 drugs targeting B cells that generate Gd-IgA1, complement system activation, and other downstream pathways activated after Gd-IgA deposition are in clinical development.

[0005] IgA is primarily produced by plasma cells located in the mucosa-associated lymphoid tissue (MALT). In the MALT, antigen-presenting cells take up antigens, triggering activation of naïve B cells and T-cell-dependent (TD) and T-cell-independent (TID) antibody class switching recombination, resulting in IgA-producing B cells. The occurrence of IgAN is likely related to abnormalities in this step. B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL, also known as CD256 or TNFSF13) drive TD and TID antibody class switching recombination and IgA production. + B cells play a crucial role (Sallustio F, et al., (2021) High levels of gut-homing immunoglobulin A +B lymphocytes support the pathogeneic role of intestinal mucosal hyperresponsiveness in immunoglobulin A nephropathy patients. Nephrol Dial Transplant 36:452-64; Cerutti A. (2021) The regulation of IgA class switching. Nat Rev Immunol (2021) 8: 421-34.; Moore JS, et al., (2007) Reactivities of N-acetylgalactos-amine-specific lectins with human IgA1 proteins. Mol Immunol 44:2598-604; Castigli E, et al., (2005) TACI and BAFFR mediate isotype switching in B cells. J Exp Med 201:35-9; Mackay F, et al., (2003) BAFF and APRIL: a tutorial on B cell survival. Annu Rev Immunol 21:231-64). Both BAFF and APRIL bind to B cell maturation antigen (BCMA) and transmembrane activation, calmodulin-cyclophilin ligand interaction factor (TACI). BCMA is mainly expressed in plasma cells, while TACI is expressed in mature B cells and activated plasma cells. BAFF supports B cell differentiation into long-lived plasma cells, which can continuously produce antibodies, including IgA1 and Gd-IgA1, without additional stimulation. APRIL can induce antibody class switching and recombinant cell formation in naïve B cells via the TACI and / or BCMA signaling pathways, forming IgA-producing B cells. In in vitro experiments, B cells from IgAN patients showed increased Gd-IgA1 production when co-incubated with APRIL, suggesting that APRIL may play a role in abnormal IgA1 glycosylation (Zhai YL, et al., (2016) Increased APRIL expression induces IgA1 aberrant glycosylation in IgA nephropathy. Medicine 95:e3099).Furthermore, polymorphisms in the APRIL and TACI genes may be associated with susceptibility to IgAN (Yu XQ, et al., (2011) A genome-wide association study in Han Chinese identifies multiple susceptibility loci for IgA nephropathy. Nat Genet. 44(2):178-82). IgAN patients have higher levels of APRIL, which is associated with disease severity and poorer prognosis (Zhai YL, et al., (2016) Increased APRIL expression induces IgA1 aberrant glycosylation in IgA nephropathy. Medicine 95:e3099; Han SS, et al., (2016) The role of TNF superfamily member 13 in the progression of IgA nephropathy. J Am Soc Nephrol 27:3430–9). Targeting APRIL may inhibit or delay the occurrence or development of IgAN by suppressing the production of Gd-IgA1 in abnormal plasma cells.

[0006] VIS649 (Sibeprenlimab) and BION-1301 (Zigakibart) are monoclonal antibodies targeting APRIL, inhibiting the binding of APRIL to its receptor. Recently released phase II clinical trial results show that both VIS649 and BION1301 significantly reduced Gd-IgA1 antibody levels and proteinuria in IgAN patients, while also observing a sustained decrease in IgA and IgM, but a smaller decrease in IgG. The lower IgG reduction may result in a lower risk of infection during treatment. Additionally, soluble TACI-Fc fusion proteins, including ataciccept and telitacicept, which simultaneously block the binding of BAFF to B cells and APRIL to plasma cells, can comprehensively inhibit the production of IgA1, IgG, and IgM. In the phase 2 clinical trial, after six months of treatment with acecept or teltascept, compared with the control group, IgA decreased by about 50%, proteinuria decreased by an average of about 40%, and the decline in renal function was also significantly slowed down.

[0007] Although novel drugs targeting APRIL in clinical trials have brought breakthrough efficacy to IgAN patients, IgAN is a chronic disease requiring long-term medication. Significant clinical challenges and needs remain in achieving rapid onset of action, reducing the frequency of medication, making administration more convenient, and better improving proteinuria and kidney function.

[0008] In addition to promoting B cell differentiation and survival, APRIL and BAFF may also induce B cell autoreactivity. Elevated levels of APRIL and BAFF have been observed in blood or tissues in many autoimmune diseases and other B cell-related diseases (Han SS, et al., (2017) BAFF and APRIL expression as an autoimmune signature of membranous nephropathy. Oncotarget. 9(3):3292-3302). Furthermore, APRIL can bind to BCMA, which is highly expressed on multiple myeloma cells, promoting the progression of multiple myeloma (Tai YT et al., (2016) APRIL and BCMA promote human multiple myeloma growth and immunosuppression in the bone marrow microenvironment. Blood 127(25):3225–36).

[0009] Reference to any document in this application is not an admission that such document is prior art.

[0010] Summary of the Invention

[0011] The inventors of this application screened for monoclonal antibodies that specifically bind to human and monkey APRIL with binding affinity comparable to or higher than that of existing antibodies. Subsequently, the inventors humanized and modified the Fc region of these antibodies, resulting in reduced immunogenicity and enhanced FcRn binding affinity in an acidic environment. In in vivo animal experiments, this antibody exhibited higher blood concentrations and a longer half-life than existing antibodies, rapidly and persistently eliminating APRIL, as well as IgG, IgA, and IgM, from the blood. Furthermore, the inventors weakened or eliminated the FcRγ binding affinity in the Fc region to avoid unnecessary antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC) in APRIL-positive cells.

[0012] Therefore, in one aspect, this application provides an isolated monoclonal antibody (e.g., mouse-derived, chimeric, or humanized antibody), or its antigen-binding portion, which is capable of specifically binding to APRIL, and may include:

[0013] i) A heavy chain variable region, which may comprise a VH-CDR1 region, a VH-CDR2 region, and a VH-CDR3 region, wherein the VH-CDR1 region, VH-CDR2 region, and VH-CDR3 region may respectively comprise amino acid sequences having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs:1, 2, and 3; and

[0014] ii) Light chain variable region, which may include VL-CDR1 region, VL-CDR2 region and VL-CDR3 region, wherein the VL-CDR1 region, VL-CDR2 region and VL-CDR3 region may respectively contain amino acid sequences having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NOs:4, 5 and 6.

[0015] In some embodiments, the heavy chain variable region may contain an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:7.

[0016] In some embodiments, the light chain variable region may contain an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:8.

[0017] In some embodiments, the heavy chain variable region and the light chain variable region may respectively contain amino acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs:7 and 8.

[0018] This application also provides an isolated monoclonal antibody (e.g., mouse-derived, chimeric, or humanized antibody) or its antigen-binding moiety, which can specifically bind to APRIL, and may include:

[0019] i) A heavy chain variable region, which may include a VH-CDR1 region, a VH-CDR2 region, and a VH-CDR3 region, wherein the VH-CDR1 region, VH-CDR2 region, and VH-CDR3 region may have at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the VH sequence containing the amino acid sequence shown in SEQ ID NO:7, respectively.

[0020] ii) A light chain variable region, which may contain a VL-CDR1 region, a VL-CDR2 region, and a VL-CDR3 region, wherein the VL-CDR1 region, VL-CDR2 region, and VL-CDR3 region may have at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the VL region, VL-CDR2 region, and VL-CDR3 region of the VL sequence containing the amino acid sequence shown in SEQ ID NO:8, respectively.

[0021] The monoclonal antibody or its antigen-binding portion thereof of this application may be mouse-derived, chimeric, or humanized. In some embodiments, the monoclonal antibody or its antigen-binding portion thereof of this application may be humanized.

[0022] The monoclonal antibody or its antigen-binding portion of this application may include a heavy chain constant region. The heavy chain constant region may be the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4, or a functional fragment thereof such as an Fc region, for example, the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, or a functional fragment thereof such as an Fc region. In some embodiments, the heavy chain variable region includes the amino acid sequence shown in SEQ ID NOs:9, 13, or 17. In some embodiments, the heavy chain constant region may contain strong FcRn binding activity, especially strong FcRn binding activity under acidic conditions. In some embodiments, the antibody of this application exhibits strong FcRn binding activity at pH 5.8. In some embodiments, the antibody of this application exhibits high binding activity to FcRn at pH 5.8, for example, K... D The value is in the range of 1.0 × 10. -9 Below M. In some embodiments, the antibody of this application exhibits strong FcRn binding affinity at pH 5.8, for example, K. D The value is 9.0 × 10 -10 Below M, further at 8.5 × 10 -10Below M. In some embodiments, the heavy chain constant region may contain the M428L / N434A, M428L / N434S, or M252Y / S254T / T256E mutation to enhance FcRn binding affinity, particularly strong FcRn binding affinity under acidic conditions. In some embodiments, the heavy chain constant region may be an IgG4 constant region containing the M428L / N434A, M428L / N434S, or M252Y / S254T / T256E mutation, which may contain, for example, the amino acid sequence shown in SEQ ID NOs:14, 15, or 16. In some embodiments, the heavy chain constant region may be an IgG1 constant region containing the M428L / N434A, M428L / N434S, or M252Y / S254T / T256E mutation, which may contain, for example, the amino acid sequence shown in SEQ ID NOs:10, 11, or 12. The heavy chain constant region may contain weak or no FcRγ binding force. In some embodiments, the heavy chain constant region may contain an L234A / L235A mutation to weaken or eliminate FcRγ binding force. In some embodiments, the heavy chain constant region may be an IgG4 constant region containing M428L / N434A, which may contain the amino acid sequence shown in SEQ ID NO:14. In some embodiments, the heavy chain constant region may be an IgG1 constant region containing L234A / L235A / M428L / N434A, which may contain the amino acid sequence shown in SEQ ID NO:10. The N-terminus of the heavy chain constant region may be linked to the C-terminus of the heavy chain variable region.

[0023] The monoclonal antibody or its antigen-binding portion of this application may include a light chain constant region. The light chain constant region may be a κ constant region or a λ constant region, or a functional fragment thereof, such as a human κ constant region or a λ constant region, or a functional fragment thereof. In some embodiments, the light chain constant region may include the amino acid sequence shown in SEQ ID NO:18. The N-terminus of the light chain constant region may be linked to the C-terminus of the light chain variable region.

[0024] In some embodiments, the antibody of this application comprises two heavy chains and two light chains, or is composed of two heavy chains and two light chains, wherein each heavy chain comprises the aforementioned heavy chain constant region sequence, heavy chain variable region sequence, and / or CDR sequence, and each light chain comprises the aforementioned light chain constant region sequence, light chain variable region sequence, and / or CDR sequence. In some embodiments, the antibody of this application may be a single-chain antibody, or may be composed of antibody fragments, such as Fab or F(ab')2 fragments.

[0025] The antibody or its antigen-binding moiety of this application, compared with prior art antibodies such as VIS649 and BION1301, possesses i) comparable or higher human APRIL binding affinity, ii) comparable or higher monkey APRIL binding affinity, iii) comparable or higher APRIL-BCMA blocking activity, iv) comparable or higher APRIL-TACI blocking activity, v) comparable or stronger FcRn binding affinity, especially comparable or stronger FcRn binding affinity in acidic environments such as pH 5.8, vi) comparable or longer half-life, vii) comparable or higher plasma concentration or bioavailability, and / or viiii) comparable or higher APRIL scavenging ability. The antibody or its antigen-binding moiety of this application does not possess mouse APRIL binding affinity. In some embodiments, the antibody or its antigen-binding portion of this application contains weak or no FcRγ binding force, thereby avoiding unnecessary antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC) on APRIL positive cells.

[0026] This application also provides a bispecific molecule containing the antibody or antigen-binding portion thereof of this application, wherein the antibody or antigen-binding portion thereof is attached to a second functional group, such as a second antibody, the second functional group having a binding specificity different from that of the antibody or antigen-binding portion thereof of this application.

[0027] This application also provides nucleic acid molecules encoding the antibody of this application or its antigen-binding portion or bispecific molecule. This application also provides an expression vector comprising the aforementioned nucleic acid molecule, and a host cell comprising the expression vector or integrating the aforementioned nucleic acid molecule into the genome. This application also provides a method for preparing the antibody of this application or its antigen-binding portion or bispecific molecule using the host cell, comprising: (i) expressing the antibody or its antigen-binding portion or bispecific molecule in the host cell, and (ii) isolating the antibody or its antigen-binding portion or bispecific molecule from the host cell or its culture.

[0028] This application also provides a composition comprising the antibody or its antigen-binding portion thereof, a bispecific molecule, a nucleic acid molecule, an expression vector, or a host cell. In some embodiments, the composition may be a pharmaceutical composition and may also comprise a pharmaceutically acceptable carrier.

[0029] On the other hand, this application provides a method for treating or alleviating APRIL-related disease in a subject, including administering a therapeutically effective amount of the pharmaceutical composition of this application to the subject.

[0030] APRIL-related diseases can be inflammatory diseases, such as autoimmune diseases. In some embodiments, APRIL-related diseases are autoimmune diseases associated with B cell dysfunction. In some embodiments, APRIL-related diseases are immunoglobulin A nephropathy (IgAN).

[0031] APRIL-related diseases can be tumors, such as B-cell-related tumors, including multiple myeloma. In some embodiments, the method may also include administering antibodies against immune checkpoints such as PD-1 or CTLA-4.

[0032] This application also provides the use of the antibody or its antigen-binding portion thereof in the preparation of a medicament for treating APRIL-related diseases.

[0033] Other features and advantages disclosed herein will become readily apparent from the following detailed description and embodiments, which should not be construed as limiting. All references, Genbank registration numbers, patents, and published patent applications cited in this specification are incorporated herein by reference. Attached Figure Description

[0034] The following detailed description, given by way of example but not intended to limit the invention to the specific embodiments described, can be better understood in conjunction with the accompanying drawings.

[0035] Figure 1 shows the binding ability of APRIL antibody to recombinant human APRIL protein (A) and monkey APRIL protein (B) in ELISA.

[0036] Figure 2 shows the blocking power of APRIL antibody against the binding of human APRIL to human BCMA (A), human APRIL to human TACI (B), and monkey APRIL to monkey BCMA (C) in ELISA.

[0037] Figure 3 shows the binding affinity of the APRIL antibody to mouse APRIL in ELISA (A), and the binding affinity of human APRIL protein to human, monkey, and mouse BCMA (B).

[0038] Figure 4 shows the rapid clearance of APRIL mediated by MIL116 antibody in C57 wild-type mice.

[0039] Figure 5 shows the serum exposure and clearance rate of MIL116 antibody in FcRn humanized mice.

[0040] Figure 6 shows the activation capacity of APRIL antibody on T cells in an in vitro immunogenicity prediction experiment, specifically the expression of T cell activation markers OX40 (A) and CD25 (B), in which the PBMC donor has a high-frequency HLA genotype in Chinese.

[0041] Figure 7 shows the changes in serum IgG (A), IgA (B), and IgM (C) levels after a single subcutaneous injection of APRIL antibody into cynomolgus monkeys.

[0042] Figure 8 shows the changes in serum APRIL antibody concentration after a single subcutaneous injection in cynomolgus monkeys. Detailed Implementation

[0043] Unless otherwise specified, the terms used herein have their common meanings as found in dictionaries, textbooks, and technical reference books, or as commonly understood by those skilled in the art. The following descriptions of some terms are for the purpose of understanding this application only and are not intended to impose any particular limitations on these terms, unless otherwise specified.

[0044] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include the plural form of the object referred to, unless the context clearly specifies otherwise.

[0045] The term "or" refers to a single element among the listed selectable elements, unless the context explicitly indicates otherwise.

[0046] The terms "comprising" or "including" mean that the stated elements, integers, or steps are included, but do not exclude the inclusion of any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise specified, they also cover combinations of the stated elements, integers, or steps. The terms "consisting of" or "comprises of" generally mean that only the stated elements, integers, or steps are included, without the addition of other elements, integers, or steps.

[0047] The term "APRIL" refers to the proliferation-inducing ligand, also known as CD256 or TNFSF13. This term includes variants, homologs, orthologs, and parallel homologs. For example, antibodies specific to human APRIL may cross-react with APRIL proteins from another species, such as monkeys, under certain conditions. In other embodiments, antibodies specific to human APRIL proteins may be completely specific to human APRIL proteins without cross-reacting with proteins from other species or other types, or may cross-react with APRIL proteins from some other species but not all others.

[0048] The term "human APRIL" refers to the APRIL protein with a human amino acid sequence, while "monkey APRIL" refers to the APRIL protein with a monkey amino acid sequence.

[0049] The term "antibody" as used in this article is intended to include full-length antibodies of IgG, IgA, IgD, IgE, and IgM, as well as any antigen-binding fragments (i.e., antigen-binding portions). Full-length antibodies are glycoproteins containing at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain is separated by a heavy chain variable region (V). H It consists of a VH domain and a heavy chain constant region. The heavy chain constant region consists of three structural domains, namely C... H1 C H2 and C H3 Each light chain consists of a light chain variable region (V for short). L It consists of a light chain constant region (or VL) and a light chain constant region. The light chain constant region consists of a structural domain C. L Composition. V H and V L The region can also be divided into highly variable regions called complementarity-determining regions (CDRs), which are separated by more conservative framework regions (FRs). Each V H and V L The antibody constant region consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The heavy chain constant region mediates the binding of immunoglobulins to host tissues or factors, including binding to various immune system cells (e.g., effector cells) and the first component (C1q) of the conventional complement system. The primary function of the light chain constant region is to enhance the structural stability of the antibody. The "functional fragment" of the antibody constant region refers to a segment within the constant region that retains certain desired functions; for example, a fragment in the heavy chain constant region that retains FcRn binding activity, such as the Fc fragment.

[0050] In this document, the term "antigen-binding moiety" (or simply antibody moiety) of an antibody refers to one or more segments of an antibody that retain the ability to specifically bind antigens. It has been demonstrated that the antigen-binding function of an antibody can be performed by segments of the full-length antibody. Examples of binding segments contained in the "antigen-binding moiety" of an antibody include (i) the Fab fragment, which is composed of V... L V H C L and C H1 (ii) F(ab')2 segment, a divalent segment containing two Fab segments connected by a disulfide bridge in the hinge region; (iii) composed of V H and C H1 (iv) Fd fragments composed of antibody single-arm V L and V H The Fv fragment comprises: (v) a separated complementarity-determining region (CDR); and (vi) a nanobody, a heavy chain variable region containing a single variable domain. Furthermore, although the two domains of the Fv fragment are V...L and V H Encoded by different genes, they can be linked via a synthetic linker that makes them single-protein chains through recombination, where V L and V H Regions pair to form monovalent molecules (called single-chain Fc (scFv)). These single-chain antibodies are also intended to be included in the terminology. These antibody fragments can be obtained using common techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.

[0051] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities. For example, an isolated antibody that specifically binds to the APRIL protein is substantially free of antibodies that specifically bind to antigens other than APRIL. However, an isolated antibody that specifically binds to human APRIL may have cross-binding to other antigens, such as APRIL proteins from other species. Furthermore, isolated antibodies are substantially free of other cellular material and / or chemicals.

[0052] The term "monoclonal antibody" or "monoclonal antibody" refers to an antibody or antibody fragment thereof that is identical to a population of antibody molecules except for naturally occurring mutations that may arise spontaneously. Monoclonal antibodies are highly specific for a single epitope of an antigen. In contrast, polyclonal antibodies typically contain two or more different antibodies that recognize multiple different epitopes of the same antigen.

[0053] The term "mouse-derived antibody" refers to an antibody whose variable region backbone and CDR region are derived from mouse germline immunoglobulin sequences. Additionally, if the antibody contains a constant region, it is also derived from mouse germline immunoglobulin sequences. Mouse-derived antibodies may contain amino acid residues not encoded by mouse germline immunoglobulin sequences, for example, mutations introduced through in vitro random or point mutations or through in vivo somatic mutations. However, the term "mouse-derived antibody" does not include antibodies in which CDR sequences derived from other mammalian species are inserted into the mouse backbone sequence.

[0054] The term "chimeric antibody" refers to an antibody obtained by combining non-human genetic material with human genetic material. Or, more broadly, a chimeric antibody is an antibody that combines genetic material from one species with genetic material from another species.

[0055] The term “humanized antibody” as used in this article refers to an antibody derived from a non-human species but whose protein sequence has been modified to increase its similarity to naturally occurring antibody variants in humans.

[0056] In this paper, antibodies that "specifically bind to human APRIL" refer to antibodies that bind to human APRIL (and APRIL from other non-human species) but substantially do not bind to non-APRIL proteins. Preferably, the antibody binds to the human APRIL protein, i.e., K, with "high affinity". D The value is 5.0 × 10 -9 Below M.

[0057] The term "basically non-binding" protein or cell refers to proteins or cells that do not bind to each other, or do not bind to them with high affinity; that is, proteins or cells that bind to K+. D 1.0×10 -6 M or more, preferably 1.0×10 -5 M or more, preferably 1.0×10 -4 M or more, 1.0×10 -3 M or more, preferably 1.0×10 -2 M and above.

[0058] The term "EC" 50 The half-maximal effect concentration (WMP) is the concentration of molecules (such as antibodies) that produce a 50% maximum effect.

[0059] The term "IC" 50 "Half-inhibition concentration" refers to the concentration of molecules (e.g., antibodies) required to inhibit a specified biological process by half.

[0060] The terms “antibody-dependent cytotoxicity,” “antibody-dependent cell-mediated cytotoxicity,” or “ADCC” refer to cell-mediated immune defense in which immune system effector cells actively bind to cell membrane surface antigens and antibodies, such as the APRIL antibody of this application, to lyse target cells.

[0061] The term "complement-dependent cytotoxicity" or "CDC" refers to complement-mediated cytotoxicity, which involves specific antibodies binding to corresponding antigens on the target cell membrane surface to form a complex that activates the classical complement pathway. The resulting membrane attack complex exerts a lytic effect on the target cell.

[0062] The term “ADCP” or “antibody-dependent phagocytosis” refers to the process by which phagocytic effector cells, such as monocytes and macrophages, endocytose target cells.

[0063] The term “subject” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, such as mammals and non-mammalians, such as non-human primates, sheep, dogs, cats, cattle, horses, chickens, amphibians, and reptiles, although mammals, such as non-human primates, sheep, dogs, cats, cattle, and horses, are preferred.

[0064] The term "therapeutic effective amount" refers to the amount of antibody in this application sufficient to prevent or alleviate symptoms associated with a disease or condition (e.g., IgAN). Therapeutic effective amount is related to the disease being treated, and the actual effective amount can be readily determined by those skilled in the art.

[0065] The term "sequence identity" as used in this article refers to the percentage of nucleotides / amino acids in a sequence that are identical to those in a reference sequence after sequence alignment. If necessary, spaces are introduced in the sequence alignment to achieve the maximum percentage of sequence similarity between the two sequences. Those skilled in the art can use various methods, such as computer software, to perform pairwise or multiple sequence alignments to determine the percentage of sequence similarity between two or more nucleic acid or amino acid sequences. Such computer software includes, for example, ClustalOmega, T-coffee, Kalign, and MAFFT.

[0066] "FcRγ" is a type of Fc receptor that is expressed on various immune cells, including monocytes, macrophages, NK cells, and neutrophils. It can recognize cells bound by immunoglobulins such as IgG and trigger phagocytosis or killing of those cells.

[0067] FcRn, or neonatal Fc receptor, is a protein that plays a crucial role in the immune system and IgG transport. It is expressed in various tissues, such as the lungs and kidneys. FcRn is responsible for the transmembrane transport of IgG, keeping IgG in circulation, and regulating (e.g., prolonging) the half-life of IgG. Studies have shown that FcRn can bind tightly to IgG in acidic environments.

[0068] "Half-life," "T1 / 2," or "plasma half-life" refers to the time required for the concentration of a drug (such as an antibody) in plasma to decrease by half, and its length can reflect the rate of drug elimination.

[0069] "Bioavailability" refers to the relative amount and rate at which a drug (such as an antibody) is absorbed into the circulation of the body. Drugs with high bioavailability can be administered at relatively low doses.

[0070] Several aspects of this application are described in more detail below.

[0071] The antibody or its antigen-binding moiety of this application, compared with prior art antibodies such as VIS649 and BION1301, possesses i) comparable or higher human APRIL binding affinity, ii) comparable or higher monkey APRIL binding affinity, iii) comparable or higher APRIL-BCMA blocking activity, iv) comparable or higher APRIL-TACI blocking activity, v) comparable or stronger FcRn binding affinity, especially comparable or stronger FcRn binding affinity in acidic environments such as pH 5.8, vi) comparable or longer half-life, vii) comparable or higher plasma concentration or bioavailability, and / or viiii) comparable or higher APRIL scavenging ability. The antibody or its antigen-binding moiety of this application does not possess mouse APRIL binding affinity. In some embodiments, the antibody or its antigen-binding portion of this application contains weak or no FcRγ binding force, thereby avoiding unnecessary antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC) on APRIL positive cells.

[0072] The exemplary antibodies or their antigen-binding portions described in this application are those whose structures and chemical properties are as described below.

[0073] The heavy chain variable region CDR and light chain variable region CDR of the antibody or its antigen-binding moiety in this application are determined using the Kabat numbering system. The heavy chain variable region CDR and light chain variable region CDR of the antibody or its antigen-binding moiety in this application can also be determined based on the sequences of the heavy chain variable region and light chain variable region using the Chothia, IMGT, AbM, or Contact numbering systems.

[0074] The antibody or its antigen-binding portion of this application may include a heavy chain constant region, such as a heavy chain constant region with high FcRn binding affinity. When it has high FcRn binding affinity, the antibody or its antigen-binding portion of this application may have a better half-life and APRIL scavenging ability, that is, after binding to the antigen and being internalized into the cell, it can rapidly dissociate from APRIL and return to the circulation system for a new round of APRIL binding. The heavy chain constant region may contain weak or no FcRγ binding affinity to avoid causing unnecessary antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC) to APRIL-positive cells. In some embodiments, the heavy chain constant region may be an IgG4 constant region containing the M428L / N434A, M428L / N434S, or M252Y / S254T / T256E mutation, which may contain, for example, the amino acid sequence shown in SEQ ID NOs:14, 15, or 16. In some embodiments, the heavy chain constant region may be an IgG1 constant region containing the M428L / N434A, M428L / N434S, or M252Y / S254T / T256E mutation, which may contain, for example, the amino acid sequence shown in SEQ ID NOs:10, 11, or 12.

[0075] The monoclonal antibody or its antigen-binding moiety of this application may include a light chain constant region. The light chain constant region may be a κ constant region or a λ constant region, or a functional fragment thereof, such as a human κ constant region or a λ constant region, or a functional fragment thereof. In some embodiments, the light chain constant region may include the amino acid sequence shown in SEQ ID NO:18.

[0076] Other APRILs that combine with human APRIL H and / or V L The sequence (or CDR sequence) can be related to the V of the antibody in this application. H and / or V L The sequences (or CDR sequences) are "mixed and paired". Specifically, when V... H and V L When (or CDRs therein) are mixed and paired, a specific V H / V L V in pairing H The sequence can be approximated by the structure of V. H Sequence substitution. Similarly, specific V is preferred. H / V L V in pairing L The sequence is approximated by V in structure L Sequence substitution.

[0077] In another embodiment, the antibody or its antigen-binding portion of the present application includes the heavy chain variable region CDR2 of an APRIL antibody and the CDRs of other antibodies that bind to human APRIL, such as the heavy chain variable regions CDR1 and / or CDR3, and / or the light chain variable regions CDR1, CDR2 and / or CDR3 of another APRIL antibody.

[0078] In another embodiment, the antibody of this application comprises one or more conserved modified heavy chain and / or light chain variable region sequences or CDR1, CDR2, and CDR3 sequences as the APRIL antibody of this application. It is known in the art that some conserved sequence modifications do not result in the loss of antigen binding.

[0079] As used herein, the term "conserved sequence modification" refers to amino acid modifications that do not significantly affect or alter antibody binding properties. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of this application using standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conserved amino acid substitutions involve replacing an amino acid residue with an amino acid residue having a similar side chain. Groups of amino acid residues with similar side chains are known in the art. These groups of amino acid residues include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), nonpolar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR region of the antibody of this application may be replaced by other amino acid residues in the same side chain group, and the resulting antibody may be tested for retaining function (i.e., the function described above) using the functional assays described herein.

[0080] In several embodiments, the antibody or its antigen-binding portion may be, for example, mouse-derived, chimeric, or humanized, particularly humanized.

[0081] The antibody of this application can be formulated with one or more V antibodies possessing the APRIL antibody of this application. H / V L Antibodies with specific sequences are used as starting materials to prepare genetically modified antibodies. Antibodies can be modified by altering one or two variable regions (i.e., V...). H and / or V LThe antibody can be genetically modified by modifying one or more residues within a region (e.g., in one or more CDR regions and / or one or more backbone regions) to improve binding affinity and / or increase similarity to naturally occurring antibodies in certain species. For example, the antibody can also be genetically modified by modifying residues in constant regions, such as altering the antibody's effector function.

[0082] In some implementations, CDR region implantation can be used to genetically modify the variable region of an antibody. Antibodies primarily interact with target antigens through amino acid residues located in the six complementarity-determining regions (CDRs) of the heavy and light chains. For this reason, the amino acid residues within the CDRs are more diverse among individual antibodies than sequences outside the CDRs. Because the CDR sequence is responsible for the main antibody-antigen interactions, recombinant antibodies that mimic the characteristics of a specific natural antibody can be expressed by constructing expression vectors containing the CDR sequence of a specific natural antibody and inserting it into the backbone sequences of different antibodies with different properties.

[0083] Therefore, another embodiment of this application relates to isolated monoclonal antibodies or their antigen-binding portions, comprising a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 having the sequences described above in this application, and the light chain variable region comprises CDR1, CDR2, and CDR3 having the sequences described above in this application. Although these antibodies contain the V of the monoclonal antibody of this application... H and V L CDR sequences can contain different backbone sequences.

[0084] Such backbone sequences can be obtained from publicly available DNA databases or references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be obtained from the Vbase Human Germline Sequence Database (www.mrc-cpe.cam.ac.uk / vbase), etc. Alternatively, germline DNA sequences for human heavy and light chain variable region genes can be obtained from the Genbank database.

[0085] The antibody protein sequence was compared with a protein sequence database using one of the sequence similarity search methods known in the art as gap BLAST.

[0086] The preferred backbone sequence used for the antibody in this application is one that is structurally similar to the backbone sequence used for the antibody in this application. HCDR1, CDR2, and CDR3 sequences can be inserted into a backbone region that has the same sequence as the germline immunoglobulin gene from which the backbone sequence is derived, or the CDR sequence can be inserted into a backbone region containing one or more mutations compared to the germline sequence. For example, in some cases, it is beneficial to mutate residues in the backbone region to maintain or enhance the antigen-binding properties of the antibody (see, for example, US Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0087] Another type of variable region modification is to modify V H and / or V L Amino acid residues within the CDR1, CDR2, and / or CDR3 regions are mutated to improve one or more binding properties (e.g., affinity) of the target antibody. Mutations can be introduced through point mutations or PCR-mediated mutations, and their effects on antibody binding or other functional properties can be evaluated using in vitro or in vivo assays known in the art. Preferably, conserved modifications known in the art are introduced. Mutations can be amino acid substitutions, additions, or deletions, but substitution is preferred. Furthermore, typically no more than one, two, three, four, or five residues within the CDR regions are altered.

[0088] The genetically modified antibody in this application includes V H and / or V L Genetic modifications are made to the backbone residues of an antibody to alter its properties, for example. Backbone modifications include mutating one or more residues in the backbone region, or even one or more CDR regions, to remove T-cell epitopes, thereby reducing the antibody's potential immunogenicity. This method is also known as "deimmunization," and is described in more detail in U.S. Patent Publication 20030153043.

[0089] In addition to modifications within the backbone or CDR region, the antibodies of this application can be genetically modified to include gene modifications in the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antibody-dependent cytotoxicity. Furthermore, the antibodies of this application can be chemically modified (e.g., by attaching one or more chemical functional groups to the antibody), or modified to alter its glycosylation, to change one or more functional properties of the antibody.

[0090] In one implementation, C H1 The hinge region is modified, altered, for example, by increasing or decreasing the number of cysteine ​​residues in the hinge region. This method is further described in U.S. Patent 5,677,425. Modification of C... H1Cysteine ​​residues in the hinge region can, for example, promote the assembly of heavy and light chains or increase / decrease antibody stability.

[0091] In another embodiment, the Fc hinge region of the antibody is mutated to increase or decrease the antibody's biological half-life. More specifically, one or more amino acid mutations are introduced into the C13C ... H2 -C H3 The linker region thus weakens the SpA binding affinity of the antibody relative to the natural Fc-hinge domain. This method is described in more detail in U.S. Patent 6,165,745.

[0092] In another embodiment, the glycosylation of the antibody is modified. For example, deglycosylated antibodies (i.e., antibodies lacking glycosylation) can be prepared. Glycosylation can be altered to, for example, increase the antibody's affinity for the antigen. Such glycosylation modification can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be made to eliminate one or more variable region backbone glycosylation sites, thereby eliminating glycosylation at that location. Such deglycosylation can increase the antibody's affinity for the antigen. See, for example, U.S. Patents 5,714,350 and 6,350,861.

[0093] Furthermore, antibodies with altered glycosylation types can be prepared, such as low-fucosylated antibodies with reduced fucose residues, or antibodies with increased bisecting GlcNac structures. The altered glycosylation forms have been shown to increase the ADCC activity of the antibodies. Such glycosylation modifications can be performed, for example, by expressing the antibody in host cells with altered glycosylation systems. Cells with altered glycosylation systems are known in the art, including, but not limited to, Slc35c1 gene knockout cell lines, FUT8 knockout cell lines, mutant CHO cell line Lec13, rat fusion tumor cell line YB2 / 0, cell lines containing small interfering RNA specifically targeting the FUT8 gene, and cell lines co-expressing β-1,4-N-acetylglucosyltransferase III and Golgi α-mannosidase II. These can be used as host cells for expressing the recombinant antibody of this application to prepare antibodies with altered glycosylation. Slc35c1 gene knockout cell lines, for example, utilize the fucose knockout platform technology independently developed by Tianguangshi, see the CHO cell line with accession number CGMCC No. 14287 in US Patent No. 10377833B2.

[0094] Another modification to the antibody described herein is polyethylene glycol (PEGylation). Antibodies can be PEGylated, for example, to increase the antibody's biological (e.g., serum) half-life. To PEGylate an antibody, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that attach one or more PEG groups to the antibody or antibody fragment. Preferably, PEGylation is carried out by an acylation or alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). The term "polyethylene glycol" as used herein includes any form of PEG used to derive other proteins, such as mono(C1-C1) PEG. 10 Alkyl- or aryl-oxy polyethylene glycol or polyethylene glycol maleimide. In some embodiments, the antibody to be PEGylated is a deglycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of this application. See, for example, EPO 154 316 and EP 0 401 384.

[0095] The antibodies of this application can be characterized by a variety of physical properties to detect and / or differentiate their classification.

[0096] For example, antibodies may contain one or more glycosylation sites in the variable region of the light or heavy chain. These glycosylation sites may cause increased antibody immunogenicity or altered antibody pK values ​​due to changed antigen binding. Glycosylation is known to occur in motifs containing NXS / T sequences. In some cases, APRIL antibodies are preferably free of variable region glycosylation. This can be achieved by selecting antibodies that do not contain glycosylated motifs in the variable region or by mutating residues in the glycosylated region.

[0097] In a preferred embodiment, the antibody does not contain an asparagine isomer site. Deamidation of asparagine may occur in the NG or DG sequence, creating isoaspartic residues that introduce kinks into the polypeptide chain and reduce its stability (isoaspartic effect).

[0098] On the other hand, this application provides nucleic acid molecules encoding heavy / light chain variable regions or CDRs of the antibody or its antigen-binding moiety of this application. The nucleic acid may be present in whole cells, in cell lysates, or in partially purified or substantially pure forms. When purified from other cellular components or other contaminants such as other cellular nucleic acids or proteins using standard techniques, the nucleic acid is "isolated" or "substantially pure." The nucleic acid of this application may be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0099] The nucleic acids used in this application can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes), the cDNA encoding the light and heavy chains of the hybridoma-prepared antibody can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), the nucleic acids encoding such antibodies can be collected from the gene library.

[0100] Preferred nucleic acid molecules of this application include V encoding an APRIL monoclonal antibody. H and V L Those sequences or CDRs. Once the encoded V is obtained... H and V L DNA fragments that can be further manipulated using standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these operations, the gene encoding V... H or V L A DNA fragment can be operatively linked to another DNA fragment encoding a different protein, such as an antibody constant region or a flexible linker. The term "operatively linked" means that two DNA fragments are joined together such that the amino acid sequences encoded by both DNA fragments are within the reading frame.

[0101] Encoding V H The isolated DNA from the region can be operatively linked to V. H Encoding DNA and Encoding Heavy Chain Constant Regions (C H1 C H2 and C H3 Another DNA molecule is transformed into a full-length heavy chain gene. The sequences of human heavy chain constant regions are known in the field, and DNA fragments including these regions can be obtained by standard PCR amplification. Heavy chain constant regions can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant regions, but are preferably IgG1 or IgG4 constant regions. For Fab fragment heavy chain genes, encoding V... H The DNA of this region can be operatively coupled with DNA that encodes only the heavy chain C. H1 Another DNA molecule is linked to the constant region.

[0102] Encoding V L The isolated DNA from the region can be operatively linked to V. L Encoding DNA and Encoding Light Chain Constant Region C LAnother DNA molecule is transformed into a full-length light chain gene. The sequences of human light chain constant regions are known in the field, and DNA fragments including these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant regions may be κ and λ constant regions.

[0103] To create the scFv gene, which encodes V H and V L The DNA fragment can be operatively linked to another fragment encoding a flexible adapter, thereby enabling V H and V L The sequence can be expressed as a continuous single-chain protein, where V H and V L The areas are connected by this flexible joint.

[0104] The monoclonal antibody of this application can be prepared using the somatic cell hybridization (hybridoma) technique described in Kohler and Milstein (1975) Nature 256:495. Other methods for preparing monoclonal antibodies include viral or oncogenic transformation of B lymphocytes and phage display techniques. Chimeric or humanized antibodies are also well known in the art. See, for example, U.S. Patents 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370.

[0105] The antibody or its antigen-binding portion of this application can also be generated in host cells transfected with tumors using, for example, recombinant DNA technology combined with gene transfection methods (e.g., Morrison, S. (1985) Science 229:1202). In one embodiment, coding portions or full-length light and heavy chain DNA obtained by standard molecular biotechnology are inserted into one or more expression vectors, thereby operatively linking the gene with transcriptional and translational regulatory sequences. In this case, the term "operatively linked" means that the antibody gene is linked to the vector so that the transcriptional and translational control sequences within the vector perform their intended functions of regulating antibody gene transcription and translation.

[0106] The term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody genes. Such regulatory sequences have been described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred regulatory sequences for mammalian host cell expression include viral elements that guide high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenoviruses such as the adenovirus major late promoter (AdMLP), and polyomaviruses. Alternatively, non-viral regulatory sequences, such as ubiquitin promoters or β-globin promoters, can be used. Additionally, regulatory elements can be composed of sequences from diverse sources, such as the SRα promoter system, which contains sequences from the SV40 early promoter and long terminal repeats from human T-cell leukemia type I virus. The expression vector and expression control sequence are selected to be compatible with the expression host cells used.

[0107] Antibody light chain genes and antibody heavy chain genes can be inserted into the same or different expression vectors. In a preferred embodiment, the variable region is used to construct a full-length antibody gene by inserting it into an expression vector that already encodes the heavy chain constant region and light chain constant region of the desired isotype, thereby V H With C in the carrier H Operable connection, V L With C in the carrier L Operable linking. Alternatively, the recombinant expression vector can encode a signal peptide that promotes the secretion of antibody chains from host cells. The antibody chain gene can be cloned into the vector, allowing the signal peptide to link to the amino terminus of the antibody chain gene within the reading frame. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin).

[0108] In addition to the antibody chain gene and regulatory sequence, the recombinant expression vector of this application may carry other sequences, such as sequences regulating vector replication in host cells (e.g., replication origin) and selectable marker genes. Selectable marker genes can be used to select host cells into which the vector has been introduced (see, for example, U.S. Patents 4,399,216; 4,634,665 and 5,179,017). For example, selectable marker genes typically confer drug resistance, such as resistance to G418, hygromycin, or methotrexate, to host cells into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for methotrexate selection / amplification in DHFR host cells) and the neo gene (for G418 selection).

[0109] For the expression of the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells using standard techniques. The term "transfection" encompasses various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, and DEAE-dextrose transfection. Although expression of the antibody described herein is theoretically feasible in prokaryotic or eukaryotic host cells, expression in eukaryotic cells is preferred, and most preferably in mammalian host cells, because eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete appropriately folded and immunologically active antibodies.

[0110] Preferred mammalian host cells for expressing the recombinant antibodies of this application include Slc35C1 knockout cell lines, FUT8 knockout cell lines, variant CHO cell line Lec13, rat fusion tumor cell line YB2 / 0, cell lines containing small interfering RNA specifically targeting the FUT8 gene, cell lines co-expressing β-1,4-N-acetylglucosidase III and Golgi α-mannosidase II, Chinese hamster ovary (CHO cells) (including dhfr-CHO cells administered with DHFR selectable markers, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, DHFR selectable markers described, for example, in RJ Kaufman and PASharp (1982) J. Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into mammalian host cells, antibodies are prepared by culturing the host cells for a period of time sufficient for antibody expression in the host cells, or preferably sufficient for antibody secretion into the culture medium in which the host cells grow. The antibodies can be recovered from the culture medium using protein purification methods.

[0111] On the other hand, this application relates to bispecific molecules comprising one or more antibodies of this application or their antigen-binding moieties linked to at least one other functional molecule, such as another peptide or protein (e.g., another antibody or receptor ligand), to generate bispecific molecules that bind to at least two different binding sites or target molecules. The term "bispecific molecule" includes molecules having three or more specificities. Bispecific molecules can appear in a variety of forms and sizes. At one end of the size spectrum, bispecific molecules retain the traditional antibody form, except that they have two binding arms, each with different specificities, instead of two binding arms with the same specificity. At the other extreme are bispecific molecules consisting of two single-chain antibody fragments (scFv) linked by peptide chains, referred to as the Bs(scFv)2 construct. Intermediate-sized bispecific molecules comprise two different F(ab) fragments linked by peptide linkers. These and other forms of bispecific molecules can be prepared by genetic engineering, somatic cell hybridization, or chemical methods.

[0112] In another aspect, this application provides a pharmaceutical composition comprising the antibody of this application or its antigen-binding moiety, a bispecific antibody, a nucleic acid molecule, an expression vector, and / or a host cell, formulated together with a pharmaceutically acceptable carrier. The composition may optionally contain one or more other pharmaceutically active ingredients.

[0113] Pharmaceutical compositions may contain any number of excipients. Excipients that may be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersants or suspending agents, solubilizers, colorants, flavoring agents, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of appropriate excipients are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003).

[0114] The pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or bolus). Depending on the route of administration, the active ingredient may be encapsulated in a material to protect it from acids and other natural conditions that may inactivate it. “Parenteral administration” refers to a method other than intestinal and topical application, typically administered by injection, including but not limited to intravenous, intramuscular, intraarterial, intramembranous, intracystic, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcystic, subarachnoid, spinal, supradural, and intrasternal injections and boluses. Alternatively, the antibody of this application may be administered via non-parenteral routes, such as topical, epidermal, or mucosal administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical application.

[0115] Pharmaceutical compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated in microemulsions, liposomes, or other ordered structures suitable for high concentrations of drugs.

[0116] The amount of active ingredient prepared together with the carrier material into a single dosage form will vary depending on the therapeutic subject and specific administration mode, and is essentially the amount of the composition that produces the therapeutic effect. In percentage terms, this amount is approximately 0.01% to approximately 99% of the active ingredient bound to a pharmaceutically acceptable carrier.

[0117] The dosing regimen is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a rapid infusion can be administered, multiple fractions can be administered over time, or the dose can be proportionally reduced or increased depending on the severity of the treatment condition. Particularly advantageous are parenteral compositions formulated in convenient and uniformly dose-unit formats. A dose-unit format refers to physically separate units suitable for a single dose to the therapeutic subject; each unit contains a predetermined amount of active ingredient calculated to produce the desired therapeutic effect when used with the pharmaceutical carrier. Alternatively, antibodies can be administered as sustained-release formulations, in which case the required dosing frequency is reduced.

[0118] For antibody administration, the dosage can be approximately 0.001-100 mg / kg of host body weight. An exemplary treatment regimen involves administration once weekly.

[0119] The "therapeutic effective amount" of the APRIL antibody in this application causes a reduction in the severity of disease symptoms and an increase in the frequency and duration of asymptomatic periods. For example, in the treatment of IgAN subjects, the "therapeutic effective amount" preferably inhibits the production of autoantibodies targeting Gd-IgA1 or proteinuria by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80%, compared to untreated subjects.

[0120] The pharmaceutical composition may be a sustained-release agent, including implants and microcapsule delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0121] Pharmaceutical compositions can be administered via medical devices, such as (1) needle-free subcutaneous injection devices (e.g., U.S. Patents 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, and 4,596,556); (2) microinfusion pumps (U.S. Patent 4,487,603); (3) transdermal drug delivery devices (U.S. Patent 4,486,194); (4) bolus injection devices (U.S. Patents 4,447,233 and 4,447,224); and (5) permeation devices (U.S. Patents 4,439,196 and 4,475,196).

[0122] In some embodiments, the monoclonal antibody of this application may be formulated to ensure suitable in vivo distribution. For example, to ensure that the therapeutic antibody of this application crosses the blood-brain barrier, the antibody may be formulated in liposomes, which may also additionally contain targeting functional groups to enhance selective delivery to specific cells or organs. See, for example, U.S. Patents 4,522,811, 5,374,548, 5,416,016, and 5,399,331.

[0123] The pharmaceutical composition of this application has various in vitro and in vitro-in vivo applications, such as in vivo treatment of APRIL-related diseases. The pharmaceutical composition can be administered to human subjects.

[0124] This application provides a method for treating or alleviating APRIL-related disease in a subject, including administering a therapeutically effective amount of the pharmaceutical composition of this application to the subject.

[0125] APRIL-related diseases can be inflammatory diseases, such as autoimmune diseases. In some embodiments, APRIL-related diseases are autoimmune diseases associated with B cell dysfunction. In some embodiments, APRIL-related diseases are immunoglobulin A nephropathy (IgAN).

[0126] APRIL-related diseases can be tumors, such as B-cell-related tumors, including multiple myeloma. In some embodiments, the method may also include administering antibodies against immune checkpoints such as PD-1 or CTLA-4.

[0127] This application provides a combination therapy in which the pharmaceutical composition of this application is administered in conjunction with one or more other antibody or non-antibody therapeutic agents. For example, it can be administered in conjunction with BAFF antibodies, or with immune checkpoint antibodies, or other anticancer agents.

[0128] The combination of therapeutic agents discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as separate compositions, wherein each agent is contained in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents can be administered sequentially.

[0129] Furthermore, if multiple combination therapies are administered and the drugs are administered sequentially, the order of administration at each time point can be reversed or kept the same, and sequential administration can be combined with simultaneous administration or any combination thereof.

[0130] This application is further described through the following embodiments, which should not be construed as limiting. All figures, all references, Genebank sequences, patents, and published patent applications cited throughout this application are incorporated herein by reference in their entirety.

[0131] Example

[0132] Example 1. Preparation of APRIL trimer-biotin

[0133] The monomeric APRIL was ligated via a linker to synthesize a human APRIL trimer protein with a higher receptor-binding affinity than the monomer. In short, the cDNA sequence encoding the human APRIL trimer (SEQ ID NO:23) was cloned into the GS plasmid via double digestion with Cla1 and Xhol to construct the expression plasmid. Using polyethyleneimine (PEI) (Cat#:24765-1, Kyfora Bio), the plasmid, culture medium, and PEI were mixed thoroughly according to the manufacturer's instructions, allowed to stand for 10 minutes, and then added dropwise to CHO cells at a cell density of 6 × 10⁶ cells / cells. 6 The DNA:PEI ratio was approximately 1:4, and the total DNA used for transfection was 1.5 μg / ml. Transfected CHO cells were cultured at 120 RPM in a 37°C, 5% CO2 incubator.

[0134] After 10-12 days, the culture supernatant was aseptically harvested for further protein purification. Specifically, the cell culture supernatant was collected, centrifuged at 3500 rpm for 5 minutes, and filtered through a 0.22 μm filter to remove cell debris. The nickel column (Cat#:17524801, Cytiva) was then equilibrated with PBS, washed with 10 column volumes, and the filtered supernatant was loaded onto the column. The column was then washed with PBS containing 25 mM imidazole, and finally eluted with elution buffer (containing 250 mM imidazole). The eluted APRIL trimer protein was concentrated and further purified using a Superdex 200 gel filtration column (Cytiva, CAT#28990944) to remove polymers and impurities. APRIL trimer protein of the correct size was collected, and its concentration was determined using a NanoDrop analyzer (Cat#:840-317400, Thermo).

[0135] Using the NHS-PEG4-Biotinylation Kit (Cat#:21455, Thermo), APRIL trimer protein and NHS-PEG4-Biotin were mixed at a molar ratio of 1:20 and reacted at room temperature for 30-60 minutes. Afterward, the mixture was added to a desalting column and centrifuged at 1000g for 2 minutes to remove excess NHS-PEG4-Biotin.

[0136] Example 2. Preparation of MIL116 antibody

[0137] A series of APRIL antibodies were obtained through mouse immunization, phage library display screening, and affinity maturation techniques. After extensive screening, a high-affinity humanized APRIL antibody, MIL116, was obtained, and its amino acid sequences of the heavy and light chain variable regions are shown in SEQ ID NO:7 and 8, respectively.

[0138] MIL116 was expressed in CHO cells using the constant region of the human IgG1 heavy chain (SEQ ID NO: 9) and the constant region of the human κ chain light chain variable region (SEQ ID NO: 18). The BION-1301 analog, VIS649 analog, and ALPN-303 (TACI domain-Fc fusion protein) analog, used as controls, were also expressed in CHO cells. The sequences of the heavy and light chain variable regions of the BION-1301 analog are shown in SEQ ID NO: 19 and 20, respectively; the constant regions of the heavy and light chains are shown in SEQ ID NO: 13 and 18, respectively. The sequences of the heavy and light chain variable regions of the VIS649 analog are shown in SEQ ID NO: 21 and 22, respectively; the constant regions of the heavy and light chains are shown in SEQ ID NO: 17 and 18, respectively. The sequence of the ALPN-303 analog is shown in SEQ ID NO: 167 of WO2024077018A2. These proteins were purified after expression.

[0139] Example 3. Binding activity of MIL116 antibody with human and monkey APRIL

[0140] The binding affinity of MIL116 to human and monkey APRIL protein was determined by ELISA.

[0141] Specifically, the human APRIL trimer-biotin protein or monkey APRIL protein (Cat#: APL-C52D2, ACRO) prepared in Example 1 was diluted with PBS to a final concentration of 1 μg / mL, and the diluted solution was added to a 96-well ELISA plate, 100 μL per well, and incubated overnight at 4°C. The ELISA plate was blocked with PBST containing 2% BSA, 200 μL / well, at 37°C for 1 hour. The ELISA plate was washed with PBST, 200 μL / well, 3 times. MIL116 and the positive control were diluted with PBST at pH 7.4, starting from 100 μg / mL, using a 3-fold or 5-fold serial dilution, for a total of 11 or 8 dilution gradients. The diluted samples were added to the blocked ELISA plate, 100 μL / well, and incubated at 37°C for 1 hour. Wells containing only the diluted PBST solution served as negative controls. Wash the ELISA plate three times with PBST (pH 7.4), 200 μL / well, for 2 minutes each time. Add 100 μL of HRP-labeled goat anti-human IgG monoclonal antibody (1:5000 dilution, Cat#:31413, Thermofisher) to the ELISA plate and incubate at 37°C for 1 hour. Wash the ELISA plate four times with PBST, 200 μL / well. Add 100 μL of TMB substrate chromogenic solution (Cat#:555214, BD) to the ELISA plate, allow to stand for color development, and then stop the color development with 100 μL of 10% sulfuric acid. Measure the absorbance at 450 nm. Analyze the data using GraphPad software.

[0142] The results are shown in Figure 1 (A and B). At pH 7.4, the MIL116 antibody showed strong binding to human (A) and monkey (B) APRIL protein, with a slightly higher maximum binding (Bmax) compared to the positive control.

[0143] Example 4. Binding affinity of MIL116 antibody to human and monkey APRIL.

[0144] The binding affinity of the MIL116 antibody to human or monkey APRIL protein was determined using BIAcore 8K.

[0145] Specifically, the his antibody (Cat#:BR100839, cytiva) was conjugated to the surface of the CM5 chip. Human (Cat#:APL-H52D1, ACRO) or monkey (Cat#:APL-C52D2, ACRO) APRIL-His protein was diluted to 1 μg / mL with HBS-EP buffer (Cat#:BR-1006-69, GE Life Sciences) at pH 7.4, ensuring that approximately 100 RU of antigen was captured by the his antibody-conjugated chip. The MIL116 antibody to be tested and the positive control were diluted to different concentrations (20, 10, 5, 2.5, 1.25, 0.625, 0.3125 μg / mL) with HBS-EP buffer at pH 7.4, and the antibody proteins of different concentrations were flowed through the surface of the stationary phase. The chip was regenerated using glycine hydrochloride solution.

[0146] Kinetic analysis was performed using the Wizard tool in the Biacore 8K control software, and the binding K of the detection antibody to APRIL was obtained by fitting the data using the Biacore 8K evaluation software. D The values ​​are shown in Table 1.

[0147] As shown in Table 1, the MIL116 antibody exhibits a similar affinity for human or monkey APRIL to the BION-1301 analogue. Furthermore, MIL116 binds to APRIL proteins of both human and monkey origin with similar affinity, making it convenient to use monkeys as subjects in preclinical experimental studies.

[0148] Table 1. Binding affinity of APRIL antibody to human or monkey APRIL

[0149] Example 5. Activity of APRIL antibody to block ligand (APRIL)-receptor (BCMA or TACI) binding / interaction.

[0150] Dilute human BCMA-HIS (Cat#: BCA-HP2H2, ACRO) to a final concentration of 1 μg / ml with PBS; dilute human TACI-Fc (Cat#: TAI-H5253, ACRO) to a final concentration of 4 μg / ml; dilute monkey BCMA-Fc (Cat#: BCA-C5253, ACRO) to a final concentration of 4 μg / ml. Coat each well with the diluent using 100 μl of the diluent and incubate overnight at 4°C. Discard the liquid and block the ELISA plates with PBST containing 2% BSA, 200 μL / well, at 37°C for 1 hour. Simultaneously with blocking, the antibody to be tested was serially diluted 3-fold, starting at a concentration of 200 μg / ml, with a total of 11 dilutions. Human APRIL-Biotin (Cat#: APL-H52D1, ACRO) or monkey APRIL-HIS (Cat#: APL-C52D2, ACRO) 200 ng / ml was added, and the plate was incubated at 37°C for 1 hour. The ELISA plate was washed with PBST 200 μL / well, three times. The antibody-APRIL mixed sample was added to the blocked ELISA plate, 100 μL / well, and incubated at 37°C for 1 hour. The ELISA plate was washed with PBST 200 μL / well, three times. Add 100 μL of 1:5000 diluted HRP-labeled streptavidin (Cat#:BIR701, Beijing Bosi Technology Co., Ltd.) or HRP-labeled HIS antibody (Cat#:CW0285, CWBIO) to the ELISA plate and incubate at 37°C for 1 hour. Wash the ELISA plate with PBST, 200 μL / well, washing 3 times. Add 100 μL of TMB substrate chromogenic solution (Cat#:555214, BD) to the ELISA plate, allow to stand for color development, and then stop the color development with 100 μL of 10% sulfuric acid. Measure the absorbance at 450 nm. Process and analyze the data using GraphPad software.

[0151] The experimental results are shown in Figure 2 (AC). The MIL116 antibody significantly inhibited the binding of human APRIL to its receptor BCMA (A) or TACI (B), with activity comparable to the positive control antibody; MIL116 also had the ability to block monkey APRIL-BCMA binding comparable to the two positive control antibodies (C).

[0152] Example 6. Fc modification of MIL116 antibody

[0153] To reduce the rate of antibody clearance in vivo, the Fc region of MIL116 was modified to enhance its binding affinity to the FcRn receptor and prolong its in vivo half-life.

[0154] To select suitable Fc mutants, the variable region sequence of the BION-1301 antibody was combined with the heavy chain constant region containing different mutation combinations M252Y / S254T / T256E (YTE), M428L / N434S (LS), or M428L / N434A (LA), followed by functional verification. Specifically, the heavy chain constant region used IgG4-wt (wild type) as shown in SEQ ID NO:13, IgG4-m1 (LA) as shown in SEQ ID NO:14, IgG4-m2 (LS) as shown in SEQ ID NO:15, or IgG4-m3 (YTS) as shown in SEQ ID NO:16, and the light chain constant region used the κ light chain constant region of SEQ ID NO:18.

[0155] The affinity of BION-1301 containing three mutant Fc cells for human FcRn at pH 5.8 was determined using a Biacore 8K instrument. The specific experimental method was the same as in Example 4, with slight adjustments. Human FcRn-his (Cat#:FCN-H52W7, ARCO) was diluted to 1 μg / mL with HBS-EP buffer (Cat#: BR-1006-69, GE Life Sciences) at pH 5.8 to ensure that approximately 10 RU of FcRn was captured by the chip coupled with the his antibody. The test antibody was diluted to different concentrations (20, 10, 5, 2.5, 1.25, 0.625, 0.3125 μg / mL) with HBS-EP buffer at pH 5.8, and the protein at different concentrations was flowed through the stationary phase surface. The chip was regenerated using glycine hydrochloride solution. Kinetic analysis was performed using the Wizard in the Biacore 8K control software.

[0156] The experimental results are shown in Table 2. Compared with the antibody containing the unmodified Fc (WT), the antibody variants containing the three modified Fcs all showed increased affinity for FcRn, and the antibodies containing LA and LS mutations showed slightly higher affinity for FcRn than the antibody containing YTE. Considering the immunogenicity and physicochemical stability of the modified antibody, the Fc containing the M428L / N434A (LA) mutation was finally selected.

[0157] Table 2. Affinity of antibodies containing different Fc groups to human FcRn

[0158] Subsequently, the Fc region of MIL116 containing IgG1 was modified to reduce the antibody clearance rate in vivo. On one hand, the L234A / L235A mutation was introduced to remove the binding between the antibody and the FcRγ receptor, reducing the binding of the antibody to peripheral lymphocytes and other cells, thus eliminating potential antibody-dependent cell-mediated killing (ADCC) and complement-dependent killing (CDC) of APRIL-positive cells. On the other hand, the M428L / N434A mutation was introduced to increase the affinity for FcRn under acidic conditions. A vector expressing MIL116 containing the heavy chain variable region of SEQ ID NO:7, the light chain variable region of SEQ ID NO:8, the heavy chain constant region IgG1-m1 containing L234A / L235A / M428L / N434A of SEQ ID NO:10, and the light chain constant region of SEQ ID NO:18 was constructed, expressed in CHO cells, and purified.

[0159] Following the above method and steps, the affinity of the modified MIL116, as well as the VIS649 analog and the BION1301 analog, for human FcRn was determined using a Biacore 8K instrument at pH 5.8.

[0160] The results are shown in Table 3. The affinity of the Fc-modified MIL116 for human FcRn was much higher than that of the two positive control antibodies. Specifically, the affinity of the Fc-modified MIL116 for FcRn was about 20 times higher than that of the VIS649 analog and about 200 times higher than that of the BION-1301 analog.

[0161] Table 3. Affinity of different APRIL antibodies to human FcRn

[0162] Example 7. Receptor-ligand species crossover experiment

[0163] The binding affinity of MIL116, which contains the heavy chain constant region IgG4-m1 (SEQ ID NO:14) and the light chain constant region SEQ ID NO:18, to mouse APRIL protein was determined by ELISA.

[0164] Specifically, referring to the method steps of Example 3, the concentration of mouse APRIL (Cat#:APR-MM113, Kaika Bio) after dilution was 1 μg / mL, and mouse BCMA-Fc (Cat#:BCA-M5258, ACRO) was used as a positive control.

[0165] As shown in Figure 3(A), MIL116 did not bind to mouse APRILs with either of the two positive control antibodies.

[0166] Furthermore, literature reports that human APRIL exhibits comparable binding activity to mouse BCMA or TACI receptors compared to mouse APRIL. In this embodiment, the binding activity of the human APRIL trimer-biotin prepared in Example 1 with human, monkey, and mouse BCMA was determined by ELISA. Specifically, human BCMA-HIS (Cat#:BCA-HP2H2, ACRO), mouse BCMA-FC (Cat#:BCA-M5258, ACRO), or monkey BCMA-FC (Cat#:BCA-C5253, ACRO) was diluted with PBS to a final concentration of 1 μg / ml, and 100 μl of the diluent was used to coat ELISA plates overnight at 4°C. The liquid was discarded, and the ELISA plates were blocked with PBST containing 2% BSA, 200 μl / well, at 37°C for 1 hour. The ELISA plates were washed three times with PBST, 200 μl / well. Human APRIL trimer-biotin (prepared in Example 1) was prepared to a concentration of 100 μg / ml and added to a sealed ELISA plate at 100 μL / well, incubated at 37°C for 1 hour. The ELISA plate was washed three times with PBST at 200 μL / well. 100 μL of HRP-labeled streptavidin (Cat#: BIR701, Beijing Bosi Technology Co., Ltd.) was added to the ELISA plate and incubated at 37°C for 1 hour. The ELISA plate was washed three times with PBST at 200 μL / well. 100 μL of TMB substrate chromogenic solution (Cat#: 555214, BD) was added to the ELISA plate, allowed to stand for color development, and then the color development was terminated with 100 μL of 10% sulfuric acid. The absorbance was measured at 450 nm. Data were processed and analyzed using GraphPad software.

[0167] The results are shown in Figure 3(B). The binding activity of the human APRIL trimer-biotin prepared in Example 1 with human, monkey, and mouse BCMA was consistent.

[0168] Based on the results in Figure 3 (A and B), which show that MIL116 does not bind to mouse APRIL, but human APRIL has comparable binding activity to human, monkey, and mouse BCMA, it is believed that human APRIL protein can be used in mouse models to evaluate the clearance effect of MIL116 on APRIL and to perform antibody pharmacokinetic (PK) assays.

[0169] Example 8. Rapid clearance of human APRIL mediated by MIL116 in wild-type C57 mice

[0170] In wild-type C57 mice, the in vivo clearance efficiency of MIL116 containing the heavy chain constant region IgG4-m1 (SEQ ID NO:14) and the light chain constant region SEQ ID NO:18 was tested against APRIL.

[0171] C57 mice (Vitalliwa) were randomly divided into 8 groups. Group 1 consisted of 7 mice, administered MIL116 (10 mg / kg) and APRIL (human APRIL trimer-biotin prepared in Example 1, 15 mg / kg); Group 2 consisted of 7 mice, administered BION1301 analog (10 mg / kg) and APRIL (15 mg / kg); Group 3 consisted of 7 mice, administered VIS649 analog (10 mg / kg) and APRIL (15 mg / kg); Group 4 consisted of 7 mice, administered APRIL (15 mg / kg); Group 5 consisted of 3 mice, administered MIL116 (10 mg / kg); Group 6 consisted of 3 mice, administered BION-1301 analog (10 mg / kg); Group 7 consisted of 3 mice, administered VIS649 analog (10 mg / kg); and Group 8 consisted of 3 mice, administered PBS, serving as the negative control group.

[0172] Using sterile capillaries, blood was collected from the orbital venous plexus of mice in each group, approximately 60-80 μL each time, recorded as 0h. Mice requiring antibody administration were subcutaneously injected with 10 mg / kg of antibody. Blood was collected from the orbital venous plexus again at 24h. At 36h, some groups of mice were subcutaneously injected with 15 mg / kg of April protein, and blood was collected from the orbital venous plexus at 38h, 40h, 42h, and 44h (i.e., 2 hours after April injection).

[0173] The collected blood samples were centrifuged to obtain serum, which was stored at -40°C. The concentration of APRIL in the blood samples at different time points was then detected using ELISA. Specifically, recombinant streptavidin protein (CAT#:STN-N5116, Bipsys) was diluted in 1×PBS buffer to a final concentration of 1 μg / ml, and then added to the microplate at a rate of 50 μl / well. The plate was incubated horizontally at 37°C for 2 hours. The microplate was then removed and washed three times with PBST. 200 μl of blocking buffer (PBS containing 3% BSA) was added to each well, and the plate was incubated horizontally at 37°C for 2 hours. The serum samples were then diluted with the blocking buffer to a dilution factor of 5. The April-Biotin standard curve concentration started at 500 ng / mL, with eight dilutions: 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.25 ng / mL, 15.625 ng / mL, 7.8125 ng / mL, and 3.90625 ng / mL. After blocking, the ELISA plate was removed and washed three times with PBST to remove any residual droplets. 50 μL of diluted serum sample or standard curve sample was added to each well, and the plate was sealed with a sealing film and incubated horizontally at 4°C overnight. The plate was then removed from the 4°C freezer and washed three times with PBST. The VIS649 analogue was diluted to 10 μg / mL, and 50 μL was added to each well. The plate was sealed with a sealing film and incubated horizontally at 37°C for 1 hour. Remove the ELISA plate, wash it three times with PBST, and add 50 μL / well of goat anti-hIgG-Fc-HRP (CAT#A0170-1ML, Sigma) diluted 5000 times with PBS containing 3% BSA. Incubate horizontally at 37°C for 1 hour. Remove the ELISA plate and wash it three times with PBST. Add 50 μL of TMB chromogenic solution to each well. After about 10 minutes, observe the color development and promptly add 50 μL of 2N H2SO4 stop solution to terminate the reaction. Read the OD value at 450 nm using an ELISA reader. Calculate the concentration values ​​and plot the APRIL clearance curve.

[0174] The results are shown in Figure 4. Compared to BION-1301 and VIS649 analogs, MIL116 rapidly and persistently reduced APRIL protein in the peripheral circulation. This is likely because MIL116 can more quickly induce endocytosis of the APRIL-antibody complex in FcRn-expressing cells, rapidly dissociate from APRIL under low pH (endosome) conditions, and subsequently re-enter the circulation.

[0175] Example 9. Study on the clearance rate of MIL116 antibody in FcRn humanized mice

[0176] The blood clearance and degradation rate of MIL116 containing the heavy chain constant region IgG1-m1 (SEQ ID NO:10) and the light chain constant region SEQ ID NO:18 were tested in B6-FcRn humanized mice (Biocytogen).

[0177] B6-FcRn humanized mice were randomly divided into 9 groups. Group 1 (6 mice) was administered MIL116 (10 mg / kg) and APRIL (human APRIL trimer-biotin prepared in Example 1, 15 mg / kg); Group 2 (6 mice) was administered BION1301 analog (10 mg / kg) and APRIL (15 mg / kg); Group 3 (6 mice) was administered VIS649 analog (10 mg / kg) and APRIL (15 mg / kg); Group 4 (5 mice) was administered... ALPN303 analog (10 mg / kg) and APRIL (15 mg / kg) were administered to mice. Group 5 (3 mice) received MIL116 (10 mg / kg); Group 6 (3 mice) received BION1301 analog (10 mg / kg); Group 7 (3 mice) received VIS649 analog (10 mg / kg); Group 8 (3 mice) received ALPN303 analog (10 mg / kg); and Group 9 (3 mice) received PBS. A total of one antibody injection and four APRIL injections were administered.

[0178] As described in Example 8, blood was collected from the orbital venous plexus of mice before antibody injection to serve as the baseline antibody concentration, designated as the 0h sample. Simultaneously, mice in groups 1-8 were subcutaneously injected with the corresponding antibody; blood was collected from the orbital venous plexus 24 hours later; thereafter, blood was collected daily (i.e., at 48h, 72h, 96h, 120h, etc.). Mice requiring antigen injection received subcutaneous injection of APRIL protein at 42h, 114h, 210h, and 282h. Each blood collection volume was approximately 60-80 μL.

[0179] The collected blood samples were centrifuged to obtain serum, which was then stored at -40°C. The antibody concentration in the blood samples at different time points was then detected using ELISA. Specifically, goat anti-hIgG Fc (CAT#:12126-1ML, Sigma) was diluted in 1×PBS buffer to a final concentration of 1 μg / ml, and 100 μl was added to each well of an ELISA plate (CAT#:9018, Corning). The plate was incubated horizontally at 37°C for 2 hours. The plate was then removed from 37°C and washed three times with PBST. 200 μl of blocking buffer (PBS containing 3% BSA) was added to each well, and the plate was blocked overnight at 4°C. The serum samples were then diluted with the blocking buffer to a dilution factor of 1500. The antibody standard curve concentration started at 500 ng / mL, diluted 10 times to obtain concentrations of 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.25 ng / mL, 15.625 ng / mL, 7.8125 ng / mL, 3.90625 ng / mL, 1.953125 ng / mL, and 0.9765625 ng / mL. After blocking, the ELISA plate was removed and washed three times with PBST. 100 μL of diluted serum sample or standard curve sample was added to each well, the plate was sealed, and incubated horizontally at 4°C for 2 hours. The ELISA plate was then removed and washed three times with PBST. 100 μL of goat anti-hIgG-full-HRP (CAT#: A8667-2ML, Sigma) diluted 5000-fold with PBS containing 3% BSA was added to each well, and the plate was incubated horizontally at 37°C for 1 hour. Remove the ELISA plate and wash it three times with PBST. Add 100 μl of TMB chromogenic buffer to each well. After approximately 10 minutes, observe the color development and terminate the reaction if necessary. Read the OD value at 450 nm using an ELISA reader. Calculate the concentration and plot the antibody clearance curve.

[0180] As shown in Figure 5, without exogenous APRIL injection, MIL116 exhibited a relatively high serum antibody concentration, higher than that of BION1301, VIS649, and ALPN303 analogs, indicating good bioavailability, no degradation due to nonspecific binding, low clearance rate, and favorable antibody properties. After the first two exogenous APRIL injections, MIL116 maintained a relatively high concentration. After the third exogenous APRIL injection, the MIL116 antibody level decreased but remained significantly higher than other positive control antibodies. Serum levels of BION1301, VIS649, and ALPN303 analogs decreased rapidly after the second APRIL injection, with the concentrations of BION1301 and VIS649 analogs approaching baseline levels at the 384-hour detection point. The data above indicate that, compared to BION1301 analogs, VIS649 analogs, and ALPN303 analogs, MIL116 can induce the APRIL-antibody complex to dissociate from APRIL under low pH (late inclusion body) conditions more quickly after endocytosis, and has a stronger interaction with human FcRn, thereby significantly reducing APRIL-mediated endocytic degradation and clearance, and enabling the reuse of the antibody itself.

[0181] It is evident that, compared to the positive control, the Fc-modified MIL116 exhibits slower clearance and degradation in FcRn-humanized mice, resulting in a longer duration of effective drug action. Therefore, this antibody may enable lower dosing frequencies and improved treatment adherence in future clinical applications.

[0182] Example 10. ADA prediction and detection of MIL116 antibody

[0183] We used the immunogenicity prediction program on the Wemole platform (https: / / wemol.wecomput.com / ui / # / frontend / home / workflow-modules) to analyze T-cell epitopes in the variable region sequence of MIL116. The results showed that there may be an HLA-binding T-cell epitope in the light chain CDR1, i.e., an immunogenicity risk sequence.

[0184] Therefore, antibody fragments digested and presented by dendritic cells (DCs) isolated from human peripheral blood mononuclear cells (PBMCs) were used to assess the immunogenicity of autologous CD4+ T cells in vitro.

[0185] In short, PBMCs containing different high-frequency HLA subtypes from Chinese individuals were collected by gradient density centrifugation (HLA subtype information is shown in Table 4) and resuspended in RPMI 1640 medium. The PBMCs were cultured at 37°C for 20 minutes, and adherent cells were collected to obtain isolated monocytes. The monocytes were cultured in RPMI 1640 medium (CAT#:11875119, Thermo) supplemented with 100 ng / ml recombinant human GM-CSF (Cat#:7954-GM, R&D), 100 ng / ml recombinant human IL-4 (Cat#:6507-IL, R&D), and 10% FBS (CAT#A5256701, Thermo). After 3 days, half of the medium was replaced with fresh medium. On day 6 of culture, the culture medium was replaced with a medium containing 100 ng / ml recombinant human GM-CSF, 100 ng / ml recombinant human IL-4, 10 ng / ml rhTNF-α (Cat#:210-TA-100, R&D), 1000 U / ml rhIL-6 (Cat#:7270-IL-025, R&D), 1 μg / ml PGE2 (Cat#363-24-6, TOCRIS), and 10 ng / ml IL-1β (Cat#:210-LB-025, R&D). Cells were incubated for another 2 days and then divided into two groups for separate treatment. Group 1 was incubated with either MIL116 or BION-1301 at a final concentration of 150 μg / ml, or KLH positive control protein (CAT#:77600, Thermo) at 300 μg / ml, for another 2 days. KLH is a commonly used strong immunogenicity control, and PBS was used as a negative control. Group 2 was incubated with medium containing 100 ng / ml recombinant human GM-CSF, 100 ng / ml recombinant human IL-4, 10 ng / ml rhTNF-α, 1000 U / ml rhIL-6, 1 μg / ml PGE2, and 10 ng / ml IL-1β for another 4 days. After the above 2 days, the DCs from the first group, after complete medium washing and centrifugation, were collected to thoroughly remove the co-incubated MIL116, BION-1301, or KLH, and were ready for use.

[0186] PBMCs from the same donor were resuspended in RPMI 1640 medium. CD4+ T cells were isolated from the PBMCs using the Invitrogen Dynabeads non-contact human CD4+ T cell isolation kit (Cat#:11346D, Thermal Fisher Scientific, USA).

[0187] In a 96-well U-shaped bottom test plate, DC from group 1 and CD4 from the same donor are tested. +T cells were seeded at densities of 200,000 cells (T cells) / well and 20,000 cells / well (DCs) in a total culture medium volume of 200 μl and co-incubated for 4 days. For the second group of DCs, after co-incubation with the first group of DCs for 2 days, 150 μg / ml of MIL116 or BION1301, or 300 μg / ml of KLH positive control protein was added, and co-incubation continued for 2 days. Then, the co-incubation antibodies or positive proteins were thoroughly removed by complete culture medium washing and centrifugation, and the cells were ready for use. The second group of DCs was then added at a density of 20,000 cells per well to the aforementioned DC-T cell mixture and incubated for 2 days before flow cytometry analysis.

[0188] Using PE mice anti-human OX40 (Cat#:350004, Biolegend) and FITC mice anti-human CD25 (Cat#:356106, Biolegend) to inhibit CD4 + T cell activation markers were stained, and the luminescence intensity of OX40 and CD25 was detected by flow cytometry. The fold change in expression between the treatment group and the PBS-negative group was calculated to describe the activation status of T cells, thereby predicting the potential risk of each antibody producing anti-drug antibodies (ADAs).

[0189] Table 4. HLA subtype information of PBMCs from 8 different donors

[0190] Figures 6 (A and B) illustrate the immunogenicity risk of MIL116 and BION1301 as tested by PBMCs from donors of the eight high-frequency HLA subtypes listed in Table 4. Figure 6 (A) shows that MIL116 and BION1301 induced T cell OX40 expression, which was comparable to the PBS group and significantly lower than the positive control KLH protein. Figure 6 (B) shows that MIL116 induced T cell CD25 expression, which was comparable to or lower than BION1301 and significantly lower than the positive control protein KLH. This indicates that MIL116 has low overall immunogenicity and a low risk of causing ADA.

[0191] Example 11. Antibody stability analysis

[0192] Differential scanning fluorescence (DSF) was used to detect the chemical and thermal stability of MIL116 molecules containing the heavy chain constant region IgG1-m1 (SEQ ID NO:10) and the light chain constant region SEQ ID NO:18 under high temperature conditions.

[0193] In short, 19 μl of 2 mg / ml APRIL antibody and 1 μL of 100×SYPRO orange dye (Cat#: S5692-500 μL, Merck) were added to an eight-tube strip and mixed thoroughly. The mixture was heated, and the changes in fluorescence signal were recorded. The changes in fluorescence signal were processed and analyzed to extract the thermostability parameters of the target molecule.

[0194] Table 5 shows the DSF test results. The Tm value of MIL116-IgG1 was higher than that of the other two antibodies, indicating that its high-temperature stability was slightly higher.

[0195] Table 5. DSF detection results of MIL116 and positive control antibody

[0196] In addition, the purified MIL116 antibody was concentrated to 2 mg / ml, placed in centrifuge tubes, and stored at high temperature (40°C) for two weeks. The changes in its aggregation were then analyzed by size exclusion-high performance liquid chromatography (SEC).

[0197] In short, the SEC-HPLC system was equilibrated using a mobile phase of 0.2 mol / L potassium phosphate buffer and 0.25 mol / L potassium chloride (pH 6.2 ± 0.1) until the baseline stabilized. The prepared sample was loaded into the chromatographic system using an autosampler. The sample was passed through the column at a constant flow rate, and proteins were eluted sequentially according to their size. The elution curves were recorded using a detector to determine the monomer content in the sample.

[0198] Table 6. Antibody aggregation after two weeks of high-temperature storage

[0199] As shown in Table 6, after two weeks of high temperature, there was no significant increase in MIL116 molecules, indicating its excellent thermal stability.

[0200] Example 12. PK / PD experiment of MIL116 in cynomolgus monkeys

[0201] The amino acid sequences of human and cynomolgus monkey APRIL share >99% homology, differing by only two amino acids, one of which is amino acid position 189 of human APRIL. Furthermore, results from Examples 4 and 5 show that MIL116 and the two positive control antibodies exhibit remarkably similar affinity and receptor-blocking activity when binding to recombinant human and cynomolgus monkey APRIL proteins. Therefore, non-human primates (NHPs) are a suitable animal model for evaluating the PD / PK and safety of MIL116. This experiment was conducted by Zhaoyan Yichuang (Suzhou) New Drug Research Co., Ltd.

[0202] The cynomolgus monkeys were randomly divided into three groups: the MIL116 group (IgG1-m1 antibody prepared in Example 6), the BION1301 analog group, and the VIS649 analog group, with two monkeys in each group (one male and one female). Animals in each group were weighed before administration, and the corresponding amount of the test substance was administered according to their body weight. All drugs were administered via a single subcutaneous injection at a dose of 100 mg / kg. The drug concentrations for MIL116 were 52.1 mg / ml, BION1301 were 60 mg / ml, and VIS649 were 57.6 mg / ml. The day of the first administration was designated D1, the second day D2, the day before administration D-1, the two days before administration D-2, and so on. Blood samples were collected from veins in the forelimbs or hindlimbs at 0h (D0), 2h, 4h, 8h, 24h (D2), 48h (D3), 72h (D4), 96h (D5), 120h (D6), 168h (D8), D15, D22, and D29. 2 mL of blood was collected from each limb and placed in blood collection tubes containing separating gel and coagulant. The samples were stored at room temperature and centrifuged within 2 hours of collection. The supernatant was collected and transferred to labeled centrifuge tubes for antibody pharmacokinetic (PK) assays. Blood samples were collected from veins of the forelimbs or hindlimbs at 0h (D0), 24h (D2), D8, D15, D22, and D29, with 2 mL collected from each limb. The samples were placed in blood collection tubes containing separating gel and coagulant and stored at room temperature. Centrifugation was completed within 2 hours after blood sample collection. The supernatant was collected after centrifugation and transferred to labeled centrifuge tubes for antibody pharmacodynamic (PD) assay. The samples were stored below -60°C.

[0203] Pharmacokinetics was primarily determined by measuring the levels of IgA, IgG, and IgM in monkey serum. Specifically, serum concentrations of IgA, IgG, and IgM were determined by immunoturbidimetry using kits (Cat#:H416, Cat#:H415, and Cat#:H417, Meikang Biotechnology) and following the manufacturer's methodological procedures.

[0204] Pharmacokinetics were determined by ELISA. Specifically, the human APRIL trimer-biotin protein prepared in Example 1 was diluted in 1× coating buffer (CAT#C1055, Solarbio) to a final concentration of 1 μg / ml, and 100 μl was added to each well of an ELISA plate (CAT#9018, CORNING) and incubated horizontally at 4°C overnight. The plate was removed from 4°C and washed three times with PBST. 250 μl of blocking buffer (PBS containing 5% skim milk powder) was added to each well, and the plate was blocked at 37°C for 1.5 hours. The standard curve of the test drug was prepared by 3-fold dilution with a maximum starting concentration of 1 μg / ml, resulting in 8 concentration points. Monkey blood samples were diluted with blocking buffer at appropriate ratios (20-10000) to ensure the detection results fell within the quantitative range. The blocked plate was then washed three times with PBST. Add 100 μL of diluted serum sample or standard curve sample to each well, seal the plate, and incubate at 37°C for 1.5 hours. Remove the plate and wash it three times with PBST. Add 100 μL of goat anti-human Fc enzyme-labeled secondary antibody (CAT#: A0170, Sigma) diluted 1:5000 with blocking buffer to each well, and incubate horizontally at 37°C for 1 hour. Remove the plate and wash it three times with PBST. Add TMB chromogenic solution (CAT#: 555214, BD), and incubate horizontally at 37°C for 15 minutes. Remove the plate, terminate the reaction, and read the values ​​using the microplate reader.

[0205] As shown in Figure 7(AC), the serum concentrations of IgG (A), IgA (B), and IgM (C) in cynomolgus monkeys decreased after administration of each antibody. Within 7 days of antibody administration, MIL116 induced a significant decrease in IgA / G / M, with a faster onset of action than the two positive control molecules. Furthermore, MIL116's ability to decrease IgA / G / M within the first 29 days was superior to that of the VIS649 and BION-1301 analogs.

[0206] Figure 8 shows the changes in serum concentration of each antibody. It can be seen that the clearance rate of MIL116 molecules in monkeys is lower than that of the two positive control antibodies.

[0207] The sequences appearing in this application are listed below.

[0208] Although the invention has been described in conjunction with one or more embodiments, it should be understood that the invention is not limited to these embodiments, and the foregoing description is intended to cover all other alternative forms, modifications, and equivalents included within the spirit and scope of the appended claims. All references cited herein are incorporated herein by reference in their entirety.

Claims

1. An isolated monoclonal antibody or its antigen-binding moiety, capable of binding to APRIL, comprising: i) Heavy chain variable regions, which include VH-CDR1, VH-CDR2, and VH-CDR3 regions, and ii) Light chain variable regions, which include VL-CDR1, VL-CDR2, and VL-CDR3 regions. The VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 regions respectively contain amino acid sequences that have at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 1, 2, 3, 4, 5, or 6.

2. The isolated monoclonal antibody or its antigen-binding portion according to claim 1, wherein the heavy chain variable region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

7.

3. The isolated monoclonal antibody or its antigen-binding portion according to claim 1, wherein the light chain variable region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

8.

4. The isolated monoclonal antibody or its antigen-binding portion according to claim 2, wherein the heavy chain variable region and the light chain variable region respectively comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 7 and 8.

5. An isolated monoclonal antibody or its antigen-binding moiety, capable of binding to APRIL, comprising: i) A heavy chain variable region comprising a VH-CDR1 region, a VH-CDR2 region, and a VH-CDR3 region, wherein the VH-CDR1 region, VH-CDR2 region, and VH-CDR3 region have at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the VH sequence containing the amino acid sequence shown in SEQ ID NO: 7, respectively. ii) A light chain variable region comprising a VL-CDR1 region, a VL-CDR2 region, and a VL-CDR3 region, wherein the VL-CDR1 region, VL-CDR2 region, and VL-CDR3 region have at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the VL region, VL-CDR2 region, and VL-CDR3 region of the VL sequence containing the amino acid sequence shown in SEQ ID NO: 8, respectively.

6. The isolated monoclonal antibody or its antigen-binding portion according to claim 1 or 5 further comprises a heavy chain constant region connected to the heavy chain variable region and a light chain constant region connected to the light chain variable region.

7. The monoclonal antibody or its antigen-binding portion according to claim 6, wherein the heavy chain constant region contains strong FcRn binding force under acidic conditions.

8. The monoclonal antibody or its antigen-binding portion according to claim 7, wherein the heavy chain constant region does not contain FcRγ binding force, or contains weak FcRγ binding force.

9. The monoclonal antibody or its antigen-binding portion according to claim 7, wherein the heavy chain constant region comprises any of the amino acid sequences shown in SEQ ID NOs: 9-17, optionally, the heavy chain constant region comprises any of the amino acid sequences shown in SEQ ID NOs: 10-16, and further optionally, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 10 or 14.

10. The monoclonal antibody or its antigen-binding portion according to claim 6, wherein the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

18.

11. A nucleic acid molecule encoding the isolated monoclonal antibody or its antigen-binding portion as described in any one of claims 1-10.

12. An expression vector comprising the nucleic acid molecule of claim 11.

13. A host cell comprising a nucleic acid molecule of claim 11 integrated into its genome, or comprising an expression vector of claim 12.

14. A composition comprising the isolated monoclonal antibody or its antigen-binding portion as described in any one of claims 1-10, the nucleic acid molecule as described in claim 11, the expression vector as described in claim 12, or the host cell as described in claim 13.

15. Use of the composition of claim 14 in the preparation of a medicament for treating APRIL-related diseases.

16. The use according to claim 15, wherein the APRIL-related disease is immunoglobulin A nephropathy.

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