An antibody binding to april and its use

Abstract

Antibodies that bind to APRIL and uses thereof. The invention relates to an isolated monoclonal antibody capable of specifically binding to APRIL or an antigen binding part thereof. The application also relates to nucleic acid molecules encoding the antibodies or antigen-binding portions thereof, expression vectors, host cells and methods for expressing the antibodies or antigen-binding portions thereof, and methods of treatment using the antibodies or antigen-binding portions thereof, nucleic acid molecules, expression vectors and / or host cells.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202411554131.4 (22) Application Date 2024.11.01 (71) Applicant Beijing Tianguangshi Biotechnology Co., Ltd. Address Room 2505, Building 1, No. 2, Ronghua South Road, Daxing Economic and Technological Development Zone, Beijing 100176 (72) Inventors Lin Guangzhong, Li Jiangmei, Zhang Lunfeng, Zhou Xuechen, Sun Mingze, Li Feng (74) Patent Agency Shanghai Duan & Duan Law Firm 31334 Patent Attorney Xia Siqiu (51) Int.Cl. C07K 16 / 28 (2006.01) C12N 15 / 13 (2006.01) A61K 39 / 395 (2006.01) A61P 13 / 12 (2006.01) A61P 37 / 02 (2006.01) A61P 35 / 00 (2006.01) (54) Title of Invention: Antibody Binding to APRIL and Its Use Thereof (57) Abstract: Antibody binding to APRIL and its use thereof. This application relates to an isolated monoclonal antibody or its antigen-binding moiety capable of specifically binding to APRIL. This application also relates to a nucleic acid molecule encoding the antibody or its antigen-binding moiety, an expression vector, host cell, and method for expressing the antibody or its antigen-binding moiety, and a treatment method using the antibody or its antigen-binding moiety, the nucleic acid molecule, the expression vector, and / or the host cell. Claims (2 pages), Description (28 pages), Sequence Listing (electronic publication), Drawings (8 pages) CN 121991226 A 2026.05.08 CN 1 21 99 12 26 A 1. An isolated monoclonal antibody or its antigen-binding portion thereof, capable of binding to APRIL, comprising: i) a heavy chain variable region comprising VH-CDR1, VH-CDR2, and VH-CDR3 regions, and ii) a light chain variable region comprising VL-CDR1, VL-CDR2, and VL-CDR3 regions, wherein the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 regions respectively contain the antigen-binding portion of SEQ ID NO. NOs: 1, 2, 3, 4, 5 or 6 have amino acid sequences with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity.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 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. 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 comprising the amino acid sequence shown in SEQ ID NO:7, respectively; and 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 VH sequence comprising the amino acid sequence shown in SEQ ID NO:8, respectively; and ii) a light chain variable region comprising a VL-CDR1 region, a VL-CDR2 region, and ... respectively; and ii) a light chain variable region comprising a VL-CDR1 region, a VL-CDR2 region, and VL-CDR3 region, respectively; and ii) a light chain variable region comprising a VL sequence comprising the amino acid sequence shown in SEQ ID NO:8, respectively; and ii) a light chain variable region comprising a VL-CDR1 region, a VL-CDR2 region, and VL-C The CDR1 region, VL-CDR2 region, and VL-CDR3 region have at least 95%, 96%, 97%, 98%, 99%, or 100% identity. 6. The isolated monoclonal antibody or its antigen-binding portion according to claim 1 or 5 further comprises a heavy chain constant region linked to the heavy chain variable region and a light chain constant region linked 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 exhibits strong FcRn binding affinity 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 according to any one of claims 1-10. Claims 1 / 2 page 2 CN 121991226 A 12. An expression vector comprising the nucleic acid molecule according to claim 11. 13. A host cell comprising the nucleic acid molecule of claim 11 integrated into its genome, or comprising the 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 of claim 11, the expression vector of claim 12, or the host cell of 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. Claims 2 / 2 Page 3 CN 121991226 A Antibody Binding to APRIL and Its Use Field of Invention

[0001] This invention relates to an antibody or its antigen-binding portion capable of specifically binding to human APRIL, its preparation and use, particularly its use in the treatment of human autoimmune diseases associated with APRIL, such as IgA nephropathy, inflammatory diseases, and other immune diseases related to B cell dysfunction. Background Art

[0002] Immunoglobulin A nephropathy (IgAN) is the most common type of primary glomerulonephritis worldwide, with a global prevalence of approximately 3 per 100,000 adults per year, of which the prevalence in East Asia is as high as 40-50%.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, which usually leads to a decline in renal function. Up to 25%-50% of patients will gradually develop end-stage renal disease within 10-20 years after the onset of the disease (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 human IgA1, there is usually an O-glucose terminal attached to the hinge region. The loss of the terminal galactose of the O-glucose in the hinge region of IgA1 (i.e., the formation of Gd-IgA1) exposes the N-acetylgalactosamine (GalNAc) residue in the O-glucose, which is recognized by anti-glucose autoantibodies in susceptible individuals, forming an immune complex that deposits in the glomerular mesangium and triggers a series of inflammatory responses. This is the "quadruple blow" hypothesis of IgAN pathogenesis (Suzuki H, et al., (2011) The pathophysiology of IgA nephropathy. J Am Soc Nephrol. 22(10):1795-803). Moreover, the co-distribution of IgA and complement C3 has been observed in most IgAN patients, indicating 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, the only first-line drugs recommended by international guidelines are RAS blockers (ACE inhibitors / ARBs), but these can only reduce a portion of proteinuria and often fail to achieve therapeutic targets. Stronger drugs for reducing proteinuria—hormones / immunosuppressants—are not very sensitive to IgAN and have significant side effects. The world's first targeted therapy for IgAN is Nefecon (budesonide delayed-release capsules) from Calliditas Therapeutics. This drug was approved by the FDA in December 2021 for the treatment of 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 mainly produced by plasma cells located in mucosa-associated lymphoid tissue (MALT).In 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 and 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) play a key role in driving TD and TID antibody class switching and recombinant and generating IgA+ B cells (Sallustio F, et al., (2021) High levels of gut-homing immunoglobulin A+B lymphocytes support the pathogenic 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 RevImmunol (2021) 8:421-34.; Moore JS, et al., (2007) Instructions 1 / 28 pages 4 CN 121991226 A 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-cyclophilic 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 recombination 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). Patients with IgAN have higher levels of APRIL, which is associated with disease severity and poor 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 against APRIL that can inhibit the binding of APRIL to its receptor. Recent results from a phase II clinical trial showed that both VIS649 and BION1301 significantly reduced Gd-IgA1 antibody levels and proteinuria in IgAN patients, and simultaneously observed a sustained decrease in IgA and IgM, but a smaller decrease in IgG. The lower decrease in IgG may result in a lower risk of infection for patients during treatment.In addition, soluble TACI-Fc fusion proteins that can simultaneously block the binding of BAFF to B cells and APRIL to plasma cells, including atacecicept and telitacicept, can comprehensively inhibit the production of IgA1, IgG, and IgM. In a phase 2 clinical study, after six months of treatment with atacecicept or telitacicept, 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 delayed.

[0007] Although novel drugs targeting APRIL in the current clinical research stage have brought breakthrough efficacy to IgAN patients, IgAN is a chronic disease requiring long-term medication. There are still huge clinical challenges and needs 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 cells to have autoreactivity. Elevated levels of APRIL and BAFF in blood or tissues have been observed 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] Any reference to any document in this application is not construed as an admission that such document is prior art. Specification 2 / 28 pages 5 CN 121991226 A Summary of the Invention

[0010] The inventors of this application screened out monoclonal antibodies that can specifically bind to human and monkey APRIL and have binding strength comparable to or higher than that of prior art antibodies. Subsequently, the inventors humanized and modified the Fc region of the antibody to reduce its immunogenicity and enhance its FcRn binding strength in an acidic environment. In in vivo animal experiments, the antibody showed higher blood drug concentrations and a longer half-life than prior art antibodies, and was able to rapidly and persistently eliminate APRIL, as well as IgG, IgA, and IgM in the blood.Furthermore, the inventors have weakened or eliminated the binding force of FcR γ in the Fc region to avoid causing unnecessary antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC) to APRIL-positive cells.

[0011] 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 comprise:

[0012] 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 an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs:1, 2, and 3; and

[0013] ii) Light chain variable region, which may include VL-CDR1, VL-CDR2 and VL-CDR3 regions, wherein the VL-CDR1, VL-CDR2 and VL-CDR3 regions 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.

[0014] In some embodiments, the heavy chain variable region may 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 NO:7.

[0015] In some embodiments, the light chain variable region may 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:8.

[0016] In some embodiments, the heavy chain variable region and the light chain variable region may each 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.

[0017] This application also provides an isolated monoclonal antibody (e.g., mouse-derived, chimeric, or humanized antibody) or its antigen-binding moiety, which is capable of specifically binding to APRIL, and may include:

[0018] 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 comprising the amino acid sequence shown in SEQ ID NO:7, respectively; and

[0019] 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 sequence containing the amino acid sequence shown in SEQ ID NO:8, respectively.

[0020] The monoclonal antibody or its antigen-binding portion of this application may be mouse-derived, chimeric, or humanized. In some embodiments, the monoclonal antibody or its antigen-binding portion of this application may be humanized.

[0021] The monoclonal antibody or its antigen-binding portion of this application may contain a heavy chain constant region. The heavy chain constant region can be the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4, or a functional fragment thereof such as the Fc region, for example, human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the heavy chain variable region comprises 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 has high binding activity to FcRn at pH 5.8, for example, a KD value below 1.0 × 10⁻⁹ M. In some embodiments, the antibody of this application has strong FcRn binding affinity at pH 5.8, for example, a KD value below 9.0 × 10⁻¹⁰ M, and more particularly below 8.5 × 10⁻¹⁰ 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 sequences 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 sequences 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.

[0022] The monoclonal antibody or its antigen-binding portion of this application may contain 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 contain the amino acid sequence shown in SEQ ID NO:18. The N-terminus of the light chain constant region can be connected to the C-terminus of the light chain variable region.

[0023] 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.

[0024] The antibody or its antigen-binding moiety of this application, compared with prior art antibodies such as VIS649 and BION1301, has 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 blood drug concentration or bioavailability, and / or viiii) comparable or higher APRIL scavenging ability. The antibody or its antigen-binding moiety of this application does not have 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) in APRIL-positive cells.

[0025] This application also provides a bispecific molecule containing the antibody or its antigen-binding portion of this application, wherein the antibody or its antigen-binding portion is linked to a second functional group, for example, a second antibody, the second functional group having a binding specificity different from that of the antibody or its binding portion of this application.

[0026] This application also provides a nucleic acid molecule encoding the antibody or its antigen-binding portion of this application, or a bispecific molecule. This application also provides an expression vector containing the above-mentioned nucleic acid molecule, and a host cell containing the expression vector or integrating the above-mentioned nucleic acid molecule into the genome. This application also provides a method for preparing the antibody or its antigen-binding portion or bispecific molecule of this application 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.

[0027] This application also provides a composition comprising the antibody or its antigen-binding portion, bispecific molecule, nucleic acid molecule, expression vector, or host cell of this application. In some embodiments, the composition may be a pharmaceutical composition and may also comprise a pharmaceutically acceptable carrier.

[0028] In another aspect, this application provides a method for treating or alleviating APRIL-related disease in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of this application to the subject.

[0029] APRIL-related disease may be an inflammatory disease, such as an autoimmune disease. In some embodiments, APRIL-related disease is an autoimmune disease associated with B cell dysfunction.In some embodiments, the APRIL-related disease is immunoglobulin A nephropathy (IgAN).

[0030] The APRIL-related disease can be a tumor, such as a B-cell-related tumor, including multiple myeloma. In some embodiments, the method may also include administering an antibody against an immune checkpoint such as PD-1 or CTLA-4.

[0031] This application also provides the use of the antibody of this application or its antigen-binding portion in the preparation of a medicament for treating APRIL-related diseases.

[0032] Other features and advantages currently disclosed will become readily apparent from the following detailed description and examples, which should not be construed as limiting. All references, Genbank registry numbers, patents and published patent applications cited in this specification are incorporated herein by reference. Brief Description of the Drawings

[0033] 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.

[0034] Figure 1 illustrates the binding ability of the APRIL antibody to recombinant human APRIL protein (A) and monkey APRIL protein (B) in an ELISA.

[0035] 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.

[0036] Figure 3 shows the binding power of APRIL antibody to mouse APRIL in ELISA (A), and the binding power of human APRIL protein to human, monkey, and mouse BCMA (B).

[0037] Figure 4 shows the rapid clearance of APRIL mediated by MIL116 antibody in C57 wild-type mice.

[0038] Figure 5 shows the serum exposure and clearance rate of MIL116 antibody in FcRn humanized mice.

[0039] Figure 6 shows the activation power of APRIL antibody on T cells in in vitro immunogenicity prediction experiments, specifically the expression of T cell activation markers OX40 (A) and CD25 (B), where the PBMC donor has a high-frequency HLA genotype from Chinese individuals.

[0040] 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.

[0041] Figure 8 shows the changes in serum APRIL antibody concentration after a single subcutaneous injection of APRIL antibody into cynomolgus monkeys. Detailed Embodiments

[0042] Unless otherwise specified, the terms used herein have their common meanings in dictionaries, textbooks, technical reference books, or as commonly understood by those skilled in the art.The following description of some terms is for the purpose of facilitating understanding of this application (page 5 / 28, CN 121991226 A) and is not intended to specifically limit these terms unless otherwise specified.

[0043] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include the plural forms of the referred object, unless the context clearly indicates otherwise.

[0044] The term “or” refers to a single element among the listed alternative elements, unless the context clearly indicates otherwise.

[0045] The terms “comprising” or “including” mean to include the stated element, integer, or step, but do not exclude the inclusion of any other element, integer, or step. In this document, when the terms “comprising” or “including” are used, they also cover combinations of the stated elements, integers, or steps, unless otherwise specified. The terms “consisting of” or “comprises” generally mean containing only the stated element, integer, or step, without the inclusion of other elements, integers, or steps.

[0046] The term “APRIL” refers to a proliferation-inducing ligand, also known as CD256 or TNFSF13. This term includes variants, homologs, orthologs, and parallel homologs. For example, an antibody specific to human APRIL may cross-react with APRIL proteins from another species, such as monkeys, under certain conditions. In other embodiments, an antibody 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.

[0047] The term “human APRIL” refers to an APRIL protein having a human amino acid sequence, while “monkey APRIL” refers to an APRIL protein having a monkey amino acid sequence.

[0048] The term “antibody” as used herein is intended to include full-length antibodies to IgG, IgA, IgD, IgE, and IgM, and 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 consists of a variable region (VH) and a constant region. The constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a variable region (VL) and a constant region. The constant region consists of one domain, CL. The VH and VL regions can also be divided into hypervariable regions called complementarity-determining regions (CDRs), which are separated by more conserved backbone regions (FRs). Each VH and VL 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 of an antibody can mediate 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 main function of the light chain constant region is to enhance the structural stability of the antibody. A “functional fragment” of the antibody constant region refers to a segment of the constant region that retains certain desired functions, such as a fragment of the heavy chain constant region that retains FcRn binding activity, such as an Fc fragment.

[0049] In this document, the term “antigen-binding portion” (or simply antibody portion) 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 fragments included in the “antigen-binding portion” of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1; (ii) F(ab')2 fragments, bivalent fragments containing two Fab fragments connected by a disulfide bridge in the hinge region; (iii) Fd fragments consisting of VH and CH1; (iv) Fv fragments consisting of VL and VH on the antibody single arm; (v) separate complementarity-determining regions (CDRs); and (vi) nanobodies, a heavy chain variable region containing a single variable domain. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be linked by recombination via a synthetic linker that makes them single-protein chains, wherein the VL and VH regions pair to form a monovalent molecule (called a single-chain Fc (scFv)). These single-chain antibodies are also intended to be included in the terminology. These antibody fragments can be obtained by techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.

[0050] As used herein, the term "isolated antibody" means 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 the APRIL protein. However, an isolated antibody that specifically binds to the human APRIL protein may have cross-binding to other antigens, such as APRIL proteins from other species. In addition, isolated antibodies are substantially free of other cellular material and / or chemicals.

[0051] The term "monoclonal antibody" or "monoclonal antibody" refers to an antibody or antibody fragment thereof derived from a population of antibody molecules that are identical 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.

[0052] The term "mouse-derived antibody" refers to an antibody whose variable region backbone and CDR region are derived from mouse germline immunoglobulin sequences. Furthermore, 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 a CDR sequence derived from another mammalian species is inserted into the mouse skeletal sequence.

[0053] The term "chimeric antibody" refers to an antibody obtained by combining non-human genetic material with human genetic material. Or more generally, a chimeric antibody refers to an antibody that combines genetic material from one species with genetic material from another species.

[0054] As used herein, the term "humanized antibody" refers to an antibody derived from a non-human species but whose protein sequence has been modified to increase similarity to naturally occurring antibody variants in humans.

[0055] In this document, an antibody that "specifically binds to human APRIL" refers to an antibody that binds to human APRIL (and APRIL from other non-human species) but substantially does not bind to non-APRIL proteins. Preferably, the antibody binds to human APRIL protein with "high affinity," i.e., a KD value of less than 5.0 × 10⁻⁹ M.

[0056] The term "fundamentally non-binding" protein or cell refers to a protein or cell that does not bind to, or does not bind to with, a high affinity, i.e., the KD of the bound protein or cell is 1.0 × 10⁻⁶ M or more, more preferably 1.0 × 10⁻⁵ M or more, more preferably 1.0 × 10⁻⁴ M or more, 1.0 × 10⁻³ M or more, and more preferably 1.0 × 10⁻² M or more.

[0057] The term "EC50," also called half-maximal effect concentration, refers to the concentration of a molecule (e.g., antibody) that causes 50% of the maximum effect.

[0058] The term "IC50" refers to the half-inhibitory concentration, i.e., the concentration of a molecule (e.g., antibody) required to inhibit a specified biological process by half.

[0059] 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.

[0060] The term "complement-dependent cytotoxicity" or "CDC" refers to complement-mediated cytotoxicity, i.e., the activation of the classical complement pathway through the binding of specific antibodies to corresponding antigens on the target cell membrane surface, forming a complex that exerts a lytic effect on the target cell.

[0061] The term "ADCP" or "antibody-dependent phagocytosis" refers to the process by which phagocytic effector cells, such as monocytes and macrophages, endocytose target cells.

[0062] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammalians, including 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.

[0063] The term "therapeutic effective amount" refers to the amount of antibody of 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.

[0064] "Sequence identity" as used herein refers to the percentage of nucleotides / amino acids in a sequence that are identical to nucleotides / amino acid residues in a reference sequence after sequence alignment, where spaces are introduced in the sequence alignment if necessary to achieve the maximum percentage of sequence similarity between the two sequences. Those skilled in the art can determine the percentage of sequence similarity between two or more nucleic acid or amino acid sequences by performing pairwise or multiple sequence alignments using various methods, such as computer software, including, for example, ClustalOmega, T-coffee, Kalign, and MAFFT.

[0065] “FcRγ” is a type of Fc receptor, expressed in various immune cells, including monocytes, macrophages, NK cells, neutrophils, etc., and can recognize cells bound by immunoglobulins such as IgG, and trigger phagocytosis or killing of these cells.

[0066] “FcRn” or “neonatal Fc receptor” is a protein that plays a key role in the immune system and IgG transport, and is expressed in various tissues such as the lungs and kidneys. FcRn is responsible for the transmembrane transport of IgG, keeping IgG in the circulation system, and regulating (e.g., prolonging) the half-life of IgG. Studies have shown that FcRn can bind tightly to IgG in an acidic environment.

[0067] “Half-life”, “T1 / 2”, or “plasma half-life” refers to the time required for the concentration of a drug (e.g., antibody) in plasma to decrease by half, and its length can reflect the rate of drug elimination.

[0068] “Bioavailability” or “bioavailability” refers to the relative amount and rate at which a drug (e.g., antibody) is absorbed into the circulation by the body. Drugs with high bioavailability can be administered at relatively low doses.

[0069] Several aspects of this application are described in more detail below.

[0070] The antibody or its antigen-binding moiety of this application, compared with prior art antibodies such as VIS649 and BION1301, has 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 blood drug concentration or bioavailability, and / or viiii) comparable or higher APRIL scavenging ability. The antibody or its antigen-binding moiety of this application does not have mouse APRIL binding affinity. In some embodiments, the antibody or 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) in APRIL-positive cells.

[0071] Exemplary antibodies or antigen-binding portions of this application are those whose structures and chemical properties are described below.

[0072] The heavy chain variable region CDR and light chain variable region CDR of the antibody or antigen-binding portion of 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 antigen-binding portion of this application can also be determined using the Chothia, IMGT, AbM, or Contact numbering system based on the sequence of the heavy chain variable region and light chain variable region.

[0073] The antibody or antigen-binding portion of this application may contain a heavy chain constant region, such as a heavy chain constant region having high FcRn binding force. When possessing high FcRn binding affinity, the antibody or its antigen-binding portion of this application can exhibit better half-life and APRIL scavenging ability. This means that 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 unnecessary antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC) in APRIL-positive cells. In some embodiments, the heavy chain constant region may be an IgG4 constant region containing mutations such as M428L / N434A, M428L / N434S, or M252Y / S254T / T256E, 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.

[0074] The monoclonal antibody of this application or its antigen-binding portion may contain 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 contain the amino acid sequence shown in SEQ ID NO: 18.

[0075] The VH and / or VL sequences (or CDR sequences) of other APRILs that bind to human APRIL may be "mixed and paired" with the VH and / or VL sequences (or CDR sequences) of the antibody of this application. Specifically, when VH and VL (or their CDRs) are mixed and paired, the VH sequence in a particular VH / VL pair may be replaced by a structurally similar VH sequence. Similarly, it is preferred that the VL sequence in a particular VH / VL pair be replaced by a structurally similar VL sequence.

[0076] In another embodiment, the antibody of this application or its antigen-binding portion includes the heavy chain variable region CDR2 of an APRIL antibody and the CDRs of other antibodies binding 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.

[0077] In another embodiment, the antibody of this application comprises heavy chain and / or light chain variable region sequences or CDR1, CDR2 and CDR3 sequences that have one or more conserved modifications to the APRIL antibody of this application. It is known in the art that some conserved sequence modifications do not cause the antigen binding to disappear.

[0078] The term “conserved sequence modification” as used herein refers to amino acid modifications that do not significantly affect or alter the antibody binding properties. Such conserved modifications include amino acid substitutions, additions, and deletions. These 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 amino acid residues with amino acid residues having similar side chains. Groups of amino acid residues with similar side chains are known in the art.These amino acid residue groups include amino acids having 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 can be replaced with other amino acid residues from the same side chain group, and the resulting antibody can be tested for retaining its function (i.e., the function described above) using the functional assays described herein.

[0079] In several embodiments, the antibody or its antigen-binding portion can be, for example, mouse-derived, chimeric, or humanized, particularly humanized.

[0080] The antibody of this application can be prepared into a genetically modified antibody using an antibody possessing one or more VH / VL sequences of the APRIL antibody of this application as starting material. The antibody can be genetically modified by modifying one or more residues in one or two variable regions (i.e., VH and / or VL) (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 of certain species. For example, the antibody can also be genetically modified by modifying residues in constant regions, for example, to change the effector function of the antibody.

[0081] In some embodiments, CDR regions can be inserted to genetically modify the variable regions of the antibody. Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementarity-determining regions (CDRs). 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 interaction, recombinant antibodies that mimic the characteristics of a specific natural antibody can be expressed by constructing an expression vector containing the CDR sequence of a specific natural antibody inserted into the backbone sequence of different antibodies with different characteristics.

[0082] Therefore, another embodiment of this application relates to isolated monoclonal antibodies or their antigen-binding portions, which comprise heavy chain variable regions and / or light chain variable regions, the heavy chain variable regions comprising CDR1, CDR2, and CDR3 having the sequences described above in this application, and the light chain variable regions comprising CDR1, CDR2, and CDR3 having the sequences described above in this application. Although these antibodies comprise the VH and VL CDR sequences of the monoclonal antibodies of this application, they may contain different backbone sequences. Specification 9 / 28 pages 12 CN 121991226 A

[0083] Such backbone sequences can be obtained from publicly available DNA databases or publicly available 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. As another embodiment, germline DNA sequences for human heavy and light chain variable region genes can be obtained from the Genbank database.

[0084] The antibody protein sequence is compared with a protein sequence database using one of the sequence similarity search methods known in the art, called gap BLAST.

[0085] Preferred backbone sequences for the antibodies of this application are those structurally similar to the backbone sequences used in the antibodies of this application. VH CDR1, CDR2, and CDR3 sequences can be inserted into backbone regions having the same sequence as the germline immunoglobulin gene from which the backbone sequence is obtained, or CDR sequences can be inserted into backbone regions 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).

[0086] Another class of variable region modifications involves mutating amino acid residues in the VH and / or VL CDR1, CDR2 and / or CDR3 regions to improve one or more binding properties (e.g., affinity) of the target antibody. Mutations can be introduced by point mutations or PCR-mediated mutations, and their effects on antibody binding or other functional properties can be evaluated in vitro or in vivo as 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 in the CDR region are altered.

[0087] The genetically modified antibodies of this application include those that make genetic modifications to the backbone residues of VH and / or VL to, for example, alter antibody properties. Backbone modifications include mutations in one or more residues of the backbone region, or even one or more CDR regions, to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This method is also known as “deimmunization”, and is described in more detail in U.S. Patent Publication 20030153043.

[0088] In addition, as an alternative to modifications within the backbone or CDR regions, the antibodies of this application may be genetically modified to include genetic 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 may be chemically modified (e.g., one or more chemical functional groups may be attached to the antibody), or modified to alter its glycosylation, to alter one or more functional properties of the antibody.

[0089] In one embodiment, the hinge region of CH1 is modified, altering, for example, increasing or decreasing the number of cysteine ​​residues in the hinge region. This method is further described in U.S. Patent 5,677,425. Modifying the cysteine ​​residues in the CH1 hinge region can, for example, promote the assembly of the heavy and light chains or increase / decrease the stability of the antibody.

[0090] In another embodiment, the Fc hinge region of the antibody is mutated to increase or decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 linker region of the Fc hinge fragment, thereby giving the antibody a weakened SpA binding affinity relative to the native Fc-hinge domain SpA binding. This method is described in more detail in U.S. Patent 6,165,745.

[0091] 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 site. Such deglycosylation can increase the antibody's affinity for the antigen. See, for example, U.S. Patents 5,714,350 and 6,350,861. Specification 10 / 28 pages 13 CN 121991226 A

[0092] 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 antibody. Such glycosylation modification 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 lines Lec13, rat fusion tumor cell lines YB2 / 0, cell lines containing small interfering RNA specifically targeting the FUT8 gene, and cell lines co-expressing β-1,4-N-acetylglucosidase III and Golgi α-mannosidase II. These can be used as host cells for expressing the recombinant antibodies of this application to prepare antibodies with altered glycosylation. The Slc35c1 gene knockout cell line, for example, utilizes the fucose knockout platform technology independently developed by Tianguangshi, see the CHO cell line in US Patent No. 10377833B2 with accession number CGMCC No. 14287.

[0093] Another modification of the antibodies described herein is polyethylene glycol (PEGylation). Antibodies can be PEGylated, for example, to increase the biological (e.g., serum) half-life of the antibody.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-C10)alkoxy- or aryloxy polyethylene glycol or polyethylene glycol maleimide. In some embodiments, the antibody requiring PEGylation 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.

[0094] The antibodies of this application can be characterized by a variety of physical properties to detect and / or differentiate their classification.

[0095] For example, the antibody 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 N-X-S / 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.

[0096] In a preferred embodiment, the antibody does not contain an asparagine isomer site. Deamidation of asparagine may occur in the N-G or D-G sequence, creating isoaspartic residues that introduce kinks into the polypeptide chain and reduce its stability (isoaspartic effect).

[0097] In another aspect, this application provides nucleic acid molecules encoding the heavy chain / light chain variable region or CDR of the antibody of this application or its antigen-binding portion. Nucleic acids may be present in whole cells, in cell lysates, or in partially purified or substantially pure forms. Nucleic acids are “isolated” or “substantially pure” when purified by standard techniques from other cellular components or other contaminants such as other cellular nucleic acids or proteins. The nucleic acid in 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.

[0098] The nucleic acid 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 acid encoding such antibodies can be collected from the gene library.

[0099] Preferred nucleic acid molecules of this application include those encoding VH and VL sequences or CDRs of the APRIL monoclonal antibody. Once the DNA fragments encoding VH and VL are obtained, these DNA fragments 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 DNA fragment encoding VH or VL is operatively ligated to another DNA fragment encoding another protein, such as the antibody constant region or a flexible linker, as described on page 11 / 28 of CN 121991226 A. The term “operatively ligated” means that two DNA fragments are ligated together such that the amino acid sequences encoded by both DNA fragments are within the reading frame.

[0100] Isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operatively ligating the VH-encoding DNA to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the field, and DNA fragments including these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but preferably an IgG1 or IgG4 constant region. For Fab fragment heavy chain genes, the DNA encoding the VH region can be operatively linked to another DNA molecule that encodes only the heavy chain CH1 constant region.

[0101] Isolated DNA encoding the VL region can be converted into a full-length light chain gene by operatively linking the VL encoding DNA to another DNA molecule that encodes the light chain constant region CL. The sequences of human light chain constant region genes 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 region can be the κ and λ constant regions.

[0102] To create the scFv gene, DNA fragments encoding VH and VL can be operatively linked to another fragment encoding a flexible adapter, so that the VH and VL sequences can be expressed as a continuous single-stranded protein, wherein the VH and VL regions are linked by the flexible adapter.

[0103] 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.

[0104] The antibody or its antigen-binding portion of this application can also be generated in a host cell transfected tumor using, for example, recombinant DNA technology combined with gene transfection methods (e.g., Morrison, S. (1985) Science 229:1202). In one embodiment, a coding portion or full-length light and heavy chain DNA obtained by standard molecular biotechnology is inserted into one or more expression vectors, thereby operatively linking the gene with transcriptional and translational regulatory sequences. In this case, the term "operatively linking" means that the antibody gene is linked to the vector, thereby allowing the transcriptional and translational control sequences within the vector to perform their intended functions of regulating antibody gene transcription and translation.

[0105] The term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control antibody gene transcription or translation. Such regulatory sequences have been described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, California. (1990)). Preferred regulatory sequences for expression in mammalian host cells 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 may consist of sequences from different 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.

[0106] Antibody light chain genes and antibody heavy chain genes may 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 subtype, such that VH is operatively linked to CH in the vector and VL is operatively linked to CL in the vector. 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 be linked 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).Specification 12 / 28 pages 15 CN 121991226 A

[0107] In addition to the antibody chain gene and regulatory sequence, the recombinant expression vector of this application may carry other sequences, such as sequences that regulate the replication of the vector in the host cell (e.g., replication origin) and selectable marker genes. Selectable marker genes can be used to select host cells that have been introduced into the vector (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 G418, hygromycin, or methotrexate resistance, to host cells that have been introduced into the vector. 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).

[0108] 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. While 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.

[0109] Preferred mammalian host cells for expressing the recombinant antibodies of this application include Slc35C1 gene 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.

[0110] 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 targeting 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 having 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 modification, somatic cell hybridization, or chemical methods.

[0111] In another aspect, this application provides a pharmaceutical composition comprising an antibody or antigen-binding moiety thereof, 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 comprise one or more other pharmaceutically effective ingredients.

[0112] The pharmaceutical composition may comprise 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 suitable excipients are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003). Instructions for Use, Pages 13 / 28, 16 CN 121991226 A

[0113] 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, usually 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 injection and bolus. Alternatively, the antibody of this application may be administered via a non-parenteral route, such as topical, epidermal, or mucosal administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical application.

[0114] The pharmaceutical compositions may be in the form of sterile aqueous solutions or dispersions. They may also be formulated in microemulsions, liposomes, or other ordered structures suitable for high concentrations of the drug.

[0115] The amount of active ingredient prepared in a single dosage form with the carrier material will vary depending on the therapeutic subject and the specific administration mode, and is essentially the amount of composition that produces the therapeutic effect. In percentage terms, this amount is about 0.01 to about 99% of the active ingredient bound to a pharmaceutically acceptable carrier.

[0116] The dosing regimen is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a rapid infusion may be administered, multiple fractions may be administered over time, or the dose may be reduced or increased proportionally to the severity of the treatment condition. It is particularly advantageous to formulate parenteral compositions in a dose-unit type that is convenient to administer and provides uniform dosage. A dose-unit type 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 with the pharmaceutical carrier. Alternatively, antibodies may be administered as a sustained-release formulation, in which case the required frequency of administration is reduced.

[0117] For antibody administration, the dose may be about 0.001-100 mg / kg host body weight. An exemplary treatment regimen involves administration once weekly.

[0118] The "therapeutic effective amount" of the present application's APRIL antibody causes a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods. For example, for the treatment of IgAN subjects, the "therapeutic effective amount" preferably inhibits the generation 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 more preferably at least about 80%, compared to untreated subjects.

[0119] 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, polyanhydride, 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.

[0120] The pharmaceutical composition 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).

[0121] In some embodiments, the monoclonal antibody of this application can 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.

[0122] The pharmaceutical compositions of this application have various in vitro and in vitro-in vivo applications, such as in vivo treatment of APRIL-related diseases. The pharmaceutical compositions may be administered to human subjects.

[0123] This application provides a method of treating or alleviating APRIL-related diseases in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of this application.

[0124] APRIL-related diseases may be inflammatory diseases, such as autoimmune diseases. In some embodiments, APRIL-related diseases are autoimmune diseases associated with B cell dysfunction. In some embodiments, the APRIL-related disease is immunoglobulin A nephropathy (IgAN).

[0125] The APRIL-related disease can be a tumor, such as a B-cell-related tumor, including multiple myeloma. In some embodiments, the method may also include administering an antibody against an immune checkpoint such as PD-1 or CTLA-4.

[0126] This application provides a combination therapy in which the pharmaceutical composition of this application is administered in combination with one or more other antibody or non-antibody therapeutic agents. For example, it may be administered in combination with BAFF antibody, or in combination with immune checkpoint antibodies, other anticancer agents.

[0127] The combination of therapeutic agents discussed herein may be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as separate compositions wherein each agent is in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents may be administered sequentially.

[0128] Furthermore, if multiple combination therapies are administered and the drugs are administered sequentially, the order of sequential 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.

[0129] This application is further described by the following examples, which should not be construed as limiting. All figures and all references, Genebank sequences, patents and published patent applications cited throughout this application are incorporated herein by reference in their entirety.

[0130] Examples

[0131] Example 1. Preparation of APRIL trimer-biotin

[0132] Human APRIL trimer protein with a higher receptor binding affinity than the monomer was synthesized by linking monomer APRIL via a adapter. In short, the cDNA sequence encoding human APRIL trimer (SEQ ID NO:23) was cloned into the GS plasmid by double digestion with Cla1 and Xhol to construct the expression plasmid. Polyethyleneimine (PEI) (Cat#:24765-1, Kyfora Bio) was used. Following the manufacturer's instructions, the plasmid, culture medium, and PEI were mixed thoroughly and added dropwise to CHO cells after standing for 10 minutes. The cell density was approximately 6 × 10⁶ / ml, the DNA:PEI ratio was 1:4, and the total DNA used for transfection was 1.5 μg / ml. The transfected CHO cells were cultured at 120 RPM in a 37°C, 5% CO₂ incubator.

[0133] 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 membrane to remove cell debris. Afterwards, the nickel column (Cat#: 17524801, Cytiva) was 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 then further purified by a Superdex200 gel filtration column (Cytiva, CAT#28990944) to remove aggregates and impurities. The APRIL trimer protein of the correct size was collected, and its concentration was determined by a NanoDrop instrument (Cat#:840-317400, Thermo).

[0134] Using the NHS-PEG4-Biotinylation Kit (Cat#:21455, Thermo), the 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. Next, the mixture was added to a desalting column and centrifuged at 1000g for 2 minutes to remove excess NHS-PEG4-biotin.

[0135] Example 2. Preparation of MIL116 antibody

[0136] A series of APRIL antibodies were obtained through mouse immunization, phage library display screening, affinity maturation and other techniques. After extensive screening, a high-affinity humanized APRIL antibody, MIL116, was obtained. The amino acid sequences of its heavy chain and light chain variable regions are shown in SEQ ID NO:7 and 8, respectively. Specification 15 / 28 pages 18 CN 121991226 A

[0137] 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 heavy and light chain variable region sequences of the BOIN1301 analog are shown in SEQ ID NO:19 and 20, respectively, and the heavy and light chain constant regions are shown in SEQ ID NO:13 and 18, respectively. The heavy and light chain variable region sequences of the VIS649 analog are shown in SEQ ID NO:21 and 22, respectively, and the heavy and light chain constant regions are shown in SEQ ID NO:17 and 18, respectively. The sequence of the APLN-303 analog is shown in SEQ ID NO:167 of WO2024077018A2. These proteins were purified after expression.

[0138] Example 3. Binding activity of MIL116 antibody with human and monkey APRIL

[0139] The binding affinity of MIL116 to human and monkey APRIL protein was determined by ELISA.

[0140] 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, and serial dilutions of 3-fold or 5-fold were performed, 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. The wells containing only the diluted PBST solution served as negative controls. Wash the ELISA plates with PBST at pH 7.4, 200 μL / well, let stand for 2 minutes, and wash 3 times.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 with PBST, 200 μL / well, 4 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. Analyze the data using GraphPad software.

[0141] The results are shown in Figure 1 (A and B). Under pH 7.4 conditions, 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.

[0142] Example 4. Binding affinity of MIL116 antibody to human and monkey APRIL

[0143] The binding affinity of MIL116 antibody to human or monkey APRIL protein was determined by BIAcore 8K.

[0144] Specifically, the his antibody (Cat#:BR100839, cytiva) was coupled to the surface of the chip CM5. The 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 to ensure that approximately 100 RU of antigen was captured by the chip coupled with the his antibody. The MIL116 antibody and 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 hydrochloric acid solution.

[0145] Kinetic analysis was performed using the Wizard in Biacore 8K control software, and the binding KD values ​​of the detection antibody and APRIL were obtained by fitting according to Biacore 8K evaluation software, as shown in Table 1.

[0146] As shown in Table 1, the affinity of the MIL116 antibody for human or monkey APRIL is comparable to that of the BION-1301 analog. Moreover, MIL116 has a similar affinity for binding to APRIL proteins from human and monkey species, thus monkeys can be conveniently used as subjects for preclinical experimental studies.

[0147] Table 1. Binding affinity of APRIL antibody to human or monkey APRIL Specification 16 / 28 pages 19 CN 121991226 A

[0148]

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

[0150] Human BCMA-HIS (Cat#: BCA-HP2H2, ACRO) was diluted with PBS to a final concentration of 1 μg / ml, human TACI-Fc (Cat#: TAI-H5253, ACRO) was diluted to a final concentration of 4 μg / ml, and monkey BCMA-Fc (Cat#: BCA-C5253, ACRO) was diluted to 4 μg / ml. ELISA plates were coated with the dilution buffer, 100 μl per well, and incubated overnight at 4°C. Discard the liquid and block the ELISA plate with PBST containing 2% BSA, 200 μL / well, at 37°C for 1 hour. Simultaneously, perform a 3-fold serial dilution of the antibody to be tested, starting at 200 μg / ml, with a total of 11 dilutions. Add 200 ng / ml of human APRIL-Biotin (Cat#: APL-H52D1, ACRO) or monkey APRIL-HIS (Cat#: APL-C52D2, ACRO), and incubate at 37°C for 1 hour. Wash the ELISA plate with PBST, 200 μL / well, 3 times. Add the antibody-APRIL mixed sample to the blocked ELISA plate, 100 μL / well, and incubate at 37°C for 1 hour. Wash the ELISA plate with PBST, 200 μL / well, 3 times. Add 100 μL of HRP-labeled streptavidin (Cat#:BIR701, Beijing Bosi Technology Co., Ltd.) or HRP-labeled HIS antibody (Cat#:CW0285, CWBIO) diluted 1:5000 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 it 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 (A-C). The MIL116 antibody significantly inhibits the binding of human APRIL to its receptor BCMA (A) or TACI (B), with activity comparable to that of the positive control antibody; MIL116 also has the ability to block monkey APRIL-BCMA binding comparable to that of the two positive control antibodies (C).

[0152] Example 6. Fc modification of MIL116 antibody

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

[0154] In order to select a suitable Fc mutant, the variable region sequence of 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), and then functional verification was performed. Specifically, the heavy chain constant region uses IgG4-wt (wild type) shown in SEQ ID NO:13, IgG4-m1 (LA) shown in SEQ ID NO:14, IgG4-m2 (LS) shown in SEQ ID NO:15, or IgG4-m3 (YTS) shown in SEQ ID NO:16, and the light chain constant region uses the κ light chain constant region of SEQ ID NO:18.

[0155] The affinity of BION-1301 containing three mutant Fcs to human FcRn at pH 5.8 was determined using a Biacore 8K instrument. The specific experimental method is 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 about 10 RU of FcRn was captured by the chip conjugated with his antibody. The antibodies to be tested were 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 proteins of different concentrations were flowed through the surface of the stationary phase. The chip was regenerated using glycine hydrochloric acid solution (page 17 / 28 of the instruction manual, 20 CN 121991226 A). 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 had slightly higher affinity for FcRn than the antibody containing YTE. Considering the immunogenicity after modification and the stability of the antibody's physicochemical properties, the Fc containing the M428L / N434A (LA) mutation was finally selected.

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

[0158]

[0159] Subsequently, the Fc region of MIL116 containing IgG1 was modified to reduce the clearance rate of the antibody in vivo.On the one hand, the L234A / L235A mutation was performed to remove the binding between the antibody and the FcR γ 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 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.

[0160] Following the above method 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.

[0161] 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.

[0162] Table 3. Affinity of different APRIL antibodies to human FcRn

[0163]

[0164] Example 7. Receptor-ligand species crossover experiment

[0165] 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.

[0166] Specifically, the method steps of Example 3 were followed, wherein the concentration of mouse APRIL (Cat#:APR-MM113, Kaika Biotechnology) after dilution was 1 μg / mL, and mouse BCMA-Fc (Cat#:BCA-M5258, ACRO) was used as a positive control.

[0167] The results are shown in Figure 3(A). MIL116 did not bind to mouse APRIL with either of the two positive control antibodies.

[0168] Furthermore, literature reports that human APRIL has 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 to human, monkey, and mouse BCMA was determined by ELISA detection.Specifically, human BCMA-HIS (Cat#: BCA-HP2H2, ACRO), mouse BCMA-FC (Cat#: BCA-M5258, ACRO), or monkey BCMA-FC (Cat#: BCA-C5253, ACRO) were diluted with PBS to a final concentration of 1 μg / ml, and 100 μl of the diluent was used to coat the ELISA plate overnight at 4°C. The liquid was discarded, and the ELISA plate was blocked with PBST containing 2% BSA, 200 μl / well, at 37°C for 1 hour. The ELISA plate was 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 the blocked ELISA plate, 100 μl / well, and incubated at 37°C for 1 hour. The ELISA plate was washed three times with PBST, 200 μl / well. Add 100 μL of HRP-labeled streptavidin (Cat#: BIR701, Beijing Bosi Technology Co., Ltd.) 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.

[0169] 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.

[0170] Based on the results in Figure 3 (A and B), that is, 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 PK detection.

[0171] Example 8. Rapid clearance of human APRIL mediated by MIL116 in wild-type C57 mice

[0172] 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 of SEQ ID NO:18 was tested for APRIL.

[0173] C57 mice (Vitalliwa) were randomly divided into 8 groups.The study included 7 mice in Group 1, administered MIL116 (10 mg / kg) and APRIL (human APRIL trimer-biotin prepared in Example 1, 15 mg / kg); 7 mice in Group 2, administered BION1301 analog (10 mg / kg) and APRIL (15 mg / kg); 7 mice in Group 3, administered VIS649 analog (10 mg / kg) and APRIL (15 mg / kg); 7 mice in Group 4, administered APRIL (15 mg / kg); 3 mice in Group 5, administered MIL116 (10 mg / kg); 3 mice in Group 6, administered BION-1301 analog (10 mg / kg); 3 mice in Group 7, administered VIS649 analog (10 mg / kg); and 3 mice in Group 8, administered PBS as a negative control group.

[0174] 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 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 April protein, and blood was collected from the orbital venous plexus at 38h, 40h, 42h, and 44h (i.e., 2 hours after April injection).

[0175] The collected blood samples were centrifuged to obtain serum, which was stored at -40℃. Then, the concentration of April in the blood samples at different time points was detected by ELISA. Specifically, recombinant streptavidin protein (CAT#:STN-N5116, Bismuth subcitrate) was diluted in 1×PBS buffer to a final concentration of 1 μg / ml, and then added to the ELISA plate at a rate of 50 μl / well, and incubated horizontally at 37℃ for 2 hours. Remove the ELISA plate and wash it three times with PBST. Add 200 μl of blocking buffer (PBS containing 3% BSA) to each well and incubate horizontally at 37°C for 2 hours. Dilute the serum sample with the blocking buffer to a dilution factor of 5. The April-Biotin standard curve concentration starts at 500 ng / mL and is diluted to 8 points: 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. Remove the blocked ELISA plate and wash it three times with PBST to remove any residual droplets. Add 50 μl of diluted serum sample or standard curve sample to each well, seal the plate, and incubate horizontally overnight at 4°C. (Instructions page 19 / 28, page 22, CN 121991226 A) Remove the ELISA plate from the 4°C cold room and wash it three times with PBST.The VIS649 analog was diluted to 10 μg / ml, and 50 μl was added to each well. The plate was sealed with a membrane and incubated horizontally at 37°C for 1 hour. The plate was then removed, washed three times with PBST, and 50 μl of goat anti-hIgG-Fc-HRP (CAT#A0170-1ML, Sigma) diluted 5000 times with PBS containing 3% BSA was added to each well. The plate was incubated horizontally at 37°C for 1 hour. The plate was then removed, washed three times with PBST. 50 μl of TMB chromogenic solution was added to each well. After about 10 minutes, the color development was observed, and 50 μl of 2N H2SO4 stop solution was added to terminate the reaction. The OD value was read at 450 nm using a microplate reader. The concentration value was then calculated, and the APRIL clearance curve was plotted.

[0176] The results are shown in Figure 4. Compared to BION-1301 and VIS649 analogs, MIL116 can rapidly and persistently reduce APRIL protein in the peripheral circulation system. This may be because MIL116 can induce faster endocytosis of the APRIL-antibody complex in FcRn-expressing cells, rapidly dissociate from APRIL under low pH (endosome) conditions, and then re-enter the circulation system.

[0177] Example 9. Study on the clearance rate of MIL116 antibody in FcRn humanized mice

[0178] The blood clearance rate 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).

[0179] B6-FcRn humanized mice were randomly divided into 9 groups. The mice were divided into several 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); Group 5 (3 mice) was administered MIL116 (10 mg / kg); Group 6 (3 mice) was administered BION1301 analog (10 mg / kg); Group 7 (3 mice) was administered VIS649 analog (10 mg / kg); and Group 8 (3 mice) was administered ALPN303 analog (10 mg / kg). Group 9 consisted of 3 mice, which were administered PBS. They were injected with antibodies once and APRIL four times.

[0180] As 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 at 24h; 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.

[0181] The collected blood samples were centrifuged to obtain serum, which was stored at -40°C. Subsequently, the antibody concentration in the blood samples at different time points was 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 the 37°C incubator 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 overnight at 4°C. Serum samples were 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-total-HRP (CAT#: A8667-2 mL, Sigma) diluted 5000 times with PBS containing 3% BSA was added to each well and 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 solution to each well. After about 10 minutes, observe the degree of color development and terminate the reaction in time. Read the OD value at 450 nm using an ELISA reader. Then calculate the concentration value and plot the antibody clearance curve.

[0182] As shown in Figure 5, without exogenous APRIL injection, MIL116 has a relatively high serum antibody concentration, which is higher than that of BION1301 analog, VIS649 analog and ALPN303 analog, indicating that MIL116 has good bioavailability and no degradation caused by non-specific binding, resulting in a low clearance rate and good antibody properties.After the first two injections of exogenous APRIL, MIL116 maintained a relatively high concentration. Following the third injection of exogenous APRIL, the MIL116 antibody level decreased but remained significantly higher than other positive control antibodies. Serum levels of BION1301 analogs, VIS649 analogs, and ALPN303 analogs decreased rapidly after the second APRIL injection. The concentrations of BION1301 and VIS649 analogs at the 384-hour mark were close to baseline levels. These data indicate that, compared to BION1301, VIS649, and ALPN303 analogs, MIL116 can induce the APRIL-antibody complex to dissociate from APRIL at low pH (late inclusion bodies) conditions more quickly after endocytosis, exhibiting stronger interaction with human FcRn. This significantly reduces APRIL-mediated endocytic degradation and clearance, enabling the reuse of the antibody itself.

[0183] It can be seen that after Fc modification, MIL116, compared with the positive control, can achieve slower clearance and degradation in FcRn humanized mice, resulting in a longer effective drug action time. Therefore, this antibody may achieve a lower dosing frequency in future clinical treatment and improve treatment compliance.

[0184] Example 10. ADA prediction and detection of MIL116 antibody

[0185] The immunogenicity prediction program of the Wemol platform of Wecomputing (https: / / wemol.wecomput.com / ui / # / frontend / home / workflow-modules) was used to analyze the T cell epitopes in the variable region sequence of MIL116. The results showed that there may be a T cell epitope that binds to HLA in the light chain CDR1, that is, there is an immunogenicity risk sequence.

[0186] 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.

[0187] 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 in a 37°C incubator for 20 minutes, and adherent cells were collected to obtain isolated mononuclear cells.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 immunogenic 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.

[0188] 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).

[0189] In 96-well U-bottom detection plates, DCs from Group 1 and CD4+ T cells from the same donor were seeded at densities of 200,000 cells (T cells) / well and 20,000 cells / well (DC), with a total culture volume of 200 μl, and incubated together for 4 days.The DCs in group 2, after co-incubating with the DC-T cells in group 1 for 2 days, were incubated with 150 μg / ml of MIL116 or BION1301, or 300 μg / ml of KLH positive control protein, and co-incubated for another 2 days. Then, the co-incubation antibodies or positive proteins were thoroughly removed by washing and centrifugation with complete culture medium (page 21 / 28, CN 121991226 A) and set aside. The DCs in group 2 were added to the aforementioned DC-T cell mixture at a rate of 20,000 per well and incubated for another 2 days for flow cytometry detection.

[0190] Activation markers of CD4+ T cells were stained with PE mouse anti-human OX40 (Cat#:350004, Biolegend) and FITC mouse anti-human CD25 (Cat#:356106, Biolegend), 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 (ADA).

[0191] Table 4. HLA subtype information of PBMCs from 8 different donor sources

[0192] Donor ID DRB1 DQB1 Y1834 01:01,09:01 05:01,03:03 T0024 04:01,15:01 06:02,03:01 T0026 13:02,14:05 05:03,06:09 Y1609 12:02,15:01 06:02,03:01 Z0313 04:05,08:03 04:01,06:01 YB3075 11:01,16:02 05:02,03:01 EZ4035 07:01,12:02 02:02,03:01 EZ4098 04:03,14:54 03:02,05:03

[0193] Figures 6 (A and B) show the immunogenicity risk of MIL116 and BION1301 as tested by PBMCs of the eight high-frequency HLA subtypes 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.

[0194] Example 11. Antibody Stability Analysis

[0195] 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.

[0196] Briefly, 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. The mixture was heated, and the changes in fluorescence signal were recorded. The changes in fluorescence signal were processed and analyzed to extract the thermal stability parameters of the target molecule.

[0197] Table 5 shows the DSF detection 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.

[0198] Table 5. DSF detection results of MIL116 and positive control antibody

[0199] Sample name Tm (°C) 350 / 330 MIL116-IgG1 70.6 BION1301 analog 63.9 VIS649 analog 68.1

[0200] 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 analyzed by size exclusion-high performance liquid chromatography (SEC).

[0201] 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 was stable. The prepared sample was loaded into the chromatographic system using an autosampler. The flow rate was constant as the chromatographic column was passed through the protein, which was eluted sequentially according to its size. The elution curve was recorded by the detector to determine the monomer content in the sample.

[0202] Table 6. Antibody aggregation after two weeks of high temperature storage

[0203] Sample name Polymer peak Monomer main peak (%) Fragment peak MIL116 No high temperature treatment 0.9 99.1 - MIL116 High temperature for two weeks 0.9 98.6 0.5

[0204] As shown in Table 6, after two weeks of high temperature, there was no significant increase in MIL116 molecules, indicating its excellent thermal stability.

[0205] Example 12. PK / PD experiment of MIL116 in cynomolgus monkeys

[0206] The amino acid sequence homology between human and cynomolgus monkey APRIL is >99%, differing by only 2 amino acids, one of which is amino acid 189 of human APRIL.Furthermore, the results of Examples 4 and 5 show that MIL116 and the two positive control antibodies exhibit extremely similar affinity and receptor blocking activity when binding to recombinant human and cynomolgus monkey APRIL protein. Therefore, non-human primates (NHPs) are a suitable animal model for evaluating the PD / PK and safety of MIL116. This experiment was commissioned to Zhaoyan Yichuang (Suzhou) New Drug Research Co., Ltd.

[0207] Cynomolgus monkeys were randomly divided into three groups: the MIL116 (IgG1-m1 antibody prepared in Example 6) group, the BION1301 analog group, and the VIS649 analog group, with two monkeys in each group, one male and one female. The animals in each group were weighed before administration, and the corresponding amount of test product 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 was 60 mg / ml, and VIS649 was 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 the veins of the forelimbs or hindlimbs at 0h (D0), 24h (D2), D8, D15, D22, and D29, 2 mL per animal, 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) detection. The samples were stored below -60°C.

[0208] Pharmacodynamics was mainly determined by detecting the amounts of IgA, IgG, and IgM in monkey serum. Specifically, the serum concentrations of IgA, IgG, and IgM were determined by immunoturbidimetry using kits (Cat#:H416, Cat#:H415, and Cat#:H417, Meikang Biotechnology) according to the manufacturer's method steps.

[0209] Pharmacokinetics were detected 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 added to an ELISA plate (CAT#9018, CORNING) at a rate of 100 μl / well. The plate was then incubated horizontally at 4°C overnight. After removing the plate from 4°C, it was washed three times with PBST.Add 250 μl of blocking buffer (PBS containing 5% skim milk powder) to each well and block at 37°C for 1.5 hours. The standard curve of the test drug is prepared by 3-fold dilution with a maximum starting concentration of 1 μg / ml, resulting in 8 concentration points. Monkey blood samples are diluted with blocking buffer at an appropriate ratio (20-10000) to ensure the detection results fall within the quantitative range. Remove the blocked plate and wash it 3 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 ELISA plate and wash it 3 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 ELISA plate and wash it 3 times with PBST. Add TMB chromogenic solution (CAT#:555214, BD), place horizontally in a 37°C incubator, incubate for 15 minutes, remove the microplate, terminate the reaction, and read the value using a microplate reader.

[0210] As shown in Figures 7 (A-C), after administration of each antibody, the serum concentrations of IgG (A), IgA (B), and IgM (C) in cynomolgus monkeys decreased. Within 7 days of antibody administration, MIL116 induced a significant decrease in IgA / G / M, with the drug taking effect faster than the two positive control molecules, and the ability of MIL116 to decrease IgA / G / M in the first 29 days was also superior to that of VIS649 analogs and BION-1301 analogs.

[0211] 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.

[0212] The sequences appearing in this application are listed below.

[0213] Specification 24 / 28 pages 27 CN 121991226 A

[0214] Specification 25 / 28 pages 28 CN 121991226 A

[0215] Specification 26 / 28 pages 29 CN 121991226 A

[0216]

[0217]

[0218] 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 documents cited herein are incorporated herein by reference in their entirety.Instruction manual, page 28 / 28, page 31, CN 121991226 A, Figure 1; Instruction manual, Figure 1 / 8, page 32, CN 121991226 A, Figure 2; Instruction manual, Figure 2 / 8, page 33, CN 121991226 A, Figure 3; Instruction manual, Figure 3 / 8, page 34, CN 121991226 A, Figure 4; Instruction manual, Figure 4 / 8, page 35, CN 121991226 A, Figure 5; Instruction manual, Figure 5 / 8, page 36, CN 121991226 A, Figure 6; Instruction manual, Figure 6 / 8, page 37, CN 121991226 A, Figure 7; Instruction manual, Figure 7 / 8, page 38, CN 121991226 A, Figure 8; Instruction manual, Figure 8 / 8, page 39, CN 121991226 A, Abstract: Abdominal ultrasound examination method, system and device. An antibody binding to APRIL and its use. The present application relates to an isolated monoclonal antibody or its antigen-binding portion that can... specifically bind to APRIL. The present application also relates to a nucleic acid molecule encoding the antibody or its antigen binding portion; an expression vector, a host cell, and a method for expressing the antibody or its antigen binding portion; as well as a therapeutic method using the antibody or its antigen binding portion, the nucleic acid molecule, the expression vector, and / or the host cell..

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-CDR1 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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