Anti-april antibody or antigen-binding fragment thereof, and use thereof

Abstract

Provided in the present invention is an anti-APRIL antibody or an antigen-binding fragment thereof. Further provided are a polynucleotide encoding the antibody, a vector and host cell for expressing the antibody, a pharmaceutical composition containing the antibody, a method for treating APRIL-associated diseases using the antibody, and the pharmaceutical use thereof.

Description

Anti-april antibody or antigen-binding fragment thereof and uses thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and specifically relates to an anti-APRIL antibody or antigen-binding fragment thereof, a pharmaceutical composition and uses thereof. BACKGROUND

[0002] APRIL, full name A Proliferation-Inducing Ligand, is a member of the tumor necrosis factor (TNF) superfamily, and its encoding gene is TNFSF13. APRIL and B cell stimulating molecule BAFF are key proteins for maintaining the stability of B cell library and humoral immunity. APRIL mainly targets TNFRSF13B / TACI (transmembrane activator and CAML interactor) and TNFRSF17 / BCMA (B cell maturation antigen) receptors, and promotes tumor cell proliferation by binding to the receptors, and plays a key role in immune regulation such as B cell antigen presentation, Ig class switching, plasma cell survival, thymus-dependent and thymus-independent responses.

[0003] APRIL plays an important role in the survival and proliferation ability of several B cell malignancies and possibly some solid tumors. APRIL also plays a key role in inflammatory or autoimmune diseases, such as systemic lupus erythematosus (SLE), IgA nephropathy (IgAN), and rheumatoid arthritis (RA). In genome-wide association study (GWAS) analysis, TNFSF13 was found to be a key candidate site associated with glomerular lesions in IgA nephropathy patients. In addition, APRIL significantly increases the production of Gd-IgA1 in vivo by promoting B cell activation, and Gd-IgA1 is the main autoantigen in IgA nephropathy, and its excessive production and abnormal polymerization is the starting factor for the subsequent formation of immune complexes and kidney damage.

[0004] In addition to theoretical support, relevant research also confirms the important role of APRIL in IgA nephropathy. Sibeprenlimab is a humanized IgG2 monoclonal antibody that prevents the production of pathogenic IgA1 by binding and neutralizing APRIL activity. Clinical research data of Sibeprenlimab shows that it can intervene in the initial stage of disease development, not only reduces the 24-hour urine protein / cratin ratio, but also effectively delays the decline of glomerular filtration rate.

[0005] In order to better meet the clinical needs, it is still necessary to provide more effective or differentiated antagonistic antibodies that can bind to APRIL. SUMMARY

[0006] In one aspect, the present application provides an anti-APRIL antibody or antigen-binding fragment thereof, which is capable of specifically binding to APRIL protein (e.g., human, monkey and / or murine APRIL) with high affinity, does not bind or substantially does not bind to BAFF, eliminates the ADCC equivalent effect of the antibody to avoid the side reaction caused thereby, and effectively blocks the binding of APRIL to receptor BCMA and / or TACI, inhibiting the level of IgA secreted by B cells.

[0007] In one aspect, the present application provides an anti-APRIL antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising HCDR1, HCDR2 and HCDR3 regions, and the VL comprising LCDR1, LCDR2 and LCDR3 regions; the HCDR1, HCDR2 and HCDR3 regions have the same sequence as the HCDR1, HCDR2 and HCDR3 of the VH as shown in any one of SEQ ID NO. 1-26, or a sequence with up to 5, 4, 3, 2 or 1 mutations compared to each of the CDRs of HCDR1-3 of the VH as shown in any one of SEQ ID NO. 1-26, and the LCDR1, LCDR2 and LCDR3 regions have the same sequence as the LCDR1, LCDR2 and LCDR3 of the VL as shown in any one of SEQ ID NO. 27-51, or a sequence with up to 5, 4, 3, 2 or 1 mutations compared to each of the CDRs of LCDR1-3 of the VL as shown in any one of SEQ ID NO. 27-51.

[0008] In some embodiments, the HCDR1, HCDR2, and HCDR3 regions and the LCDR1, LCDR2, and LCDR3 regions have the same sequence as the HCDR1-3 of a VH and the LCDR1-3 of a VL selected from the group consisting of: the VH as shown in SEQ ID NO. 1 and the VL as shown in SEQ ID NO. 27; the VH as shown in SEQ ID NO. 2 and the VL as shown in SEQ ID NO. 28; the VH as shown in SEQ ID NO. 3 and the VL as shown in SEQ ID NO. 29; the VH as shown in SEQ ID NO. 4 and the VL as shown in SEQ ID NO. 30; the VH as shown in SEQ ID NO. 5 and the VL as shown in SEQ ID NO. 31; the VH as shown in SEQ ID NO. 6 and the VL as shown in SEQ ID NO. 32; the VH as shown in SEQ ID NO. 7 and the VL as shown in SEQ ID NO. 33; the VH as shown in SEQ ID NO. 8 and the VL as shown in SEQ ID NO. 34; the VH as shown in SEQ ID NO. 9 and the VL as shown in SEQ ID NO. 35; the VH as shown in SEQ ID NO. 10 and the VL as shown in SEQ ID NO. 36; the VH as shown in SEQ ID NO. 11 and the VL as shown in SEQ ID NO. 37; the VH as shown in SEQ ID NO. 12 and the VL as shown in SEQ ID NO. 38; the VH as shown in SEQ ID NO. 13 and the VL as shown in SEQ ID NO. 39; the VH as shown in SEQ ID NO. 14 and the VL as shown in SEQ ID NO. 40; the VH as shown in SEQ ID NO. 15 and the VL as shown in SEQ ID NO. 41; the VH as shown in SEQ ID NO. 16 and the VL as shown in SEQ ID NO. 42; the VH as shown in SEQ ID NO. 17 and the VL as shown in SEQ ID NO. 42; the VH as shown in SEQ ID NO. 18 and the VL as shown in SEQ ID NO. 43; the VH as shown in SEQ ID NO. 19 and the VL as shown in SEQ ID NO. 44; the VH as shown in SEQ ID NO. 20 and the VL as shown in SEQ ID NO. 45; the VH as shown in SEQ ID NO. 21 and the VL as shown in SEQ ID NO. 46; the VH as shown in SEQ ID NO. 22 and the VL as shown in SEQ ID NO. 47; the VH as shown in SEQ ID NO. 23 and the VL as shown in SEQ ID NO. 48; the VH as shown in SEQ ID NO. 24 and the VL as shown in SEQ ID NO. 49; the VH as shown in SEQ ID NO. 25 and the VL as shown in SEQ ID NO. 50; the VH as shown in SEQ ID NO. 26 and the VL as shown in SEQ ID NO. 51; or have a sequence that differs from each of the CDRs in the HCDR1-3 of a VH and the LCDR1-3 of a VL selected from the group consisting of: the VH as shown in SEQ ID NO. 1 and the VL as shown in SEQ ID NO. 27; the VH as shown in SEQ ID NO. 2 and the VL as shown in SEQ ID NO. 28; the VH as shown in SEQ ID NO. 3 and the VL as shown in SEQ ID NO. 29; the VH as shown in SEQ ID NO. 4 and the VL as shown in SEQ ID NO. 30; the VH as shown in SEQ ID NO. 5 and the VL as shown in SEQ ID NO. 31; the VH as shown in SEQ ID NO. 6 and the VL as shown in SEQ ID NO. 32; the VH as shown in SEQ ID NO. 7 and the VL as shown in SEQ ID NO. 33; the VH as shown in SEQ ID NO. 8 and the VL as shown in SEQ ID NO. 34; the VH as shown in SEQ ID NO. 9 and the VL as shown in SEQ ID NO. 35; the VH as shown in SEQ ID NO. 10 and the VL as shown in SEQ ID NO. 36; the VH as shown in SEQ ID NO. 11 and the VL as shown in SEQ ID NO. 37; the VH as shown in SEQ ID NO. 12 and the VL as shown in SEQ ID NO. 38; the VH as shown in SEQ ID NO. 13 and the VL as shown in SEQ ID NO. 39; the VH as shown in SEQ ID NO. 14 and the VL as shown in SEQ ID NO. 40; the VH as shown in SEQ ID NO. 15 and the VL as shown in SEQ ID NO. 41; the VH as shown in SEQ ID NO. 16 and the VL as shown in SEQ ID NO. 42; the VH as shown in SEQ ID NO. 17 and the VL as shown in SEQ ID NO. 42; the VH as shown in SEQ ID NO. 18 and the VL as shown in SEQ ID NO. 43; the VH as shown in SEQ ID NO. 19 and the VL as shown in SEQ ID NO. 44; the VH as shown in SEQ ID NO. 20 and the VL as shown in SEQ ID NO. 45; the VH as shown in SEQ ID NO. 21 and the VL as shown in SEQ ID NO. 46; the VH as shown in SEQ ID NO. 22 and the VL as shown in SEQ ID NO. 47; the VH as shown in SEQ ID NO. 23 and the VL as shown in SEQ ID NO. 48; the VH as shown in SEQ ID NO. 24 and the VL as shown in SEQ ID NO. 49; the VH as shown in SEQ ID NO. 25 and the VL as shown in SEQ ID NO. 50; the VH as shown in SEQ ID NO. 26 and the VL as shown in SEQ ID NO. 51; up to 5, 4, 3, 2, or 1 mutations.23 and a VL as set forth in SEQ ID NO. 48; a VH as set forth in SEQ ID NO. 24 and a VL as set forth in SEQ ID NO. 49; a VH as set forth in SEQ ID NO. 25 and a VL as set forth in SEQ ID NO. 50; or, a VH as set forth in SEQ ID NO. 26 and a VL as set forth in SEQ ID NO. 51.

[0009] said mutations are selected from the group consisting of insertions, deletions and / or substitutions which do not affect the function, said substitutions being preferably conservative amino acid substitutions.

[0010] In some embodiments, the VH has an HCDR1, HCDR2 and HCDR3 of the same sequence as the VH as set forth in SEQ ID NO. 1 and the VL has an LCDR1, LCDR2 and LCDR3 of the same sequence as the VL as set forth in SEQ ID NO. 27.

[0011] In some embodiments, the VH has an HCDR1, HCDR2 and HCDR3 of the same sequence as the VH as set forth in SEQ ID NO. 2 and the VL has an LCDR1, LCDR2 and LCDR3 of the same sequence as the VL as set forth in SEQ ID NO. 28.

[0012] In some embodiments, the VH has an HCDR1, HCDR2 and HCDR3 of the same sequence as the VH as set forth in SEQ ID NO. 3 and the VL has an LCDR1, LCDR2 and LCDR3 of the same sequence as the VL as set forth in SEQ ID NO. 29.

[0013] In some embodiments, the VH has an HCDR1, HCDR2 and HCDR3 of the same sequence as the VH as set forth in SEQ ID NO. 4 and the VL has an LCDR1, LCDR2 and LCDR3 of the same sequence as the VL as set forth in SEQ ID NO. 30.

[0014] In some embodiments, the VH has an HCDR1, HCDR2 and HCDR3 of the same sequence as the VH as set forth in SEQ ID NO. 5 and the VL has an LCDR1, LCDR2 and LCDR3 of the same sequence as the VL as set forth in SEQ ID NO. 31.

[0015] In some embodiments, the VH has an HCDR1, HCDR2 and HCDR3 of the same sequence as the VH as set forth in SEQ ID NO. 6 and the VL has an LCDR1, LCDR2 and LCDR3 of the same sequence as the VL as set forth in SEQ ID NO. 32.

[0016] In some embodiments, the VH has HCDR1, HCDR2, and HCDR3 having the same sequence as the VH set forth in any one of SEQ ID NO. 54-57, 61-64, 67, 72-73, and / or the VL has LCDR1, LCDR2, and LCDR3 having the same sequence as the VL set forth in any one of SEQ ID NO. 58-60, 65-66, 68-71, 74-75.

[0017] In some preferred embodiments, the VH has HCDR1, HCDR2, and HCDR3 having the same sequence as the VH set forth in any one of SEQ ID NO. 54-57, and the VL has LCDR1, LCDR2, and LCDR3 having the same sequence as the VL set forth in any one of SEQ ID NO. 58-60. In some specific embodiments, the HCDR1, HCDR2, and HCDR3 regions and the LCDR1, LCDR2, and LCDR3 regions have the same sequence as the HCDR1-3 of a VH and the LCDR1-3 of a VL selected from the group consisting of: the VH set forth in SEQ ID NO. 54 and the VL set forth in SEQ ID NO. 58; the VH set forth in SEQ ID NO. 55 and the VL set forth in SEQ ID NO. 58; the VH set forth in SEQ ID NO. 56 and the VL set forth in SEQ ID NO. 58; the VH set forth in SEQ ID NO. 54 and the VL set forth in SEQ ID NO. 59; the VH set forth in SEQ ID NO. 55 and the VL set forth in SEQ ID NO. 59; the VH set forth in SEQ ID NO. 54 and the VL set forth in SEQ ID NO. 60; the VH set forth in SEQ ID NO. 55 and the VL set forth in SEQ ID NO. 60; the VH set forth in SEQ ID NO. 56 and the VL set forth in SEQ ID NO. 60; or, the VH set forth in SEQ ID NO. 57 and the VL set forth in SEQ ID NO. 60.

[0018] In some preferred embodiments, the VH has HCDR1, HCDR2, and HCDR3 of the same sequence as the VH set forth in any one of SEQ ID NO. 61-64, and the VL has LCDR1, LCDR2, and LCDR3 of the same sequence as the VL set forth in any one of SEQ ID NO. 65-66. In some specific embodiments, the HCDR1, HCDR2, and HCDR3 regions and the LCDR1, LCDR2, and LCDR3 regions have the same sequence as the HCDR1-3 of a VH and the LCDR1-3 of a VL selected from the group consisting of: a VH set forth in SEQ ID NO. 61 and a VL set forth in SEQ ID NO. 65; a VH set forth in SEQ ID NO. 62 and a VL set forth in SEQ ID NO. 65; a VH set forth in SEQ ID NO. 63 and a VL set forth in SEQ ID NO. 65; or a VH set forth in SEQ ID NO. 64 and a VL set forth in SEQ ID NO. 66.

[0019] In some preferred embodiments, the VH has HCDR1, HCDR2, and HCDR3 of the same sequence as the VH set forth in SEQ ID NO. 67, and the VL has LCDR1, LCDR2, and LCDR3 of the same sequence as the VL set forth in any one of SEQ ID NO. 68-71. In some specific embodiments, the HCDR1, HCDR2, and HCDR3 regions and the LCDR1, LCDR2, and LCDR3 regions have the same sequence as the HCDR1-3 of a VH and the LCDR1-3 of a VL selected from the group consisting of: a VH set forth in SEQ ID NO. 67 and a VL set forth in SEQ ID NO. 68; a VH set forth in SEQ ID NO. 67 and a VL set forth in SEQ ID NO. 69; a VH set forth in SEQ ID NO. 67 and a VL set forth in SEQ ID NO. 70; or a VH set forth in SEQ ID NO. 67 and a VL set forth in SEQ ID NO. 71.

[0020] In some preferred embodiments, the VH has the same sequence of HCDR1, HCDR2, and HCDR3 as set forth in any one of SEQ ID NO. 72-73, and the VL has the same sequence of LCDR1, LCDR2, and LCDR3 as set forth in any one of SEQ ID NO. 74-75. In some particular embodiments, the HCDR1, HCDR2, and HCDR3 regions and the LCDR1, LCDR2, and LCDR3 regions have the same sequence as the HCDR1-3 of a VH and the LCDR1-3 of a VL selected from the group consisting of: a VH as set forth in SEQ ID NO. 72 and a VL as set forth in SEQ ID NO. 74; a VH as set forth in SEQ ID NO. 72 and a VL as set forth in SEQ ID NO. 75; a VH as set forth in SEQ ID NO. 73 and a VL as set forth in SEQ ID NO. 74; or, a VH as set forth in SEQ ID NO. 73 and a VL as set forth in SEQ ID NO. 75.

[0021] In some embodiments, the HCDR1, HCDR2, and HCDR3 of the VH, and the LCDR1, LCDR2, and LCDR3 of the VL are defined according to IMGT, Kabat, Chothia, AbM, Contact, or any combination thereof.

[0022] In some embodiments, the HCDR1 comprises an amino acid sequence selected from any one of Table 4.1 or Table 4.2, and / or the HCDR2 comprises an amino acid sequence selected from any one of Table 4.1 or Table 4.2, and / or the HCDR3 comprises an amino acid sequence selected from any one of Table 4.1 or Table 4.2, and / or the LCDR1 comprises an amino acid sequence selected from any one of Table 4.1 or Table 4.2, and / or the LCDR2 comprises an amino acid sequence selected from any one of Table 4.1 or Table 4.2, and / or the LCDR3 comprises an amino acid sequence selected from any one of Table 4.1 or Table 4.2.

[0023] In some embodiments, the HCDR1-3 and LCDR1-3 are selected from any one of the groups set forth in Table 4.1.

[0024] In some embodiments, the HCDR1-3 and LCDR1-3 are selected from any one of the groups set forth in Table 4.2.

[0025] In some embodiments, the HCDR1-3 and LCDR1-3 are selected from any one of the following groups:

[0026] In some embodiments, the HCDR1-3 and LCDR1-3 are selected from any one of the following groups:

[0027] In some embodiments, the VH has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NO. 1-26, 54-57, 61-64, 67, 72-73, and / or the VL has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NO. 27-51, 58-60, 65-66, 68-71, 74-75.

[0028] In some embodiments, the VH has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NO. 1-26, and / or the VL has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NO. 27-51.

[0029] In some embodiments, the VH has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NO. 54-57, 61-64, 67, 72-73, and / or the VL has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NO. 58-60, 65-66, 68-71, 74-75.

[0030] In some preferred embodiments, the VH has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NO. 54-57, and / or the VL has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NO. 58-60. In some preferred embodiments, the VH has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NO. 61-64, and / or the VL has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NO. 65-66. In some preferred embodiments, the VH has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO. 67, and / or the VL has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NO. 68-71. In some embodiments, the VH has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NO. 72-73, and / or the VL has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NO. 74-75.

[0031] In some preferred embodiments, the VH and VL comprise a sequence selected from the group consisting of, or comprising a sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of: the VL set forth in SEQ ID NO. 1 and 27; SEQ ID NO. 2 and 28; SEQ ID NO. 3 and 29; SEQ ID NO. 4 and 30; SEQ ID NO. 5 and 31; SEQ ID NO. 6 and 32; SEQ ID NO. 7 and 33; SEQ ID NO. 8 and 34; SEQ ID NO. 9 and 35; SEQ ID NO. 10 and 36; SEQ ID NO. 11 and 37; SEQ ID NO. 12 and 38; SEQ ID NO. 13 and 39; SEQ ID NO. 14 and 40; SEQ ID NO. 15 and 41; SEQ ID NO. 16 and 42; SEQ ID NO. 17 and 42; SEQ ID NO. 18 and 43; SEQ ID NO. 19 and 44; SEQ ID NO. 20 and 45; SEQ ID NO. 21 and 46; SEQ ID NO. 22 and 47; SEQ ID NO. 23 and 48; SEQ ID NO. 24 and 49; SEQ ID NO. 25 and 50; or, SEQ ID NO. 26 and 51.

[0032] In some preferred embodiments, the VH and VL comprise a sequence selected from the group consisting of, or comprising a sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of: the VL set forth in SEQ ID NO. 1 and 27; SEQ ID NO. 2 and 28; SEQ ID NO. 3 and 29; SEQ ID NO. 4 and 30; SEQ ID NO. 5 and 31; SEQ ID NO. 6 and 32; SEQ ID NO. 7 and 33; SEQ ID NO. 8 and 34; SEQ ID NO. 9 and 35; SEQ ID NO. 10 and 36; SEQ ID NO. 11 and 37; SEQ ID NO. 12 and 38; SEQ ID NO. 13 and 39; SEQ ID NO. 14 and 40; SEQ ID NO. 15 and 41; SEQ ID NO. 16 and 42; SEQ ID NO. 17 and 42; SEQ ID NO. 18 and 43; SEQ ID NO. 19 and 44; SEQ ID NO. 20 and 45; SEQ ID NO. 21 and 46; SEQ ID NO. 22 and 47; SEQ ID NO. 23 and 48; SEQ ID NO. 24 and 49; SEQ ID NO. 25 and 50; or, SEQ ID NO. 26 and 51.

[0033] In some preferred embodiments, the VH and VL comprise or consist of the sequences set forth in SEQ ID NO. 1 and 27, respectively; the VH and VL comprise or consist of the sequences set forth in SEQ ID NO. 2 and 28, respectively; the VH and VL comprise or consist of the sequences set forth in SEQ ID NO. 3 and 29, respectively; the VH and VL comprise or consist of the sequences set forth in SEQ ID NO. 4 and 30, respectively; the VH and VL comprise or consist of the sequences set forth in SEQ ID NO. 5 and 31, respectively; or the VH and VL comprise or consist of the sequences set forth in SEQ ID NO. 6 and 32, respectively.

[0034] In some preferred embodiments, the VH and VL comprise or consist of the sequences set forth in SEQ ID NO. 56 and 60, respectively; the VH and VL comprise or consist of the sequences set forth in SEQ ID NO. 57 and 60, respectively; or the VH and VL comprise or consist of the sequences set forth in SEQ ID NO. 63 and 65, respectively.

[0035] In some embodiments, the anti-APRIL antibody provided herein further comprises a heavy chain constant region selected from the constant region of IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE, or a variant thereof, preferably from the constant region of, e.g., human IgGl, IgG2, IgG3, and IgG4, or a variant thereof; and a light chain constant region selected from the constant region of kappa and lambda chains, or a variant thereof, preferably from the constant region of human kappa and lambda chains, or a variant thereof. The variant has altered effector function (e.g., ADCC and / or CDC activity, affinity for FcyRIIIa and / or Clq), prolonged half-life, and / or increased therapeutic efficacy mediated by the Fc region, but does not alter the function of the antibody variable region. In certain embodiments, the variant Fc region has at least one amino acid substitution compared to the wild-type Fc region, e.g., about 1-10 amino acid substitutions, or about 1-5 amino acid substitutions, or about 1-3 amino acid substitutions in the wild-type Fc region. The variant Fc region can have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identity to the wild-type Fc region. In some embodiments, the variant reduces or eliminates ADCC and / or CDC effect. In some embodiments, the variant enhances FcRn binding and / or prolongs half-life and / or improves pharmacokinetics. In certain embodiments, the antibody or antigen-binding fragment thereof comprises the constant region of human IgG4 or IgGl, or a variant thereof, preferably the variant has one or more than one of the following substitutions compared to the wild-type sequence from which it is derived: S228P, L234A / L235A / P329G (LALAPG), M428L / N434S (LS) (positions according to the EU numbering system). In some specific embodiments, the variant has LALAPG and LS mutations. In some preferred embodiments, the heavy chain constant region comprises a sequence as set forth in SEQ ID NO. 52 or 76, or a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; and the light chain constant region comprises a sequence as set forth in SEQ ID NO. 53, or a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0036] In some embodiments, the anti-APRIL antibody comprises a heavy chain comprising an amino acid sequence which differs from the sequence as set forth in SEQ ID NO. 77 or 79 by up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 mutations (e.g., substitutions, deletions, or additions of amino acids, preferably conservative amino acid substitutions) or comprises an amino acid sequence which is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the sequence as set forth in SEQ ID NO. 77 or 79; and / or,

[0037] the light chain comprises an amino acid sequence which differs from the sequence as set forth in SEQ ID NO. 78 by up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 mutations (e.g., substitutions, deletions, or additions of amino acids, preferably conservative amino acid substitutions) or comprises an amino acid sequence which is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the sequence as set forth in SEQ ID NO. 78.

[0038] In some embodiments, the anti-APRIL antibody comprises a heavy chain comprising an amino acid sequence which differs from the sequence as set forth in SEQ ID NO. 80 by up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 mutations (e.g., substitutions, deletions, or additions of amino acids, preferably conservative amino acid substitutions) or comprises an amino acid sequence which is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the sequence as set forth in SEQ ID NO. 80; and / or,

[0039] the light chain comprises an amino acid sequence which differs from the sequence as set forth in SEQ ID NO. 81 by up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 mutations (e.g., substitutions, deletions, or additions of amino acids, preferably conservative amino acid substitutions) or comprises an amino acid sequence which is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the sequence as set forth in SEQ ID NO. 81.

[0040] In some embodiments, the heavy chain constant region lacks a C-terminal lysine. In some embodiments, the N-terminal amino acid of the antibody or antigen-binding fragment thereof (e.g., glutamine or glutamic acid) can cyclize to pyroglutamic acid or pyroglutamate.

[0041] In some embodiments, the antibody or antigen-binding fragment thereof can include post-translational modifications (e.g., C-terminal lysine clipping in the heavy chain, N-terminal glutamine or glutamic acid cyclization to pyroglutamic acid or pyroglutamate in the heavy chain or light chain), which can occur upon recombinant expression in a host cell (e.g., a CHO cell) or during purification / storage.

[0042] In some specific embodiments, the anti-APRIL antibody or antigen-binding fragment thereof provided herein is selected from any one of the antibodies shown in Table 4.3, Table 5, Table 8.1, Table 8.2, Table 8.3, Table 8.4.

[0043] In one aspect, the present application provides an anti-APRIL antibody or antigen-binding fragment thereof that binds to the same epitope of APRIL or competes for binding to an epitope of APRIL as any of the foregoing anti-APRIL antibodies or antigen-binding fragments thereof.

[0044] In some embodiments, the anti-APRIL antibody or antigen-binding fragment thereof provided herein has at least one of the following functions:

[0045] (1) the antibody or antigen-binding fragment thereof binds to human or monkey APRIL with a KD of about 10 -8 M, 10 -9 M, about 10 -10 M, about 10 -11 M, about 10 -12 M or lower, for example, about 10 nM to 0.01 nM, about 5 nM to 0.01 nM, about 3 nM to 0.01 nM, about 1 nM to 0.01 nM, about 0.05 nM to 0.01 nM, about 0.01 nM to 0.001 nM, about 0.005 to 0.001 nM, or for example, less than about 80 nM, about 70 nM, about 60 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM, about 10 nM, about 8 nM, about 6 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.5 nM, about 0.2 nM, about 0.1 nM, about 0.08 nM, about 0.05 nM, about 0.03 nM, about 0.01 nM, about 0.005 nM, or about 0.001 nM;

[0046] or, an EC50of about 1 nM to about 0.001 nM, about 0.5 nM to about 0.001 nM, about 0.3 nM to about 0.001 nM, about 0.2 nM to about 0.001 nM, about 0.1 nM to about 0.001 nM, about 0.08 nM to about 0.01 nM, about 0.05 nM to about 0.01 nM, or lower 50 binds to human or monkey APRIL with an IC50of less than about 2 nM, about 1.8 nM, about 1.6 nM, about 1.4 nM, about 1.2 nM, about 1 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM, about 0.1 nM;

[0047] (2) the antibody or antigen-binding fragment thereof binds to human or monkey APRIL with a KDsubstantially the same as or better than that of a reference APRIL antibody;

[0048] (3) the antibody or antigen-binding fragment thereof blocks (or inhibits, neutralizes) the binding of human APRIL to human BCMA, and / or blocks (or inhibits, neutralizes) the binding of human APRIL to human TACI, for example, blocks the binding of human APRIL to human BCMA and / or human TACI at the cellular level;

[0049] Preferably, the antibody or antigen-binding fragment thereof binds to human or monkey APRIL with an IC50of about 10 nM to about 0.01 nM, about 5 nM to about 0.01 nM, about 2 nM to about 0.01 nM, about 1.5 nM to about 0.01 nM, about 1 nM to about 0.01 nM, about 1 nM to about 0.5 nM, about 0.8 nM to about 0.4 nM, about 0.5 nM to about 0.1 nM, or lower 50 blocks the binding of human APRIL to human BCMA, for example, less than about 2 nM, about 1.8 nM, about 1.6 nM, about 1.4 nM, about 1.2 nM, about 1 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM, about 0.1 nM;

[0050] and / or, the antibody or antigen-binding fragment thereof blocks the binding of human APRIL to human TACI with an IC50of about 10 nM to about 0.1 nM, about 5 nM to about 0.1 nM, about 2 nM to about 0.1 nM, about 1 nM to about 0.1 nM, about 5 nM to about 1 nM, about 3 nM to about 1 nM, about 2 nM to about 1 nM, about 1.5 nM to about 1 nM, or lower 50 blocks the binding of human APRIL to human TACI, for example, less than about 4.5 nM, about 4.2 nM, about 4 nM, about 3.5 nM, about 3 nM, about 2.5 nM, about 2.3 nM, about 2 nM, about 1.8 nM, about 1.5 nM, about 1 nM, about 0.5 nM, about 0.1 nM;

[0051] (4) the antibody or antigen-binding fragment thereof binds human APRIL with an IC50 that is substantially the same as or lower than that of a reference APRIL antibody 50 blocks (or inhibits, neutralizes) the binding of human APRIL to human BCMA, and / or blocks (or inhibits, neutralizes) the binding of human APRIL to human TACI;

[0052] (5) the antibody or antigen-binding fragment thereof reduces (e.g., inhibits, blocks, or neutralizes) one or more biological activities of APRIL (e.g., human APRIL, monkey APRIL, murine APRIL, or two or more thereof, e.g., human and monkey APRIL, human and murine APRIL, or human, monkey, and murine APRIL, with murine APRIL preferably being rat APRIL) in vitro, ex vivo, or in vivo;

[0053] (6) the antibody or antigen-binding fragment thereof binds human APRIL, monkey APRIL, murine APRIL, or two or more thereof, e.g., human and monkey APRIL, human and murine APRIL, or human, monkey, and murine APRIL, with murine APRIL preferably being rat APRIL;

[0054] (7) the antibody or antigen-binding fragment thereof inhibits B cell (e.g., human B cell) proliferation;

[0055] (8) the antibody or antigen-binding fragment thereof reduces or inhibits the level of IgA, e.g., IgA secretion by B cells;

[0056] (9) the antibody or antigen-binding fragment thereof blocks (or inhibits, neutralizes) the binding of monkey APRIL to BCMA;

[0057] (10) the antibody or antigen-binding fragment thereof does not bind or substantially bind BAFF, e.g., as determined by ELISA.

[0058] In some preferred embodiments, the antibody or antigen-binding fragment thereof binds human APRIL with a KD of about 1 nM to 0.01 nM or about 0.05 nM to 0.01 nM, or, e.g., less than about 1 nM, less than about 0.1 nM, less than about 0.05 nM.

[0059] In some preferred embodiments, the antibody or antigen-binding fragment thereof binds human APRIL with an IC50 that is about 1 nM to 0.01 nM or lower, or, e.g., less than about 1 nM, less than about 0.8 nM, less than about 0.7 nM, less than about 0.6 nM, less than about 0.5 nM, less than about 0.4 nM, less than about 0.3 nM, less than about 0.2 nM, less than about 0.1 nM. 50 blocks the binding of human APRIL to human BCMA, e.g., less than about 1 nM, less than about 0.8 nM, less than about 0.7 nM, less than about 0.6 nM, less than about 0.5 nM, less than about 0.4 nM, less than about 0.3 nM, less than about 0.2 nM, less than about 0.1 nM.

[0060] In some preferred embodiments, the antibody or antigen binding fragment thereof has an IC 50 blocks the binding of human APRIL to human TACI, for example less than about 2 nM, less than about 1.8 nM, less than about 1.5 nM, less than about 1 nM, less than about 0.5 nM, less than about 0.1 nM.

[0061] In some embodiments, the monkey APRIL is preferably cynomolgus monkey APRIL.

[0062] The KD can be determined using methods known to those skilled in the art, for example using a Fortebio Octet®molecular interaction instrument assay, surface plasmon resonance (SPR), bio-layer interferometry (BLI), for example using the methods provided in Example 5 or Example 6 herein.

[0063] The EC 50 or IC 50 can be determined using methods known to those skilled in the art, for example using an ELISA assay, for example using the methods provided in Example 2 herein.

[0064] In some embodiments, the reference APRIL antibody can be prepared with reference to patent CN109089419B, WO2010100056A3, CN107207602B, which are incorporated herein in their entirety. In some specific embodiments, the reference APRIL antibody is selected from Sibeprenlimab, Zigakibart.

[0065] Sibeprenlimab, also known as VIS649, can be cloned and prepared de novo with reference to the sequence disclosed in WHO Drug Information, Volume 34. Number 4. 2020. Proposed INN: List 124, and also with reference to patent document CN109089419B.

[0066] Zigakibart, also known as BION-1301, can be cloned and prepared de novo with reference to the sequence disclosed in WHO Drug Information, Volume 36. Number 2. 2022. Proposed INN: List 127, and also with reference to patent document CN107207602B.

[0067] In some embodiments, the anti-APRIL antibody or antigen-binding fragment thereof provided herein is a murine, chimeric, humanized, or fully human antibody. In some embodiments, the variable region of the antibody or antigen-binding fragment thereof is human. In some embodiments, the anti-APRIL antibody is a monoclonal antibody.

[0068] In some embodiments, the antibody or antigen-binding fragment thereof herein is selected from the group consisting of: Fab, F(ab')2, Fab', Fd, Fv, dsFv, scFv, and diabody.

[0069] In one aspect, the present application provides a conjugate comprising the antibody or antigen-binding fragment thereof of the present application. In one embodiment, the conjugate further comprises an effector molecule. In another embodiment, the conjugate further comprises a linker linking the antibody or antigen-binding fragment thereof to the effector molecule.

[0070] In some embodiments, the effector molecule is selected from the group consisting of an anti-tumor agent, a drug, a toxin, a biologically active protein (e.g., an enzyme), other antibodies or antibody fragments, a synthetic or naturally occurring polymer, a nucleic acid and fragments thereof such as DNA, RNA and fragments thereof, a radionuclide (e.g., radioiodide), a radioisotope, a chelated metal, a nanoparticle, and a reporter group (e.g., a fluorescent compound), or a compound detectable by NMR or ESR spectroscopy, a detectable label (e.g., horseradish peroxidase, a fluorescent dye, a luminescent substance, or biotin).

[0071] In one aspect, the present application provides a bispecific or multispecific antibody comprising the anti-APRIL antibody or antigen-binding fragment thereof of the present application.

[0072] In some embodiments, the bispecific or multispecific antibody comprises a first antigen binding domain against APRIL and at least one second antigen binding domain against other targets, wherein the first antigen binding domain comprises the anti-APRIL antibody or antigen-binding fragment thereof of the present application. In some embodiments, each antigen binding domain of the bispecific or multispecific antibody retains the respective original binding specificity. In certain embodiments, the multispecific antibody is a trispecific antibody or a tetraspecific antibody.

[0073] In one aspect, the present application provides a chimeric antigen receptor comprising the anti-APRIL antibody or antigen-binding fragment thereof of the present application, preferably the antibody or antigen-binding fragment thereof (e.g., ScFv) as an extracellular antigen binding domain that specifically binds to APRIL in the chimeric antigen receptor.

[0074] In one aspect, the present application provides a polynucleotide encoding an anti-APRIL antibody or antigen-binding fragment thereof of the present application, or a conjugate of the present application, or a bispecific or multispecific antibody of the present application, or a chimeric antigen receptor of the present application. The polynucleotide of the present application can be, for example, DNA or RNA, and can or can not contain intron sequences. In a preferred embodiment, the polynucleotide is a cDNA molecule. The polynucleotide of the present application can be prepared or obtained by known means based on the information of the amino acid sequences of the present application, for example, by automated DNA synthesis and / or recombinant DNA technology.

[0075] As is well known in the art, multiple codons can encode the same amino acid. Thus, nucleic acids encoding a protein sequence include nucleic acids with codon degeneracy. The amino acid sequences described in the present application can be encoded by a variety of nucleic acids. The genetic code is universal and well known. Nucleic acids encoding any of the amino acid sequences described in the present application can be readily conceived based on the common general knowledge in the art, and can be optimized for production. Although the number of possible nucleic acid sequences encoding a given amino acid is large, under the standard table of the given genetic code, and with the aid of a computer, one of skill in the art can readily generate every possible combination of nucleic acid sequences encoding a given amino acid.

[0076] In one aspect, the present application provides a vector comprising a polynucleotide of the present application. The vector is capable of delivering and preferably expressing a construct of one or more genes or sequences of interest in a host cell. The vector includes eukaryotic expression vectors, prokaryotic expression vectors, viral vectors, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0077] In some embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain or a heavy chain variable region of an antibody or antigen-binding fragment thereof of the present application and a second nucleotide sequence encoding a light chain or a light chain variable region thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector of the present application comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

[0078] In one aspect, the present application provides a host cell comprising a polynucleotide or a vector of the present application. The host cell is capable of producing or secreting an intact antibody or antigen-binding fragment thereof, conjugate, bispecific or multispecific antibody, or chimeric antigen receptor of the present application. The host cell comprises a prokaryotic cell, a fungal cell, or a mammalian cell, such as a CHO cell, NS0 cell, or other mammalian cell, an E. coli or other prokaryotic cell, a yeast cell or other fungal cell. In some embodiments, the host cell is an immune cell, such as a T cell, an NK cell, a DC cell, a macrophage.

[0079] In one aspect, the present application provides a pharmaceutical composition comprising an anti-APRIL antibody or antigen-binding fragment thereof, conjugate, bispecific or multispecific antibody, chimeric antigen receptor, polynucleotide, vector, or host cell of the present application, and one or more pharmaceutically acceptable carriers.

[0080] In some preferred embodiments, the pharmaceutical composition contains a therapeutically effective amount of the foregoing anti-APRIL antibody or antigen-binding fragment thereof, the foregoing conjugate, the foregoing bispecific or multispecific antibody, or the foregoing chimeric antigen receptor, or the foregoing polynucleotide, or the foregoing vector, or the foregoing host cell, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.

[0081] In some embodiments, the pharmaceutical composition can comprise any number of excipients. Excipients that can be used include carriers, surface active agents, thickening or emulsifying agents, solid binding agents, dispersing or suspending aids, solubilizing agents, coloring, flavoring, coating agents, disintegrating agents, lubricants, sweetening agents, preservatives, isotonic agents, or combinations thereof. The selection and use of suitable excipients are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20thedition (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

[0082] In some embodiments, the pharmaceutical composition further comprises one or more other therapeutic agents. In some embodiments, the other therapeutic agents include, but are not limited to, drugs with anti-tumor activity, drugs for treating autoimmune diseases (e.g., IgA nephropathy).

[0083] In one aspect, the present application provides a kit comprising the foregoing anti-APRIL antibody or antigen-binding fragment thereof, the foregoing conjugate, or the foregoing bispecific or multispecific antibody.

[0084] In some embodiments, the kit further comprises one or more additional therapeutic or detection reagents (e.g., ELISA detection reagents).

[0085] In one aspect, the present application provides a method of preventing and / or treating a disease or disorder associated with APRIL in a subject, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an anti-APRIL antibody or antigen-binding fragment thereof, conjugate, bispecific or multispecific antibody, chimeric antigen receptor, polynucleotide, vector, host cell, or pharmaceutical composition of the present application.

[0086] In one embodiment, when the antibody or antigen-binding fragment thereof is administered to the subject, the subject will experience less severe symptoms and / or recover more quickly compared to not administering the antibody or antigen-binding fragment thereof. In one embodiment, in treating IgA nephropathy, after effective treatment of IgA nephropathy, a kidney biopsy will show less or no IgA deposition, for example, in the form of immune complexes in the mesangium. For example, after effective treatment of IgA nephropathy by administering an antibody or antigen-binding fragment thereof of the present application, less IgA deposits can be detected in a biological sample of the subject using a diagnostic assay of immunofluorescence or electron microscopy. After treatment of IgA nephropathy in a subject, other assays, urine tests, blood tests, iodopyracet clearance tests, or kidney imaging (e.g., ultrasound, X-ray, or cystoscopy) can also be used to monitor treatment of the subject, or to detect the presence (or absence) of symptoms of lgA nephropathy. The treatment may, for example, partially or completely alleviate, ameliorate, relieve, inhibit, or reduce the severity and / or incidence of, and optionally delay the onset of, one or more manifestations of the effects or symptoms, characteristics, and / or causes of the disease, e.g., lgA nephropathy. In one embodiment, the treatment refers to treatment of a subject who does not exhibit signs of certain disorders, e.g., signs of IgA nephropathy, and / or who exhibits early symptoms of a disorder, e.g., kidney disease. In one embodiment, the treatment refers to treatment of a subject who exhibits one or more established signs of a disorder, e.g., IgA nephropathy. In one embodiment, the treatment refers to treatment of a subject who is diagnosed with a disorder, e.g., IgA nephropathy.

[0087] In one aspect, the present application provides use of the foregoing anti-APRIL antibody or antigen-binding fragment thereof, the foregoing conjugate, the foregoing bispecific or multispecific antibody, the foregoing chimeric antigen receptor, the foregoing polynucleotide, the foregoing vector, the foregoing host cell, the foregoing pharmaceutical composition, or the foregoing kit in the manufacture of a medicament for preventing and / or treating a disease or disorder associated with APRIL in a subject.

[0088] In some embodiments, the APRIL-related disease or disorder is a disease or disorder caused by abnormal expression of APRIL (e.g., overexpression of APRIL). In some embodiments, the APRIL-related disease or disorder is selected from a disease or disorder mediated by IgA secretion, or a disease or disorder caused by B cell proliferation.

[0089] In some embodiments, the preventing and / or treating a subject from a disease or disorder related to APRIL comprises inhibiting immune cell proliferation and / or immune cell survival, preferably, the immune cell is a B cell; reducing the level of IgA; or, inhibiting the binding of APRIL to BCMA and / or TACI.

[0090] In some preferred embodiments, the APRIL-related disease or disorder comprises cancer, autoimmune diseases, such as B-cell related cancers, hematological cancers, IgA nephropathy, multiple sclerosis, rheumatoid arthritis, type 1 diabetes, psoriasis, Crohn's disease, and other inflammatory bowel diseases such as ulcerative colitis, systemic lupus erythematosus (SLE), autoimmune encephalomyelitis, myasthenia gravis (MG), Hashimoto's thyroiditis, Goodpasture's syndrome, pemphigus, Graves disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, anti- collagen antibody containing scleroderma, mixed connective tissue disease, polymyositis, pernicious anemia, idiopathic Addison's disease, autoimmune-associated infertility, glomerulonephritis, crescentic glomerulonephritis, proliferative glomerulonephritis, bullous pemphigoid, Sjogren's syndrome, psoriatic arthritis, insulin resistance, autoimmune diabetes, autoimmune hepatitis, autoimmune hemophilia, autoimmune lymphoproliferative syndrome (ALPS), autoimmune hepatitis, autoimmune hemophilia, autoimmune lymphoproliferative syndrome, autoimmune uveitis, Guillain-Bare syndrome, arteriosclerosis and Alzheimer's disease, leukemia, acute lymphoblastic leukemia, acute myelocytic leukemia, myeloblasts promyelocytic, myelomonocytic monocytic erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt's lymphoma and marginal zone B-cell lymphoma, polycythemia vera lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumors, sarcomas and carcinomas.fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon sarcoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma, esophageal cancer, basal cell carcinoma, biliary duct carcinoma, bladder cancer, bone cancer, brain and central nervous system (CNS) cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer, intraepithelial neoplasm, kidney cancer, laryngeal cancer, liver cancer, lung cancer (small cell lung cancer, large cell lung cancer), melanoma, neuroblastoma; oral cancer (e.g., lip cancer, tongue cancer, mouth cancer, and pharynx cancer), ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer; respiratory system cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and urinary system cancer.

[0091] In one aspect, the present application provides a method of inhibiting cell proliferation, comprising contacting the cell with the aforementioned anti-APRIL antibody or antigen-binding fragment thereof, the aforementioned conjugate, the aforementioned bispecific or multispecific antibody, the aforementioned chimeric antigen receptor, the aforementioned polynucleotide, the aforementioned vector, the aforementioned host cell, the aforementioned pharmaceutical composition, or the aforementioned kit; preferably, the cell is a B cell or a tumor cell; more preferably, the cell is ex vivo.

[0092] In one aspect, the present application provides a method of reducing IgA level in a cell, comprising contacting the cell with the aforementioned anti-APRIL antibody or antigen-binding fragment thereof, the aforementioned conjugate, the aforementioned bispecific or multispecific antibody, the aforementioned chimeric antigen receptor, the aforementioned polynucleotide, the aforementioned vector, the aforementioned host cell, the aforementioned pharmaceutical composition, or the aforementioned kit; preferably, the cell is a B cell; more preferably, the cell is ex vivo.

[0093] In one aspect, the present application provides a method of inhibiting binding of APRIL to an APRIL receptor on a cell, comprising contacting the cell with the aforementioned anti-APRIL antibody or antigen-binding fragment thereof, the aforementioned conjugate, the aforementioned bispecific or multispecific antibody, the aforementioned chimeric antigen receptor, the aforementioned polynucleotide, the aforementioned vector, the aforementioned host cell, the aforementioned pharmaceutical composition, or the aforementioned kit; preferably, the cell is a B cell, or, the APRIL receptor is BCMA and / or TACI; more preferably, the cell is ex vivo.

[0094] In one aspect, the present application provides use of the aforementioned anti-APRIL antibody or antigen-binding fragment thereof, the aforementioned conjugate, or the aforementioned kit in detecting APRIL or in preparing a reagent for detecting APRIL. The detection of APRIL can be in vivo or in vitro, and can be for non-disease diagnostic purposes. The detection of APRIL can be detecting the presence or level of APRIL in a sample, and the APRIL can be human APRIL or monkey (e.g., cynomolgus monkey) APRIL.

[0095] The present application also provides a method of producing an anti-APRIL antibody or antigen-binding fragment thereof of the present application.

[0096] The anti-APRIL antibody or antigen-binding fragment thereof of the present application can be obtained using conventional techniques, such as the Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., Chapters 5-8 and 15. For example, a mouse can be immunized with human APRIL or a fragment thereof, and the resulting antibodies can be recovered, purified, and subjected to amino acid sequencing using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibody or antigen-binding fragment of the present application can be genetically engineered to add one or more human FR regions to the CDR regions of a non-human source. Human FR germline sequences can be obtained from the website of ImMunoGeneTics (IMGT) by alignment of the IMGT human antibody variable germline genes database and MOE software, or from Immunoglobulin Journal, 2001 ISBN 012441351. Alternatively, for example, cDNA sequences encoding the heavy and light chains can be cloned into expression vectors. The recombinant immunoglobulin expression vectors can be stably transfected into CHO cells. Stable clones expressing antibodies that specifically bind to human APRIL are obtained. Positive clones are expanded in serum-free media in a bioreactor to produce the antibodies.

[0097] The anti-APRIL antibody or antigen-binding fragment thereof of the present application can be expressed intracellularly, on the cell membrane, or secreted outside the cell. If desired, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Generally, the transformed host cell is cultured under conditions suitable for expression of the antibody of the present application, and then the anti-APRIL antibody or antigen-binding fragment thereof of the present application is purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose affinity chromatography, ion exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, and other conventional separation procedures, alone or in combination. The antibody can be concentrated by filtration.

[0098] As a preferred embodiment of the method for preparing the anti-APRIL antibody or antigen-binding fragment thereof of the present application, the method for isolating and purifying the anti-APRIL antibody or antigen-binding fragment thereof is protein A affinity chromatography, cation exchange, or anion exchange.

[0099] The resulting monoclonal or bispecific antibodies can be identified using conventional means. For example, the binding specificity of the antibodies can be determined using immunoprecipitation or an in vitro binding assay, such as an enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay (RIA). The binding affinity of the antibodies can be determined, for example, using the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980), or surface plasmon resonance (SPR).

[0100] For clarity of disclosure, the general terms used herein are defined in the description of the compounds. Unless defined otherwise, the following terms and phrases used herein are intended to have the following meanings.

[0101] A particular term or phrase should not be considered indefinite or unclear if not specifically defined, but should be construed in accordance with its ordinary meaning. When a trade name appears herein, what is meant is the corresponding product or active ingredient thereof. The term "pharmaceutically acceptable" is employed herein to describe those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0102] The term "APRIL" refers to A proliferation-inducing ligand, which is a proliferation-inducing ligand, also known as CD256, TNF- and APOL-related leukocyte-expressed ligand 2 (TALL-2) or TNF-related death ligand 1 (TRDL-1), consisting of 250 amino acid residues (NCBI Reference Sequence: NP_003799.1), and belongs to the TNF superfamily. Unless otherwise indicated, APRIL includes, but is not limited to, that derived from human, rodent, mouse, rat, primate, monkey, and guinea pig. The term also refers to fragments or variants of native APRIL that retain at least one in vivo or in vitro activity of native APRIL. The term includes the full-length, unprocessed precursor form as well as the mature form that results from post-translational cleavage of the signal sequence. APRIL as used herein also refers to specific polypeptides expressed in cells via naturally occurring DNA sequence variations of the APRIL-encoding gene, such as single nucleotide polymorphisms of the APRIL-encoding gene. APRIL sequences are known in the art, additional examples are available using, e.g., GenBank, UniProt, OMIM, e.g., Genbank Accession Nos. NP_742084.1, NP_742085.1, etc., or UniPort Accession Nos. 075888-1, 075888-2, 075888-3, 075888-4, 075888-5, 043508-2, etc. As used in the present application, when a described anti-APRIL antibody or antigen-binding fragment thereof binds or substantially binds to human APRIL, it binds or substantially binds to one or more isoforms of human APRIL; when a described anti-APRIL antibody or antigen-binding fragment thereof binds or substantially binds to monkey APRIL, it binds or substantially binds to one or more isoforms of monkey APRIL, preferably cynomolgus monkey APRIL; when a described anti-APRIL antibody or antigen-binding fragment thereof binds or substantially binds to murine APRIL, it binds or substantially binds to one or more isoforms of murine APRIL, preferably rat APRIL.

[0103] The term "antibody" as referred to herein includes intact antibodies and any antigen binding fragment (i.e., "antigen binding portion") or single chains thereof. Intact antibodies are glycoproteins comprising two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of a light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of a heavy chain are referred to as HCDR1, HCDR2, and HCDR3. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0104] For the determination or definition of CDRs, a definitive delineation of CDRs and identification of residues comprising the binding site of an antibody can be accomplished by resolving the structure of the antibody and / or resolving the structure of an antibody-ligand complex. This can be accomplished by any of a variety of techniques known to those of skill in the art, e.g., X-ray crystallography. A variety of analytical methods, including combinations of methods, can be used to identify CDRs, including but not limited to the Kabat definition, the Chothia definition, the AbM definition, the IMGT definition, the Contact definition, the conformational definition. All of these are well-known techniques in the art, see, e.g., Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al. (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17: 132-143 (2004). Depending on the method used for identification, the boundaries of a given CDR can vary. Thus, unless otherwise noted, the terms “CDR” of a given antibody or region thereof, such as a variable region, and individual CDRs of an antibody or region thereof (e.g., HCDR1, HCDR2) are understood to encompass the complementarity determining regions as defined above by any of the known methods described herein. In some cases, a protocol for identifying a particular CDR or CDRs, such as those defined by the IMGT, Kabat, Chothia, or Contact methods, is set forth, and CDRs can also be identified with the aid of software programs, including but not limited to AbRSA (http: / / cao.labshare.cn / AbRSA / cdrs.php), abYsis (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and IMGT (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). In other cases, a particular amino acid sequence of a CDR is given. It should be noted that CDR regions can also be defined by a combination of various numbering systems (e.g., a combination of Kabat and Chothia or a combination of Kabat and IMGT.Thus, once a variable region (e.g., VH or VL) is given, one of skill in the art will understand that the CDRs within that region can be defined by different numbering systems or combinations thereof.

[0105] An exemplary defined CDRs include the following positions of amino acid residues:

[0106] Note 1 : The definitions vary slightly in the literature, particularly the Chothia definition scheme;

[0107] Note 2: Except for the Contact definition, which uses the Chothia numbering or the Martin numbering scheme, the other definition schemes are compatible with various numbering schemes;

[0108] Note 3: When using Kabat Numbering, the end of Chothia HCDR1 varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B).

[0109] Note: The amino acid numbering on the heavy chain is indicated by "H + number" and the amino acid numbering on the light chain is indicated by "L + number"; for example, L24-L34 in the second column of the second row in the table indicates the amino acid sequence from the 24th to the 34th residue from the N-terminus of the variable region of the light chain according to the Kabat coding scheme; the others are similarly defined.

[0110] The term "antigen binding fragment" (or simply "antibody portion") refers to one or more fragments of an antibody that specifically bind to an antigen (e.g., APRIL). It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; (vi) an isolated complementarity determining region (CDR); and (vii) a nanobody, a heavy chain variable region comprising a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those with ordinary skill in the art, and the

[0111] The terms "Fc fragment," "Fc region," or "Fc" are used interchangeably herein and refer to a fragment of an antibody that is generated by digestion with the enzyme pepsin, which cleaves the polypeptide chain at the hinge region between the first and second constant regions of the heavy chain. The Fc fragment of an antibody has several distinct functions, but is not involved in antigen binding. Methods are known in the art for altering the effector functions of an antibody, including but not limited to Fc receptor binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), half-life / clearance of antibodies and antigen-antibody complexes, etc., by introducing mutations in the Fc region.

[0112] As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. The monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

[0113] The term "murine antibody" refers to a monoclonal antibody against human APRIL prepared according to the knowledge and skill in the art. The preparation can be performed by injecting a test subject with an APRIL antigen, and then isolating a hybridoma expressing an antibody having a desired sequence or functional property. The murine anti-APRIL antibody or antigen-binding fragment thereof can further comprise a light chain constant region of murine kappa, lambda chain or variant thereof, or further comprise a heavy chain constant region of murine IgGl, IgG2, IgG3 or variant thereof.

[0114] The term "chimeric antibody" is an antibody in which the variable region of a heterologous (e.g., murine) antibody is fused with the constant region of a parent antibody (e.g., human antibody) to reduce the immune response induced by the heterologous antibody. For example, a human-murine chimeric antibody, to construct a chimeric antibody, a hybridoma secreting a murine-specific monoclonal antibody is first constructed, and then the variable region gene is cloned from the murine hybridoma cell, and the constant region gene of a human antibody is cloned as needed, and the murine variable region gene is linked to the human constant region gene to form a chimeric gene, which is inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system.

[0115] The term "humanized antibody" is also referred to as a CDR-grafted antibody, which refers to an antibody in which the CDR sequences of a murine antibody are grafted into a human antibody variable region framework, i.e., an antibody generated by grafting different types of human germline antibody framework sequences. This can overcome the heterologous reaction induced by the chimeric antibody carrying a large amount of murine protein component. Such framework sequences can be obtained from public DNA databases or published references including germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be obtained from the "VBase" human germline sequence database, and from Kabat, E. A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in immunogenicity while causing a decrease in activity, the human antibody variable region framework sequence can be subjected to a minimum of reverse mutations or back mutations to maintain activity.

[0116] The terms "fully human antibody" and "fully human antibody" are used interchangeably and generally refer to an antibody in which all parts (including the variable region and the constant region of the antibody) are encoded by genes of human origin. A fully human antibody can greatly reduce the immune side effects caused by heterologous antibody parts on the human body. Methods for obtaining fully human antibodies in the art can include phage display technology, transgenic mouse technology, ribosome display technology, etc.

[0117] The term "bispecific" refers to an antibody and / or antigen binding molecule that is capable of specifically binding to two different antigenic determinants, typically, a bispecific antibody or antigen binding molecule comprises two antigen binding sites, each of which is specific for a different antigenic determinant. In certain embodiments, the bispecific antibody or antigen binding molecule is capable of simultaneously binding to two antigenic determinants, particularly two antigenic determinants expressed on two different cells.

[0118] The term "conjugate" refers to an antibody or antigen binding fragment thereof linked to an effector molecule, such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, a biologically active protein, an imaging probe, and the like. The linkage can be a covalent bond or a non-covalent interaction, such as by way of electrostatic forces. The linkage can be by chemical or recombinant means. Various linkers known in the art can be employed in order to form the conjugate.

[0119] In one embodiment, the linkage is chemical, wherein a reaction between the antibody or antigen binding fragment thereof and the effector molecule results in a covalent bond formed between the two molecules to form one molecule. Polypeptides typically contain a variety of functional groups; such as carboxyl (COOH), free amine (-NH2) or thiol (-SH) groups, which can be used to react with suitable functional groups on the antibody to result in the attachment of the chemical moiety. Where the antibody and the effector molecule are polypeptides, the linker can be attached to a constituent amino acid through a side group (such as through a disulfide bond to a cysteine) or to the alpha carbon amino and carboxyl groups of terminal amino acids.

[0120] In one embodiment, the linkage is recombinant, the conjugate can be provided as a fusion protein, which can be expressed from a polynucleotide encoding the conjugate. As used herein, "fusion protein" refers to a protein produced by joining two or more genes or gene fragments that originally coded for separate proteins (including peptides and polypeptides). Translation of the fusion gene results in a single protein with functional properties derived from each of the original proteins.

[0121] The term "variant" of a heavy chain constant region and a light chain constant region refers to variants of a heavy chain constant region or a light chain constant region derived from humans that have been disclosed in the prior art that do not alter the structure and function of the antibody variable region, exemplary variants include IgGl, IgG2, IgG3, or IgG4 heavy chain constant region variants that have been subjected to site-directed engineering and amino acid substitutions, particular substitutions such as the YTE mutation, the L234A and / or L235A mutation, the S228P mutation, and / or mutations to obtain a knob-into-hole structure (such that the antibody heavy chain has a combination of knob-Fc and hole-Fc), which have been demonstrated to impart new properties to the antibody, but do not alter the function of the antibody variable region.

[0122] The term "variant," in the context of polypeptides, refers to a polypeptide or peptide that comprises an amino acid sequence that has been altered by the introduction of an amino acid residue substitution, deletion, or addition. In certain instances, a variant has similar, the same, or improved function as the polypeptide or peptide from which it is derived.

[0123] The term "affinity" refers to the strength of the sum total of noncovalent interactions between individual binding sites of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1 : 1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can be represented generally by the dissociation constant (KD). Affinity can be measured by routine methods known in the art, including those described herein.

[0124] As used herein, the terms "comprising," "including," and "having" are interchangeable terms, and are intended to mean the inclusion of the elements whose presence is recited, but not excluding the presence of other elements. It is also to be understood that the description herein using "comprising," "including," and "having" also provides "consisting of" schemes.

[0125] The terms "about" and "approximately" generally mean an acceptable degree of error for the quantity measured considering the nature of the measurement and the precision of the measuring instrument. An exemplary degree of error is within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values.

[0126] The term "does not substantially bind" means that an antibody or antigen binding fragment of the disclosure does not exhibit detectable binding to a given target, e.g., reactivity with a given target of no more than 30%, no more than 20%, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, or no more than 3%.

[0127] The term "antibody" herein includes, but is not limited to, a monoclonal antibody, a polyclonal antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, an intact antibody, an antigen binding fragment, a naked antibody, a conjugated antibody, a humanized antibody, or a fully human antibody.

[0128] It is to be understood that the antibodies or antigen binding fragments thereof described herein can have additional conservative or non-essential amino acid substitutions that do not substantially affect their function.

[0129] The term "amino acid" is intended to encompass all molecules, whether natural or synthetic, which contain both an amino functional group and an acid functional group and which are capable of being incorporated into a polymer of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; analogs, derivatives, and homologs thereof; amino acid analogs with derived side chains; and all stereoisomers of any of the foregoing. As used in the present application, the term "amino acid" includes D- or L-optical isomers and peptidomimetics.

[0130] The term "conservative amino acid" is used herein to refer to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity).

[0131] Exemplary, the amino acids within each of the following groups are conservative amino acid residues with respect to each other, substitution of an amino acid residue within a group is a substitution of a conservative amino acid:

[0132] 1) Alanine (A), Serine (S), Threonine (T);

[0133] 2) Aspartic acid (D), Glutamic acid (E);

[0134] 3) Asparagine (N), Glutamine (Q);

[0135] 4) Arginine (R), Lysine (K), Histidine (H);

[0136] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0137] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0138] The terms "identity" and "sequence identity" are used interchangeably herein and are calculated by determining the number of positions at which the amino acid residues or nucleotides of two amino acid sequences or two nucleic acid sequences are identical, out of the total number of positions in the sequences. The percentage of sequence identity is then calculated by comparing the two sequences at the positions identified above. The two sequences are identical at a position if the amino acid residue or nucleotide is the same at that position in both sequences.

[0139] The term "epitope" refers to a region or site of an antigen to which an antibody specifically binds, e.g., a region or site that comprises contact residues that interact with the antibody. Thus, the term "epitope" refers to the portion of a molecule that is recognized and bound by an antibody at one or more antigen combining sites of the antibody. An epitope is usually defined by the molecular interaction between an antibody, or antigen-binding fragment thereof, and its corresponding antigen. Epitopes usually consist of contiguous groups of surface residues of molecules such as amino acid or sugar side chains and have specific three-dimensional structural characteristics, as well as specific charge characteristics. In some embodiments, an epitope can be a protein epitope. A protein epitope can be linear or conformational. In a linear epitope, all points of interaction between the protein and the interacting molecule, such as an antibody, exist linearly along the primary amino acid sequence of the protein. A "non-linear epitope" or "conformational epitope" comprises non-contiguous polypeptides (or amino acids) within the antigenic protein that are bound by an antibody specific for that epitope. As used herein, the term "antigenic epitope" is defined as a portion of an antigen that can specifically bind to an antibody as determined by any method known in the art, e.g., by routine immunoassay. Alternatively, during the discovery process, the generation and characterization of antibodies can reveal information about the desired epitope. From this information, it is then possible to competitively screen for antibodies that bind to the same epitope. Methods to accomplish this are to conduct competition and cross-competition studies to find antibodies that compete or cross-compete for binding to APRIL, e.g., antibodies that compete for binding to the antigen.

[0140] When the term "compete" is used in the context of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope, it is meant to compete between antigen binding proteins as determined by an assay in which the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) to be tested prevents or inhibits (e.g., reduces) the specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen (e.g., an APRIL antigen or fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen binding protein competes with another, such as: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid phase direct label assay, solid phase direct label sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA with 1-125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct label RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically the assay involves the use of purified antigen bound to a solid surface or cells bearing either the unlabeled test antigen binding protein and the labeled reference antigen binding protein. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cells in the presence of the test antigen binding protein. Typically the test antigen binding protein is present in excess. Antigen binding proteins identified by competitive assays (competing antigen binding proteins) include: antigen binding proteins that bind to the same epitope as the reference antigen binding protein; and antigen binding proteins that bind to an adjacent epitope in sufficient proximity to the binding epitope of the reference antigen binding protein that the two epitopes sterically hinder each other from binding. Typically when competing antigen binding proteins are present in excess, they will inhibit (e.g., reduce) specific binding of the reference antigen binding protein to the common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0141] The term "antagonist" refers to inhibition of receptor signaling to inhibit a biological response associated with activation of the receptor. The term antagonist is used in the broadest sense, and includes an antibody that partially or completely blocks, inhibits or neutralizes a biological activity of the epitope, polypeptide or cell to which it specifically binds. Methods of identifying an antagonist antibody can include contacting a polypeptide or cell to which a candidate antagonist antibody specifically binds with the candidate antagonist antibody and measuring a detectable change in one or more biological activities normally associated with the polypeptide or cell.

[0142] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, rodents, rabbits, pigs, dogs, cats, chickens, amphibians, and reptiles, although mammals such as non-human primates, rodents are preferred.

[0143] The term "therapeutically effective amount" refers to the amount of an anti-APRIL antibody or antigen-binding fragment thereof of the present application that is sufficient to prevent or ameliorate symptoms associated with a disease or condition (e.g., IgA nephropathy) and / or reduce the severity of the disease or condition. A therapeutically effective amount is understood in the context of the condition being treated, where one of skill in the art can readily identify the actual effective amount.

[0144] The antibodies of the present application are monoclonal antibodies that are structurally and chemically characterized as described below and in the Examples below. The amino acid sequence ID numbers for the heavy chain / light chain variable regions of exemplary antibodies are summarized in Tables 1-2 (the italicized portions are the CDR regions as defined by Kabat, identified by abYsis), and the heavy chain / light chain constant region sequence ID numbers are summarized in Table 3.

[0145] Table 1 Amino acid sequences of heavy chain variable regions of murine anti-APRIL antibodies

[0146] Table 2 Amino acid sequences of light chain variable regions of murine anti-APRIL antibodies

[0147] Table 3 Amino acid sequences of antibody heavy / light chain constant regions

[0148] The heavy chain variable region and light chain variable region CDRs in Tables 1-2, 7.1-7.4 can be determined according to the Kabat, Chothia, IMGT, AbM, Contact definitions, or a combination thereof. Tables 4.1 and 4.2 provide the amino acid sequences of the heavy chain / light chain CDRs of exemplary anti-APRIL antibodies of the present application identified using abYsis. Antibodies having the same heavy chain and light chain CDR1, CDR2, and CDR3 regions as the anti-APRIL antibodies of the present application are also within the scope of the present application.

[0149] Table 4.1

[0150] Table 4.2

[0151] The full length amino acid sequences of the heavy chain and light chain of the exemplary antibodies provided herein are listed in Table 4.3.

[0152] Table 4.3 Heavy and light chain amino acid sequences of anti-APRIL antibodies

[0153] Other features and advantages of the present application will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which should be construed as illustrative only, not limiting. The contents of all references, Genbank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. BRIEF DESCRIPTION OF DRAWINGS

[0154] Figure 1 shows the inhibition of APRIL-mediated BCMA signaling by recombinant antibodies in HEK293 / NFκB-Luc / BCMA cells.

[0155] Figure 2 shows the inhibitory activity of recombinant antibodies on B cell proliferation.

[0156] Figure 3 shows the inhibitory activity of recombinant antibodies on IgA secretion.

[0157] Figure 4 shows the inhibition of APRIL-mediated BCMA signaling by partially humanized antibodies in HEK293 / NFκB-Luc / BCMA cells.

[0158] Figure 5 shows the inhibition of APRIL-mediated TACI signaling by partially humanized antibodies in HEK293 / NFκB-Luc / TACI cells.

[0159] Figure 6 shows the inhibitory activity of partially humanized antibodies on B cell proliferation.

[0160] Figure 7 shows the inhibitory activity of partially humanized antibodies on IgA secretion.

[0161] Figure 8 shows the blocking of monkey APRIL binding to BCMA by partially humanized antibodies.

[0162] Figure 9 shows the ELISA results of partially humanized antibodies on BAFF.

[0163] Figure 10 shows the detection of free APRIL in the administration group, and the administration is indicated by ▲. DETAILED DESCRIPTION

[0164] Example 1 Preparation of Anti-APRIL Monoclonal Antibodies

[0165] Various antibody development methods were used to identify anti-APRIL monoclonal antibodies as disclosed herein. To generate a diverse set of antibodies, mice were immunized. Briefly, several female Balb / c and SJL mice were immunized with APRIL protein and mRNA encoding APRIL protein as antigens, respectively, and at certain time points after the start of immunization, the reactivity of mouse sera to APRIL protein was detected, and mice with strong titers were selected, and then subclones capable of binding human, monkey, and mouse APRIL were obtained by hybridoma method, or mouse B cells binding human, monkey, and mouse APRIL were screened using Beacon.

[0166] For the hybridoma method, the mouse spleen was extracted and homogenized to produce a single cell suspension, and a single cell suspension of myeloma cells (SP2 / 0) was prepared. The plasma cells were enriched using a CD138 plasma cell enrichment kit, and the enriched plasma cells were electrofused with SP2 / 0 mouse myeloma cells, incubated, and then tested for the presence of antibodies specific to APRIL by ELISA binding and blocking. The preferred hybridoma clones selected were subcloned using limited dilution. After the hybridoma cells were cultured at 37°C in 5% CO2 for 1 week, the supernatant was tested for ELISA binding and blocking, and candidate anti-APRIL antibodies were selected based on the binding and functional blocking of murine antibodies to APRIL protein.

[0167] For Beacon screening, the mouse spleen was extracted and homogenized to produce a single cell suspension, the plasma cells were enriched using a CD138 plasma cell enrichment kit, the cell density was adjusted, and introduced into a BEACON instrument for single B cell screening to identify anti-APRIL antibodies exhibiting human, monkey, or mouse species cross-reactivity.

[0168] The RNA of positive hybridomas or single B cells was extracted, and NGS or Sanger sequencing was performed, and the amino acid sequence information of the candidate antibodies screened in this example is shown in Table 5.

[0169] Table 5. Variable region amino acid sequences of murine anti-APRIL antibodies

[0170] The DNA encoding sequences of the antibody variable regions shown in Table 5 were synthesized and constructed into pcDNA3.4 expression vectors containing human IgG4_S228P or Kappa chain, respectively. Expi-CHO-S cells were transfected, and after 7-10 days of growth, cell supernatants were collected and subjected to Protein-A purification to produce recombinant antibodies. The heavy chain and light chain constant region sequences of each recombinant antibody are shown in SEQ ID NO. 52-53, respectively. The above-mentioned VH sequences were directly spliced with the heavy chain constant region sequence to obtain the full-length sequence of the heavy chain of each antibody, and the above-mentioned VL sequences were directly spliced with the light chain constant region sequence to obtain the full-length sequence of the light chain of the antibody. The control antibody Sibeprenlimab (VIS649) was prepared according to the literature CN109089419B, and Zigakibart (BION-1301) was prepared according to the literature CN107207602B.

[0171] Example 2. Affinity and receptor blocking activity test of recombinant antibodies

[0172] 2.1 Affinity test of recombinant antibodies to APRIL protein

[0173] The binding ability of recombinant antibodies to antigens was evaluated by ELISA. Human APRIL protein (hAPRIL) or monkey APRIL protein (cynoAPRIL (ACRO, item number APL-C52D2)) was coated at a concentration of 0.5 μg / mL, 100 μL / well, and the coated enzyme-labeled plate was incubated at 4°C overnight. The plate was washed with 0.05% PBST, and then 150 μL of blocking solution (1% BSA in PBS) was added to each well, and incubated at 37°C for 1 h. The blocking solution was discarded, and 100 μL / well of recombinant antibody was added, with the first well at a concentration of 10 nM, 3-fold gradient dilution to the 10th well, and the remaining 2 wells added with diluent (PBS) as blank controls, incubated at 37°C for 1 h. The plate was washed, and 100 μL of diluted goat anti-human IgG (H&L) antibody peroxidase conjugate (Rockland, 609-103-123) was added to each well and incubated at 37°C. The enzyme-labeled plate was removed, washed, and 100 μL of color developing solution (TMB) was added, and reacted at 25°C for 15 min. The reaction was terminated by adding 50 μL of 1M HCl, and the OD450 nm reading was taken on a microplate reader. The EC 50 value was calculated, and the results are shown in Table 6.

[0174] 2.2 Competitive binding test of recombinant antibodies to receptors

[0175] The binding ability of the recombinant antibody to the antigen was evaluated by competitive ELISA. Competition with human BCMA: 1 pg / mL of BCMA-Fc was coated in 100 pL / well, and then the plate washing and blocking steps were completed as described in 2.1, 50 pL / well of the recombinant antibody was added, the first well was diluted at a concentration of 200 nM, and 3-fold gradient dilution was performed to the 11th well, 1 well of the remaining was added with diluent as a blank control, and 50 pL of 150 ng / mL biotinylated hAPRIL-Fc was added to each well for incubation; the plate was washed, the enzyme-labeled secondary antibody (GenScript, M00091) was diluted to 50 ng / mL, and then mixed and added to the 96-well plate at 100 pL / well for incubation; the plate was washed, 100 pL of TMB substrate was added to each well for incubation at room temperature, and then 50 pL of 1M HCl was added to each well to terminate the reaction, and the OD450 nm reading was performed by the enzyme-labeled instrument. Competition with human TACI: 2 pg / mL of hAPRIL-Fc was coated in 100 pL / well, and then the above steps were repeated, except that 50 pL of the diluted antibody was added to each well to compete with 350 ng / mL of biotinylated TACI-Fc. The IC50 value was calculated, and the results are shown in Table 6.

[0176] Table 6

[0177] According to the results in Table 6, the recombinant antibody of the present application can bind to the APRIL protein with high affinity, and effectively block the binding of APRIL to the receptors BCMA and TACI.

[0178] Example 3 Test of cell functional activity of the recombinant antibody

[0179] 3.1 Blocking of the binding of recombinant antibody to BCMA and APRIL

[0180] HEK293 / NFκB-Luc reporter cells were infected with a lentiviral vector expressing human BCMA (GenBank ID: NM_001192.3) and cultured in DMEM complete medium containing 10% FBS. Positive cells were selected using puromycin. The cell BCMA expression was evaluated by flow cytometry using PE-labeled anti-BCMA antibody (BioLegend, 357504), and the reporter cell strain HEK293 / NFκB-Luc / BCMA capable of stably expressing human BCMA was obtained to evaluate the effect of the recombinant antibody on the binding of APRIL to BCMA.

[0181] HEK293 / NFκB-Luc / BCMA cells were seeded at a density of 3 x 104cells / well in 96-well plates and cultured overnight. The next day, 50 pL of 10 ng / mL hAPRIL-Fc was added to each well, and the plate was incubated for 30 min. Then, 50 pL of the recombinant antibody was added to each well, and the plate was incubated for 2 h. The plate was washed, and 100 pL of TMB substrate was added to each well for incubation at room temperature. Then, 50 pL of 1M HCl was added to each well to terminate the reaction, and the OD450 nm reading was performed by the enzyme-labeled instrument. 5HEK293 / NFκB-Luc / BCMA cells at 2.5 x 105cells / mL were seeded in 96-well plates at 100 μL / well, and the cells were incubated at 37 °C, 5% CO2 in a cell incubator overnight. Recombinant antibodies and human APRIL protein (ACROBiosystems, APL-H52D1) were diluted in DMEM complete medium containing 10% FBS. The recombininant antibodies were diluted from 66 nM or 132 nM, and then 4-fold dilution was performed for 7 points. The APRIL protein was diluted to 40 ng / ml. The diluted recombinant antibodies and the APRIL protein were mixed at a ratio of 1:1, and incubated at room temperature for 30 min. After the medium was removed, 100 μL of the antigen-antibody mixture was added to the HEK293 / NFκB-Luc / BCMA cells, which were incubated at 37 °C, 5% CO2 in a cell incubator for 6 h. Then, 100 μL of luciferase detection reagent (Vazyme, DD1201-02) was added, and the cells were incubated at room temperature for 5 min. The fluorescence value was measured by a microplate reader, and the dose-effect curve was fitted and analyzed using GraphPad Prism software.

[0182] The results are shown in FIG. 1. It can be seen that the recombinant antibody of the application can effectively antagonize the activation of the HEK293 / NFκB-Luc / BCMA reporter cell line by the APRIL protein in a dose-dependent manner, and the activity is significantly better than that of the control antibody BION-1301.

[0183] 3.2 Recombinant antibody blocking the binding of APRIL to TACI

[0184] HEK293 / NFκB-Luc reporter cells were infected with a lentiviral vector expressing human TACI (GenBank ID: NM_012452.3) and cultured in DMEM complete medium containing 10% FBS. Positive cells were selected using puromycin and hygromycin B. The TACI expression of the cells was evaluated by flow cytometry using PE-labeled anti-TACI antibody (BioLegend, 311906), and the reporter cell strain HEK293 / NFκB-Luc / TACI capable of stably expressing human TACI was obtained, which was used to evaluate the effect of the recombinant antibody on the binding of APRIL to TACI.

[0185] The HEK293 / NFκB-Luc / TACI reporter cell line was used to evaluate the blocking of the binding of APRIL to TACI by molecules. HEK293 / NFκB-Luc / TACI cells at a density of 2 x 105cells / mL were seeded in 96-well plates at 100 μL / well, and the cells were incubated at 37 °C, 5% CO2 in a cell incubator overnight. Recombinant antibodies and human APRIL protein (ACROBiosystems, APL-H52D1) were diluted in DMEM complete medium containing 10% FBS. The recombininant antibodies were diluted from 66 nM or 132 nM, and then 4-fold dilution was performed for 7 points. The APRIL protein was diluted to 40 ng / ml. The diluted recombinant antibodies and the APRIL protein were mixed at a ratio of 1:1, and incubated at room temperature for 30 min. After the medium was removed, 100 μL of the antigen-antibody mixture was added to the HEK293 / NFκB-Luc / TACI cells, which were incubated at 37 °C, 5% CO2 in a cell incubator for 6 h. Then, 100 μL of luciferase detection reagent (Vazyme, DD1201-02) was added, and the cells were incubated at room temperature for 5 min. The fluorescence value was measured by a microplate reader, and the dose-effect curve was fitted and analyzed using GraphPad Prism software. 5HEK293 / NFkB-Luc / TACI cells were diluted with DMEM complete medium containing 10% FBS, and the recombinant antibodies were diluted from 10 nM, 2-fold dilution of 7 points. The diluted recombinant antibodies were mixed with the APRIL protein (ACROBiosystems, APL-H52D1) at a ratio of 1:1 for pre-incubation. After the pre-incubation, the medium was removed, and 100 μL of the antigen-antibody mixture was added to the HEK293 / NFkB-Luc / TACI cells, which were incubated at 37°C in a 5% CO2 incubator for 6 h. Then, 100 μL of luciferase detection reagent (Vazyme, DD1201-02) was added, and the mixture was incubated at room temperature for 5 min. The fluorescence value was measured, and the dose-effect curve was fitted and analyzed using GraphPad Prism software.

[0186] The recombinant antibodies of the present application can effectively antagonize the activation of the HEK293 / NFkB-Luc / TACI reporter gene cell line by the APRIL protein in a dose-dependent manner.

[0187] 3.3 Recombinant antibodies inhibit human B cell proliferation and IgA secretion

[0188] The human PBMC cells were resuspended with RPMI1640 medium + 10% FBS + 100 IU / mL IL-2 and cultured overnight. The PBMC cells were collected by centrifugation, and B cells were separated using a B cell sorting kit (Stemcell, 17954). The separated B cells were resuspended with complete medium (IMDM medium + 10% FBS + 50 μg / mL human transferrin + 5 μg / mL human insulin) and adjusted to a cell density of 2×10 6 The human PBMC cells were resuspended with RPMI1640 medium + 10% FBS + 100 IU / mL IL-2 and cultured overnight. The PBMC cells were collected by centrifugation, and B cells were separated using a B cell sorting kit (Stemcell, 17954). The separated B cells were resuspended with complete medium (IMDM medium + 10% FBS + 50 μg / mL human transferrin + 5 μg / mL human insulin) and adjusted to a cell density of 2×10

[0189] The results are shown in Figures 2 and 3. It can be seen that the recombinant antibodies of the application can effectively antagonize the activation of B cells by APRIL protein in a dose-dependent manner, inhibit the proliferation of human B cells and inhibit the secretion of human IgA, and the inhibitory effect is better than that of the control antibody BION-1301.

[0190] Example 4 Antibody humanization and functional characterization

[0191] The CDR grafting method was used to modify the variable region sequences of the murine antibodies to generate the corresponding humanized antibodies, to reduce the immunogenicity of the antibodies. Briefly, the VH and VL sequences of the murine antibodies were compared with the available human Ig gene sequence database (http: / / www.ncbi.nlm.nih.gov / igblast / ) to find the overall most matching human germline Ig gene sequence as the framework for humanization modification, and a different number of back mutations were introduced into the selected human framework to maintain the structure and / or function of the resulting antibodies. Further, a series of optimization modifications were made to the humanized antibodies, to obtain the humanized VH and VL sequences as shown in Tables 7.1 to 7.4 (the italicized part is the CDR region defined by Kabat, identified by abYsis).

[0192] Table 7.1 Humanized variable region sequences of m5O13

[0193] Table 7.2 Humanized variable region sequences of m24D1

[0194] Table 7.3 Humanized variable region sequences of m1P34

[0195] Table 7.4 Humanized variable region sequences of m4G11

[0196] The VH, VL sequences shown in Tables 7.1-7.4 were combined into the optimized humanized antibodies, the variable region sequences of each antibody are shown in Tables 8.1-8.4, and the constant region required by the antibody is from human IgG1. The constant region sequence is directly spliced with the VH and VL shown in Tables 8.1-8.4 to obtain the complete sequence of each antibody. The humanized antibodies were expressed and purified according to the conventional procedures, for example, referring to the method steps of Example 1, i.e. the DNA coding sequence of the antibody variable region shown in Tables 8.1-8.4 is genetically synthesized, the DNA of VH is inserted into a mammalian cell expression vector containing the heavy chain constant region coding gene shown in SEQ ID NO. 76, and the DNA of VL is inserted into a mammalian cell expression vector containing the light chain constant region coding gene shown in SEQ ID NO. 53, transfected into mammalian cells capable of expressing the aforementioned vector, and after culturing for several days, the cell supernatant is collected and purified to obtain the antibody.​

[0197] Table 8.1

[0198] Table 8.2

[0199] Table 8.3

[0200] Table 8.4

[0201] Each of the humanized antibodies was tested for its effect on the binding between APRIL and BCMA according to the method described in Example 3.1. Figure 4 shows that the humanized antibodies were able to block the binding of APRIL to BCMA (IC 50 < 0.5 nM) and the blocking effect was substantially comparable to or superior to the parent antibody or the positive control.

[0202] Each of the humanized antibodies was tested for its effect on the binding between APRIL and TACI according to the method described in Example 3.2. Figure 5 shows that the humanized antibodies were able to block the binding of APRIL to TACI (IC 50 < 0.5 nM) and the blocking effect was superior to the positive control.

[0203] Each of the humanized antibodies was tested for its effect on the proliferation of human B cells and the secretion of IgA according to the method described in Example 3.3. Figures 6 and 7 show that the humanized antibodies were able to effectively antagonize the activating effect of APRIL protein on B cells, inhibit the proliferation of human B cells and inhibit the secretion of human IgA in a dose-dependent manner, and the effect was comparable to or significantly superior to the positive control. The IC 50 of each of the humanized antibodies for inhibiting the proliferation of B cells was less than 2 nM; and the IC 50 of each of the humanized antibodies for inhibiting the secretion of IgA was less than 2 nM. The relative activities of each of the humanized antibodies for inhibiting the proliferation of B cells and the secretion of IgA are summarized in Table 8.5.

[0204] Table 8.5 Note: The relative activity for inhibiting the proliferation of B cells is the ratio of the IC50 of the control antibody VIS649 to that of each of the humanized antibodies; and the relative activity for inhibiting the secretion of IgA is the ratio of the IC50 of the control antibody VIS649 to that of each of the humanized antibodies

[0205] Example 5 Surface Plasmon Resonance (SPR) Assay for Affinity

[0206] The binding affinity of the antibodies to human APRIL protein was determined by SPR. Briefly, antibodies were captured onto a protein A sensor chip, with a capture time of 12 seconds, a flow rate of 30 pl / min, and the chip was rinsed with buffer until the baseline was stable. A gradient dilution of the antigen (10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625 nM) was then flowed over the chip at a flow rate of 30 pl / min, with an association time of 120 seconds and a dissociation time of 360 seconds. The dissociation rate (kd) and the association rate (ka) were obtained using Biacore T200 analysis software 3.2.1, and the equilibrium dissociation constant (KD) was calculated from the ratio of kd to ka. The results show that the dissociation constant KD of some of the humanized antibodies of the application is lower than 0.1 nM (Table 9), indicating that they are able to bind to human APRIL protein with high affinity.

[0207] Table 9

[0208] Example 6 Cross-reactivity of humanized antibodies

[0209] The reactivity of the humanized antibodies with cynomolgus monkey APRIL was verified. Using the Bio-Layer Interferometry technique (BLI), the equilibrium dissociation constant (KD) of the antibodies binding to monkey APRIL was determined. The affinity of the antibodies to monkey APRIL (ACRO, cat. no. APL-C52D2) protein was determined using Octet (Sartorius), using Protein A biosensors to immobilize the antibodies, and reacting them with a gradient dilution of monkey APRIL protein, with an association time of 60 s and a dissociation time of 360 s. The Octet analysis software was used to select the model to fit the data, and the affinity KD, kon (association rate constant), and kdis (dissociation rate constant) of the binding of the two were measured, as shown in Table 10.

[0210] Table 10

[0211] The reporter cell line HEK293 / NFkB-Luc / BCMA expressing human BCMA was washed and the supernatant was discarded. 1 mL of trypsin was added and the digestion was carried out for 30 s. Immediately after the digestion was stopped by adding 4 mL of complete medium (DMEM + 10% FBS), the cells were centrifuged at 180 g for 3 min, the supernatant was discarded, and 2 mL of complete medium was added to resuspend and count the cells. The treated cells were adjusted to a density of 3 x 105cells / mL in complete medium, and 100 pL of the cell suspension was added to each well of a 96-well plate. The plate was incubated at 37°C in a 5% CO2 atmosphere for 24 h. 5HEK293 / NFκB-Luc / BCMA cells were seeded at 1 x 105cells / mL, 100 μL / well into cell culture plates and incubated at 37 °C, 5% CO2for 16-24 h. The test antibody was gradient diluted using complete medium, cynoAPRIL protein (ACRO, Cat. APL-C52D2) was diluted to 40 ng / ml. The diluted recombinant antibody was mixed with the APRIL protein at 1:1 and incubated at room temperature for 30 min. The cell culture medium in the cell wells was removed, 100 μL of the antigen-antibody mixture was added to the HEK293 / NFκB-Luc / BCMA cells and incubated at 37 °C, 5% CO2in an incubator for 6 h. The cell culture plate was taken out of the incubator and placed at room temperature for 30 min, and luciferase detection reagent (Promega, DD1201-03) was added, and the fluorescence signal was detected by a microplate reader. The results are shown in Figure 8, which shows that the antibody of the application can block the binding of monkey APRIL to the receptor BCMA at the cellular level.

[0212] ELISA was used to detect whether the humanized antibody had non-specific binding with BAFF. Human BAFF protein (ACRO, Cat. BAF-H52D4) was diluted with 1xPBS to 2 μg / ml, added to a 96-well high adsorption enzyme-labeled plate at 100 μl / well, and incubated at 4 °C for 16 h; the plate was washed with 0.05% PBST for 3 times, 1% BSA solution was added at 200 μl / well, and incubated at 37 °C for 2 h; the plate was washed with 0.05% PBST for 3 times, the test antibody was gradient diluted with 1xPBS, and the antibody diluent at each concentration gradient was added to the enzyme-labeled plate, and incubated at 37 °C for 1 h; the plate was washed with 0.05% PBST for 3 times, 1% BSA solution was added at 1:5000 dilution of enzyme-labeled secondary antibody (JACKSON, 109-035-170), 100 μl / well, and incubated at room temperature in the dark for 1 h, TMB was used for color development, and the OD450 absorbance was measured by a microplate reader after the reaction was terminated. The results are shown in Figure 9, which shows that the antibody of the application does not recognize BAFF, and it only specifically binds to APRIL.

[0213] Example 7 FcRn affinity of humanized antibody

[0214] The equilibrium dissociation constant (KD) of the antibody binding to human neonatal receptor (FcRn) was determined using bio-layer interference technology (BLI). The affinity of the antibody to human FcRn protein (Acro, item number FCM-H82W4) was determined using Octet (Sartorius), using streptavidin biosensor to immobilize human FcRn protein, reacting with gradient-diluted antibody, binding time 60 s, dissociation time 300 s, using Octet analysis software to select model to fit the data, and the affinity KD, kon (binding rate constant), and kdis (dissociation rate constant) of the binding of the two were determined, as shown in Table 11. It can be seen that the antibody of the application has a better FcRn affinity than the control antibody, and is expected to have a better half-life in the human body.

[0215] Table 11

[0216] Example 8 Animal model pharmacodynamic verification

[0217] A number of male cynomolgus monkeys were selected for routine physical examination, and serum IgA levels were detected. In combination with animal age and weight data, 12 animals were selected for the experiment. According to the weight, age, and serum IgA level, the animals were randomly divided into 3 groups, 4 animals in each group. Among them, Group 1 was administered 5 mg / kg of VIS649, Group 2 was administered 5 mg / kg of h5O13.I, and Group 3 was administered 5 mg / kg of h24D1.C. The above antibodies were administered by intravenous bolus injection, and the administration frequency was once a week, for a total of 4 administrations. The clinical symptoms of the experimental animals were recorded, and the weight was measured at specific time points, and serum samples of the animals were used. The following indicators in the serum samples were detected using a fully automatic biochemical analyzer (SYSMEX BX-3010, SYSMEX): immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin G (IgG). The percentage change of each indicator was calculated based on the baseline IgA, IgM, and IgG of the animals at the start of administration (D0).

[0218] The results showed that the animals in each group were well tolerated to the test substance, and no weight loss was shown. The IgA levels of the animals in the 3 groups were significantly reduced after administration. At week 15, the h5O13.I and h24D1.C groups had a greater IgA reduction than the VIS649 group, and the difference between the two and the IgA percentage change of the VIS649 group was about 10%.

[0219] The sandwich ELISA method was further used to detect the level of free APRIL (fAPRIL) in serum samples. Briefly, the ruthenium-labeled VIS649, h5O13.I and h24D1.C were used as capture antibodies, and the biotin-labeled m2P38 antibody was used as detection antibody. The content of free APRIL in monkey serum was detected by electrochemiluminescence analyzer. The results are shown in Figure 10. The serum fAPRIL level of each group of animals relative to the baseline level showed a downward trend 1 week after the first administration, and continued to the 8th week. The results showed that the serum fAPRIL level of the VIS649 group returned to the baseline level before administration, while the h5O13.I and h24D1.C did not return to the baseline level until the 13th week, indicating that they had better inhibition of fAPRIL than VIS649.

[0220] The method for determining the serum drug concentration of the antibody is as follows: the antigen human APRIL protein (Acro Biosystems, item number APL-H52D1) is diluted to 1 μg / mL with 1 × PBS, added to a 96-well microplate (Corning, item number 42592), 100 μL per well, and incubated at 4°C overnight. After discarding the coating solution, the plate is washed 3 times with 1 × PBST. Add 300 μL of blocking solution (1% BSA in PBST solution) per well, and incubate at room temperature for 2 hours. Discard the blocking solution, wash the plate 3 times with 1 × PBST, and add the diluted monkey serum, and incubate at room temperature for 2 hours. Discard the solution in the microplate, and wash 3 times with 1 × PBST. Dilute Goat anti-Human IgG-h+l HRP (Bethyl, Cat: A80-319P) 20,000 times with diluent (1% BSA in PBST solution), and add 100 μl per well to the enzyme-labeled plate, and incubate at room temperature for 1 hour. Discard the solution in the microplate, and wash 3 times with 1 × PBST. Add 100 μL of TMB color developing solution per well, and develop color after incubation for 5-10 minutes. Add 50 μL of stop solution to stop the reaction. Measure the absorbance value at OD450nm. The pharmacokinetic parameters of the antibody to be tested are calculated using Winnolin software.

[0221] Although the present application has been described in connection with one or more embodiments, it will be understood that the application is not limited to such embodiments, and that it describes in its specification all alternatives, modifications and variations that fall within the spirit and scope of the appended claims. All references cited by the present application are incorporated by reference in their entirety.

Claims

1. An anti-APRIL antibody or antigen-binding fragment thereof, characterized in that, The anti-APRIL antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 regions, and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 regions; the HCDR1, HCDR2, and HCDR3 regions and the LCDR1, LCDR2, and LCDR3 regions have the same sequence as the HCDR1-3 and LCDR1-3 of a VH and VL selected from the group consisting of: VH as shown in SEQ ID NO. 1 and VL as shown in SEQ ID NO. 27; VH as shown in SEQ ID NO. 2 and VL as shown in SEQ ID NO. 28; VH as shown in SEQ ID NO. 3 and VL as shown in SEQ ID NO. 29; VH as shown in SEQ ID NO. 4 and VL as shown in SEQ ID NO. 30; VH as shown in SEQ ID NO. 5 and VL as shown in SEQ ID NO. 31 ; VH as shown in SEQ ID NO. 6 and VL as shown in SEQ ID NO. 32; VH as shown in SEQ ID NO. 7 and VL as shown in SEQ ID NO. 33; VH as shown in SEQ ID NO. 8 and VL as shown in SEQ ID NO. 34; VH as shown in SEQ ID NO. 9 and VL as shown in SEQ ID NO. 35; VH as shown in SEQ ID NO. 10 and VL as shown in SEQ ID NO. 36; VH as shown in SEQ ID NO. 11 and VL as shown in SEQ ID NO. 37; VH as shown in SEQ ID NO. 12 and VL as shown in SEQ ID NO. 38; VH as shown in SEQ ID NO. 13 and VL as shown in SEQ ID NO. 39; VH as shown in SEQ ID NO. 14 and VL as shown in SEQ ID NO. 40; VH as shown in SEQ ID NO. 15 and VL as shown in SEQ ID NO. 41 ; VH as shown in SEQ ID NO. 16 and VL as shown in SEQ ID NO. 42; VH as shown in SEQ ID NO. 17 and VL as shown in SEQ ID NO. 42; VH as shown in SEQ ID NO. 18 and VL as shown in SEQ ID NO. 43; VH as shown in SEQ ID NO. 19 and VL as shown in SEQ ID NO. 44; VH as shown in SEQ ID NO. 20 and VL as shown in SEQ ID NO. 45; VH as shown in SEQ ID NO. 21 and VL as shown in SEQ ID NO. 46; VH as shown in SEQ ID NO. 22 and VL as shown in SEQ ID NO. 47; VH as shown in SEQ ID NO. 23 and VL as shown in SEQ ID NO. 48; VH as shown in SEQ ID NO. 24 and VL as shown in SEQ ID NO. 49; VH as shown in SEQ ID NO. 25 and VL as shown in SEQ ID NO. 50; or, VH as shown in SEQ ID NO. 26 and VL as shown in SEQ ID NO. 51 ; or the VH has HCDR1, HCDR2 and HCDR3 identical to those of a VH as shown in SEQ ID NO. 2, and the VL has LCDR1, LCDR2 and LCDR3 identical to those of a VL as shown in SEQ ID NO. 28; or the VH has HCDR1, HCDR2 and HCDR3 identical to those of a VH as shown in SEQ ID NO. 3, and the VL has LCDR1, LCDR2 and LCDR3 identical to those of a VL as shown in SEQ ID NO. 29; or the VH has HCDR1, HCDR2 and HCDR3 identical to those of a VH as shown in SEQ ID NO. 5, and the VL has LCDR1, LCDR2 and LCDR3 identical to those of a VL as shown in SEQ ID NO. 31; or the VH has HCDR1, HCDR2 and HCDR3 identical to those of a VH as shown in SEQ ID NO. 6, and the VL has LCDR1, LCDR2 and LCDR3 identical to those of a VL as shown in SEQ ID NO. 32; or the VH has HCDR1, HCDR2 and HCDR3 identical to those of a VH as shown in SEQ ID NO. 67, and the VL has LCDR1, LCDR2 and LCDR3 identical to those of a VL as shown in any one of SEQ ID NO. 68-71; or the VH has HCDR1, HCDR2 and HCDR3 identical to those of a VH as shown in any one of SEQ ID NO. 54-57, and the VL has LCDR1, LCDR2 and LCDR3 identical to those of a VL as shown in any one of SEQ ID NO. 58-60; or the VH has HCDR1, HCDR2 and HCDR3 identical to those of a VH as shown in any one of SEQ ID NO. 61-64, and the VL has LCDR1, LCDR2 and LCDR3 identical to those of a VL as shown in any one of SEQ ID NO. 65-66; or the VH has HCDR1, HCDR2 and HCDR3 identical to those of a VH as shown in any one of SEQ ID NO. 72-73, and the VL has LCDR1, LCDR2 and LCDR3 identical to those of a VL as shown in any one of SEQ ID NO. 74-75; or the HCDR1, HCDR2, and HCDR3 regions and the LCDR1, LCDR2, and LCDR3 regions have the same sequence as the HCDR1-3 of a VH and the LCDR1-3 of a VL selected from the group of: a VH as set forth in SEQ ID NO. 54 and a VL as set forth in SEQ ID NO. 58; a VH as set forth in SEQ ID NO. 55 and a VL as set forth in SEQ ID NO. 58; a VH as set forth in SEQ ID NO. 56 and a VL as set forth in SEQ ID NO. 58; a VH as set forth in SEQ ID NO. 54 and a VL as set forth in SEQ ID NO. 59; a VH as set forth in SEQ ID NO. 55 and a VL as set forth in SEQ ID NO. 59; a VH as set forth in SEQ ID NO. 54 and a VL as set forth in SEQ ID NO. 60; a VH as set forth in SEQ ID NO. 55 and a VL as set forth in SEQ ID NO. 60; a VH as set forth in SEQ ID NO. 56 and a VL as set forth in SEQ ID NO. 60; or, a VH as set forth in SEQ ID NO. 57 and a VL as set forth in SEQ ID NO. 60; or the HCDR1, HCDR2, and HCDR3 regions and the LCDR1, LCDR2, and LCDR3 regions have the same sequence as the HCDR1-3 of a VH and the LCDR1-3 of a VL selected from the group of: a VH as set forth in SEQ ID NO. 61 and a VL as set forth in SEQ ID NO. 65; a VH as set forth in SEQ ID NO. 62 and a VL as set forth in SEQ ID NO. 65; a VH as set forth in SEQ ID NO. 63 and a VL as set forth in SEQ ID NO. 65; or, a VH as set forth in SEQ ID NO. 64 and a VL as set forth in SEQ ID NO. 66; or the HCDR1, HCDR2, and HCDR3 regions and the LCDR1, LCDR2, and LCDR3 regions have the same sequence as the HCDR1-3 of a VH and the LCDR1-3 of a VL selected from the group of: a VH as set forth in SEQ ID NO. 67 and a VL as set forth in SEQ ID NO. 68; a VH as set forth in SEQ ID NO. 67 and a VL as set forth in SEQ ID NO. 69; a VH as set forth in SEQ ID NO. 67 and a VL as set forth in SEQ ID NO. 70; or, a VH as set forth in SEQ ID NO. 67 and a VL as set forth in SEQ ID NO. 71; or the HCDR1, HCDR2, and HCDR3 regions of the VH and the LCDR1, LCDR2, and LCDR3 regions of the VL have the same sequence as the HCDR1-3 of a VH and the LCDR1-3 of a VL selected from the group consisting of: a VH as set forth in SEQ ID NO. 72 and a VL as set forth in SEQ ID NO. 74; a VH as set forth in SEQ ID NO. 72 and a VL as set forth in SEQ ID NO. 75; a VH as set forth in SEQ ID NO. 73 and a VL as set forth in SEQ ID NO. 74; or, a VH as set forth in SEQ ID NO. 73 and a VL as set forth in SEQ ID NO.

75.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, the HCDR1, HCDR2, and HCDR3 of the VH and the LCDR1, LCDR2, and LCDR3 of the VL are defined according to IMGT, Kabat, Chothia, AbM, Contact, or any combination thereof.

3. The antibody or antigen-binding fragment thereof of any one of claims 1-2, wherein, the VH has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NO. 1-26, 54-57, 61-64, 67, 72-73, and / or the VL has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NO. 27-51, 58-60, 65-66, 68-71, 74-75; or the VH has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NO. 54-57, and / or the VL has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NO. 58-60; or the VH has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NO. 61-64, and / or the VL has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NO. 65-66; or the VH has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO. 67, and / or the VL has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOs. 68-71; or the VH has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOs. 72-73, and / or the VL has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOs. 74-75; or the VH has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOs. 72-73, and / or the VL has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOs. 74-75; or, the VH and VL comprise a sequence selected from the group consisting of: VL set forth in SEQ ID NO. 1 and 27; SEQ ID NO. 2 and 28; SEQ ID NO. 3 and 29; SEQ ID NO. 4 and 30; SEQ ID NO. 5 and 31; SEQ ID NO. 6 and 32; SEQ ID NO. 7 and 33; SEQ ID NO. 8 and 34; SEQ ID NO. 9 and 35; SEQ ID NO. 10 and 36; SEQ ID NO. 11 and 37; SEQ ID NO. 12 and 38; or a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of: VL set forth in SEQ ID NO. 1 and 27; SEQ ID NO. 2 and 28; SEQ ID NO. 3 and 29; SEQ ID NO. 4 and 30; SEQ ID NO. 5 and 31; SEQ ID NO. 6 and 32; SEQ ID NO. 7 and 33; SEQ ID NO. 8 and 34; SEQ ID NO. 9 and 35; SEQ ID NO. 10 and 36; SEQ ID NO. 11 and 37; SEQ ID NO. 12 and 38; or, the VH and VL comprise a sequence selected from the group consisting of: VH set forth in SEQ ID NO. 13 and 39; SEQ ID NO. 14 and 40; SEQ ID NO. 15 and 41; SEQ ID NO. 16 and 42; SEQ ID NO. 17 and 42; SEQ ID NO. 18 and 43; SEQ ID NO. 19 and 44; SEQ ID NO. 20 and 45; SEQ ID NO. 21 and 46; SEQ ID NO. 22 and 47; SEQ ID NO. 23 and 48; SEQ ID NO. 24 and 49; SEQ ID NO. 25 and 50; SEQ ID NO. 26 and 51; SEQ ID NO. 54 and 58; SEQ ID NO. 55 and 58; SEQ ID NO. 56 and 58; SEQ ID NO. 54 and 59; SEQ ID NO. 55 and 59; SEQ ID NO. 54 and 60; SEQ ID NO. 55 and 60; SEQ ID NO. 56 and 60; SEQ ID NO. 57 and 60; SEQ ID NO. 61 and 65; SEQ ID NO. 62 and 65; SEQ ID NO. 63 and 65; SEQ ID NO. 64 and 66; SEQ ID NO. 67 and 68; SEQ ID NO. 67 and 69; SEQ ID NO. 67 and 70; SEQ ID NO. 67 and 71; SEQ ID NO. 72 and 74; SEQ ID NO. 72 and 75; SEQ ID NO. 73 and 74; or, SEQ ID NO. 73 and 75; or, a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of: VH set forth in SEQ ID NO. 13 and 39; SEQ ID NO. 14 and 40; SEQ ID NO. 15 and 41; SEQ ID NO. 16 and 42; SEQ ID NO. 17 and 42; SEQ ID NO. 18 and 43; SEQ ID NO. 19 and 44; SEQ ID NO. 20 and 45; SEQ ID NO. 21 and 46; SEQ ID NO. 22 and 47; SEQ ID NO. 23 and 48; SEQ ID NO. 24 and 49; SEQ ID NO. 25 and 50; SEQ ID NO. 26 and 51; SEQ ID NO. 54 and 58; SEQ 4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, the VH and VL comprise or consist of the sequences of SEQ ID NO. 1 and 27, respectively; the VH and VL comprise or consist of the sequences of SEQ ID NO. 2 and 28, respectively; the VH and VL comprise or consist of the sequences of SEQ ID NO. 3 and 29, respectively; the VH and VL comprise or consist of the sequences of SEQ ID NO. 4 and 30, respectively; the VH and VL comprise or consist of the sequences of SEQ ID NO. 5 and 31, respectively; the VH and VL comprise or consist of the sequences of SEQ ID NO. 6 and 32, respectively; the VH and VL comprise or consist of the sequences of SEQ ID NO. 56 and 60, respectively; the VH and VL comprise or consist of the sequences of SEQ ID NO. 57 and 60, respectively; or the VH and VL comprise or consist of the sequences of SEQ ID NO. 63 and 65, respectively.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, further comprising a heavy chain constant region selected from the constant region of IgG4 or IgG1 or a variant thereof, and a light chain constant region selected from a kappa chain constant region or a variant thereof; and / or the variant reduces or abrogates ADCC and / or CDC effects, and / or enhances FcRn binding and / or prolongs half-life and / or improves pharmacokinetics; and / or the heavy chain constant region comprises the sequence of SEQ ID NO. 52 or 76, or a sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; and / or the light chain constant region comprises the sequence of SEQ ID NO. 53, or a sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

6. The antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein, The anti-APRIL antibody comprises a heavy chain comprising an amino acid sequence that differs from the sequence as set forth in SEQ ID NO. 77 or 79 by up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 mutations, or comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the sequence as set forth in SEQ ID NO. 77 or 79; and / or, a light chain comprising an amino acid sequence that differs from the sequence as set forth in SEQ ID NO. 78 by up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 mutations, or comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the sequence as set forth in SEQ ID NO. 78; or The heavy chain comprises an amino acid sequence that differs from the sequence as set forth in SEQ ID NO. 80 by up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 mutations (e.g., substitutions, deletions, or additions of amino acids), or comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the sequence as set forth in SEQ ID NO. 80; and / or, the light chain comprises an amino acid sequence that differs from the sequence as set forth in SEQ ID NO. 81 by up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 mutations (e.g., substitutions, deletions, or additions of amino acids), or comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the sequence as set forth in SEQ ID NO. 81; and / or, the heavy chain constant region lacks a C-terminal lysine, and / or the N-terminal amino acid of the antibody or antigen-binding fragment thereof is cyclized to a pyroglutamic acid or pyroglutamic acid salt.

7. An anti-APRIL antibody or antigen-binding fragment thereof that binds to the same APRIL epitope or competes for binding to an APRIL epitope as the anti-APRIL antibody or antigen-binding fragment thereof of any one of claims 1-6.

8. The antibody or antigen-binding fragment thereof of any one of claims 1-7, which has at least one of the following functions: (1) the antibody or antigen-binding fragment thereof binds human or monkey APRIL with a KD of about 10 -8 M, 10 -9 M, about 10 -10 M, about 10 -11 M, about 10 -12 M or lower. or, an EC50of about 1 nM to 0.001 nM, about 0.5 nM to 0.001 nM, about 0.3 nM to 0.001 nM, about 0.2 nM to 0.001 nM, about 0.1 nM to 0.001 nM, about 0.08 nM to 0.01 nM, about 0.05 nM to 0.01 nM or lower 50 binds human APRIL; (2) the antibody or antigen-binding fragment thereof binds human or monkey APRIL with substantially the same or a better KD than a reference APRIL antibody; (3) the antibody or antigen-binding fragment thereof blocks (or inhibits, neutralizes) the binding of human APRIL to human BCMA, and / or blocks (or inhibits, neutralizes) the binding of human APRIL to human TACI; (4) the antibody or antigen-binding fragment thereof has an IC50 for binding to human APRIL that is substantially the same or better than that of a reference APRIL antibody 50 blocks (or inhibits, neutralizes) the binding of human APRIL to human BCMA, and / or blocks (or inhibits, neutralizes) the binding of human APRIL to human TACI; (5) the antibody or antigen-binding fragment thereof reduces (e.g., inhibits, blocks, or neutralizes) one or more biological activities of APRIL in vitro, ex vivo, or in vivo; (6) the antibody or antigen-binding fragment thereof binds human APRIL, monkey APRIL, murine APRIL, or two or more thereof, e.g., human and monkey APRIL, human and murine APRIL, or human, monkey, and murine APRIL; (7) the antibody or antigen-binding fragment thereof inhibits B cell (e.g., human B cell) proliferation; (8) the antibody or antigen-binding fragment thereof reduces or inhibits the level of IgA, e.g., IgA secretion by B cells; (9) the antibody or antigen-binding fragment thereof blocks (or inhibits, neutralizes) the binding of monkey APRIL to BCMA; (10) the antibody or antigen-binding fragment thereof does not bind or does not substantially bind BAFF; and / or, the reference APRIL antibody is selected from Sibeprenlimab, Zigakibart. The anti-APRIL antibody is a murine, chimeric, or humanized antibody.

9. The antibody or antigen-binding fragment thereof of any one of claims 1-8, wherein, 10. A conjugate or bispecific or multispecific antibody or chimeric antigen receptor comprising the anti-APRIL antibody or antigen-binding fragment thereof of any one of claims 1-9.

11. A polynucleotide encoding the anti-APRIL antibody or antigen-binding fragment thereof of any one of claims 1-10, or the conjugate or bispecific or multispecific antibody or chimeric antigen receptor of claim 11.

12. A vector comprising the polynucleotide of claim 12.

13. A host cell comprising the polynucleotide of claim 11 or the vector of claim 12.

14. A pharmaceutical composition comprising the anti-APRIL antibody or antigen-binding fragment thereof of any one of claims 1-9, or the conjugate or bispecific or multispecific antibody or chimeric antigen receptor of claim 10, or the polynucleotide of claim 11, or the vector of claim 12, or the host cell of claim 13, and one or more pharmaceutically acceptable carriers.

15. A kit comprising the anti-APRIL antibody or antigen-binding fragment thereof of any one of claims 1-9, or the conjugate or bispecific or multispecific antibody or chimeric antigen receptor of claim 10, or the polynucleotide of claim 11, or the vector of claim 12, or the host cell of claim 13, or the pharmaceutical composition of claim 15. ​ 16. Use of the anti-APRIL antibody or antigen-binding fragment thereof of any one of claims 1-9, or the conjugate or bispecific antibody or multispecific antibody or chimeric antigen receptor of claim 10, or the polynucleotide of claim 11, or the vector of claim 12, or the host cell of claim 13, or the pharmaceutical composition of claim 14, or the kit of claim 15 in the manufacture of a medicament for preventing and / or treating a disease or disorder associated with APRIL in a subject; and / or, the disease or disorder associated with APRIL is selected from an IgA secretion-mediated disease or disorder, or a disease or disorder caused by B cell proliferation; and / or, the disease or disorder associated with APRIL is selected from IgA nephropathy.

17. A method of inhibiting cell proliferation, comprising contacting the cell with the anti-APRIL antibody or antigen-binding fragment thereof of any one of claims 1-9, or the conjugate or bispecific antibody or multispecific antibody or chimeric antigen receptor of claim 10, or the polynucleotide of claim 11, or the vector of claim 12, or the host cell of claim 13, or the pharmaceutical composition of claim 14, or the kit of claim 15.

18. A method of reducing IgA level in a cell, comprising contacting the cell with the anti-APRIL antibody or antigen-binding fragment thereof of any one of claims 1-9, or the conjugate or bispecific antibody or multispecific antibody or chimeric antigen receptor of claim 10, or the polynucleotide of claim 11, or the vector of claim 12, or the host cell of claim 13, or the pharmaceutical composition of claim 14, or the kit of claim 15.

19. A method of inhibiting the binding of APRIL to an APRIL receptor on a cell, comprising contacting the cell with the anti-APRIL antibody or antigen-binding fragment thereof of any one of claims 1-9, or the conjugate or bispecific antibody or multispecific antibody or chimeric antigen receptor of claim 10, or the polynucleotide of claim 11, or the vector of claim 12, or the host cell of claim 13, or the pharmaceutical composition of claim 14, or the kit of claim 15.

20. Use of the anti-APRIL antibody or antigen-binding fragment thereof of any one of claims 1-9, or the conjugate or bispecific antibody or multispecific antibody or chimeric antigen receptor of claim 10, or the pharmaceutical composition of claim 14, or the kit of claim 15 in detecting APRIL or in the manufacture of a reagent for detecting APRIL.

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