Anti-BK virus antibody molecule

Abstract

Disclosed are anti-BK virus antibody molecules or binding fragments thereof that can be used to treat or prevent BK virus infection and / or BK virus-related disease.

Description

[Technical Field]

[0001] This disclosure relates to an anti-BK virus antibody molecule or its conjugate fragment. The disclosure further relates to nucleic acids encoding the antibody molecule or its conjugate fragment, expression vectors, host cells, and methods for producing the antibody molecule or its conjugate fragment. Pharmaceutical compositions comprising this antibody molecule or its conjugate fragment are also provided. The anti-BK virus antibody molecule or its conjugate fragment of this disclosure can be used (alone or in combination with other agents or modes of treatment) to treat or prevent BK virus infection and / or BK virus-related diseases. Therefore, the disclosure further relates to an anti-BK virus antibody molecule or its conjugate fragment, or a pharmaceutical composition comprising an anti-BK virus antibody molecule or its conjugate fragment, for use in the treatment or prevention of BK virus infection and / or BK virus-related diseases. Diagnostic compositions comprising such antibody molecule or its conjugate fragment are also provided. [Background technology]

[0002] Immunosuppressants are standard treatments to enable graft survival in transplant patients and prevent graft rejection. Immunosuppression can induce reactivation of human BK polyomavirus in 40-50% of kidney transplant recipients (Hurdiss et al., Structure, 2016 Apr 5;24(4):528-536), and in up to 10% of cases, it can lead to BK virus (BKV)-associated nephropathy (BKVAN) (Bennett et al., Microbes Infect, 2012 Aug;14(9):672-83, Rinaldo et al, APMIS, 2013 Aug;121(8):728-45). BKVAN is a serious threat and can lead to decreased graft function or graft loss (Ramos et al, Transplantation, 2009 Mar 15;87(5):621-30). The use of antiviral agents has not yielded consistent efficacy results and is not considered a worthwhile treatment (Santeusanio et al. Am J Health Syst Pharm, 2017 Dec 15;74(24):2037-2045; Kable et al., Transplant Direct, 2017 Mar 10;3(4):e142).

[0003] Therefore, the standard treatment for acute viremia is to reduce immunosuppression so that the immune system can suppress the virus. However, in this case, there is a significant risk that the graft may become dysfunctional in the short and long term due to the immune response between the host and the graft, such as the formation of donor-specific antibodies.

[0004] Given the need for improved treatments for prophylactic or curative therapy in immunocompromised patients, there is a great demand for novel compositions to neutralize BK virus activity. [Overview of the project]

[0005] Aspect A In one aspect A1, the present disclosure relates to an anti-BK virus antibody molecule or an anti-BK virus conjugated fragment thereof.

[0006] Structural properties In some embodiments of aspect A1, the disclosure relates to an anti-BK virus antibody molecule or an anti-BK virus binding fragment thereof, wherein the antibody molecule or binding fragment comprises at least one, two, three, four, five, or six complementarity-determining regions (CDRs) (or collectively all of the CDRs) from a heavy chain variable region (VH) and / or light chain variable region (VL) comprising the amino acid sequences shown in Table 3, wherein one or more of the CDRs (or collectively all of the CDRs) may have one, two, three, four, five, six or more variations, for example, amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions with respect to the amino acid sequences shown in Table 3.

[0007] In some embodiments of aspect A1, this disclosure relates to an anti-BK virus antibody molecule or an anti-BK virus conjugate fragment thereof, comprising: Heavy chain variable regions (VH) including one, two, or three of the following: the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 21 or a sequence with one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions; the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 22 or a sequence with one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions; and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 23 or a sequence with one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions; and / or Light chain variable regions (VLs) including one, two, or three of the following: the light chain complementarity determination region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 24, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions; the light chain complementarity determination region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 25, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions; and the light chain complementarity determination region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 26, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions.

[0008] In some embodiments of phase A1, this disclosure provides, Heavy chain variable region (VH) containing the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 23, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions); and / or This invention relates to an anti-BK virus antibody molecule or an anti-BK virus binding fragment thereof, comprising a light chain variable region (VL) containing the light chain complementarity determination region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 26, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conserved amino acid substitutions).

[0009] In some embodiments of aspect A1, this disclosure relates to an anti-BK virus antibody molecule or an anti-BK virus conjugate fragment thereof, comprising: Heavy chain variable regions (VH) including the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 21, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions), the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 22, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions), and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 23, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions); and / or A light chain variable region (VL) comprising the light chain complementarity determination region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 24, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions), the light chain complementarity determination region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 25, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions), and the light chain complementarity determination region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 26, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions).

[0010] In some embodiments of aspect A1, this disclosure relates to an anti-BK virus antibody molecule or an anti-BK virus conjugate fragment thereof, comprising: Heavy chain variable region (VH) including the amino acid sequence of heavy chain complementarity determination region 1 (VHCDR1) of SEQ ID NO: 21, the amino acid sequence of heavy chain complementarity determination region 2 (VHCDR2) of SEQ ID NO: 22, or a sequence having 1, 2, or 3 amino acid substitutions, and the amino acid sequence of heavy chain complementarity determination region 3 (VHCDR3) of SEQ ID NO: 23; and / or A light chain variable region (VL) comprising the amino acid sequence of light chain complementarity determination region 1 (VLCDR1) of SEQ ID NO: 24, or a sequence having one or two amino acid substitutions, the amino acid sequence of light chain complementarity determination region 2 (VLCDR2) of SEQ ID NO: 25, and the amino acid sequence of light chain complementarity determination region 3 (VLCDR3) of SEQ ID NO: 26, or a sequence having one, two, three, or four amino acid substitutions.

[0011] In some embodiments of surface A1, the anti-BK virus antibody molecule or its anti-BK virus conjugated fragment includes: The heavy chain variable region (VH) includes the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 21, the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 22, and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 23; and The light chain variable region (VL) includes the amino acid sequence of light chain complementarity determination region 1 (VLCDR1) of SEQ ID NO: 24, the amino acid sequence of light chain complementarity determination region 2 (VLCDR2) of SEQ ID NO: 25, and the amino acid sequence of light chain complementarity determination region 3 (VLCDR3) of SEQ ID NO: 26. Here, one or two amino acids in the CDR are inserted, deleted, or substituted.

[0012] In some embodiments of surface A1, the anti-BK virus antibody molecule or its anti-BK virus conjugated fragment includes: The heavy chain variable region (VH) includes the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 21, the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 22, and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 23; and The light chain variable region (VL) includes the amino acid sequence of light chain complementarity determination region 1 (VLCDR1) of SEQ ID NO: 24, the amino acid sequence of light chain complementarity determination region 2 (VLCDR2) of SEQ ID NO: 25, and the amino acid sequence of light chain complementarity determination region 3 (VLCDR3) of SEQ ID NO: 26.

[0013] In some embodiments of aspect A1, the anti-BK virus antibody molecule or its anti-BK virus binding fragment includes a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 27.

[0014] In some embodiments of surface A1, the anti-BK virus antibody molecule or its anti-BK virus conjugated fragment includes: (i) A light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 28; or (ii) A light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 31.

[0015] In some embodiments of Aspect A1, the anti-BK virus antibody molecule or its anti-BK virus binding fragment comprises: (i) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 27, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 28, or (ii) a heavy chain variable region (VH) consisting of the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 27, and a light chain variable region (VL) consisting of the amino acid sequence of SEQ ID NO: 31 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 31.

[0016] In some embodiments of Aspect A1, the anti-BK virus antibody molecule or its anti-BK virus binding fragment comprises: (i) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 27 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 28; or (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 27 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 31.

[0017] Functional characteristics In some embodiments of Aspect A1, the anti-BK virus antibody molecule or its anti-BK virus binding fragment comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) of the following characteristics: (i) When an antibody molecule or its binding fragment binds to BK virus serotype I VP1 and is tested as a bivalent molecule using ELISA, for example as described in Example 2, the EC50 is less than approximately 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.19 nM, 0.18 nM, 0.17 nM, 0.16 nM, 0.15 nM, 0.14 nM, 0.13 nM, 0.12 nM, 0.11 nM, 0.10 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, or 0.02 nM; (ii) When an antibody molecule or its binding fragment binds to BK virus serotype II VP1 and is tested as a bivalent molecule using ELISA, for example as described in Example 2, the EC50 is less than approximately 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.19 nM, 0.18 nM, 0.17 nM, 0.16 nM, 0.15 nM, 0.14 nM, 0.13 nM, 0.12 nM, 0.11 nM, 0.10 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, or 0.02 nM; (iii) When an antibody molecule or its conjugate fragment binds to BK virus serotype III VP1 and is tested as a bivalent molecule using ELISA, for example as described in Example 2, the EC50 is less than approximately 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.19 nM, 0.18 nM, 0.17 nM, 0.16 nM, 0.15 nM, 0.14 nM, 0.13 nM, 0.12 nM, 0.11 nM, 0.10 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, or 0.02 nM; (iv) binds to BK virus serotype IV VP1 and has an EC50 of less than about 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.19 nM, 0.18 nM, 0.17 nM, 0.16 nM, 0.15 nM, 0.14 nM, 0.13 nM, 0.12 nM, 0.11 nM, 0.10 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, or 0.02 nM when, for example, an antibody molecule or a binding fragment thereof is tested as a bivalent molecule using ELISA as described in Example 2; (v) does not bind to JC virus VP1 as described, for example, in Example 6; (vi) neutralizes BK virus serotype I; (vii) neutralizes BK virus serotype II; (viii) neutralizes BK virus serotype III; (ix) neutralizes BK virus serotype IV.

[0018] In one aspect A2, the disclosure relates to an antibody molecule or a binding fragment thereof that competes with the antibody molecules or binding fragments thereof described herein for binding to BK virus serotype I VP1, BK virus serotype II VP1, BK virus serotype III VP1, and / or BK virus serotype IV VP1. In some embodiments of aspect A2, the disclosure relates to an antibody molecule or a binding fragment thereof that competes with an antibody molecule or a binding fragment thereof comprising: a heavy chain variable region (VH) comprising the amino acid sequence of heavy chain complementarity determining region 1 (VHCDR1) of SEQ ID NO: 21, and the amino acid sequence of heavy chain complementarity determining region 2 (VHCDR2) of SEQ ID NO: 22, and the amino acid sequence of heavy chain complementarity determining region 3 (VHCDR3) of SEQ ID NO: 23 for binding to BK virus genotype I VP1, BK virus genotype II VP1, BK virus genotype III VP1, and / or BK virus genotype IV VP1; and The light chain variable region (VL) includes the amino acid sequence of light chain complementarity determination region 1 (VLCDR1) of SEQ ID NO: 24, the amino acid sequence of light chain complementarity determination region 2 (VLCDR2) of SEQ ID NO: 25, and the amino acid sequence of light chain complementarity determination region 3 (VLCDR3) of SEQ ID NO: 26.

[0019] In some embodiments of aspect A2, the disclosure relates to antibody molecules or their binding fragments that compete for binding to BK virus genotype I VP1, BK virus genotype II VP1, BK virus genotype III VP1, and / or BK virus genotype IV VP1 with antibody molecules or their binding fragments, including: (i) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 27, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 28; or (ii) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 27, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 31.

[0020] In aspect A3, the present disclosure relates to a pharmaceutical composition comprising an antibody molecule or a conjugated fragment thereof as described herein, and a pharmaceutically acceptable carrier, excipient, or stabilizer.

[0021] In one aspect A4, the present disclosure relates to an anti-BK virus antibody molecule or its conjugate fragment described herein, or a pharmaceutical composition comprising an anti-BK virus antibody molecule or its conjugate fragment, for use in the treatment or prevention of BK virus infection and / or BK virus-related disease. In some embodiments of aspect A4, the BK virus-related disease is selected from the group consisting of nephropathy, BK virus-associated nephropathy (BKVAN), and hemorrhagic cystitis (HC).

[0022] In one aspect, A5, the present disclosure relates to nucleic acids encoding antibody heavy chain and / or light chain variable regions of antibody molecules or their binding fragments as described herein.

[0023] In one aspect, A6, this disclosure relates to an expression vector comprising nucleic acid as described herein.

[0024] In one aspect, A7, this disclosure relates to a host cell comprising a nucleic acid or an expression vector as described herein.

[0025] In one aspect A8, the present disclosure relates to a method for producing an antibody molecule, the method comprising culturing host cells described herein under conditions suitable for gene expression.

[0026] In one aspect A9, this disclosure relates to a diagnostic composition comprising an antibody molecule or a conjugated fragment thereof as described herein.

[0027] Phase B In one aspect B1, the present disclosure relates to an anti-BK virus antibody molecule or an anti-BK virus conjugated fragment thereof.

[0028] Structural properties In some embodiments of Section B1, the Disclosure relates to an anti-BK virus antibody molecule or an anti-BK virus binding fragment thereof, wherein the antibody molecule or binding fragment comprises at least one, two, three, four, five, or six complementarity-determining regions (CDRs) (or all of the CDRs collectively) from heavy chain variable regions (VH) and / or light chain variable regions (VL) containing the amino acid sequences shown in Table 4, where one or more of the CDRs (or all of the CDRs collectively) may have one, two, three, four, five, six or more variations, for example, amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions with respect to the amino acid sequences shown in Table 4.

[0029] In some embodiments of aspect B1, this disclosure relates to an anti-BK virus antibody molecule or an anti-BK virus conjugate fragment thereof, comprising: (i) Heavy chain variable region (VH) including one, two, or three of the following: the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 34, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions; the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 35, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions; and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 36, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions; and / Or a light chain variable region (VL) containing one, two, or three of the following: the light chain complementarity determination region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 37, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions; the light chain complementarity determination region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 38, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions; and the light chain complementarity determination region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 39, or a sequence having one, two, or three of the following amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions; or (ii) The amino acid sequence of the heavy chain complementarity determination region 1 (VHCDR1) of SEQ ID NO: 34 or a sequence having 1, 2, 3 or 4 amino acid substitutions (e.g., conservative amino acid substitutions), insertions or deletions, the amino acid sequence of the heavy chain complementarity determination region 2 (VHCDR2) of SEQ ID NO: 44 or a sequence having 1, 2, 3 or 4 amino acid substitutions (e.g., conservative amino acid substitutions), insertions or deletions, and the amino acid sequence of the heavy chain complementarity determination region 3 (VHCDR3) of SEQ ID NO: 36 or a sequence having 1, 2, 3 or 4 amino acid substitutions (e.g., conservative amino acid substitutions). The light chain variable region (VL) containing one, two, or three sequences having one, two, or three of the following: sequences with conservative amino acid substitutions, insertions, or deletions; and / or the light chain complementarity determination region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 37, or sequences having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions, the light chain complementarity determination region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 38, or sequences having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions, and the light chain complementarity determination region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 39, or the light chain variable region (VL) containing one, two, or three of the following sequences having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions, or (iii) The amino acid sequence of the heavy chain complementarity determination region 1 (VHCDR1) of SEQ ID NO: 34 or a sequence having 1, 2, 3 or 4 amino acid substitutions (e.g., conservative amino acid substitutions), insertions or deletions; the amino acid sequence of the heavy chain complementarity determination region 2 (VHCDR2) of SEQ ID NO: 44 or a sequence having 1, 2, 3 or 4 amino acid substitutions (e.g., conservative amino acid substitutions), insertions or deletions; and the amino acid sequence of the heavy chain complementarity determination region 3 (VHCDR3) of SEQ ID NO: 36 or a sequence having 1, 2, 3 or 4 amino acid substitutions (e.g., conservative amino acid substitutions). A light chain variable region (VL) comprising one, two, or three sequences having one, two, or three of the following: the light chain complementarity determination region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 37, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions, the light chain complementarity determination region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 38, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions, and the light chain complementarity determination region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 48, or one, two, or three of the following sequences having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions.

[0030] In some embodiments of aspect B1, this disclosure relates to an anti-BK virus antibody molecule or an anti-BK virus conjugate fragment thereof, comprising: (i) The heavy chain variable region (VH) containing the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 36, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions); and / or A light chain variable region (VL) containing the light chain complementarity determination region 3 (VLCDR3) amino acid sequence of Sequence ID No. 39, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions). (ii) The heavy chain variable region (VH) containing the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 36, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions); and / or A light chain variable region (VL) containing the light chain complementarity determination region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 48, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions).

[0031] In some embodiments of aspect B1, this disclosure relates to an anti-BK virus antibody molecule or an anti-BK virus conjugate fragment thereof, comprising: (i) Heavy chain variable regions (VH) including the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 34, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions), the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 35, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions), and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 36, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions); and / or Light chain variable region (VL) including the light chain complementarity determination region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 37, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions), the light chain complementarity determination region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 38, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions), and the light chain variable region (VL) including the light chain complementarity determination region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 39, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions); or (ii) Heavy chain variable regions (VH) including the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 34, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions), the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 44, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions), and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 36, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions); and / or Light chain variable region (VL) including the light chain complementarity determination region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 37, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions), the light chain complementarity determination region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 38, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions), and the light chain variable region (VL) including the light chain complementarity determination region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 39, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions); or (iii) Heavy chain variable regions (VH) including the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 34, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions), the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 44, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions), and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 36, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions); and / or A light chain variable region (VL) comprising the light chain complementarity determination region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 37, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions), the light chain complementarity determination region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 38, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions), and the light chain variable region (VL) comprising the light chain complementarity determination region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 48, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions).

[0032] In some embodiments of aspect B1, this disclosure relates to an anti-BK virus antibody molecule or an anti-BK virus conjugate fragment thereof, comprising: (i) A heavy chain variable region (VH) comprising the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 34, the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 35 or a sequence having one, two, or three amino acid substitutions, and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 36, and / or Light chain variable region (VL) including the amino acid sequence of light chain complementarity determination region 1 (VLCDR1) of SEQ ID NO: 37 or a sequence having one or two amino acid substitutions, the amino acid sequence of light chain complementarity determination region 2 (VLCDR2) of SEQ ID NO: 38, the amino acid sequence of light chain complementarity determination region 3 (VLCDR3) of SEQ ID NO: 39 or a sequence having one, two, three, or four amino acid substitutions, or (ii) A heavy chain variable region (VH) comprising the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 34, the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 44 or a sequence having one, two, or three amino acid substitutions, and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 36, and / or Light chain variable regions (VL) including the amino acid sequence of light chain complementarity determination region 1 (VLCDR1) of SEQ ID NO: 37 or a sequence having one or two amino acid substitutions, the amino acid sequence of light chain complementarity determination region 2 (VLCDR2) of SEQ ID NO: 38, the amino acid sequence of light chain complementarity determination region 3 (VLCDR3) of SEQ ID NO: 39, or a sequence having one, two, three, or four amino acid substitutions, or (iii) A heavy chain variable region (VH) including the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 34, the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 44 or a sequence having one, two, or three amino acid substitutions, and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 36, and / or A light chain variable region (VL) containing the amino acid sequence of light chain complementarity determination region 1 (VLCDR1) of SEQ ID NO: 37 or a sequence having one or two amino acid substitutions, the amino acid sequence of light chain complementarity determination region 2 (VLCDR2) of SEQ ID NO: 38, the amino acid sequence of light chain complementarity determination region 3 (VLCDR3) of SEQ ID NO: 48, or a sequence having one, two, three, or four amino acid substitutions.

[0033] In some embodiments of surface B1, the anti-BK virus antibody molecule or its anti-BK virus binding fragment includes: (i) Heavy chain variable region (VH) including the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 34, the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 35, and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 36; and Light chain variable region (VL) including the amino acid sequence of light chain complementarity determination region 1 (VLCDR1) of SEQ ID NO: 37, the amino acid sequence of light chain complementarity determination region 2 (VLCDR2) of SEQ ID NO: 38, and the amino acid sequence of light chain complementarity determination region 3 (VLCDR3) of SEQ ID NO: 39; Here, one or two amino acids in the CDR are inserted, deleted, or substituted, (ii) Heavy chain variable region (VH) including the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 34, the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 44, and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 36; and Light chain variable region (VL) including the amino acid sequence of light chain complementarity determination region 1 (VLCDR1) of SEQ ID NO: 37, the amino acid sequence of light chain complementarity determination region 2 (VLCDR2) of SEQ ID NO: 38, and the amino acid sequence of light chain complementarity determination region 3 (VLCDR3) of SEQ ID NO: 39; Here, one or two amino acids in the CDR are inserted, deleted, or substituted. or (iii) Heavy chain variable region (VH) including the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 34, the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 44, and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 36; and Light chain variable region (VL) including the amino acid sequence of light chain complementarity determination region 1 (VLCDR1) of SEQ ID NO: 37, the amino acid sequence of light chain complementarity determination region 2 (VLCDR2) of SEQ ID NO: 38, and the amino acid sequence of light chain complementarity determination region 3 (VLCDR3) of SEQ ID NO: 48; Here, one or two amino acids in the CDR are inserted, deleted, or substituted.

[0034] In some embodiments of surface B1, the anti-BK virus antibody molecule or its anti-BK virus binding fragment includes: (i) Heavy chain variable region (VH) including the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 34, the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 35, and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 36; and Light chain variable region (VL) including the amino acid sequence of light chain complementarity determination region 1 (VLCDR1) of SEQ ID NO: 37, the amino acid sequence of light chain complementarity determination region 2 (VLCDR2) of SEQ ID NO: 38, and the amino acid sequence of light chain complementarity determination region 3 (VLCDR3) of SEQ ID NO: 39; or (ii) Heavy chain variable region (VH) including the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 34, the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 44, and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 36; and Light chain variable region (VL) including the amino acid sequence of light chain complementarity determination region 1 (VLCDR1) of SEQ ID NO: 37, the amino acid sequence of light chain complementarity determination region 2 (VLCDR2) of SEQ ID NO: 38, and the amino acid sequence of light chain complementarity determination region 3 (VLCDR3) of SEQ ID NO: 39; or (iii) Heavy chain variable region (VH) including the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 34, the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 44, and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 36; and The light chain variable region (VL) includes the amino acid sequence of light chain complementarity determination region 1 (VLCDR1) of SEQ ID NO: 37, the amino acid sequence of light chain complementarity determination region 2 (VLCDR2) of SEQ ID NO: 38, and the amino acid sequence of light chain complementarity determination region 3 (VLCDR3) of SEQ ID NO: 48.

[0035] In some embodiments of surface B1, the anti-BK virus antibody molecule or its anti-BK virus binding fragment includes: (i) The amino acid sequence of SEQ ID NO: 40, or a heavy chain variable region (VH) containing an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 40, (ii) The amino acid sequence of SEQ ID NO: 45, or a heavy chain variable region (VH) containing an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 45, (iii) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 49.

[0036] In some embodiments of surface B1, the anti-BK virus antibody molecule or its anti-BK virus binding fragment includes: (i) The amino acid sequence of SEQ ID NO: 41, or a light chain variable region (VL) containing an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 41, (ii) A light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 50.

[0037] In some embodiments of surface B1, the anti-BK virus antibody molecule or its anti-BK virus binding fragment includes: (i) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 40, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 41, or an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 41, or (ii) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 45, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 41, or an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 41, or (iii) A heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 49, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 50.

[0038] In some embodiments of surface B1, the anti-BK virus antibody molecule or its anti-BK virus binding fragment includes: (i) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 40, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 41. (ii) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 45, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 41. (iii) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 49, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 50.

[0039] Functional characteristics In some embodiments of surface B1, the anti-BK virus antibody molecule or its anti-BK virus binding fragment includes one or more of the following properties (e.g., 2, 3, 4, 5, 6, 7, 8, or 9): (i) When an antibody molecule or its binding fragment binds to BK virus serotype I VP1 and is tested as a bivalent molecule using ELISA, for example as described in Example 2, the EC50 is less than approximately 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.19 nM, 0.18 nM, 0.17 nM, 0.16 nM, 0.15 nM, 0.14 nM, 0.13 nM, 0.12 nM, 0.11 nM, 0.10 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, or 0.02 nM; (ii) When an antibody molecule or its binding fragment binds to BK virus serotype II VP1 and is tested as a bivalent molecule using ELISA, for example as described in Example 2, the EC50 is less than approximately 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.19 nM, 0.18 nM, 0.17 nM, 0.16 nM, 0.15 nM, 0.14 nM, 0.13 nM, 0.12 nM, 0.11 nM, 0.10 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, or 0.02 nM; (iii) When the antibody molecule or its conjugate fragment binds to BK virus serotype III VP1 and is tested as a bivalent molecule using ELISA, for example as described in Example 2, the EC50 is less than approximately 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.19 nM, 0.18 nM, 0.17 nM, 0.16 nM, 0.15 nM, 0.14 nM, 0.13 nM, 0.12 nM, 0.11 nM, 0.10 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, or 0.02 nM; (iv) When an antibody molecule or its conjugate fragment binds to BK virus serotype IV VP1 and is tested as a bivalent molecule using ELISA, for example as described in Example 2, the EC50 is less than approximately 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.19 nM, 0.18 nM, 0.17 nM, 0.16 nM, 0.15 nM, 0.14 nM, 0.13 nM, 0.12 nM, 0.11 nM, 0.10 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, or 0.02 nM; (v) For example, as described in Example 6, it does not bind to JC virus VP1; (vi) Neutralize BK virus serotype I; (vii) Neutralize BK virus serotype II; (viii) Neutralize BK virus serotype III; (ix) Neutralize BK virus serotype IV.

[0040] In one aspect B2, the disclosure relates to an antibody molecule or its binding fragment that competes with the antibody molecules or their binding fragments described herein for binding to BK virus serotype I VP1, BK virus serotype II VP1, BK virus serotype III VP1, and / or BK virus serotype IV VP1. In some embodiments of aspect B2, the disclosure relates to an antibody molecule or its binding fragment that competes with the antibody molecules or their binding fragments for binding to BK virus genotype I VP1, BK virus genotype II VP1, BK virus genotype III VP1, and / or BK virus genotype IV VP1, including: (i) Heavy chain variable region (VH) including the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 34, the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 35, and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 36; and Light chain variable region (VL) including the amino acid sequence of light chain complementarity determination region 1 (VLCDR1) of SEQ ID NO: 37, the amino acid sequence of light chain complementarity determination region 2 (VLCDR2) of SEQ ID NO: 38, and the amino acid sequence of light chain complementarity determination region 3 (VLCDR3) of SEQ ID NO: 39; or (ii) Heavy chain variable region (VH) including the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 34, the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 44, and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 36; and Light chain variable region (VL) including the amino acid sequence of light chain complementarity determination region 1 (VLCDR1) of SEQ ID NO: 37, the amino acid sequence of light chain complementarity determination region 2 (VLCDR2) of SEQ ID NO: 38, and the amino acid sequence of light chain complementarity determination region 3 (VLCDR3) of SEQ ID NO: 39; or (iii) Heavy chain variable region (VH) including the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 34, the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 44, and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 36; and The light chain variable region (VL) includes the amino acid sequence of light chain complementarity determination region 1 (VLCDR1) of SEQ ID NO: 37, the amino acid sequence of light chain complementarity determination region 2 (VLCDR2) of SEQ ID NO: 38, and the amino acid sequence of light chain complementarity determination region 3 (VLCDR3) of SEQ ID NO: 48.

[0041] In some embodiments of aspect B2, the disclosure relates to antibody molecules or their binding fragments that compete for binding to BK virus genotype I VP1, BK virus genotype II VP1, BK virus genotype III VP1, and / or BK virus genotype IV VP1 with antibody molecules or their binding fragments, including: (i) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 40, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 41. (ii) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 45, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 41. (iii) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 49, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 50.

[0042] In aspect B3, the present disclosure relates to a pharmaceutical composition comprising an antibody molecule or a conjugated fragment thereof as described herein, and a pharmaceutically acceptable carrier, excipient, or stabilizer.

[0043] In one aspect B4, the present disclosure relates to an anti-BK virus antibody molecule or its conjugate fragment described herein, or a pharmaceutical composition comprising an anti-BK virus antibody molecule or its conjugate fragment, for use in the treatment or prevention of BK virus infection and / or BK virus-related disease. In some embodiments of aspect B4, the BK virus-related disease is selected from the group consisting of nephropathy, BK virus-associated nephropathy (BKVAN), and hemorrhagic cystitis (HC).

[0044] In one aspect, B5, the present disclosure relates to nucleic acids encoding antibody heavy chain and / or light chain variable regions of antibody molecules or their binding fragments as described herein.

[0045] In one aspect, B6, this disclosure relates to an expression vector comprising nucleic acid as described herein.

[0046] In one aspect, B7, this disclosure relates to a host cell comprising a nucleic acid or an expression vector as described herein.

[0047] In one aspect B8, the present disclosure relates to a method for producing an antibody molecule, the method comprising culturing host cells described herein under conditions suitable for gene expression.

[0048] In one aspect B9, this disclosure relates to a diagnostic composition comprising an antibody molecule or a conjugated fragment thereof as described herein. [Brief explanation of the drawing]

[0049] [Figure 1]The binding of anti-BK virus antibodies to BKV-VP1 serotypes I-IV using the ELISA method. Based on this data, a three-parameter analysis was performed using GraphPad Prism Software to determine the EC50E value.

[0050] [Figure 2] This study quantitatively evaluates antibody-mediated neutralization of BKV strains (serotypes Ia, Ib, II, III, and IV). 293TT cells were used as infection targets for luciferase-expressing BK pseudoviruses formed with VP1 cells of serotype Ia, Ib, II, III, or IV. Anti-BK virus antibodies were used at the indicated concentrations.

[0051] [Figure 3] Long-term viral neutralization of wild-type BKV on human primary tubular epithelial cells by anti-BK virus antibodies. The residual viral load is shown after long-term co-culture of cells, BK virus, and anti-BK virus antibodies at the indicated concentrations. Antibody concentrations correspond to EC95 (1.500), EC50 (0.15), and EC5 (0.015). P8D11 is denoted as "BM2" in this figure.

[0052] [Figure 4] Binding of anti-BK virus antibodies to conformational epitopes.

[0053] [Figure 5] The binding strength of antibodies 319C07 and 336F07 to VP1 variants and JC-VP1 was evaluated by ELISA compared to antibody P8D11. EC50 values ​​for various VP1 and JC-VP1 variants were determined compared to wild-type VP1. VP1 variants are specified in a standard format indicating the position of change in the VP1-STI sequence, from the amino acid to the right of the number.

[0054] The graph shows the binding of antibodies 319C07, 336F07, isotype control antibody 24C03, and P8D11 to the selected BKV-VP1 variant, JCV-VP1, and the CMV-gH pentamer as an unrelated antigen.

[0055] The table shows data for all BKV-VP1 variants and JCV-VP1 tested. "+" indicates no difference in binding affinity between the original and mutated sequences. "red" indicates decreased binding. "-" indicates no binding was observed.

[0056] [Figure 6] Suppression of BKV diffusion on HRPTEC by anti-BK virus antibodies (in this figure, P8D11 is labeled "BM2").

[0057] [Figure 7] ADCC activity of anti-BKV antibodies against infectious and non-infectious HPRTECs.

[0058] [Figure 8] CDC activity of anti-BKV antibodies against infected and non-infectious HPRTECs in the presence of human serum.

[0059] (Detailed explanation) The present invention, as illustrated below, can also be suitably implemented without any elements or limitations not specifically disclosed herein.

[0060] The present invention will be described below with reference to specific embodiments and specific figures, but the present invention is not limited thereto and is limited only by the claims.

[0061] Where the term “including” is used in this specification and in the claims, it does not exclude other elements. For the purposes of the present invention, the term “consisting of” is considered a preferred embodiment of the term “including.” Hereinafter, where a group is defined as containing at least a certain number of embodiments, this is also understood to mean disclosing a group that preferably consists only of these embodiments.

[0062] When an indefinite or definite article is used with a singular noun, such as "a," "an," or "the," it includes the plural form of that noun unless something else is explicitly stated. In the context of this invention, the terms "about" or "approximately" indicate an interval of precision that a person skilled in the art would understand in order to still ensure the technical effect of the feature. This represents a deviation of ±20%, preferably ±10%, and more preferably ±5% from the indicated number.

[0063] Technical terms are used in their general sense. Where a specific term has a specific meaning, its definition is provided below in the context in which it is used.

[0064] Certain aspects of this disclosure are based, at least in part, on the identification of the following anti-BK virus antibody molecules or their conjugated fragments. - Binds to and neutralizes BK virus serotype I; and / or - Binds to and neutralizes BK virus serotype II; and / or - Binds to and neutralizes BK virus serotype III; and / or - Binds to and neutralizes BK virus serotype IV; and / or - Does not bind to JC virus VP1.

[0065] In preferred embodiments, the anti-BK virus antibody molecule or its conjugated fragment neutralizes BK virus serotypes I, II, III, and IV.

[0066] In another preferred embodiment, the anti-BK virus antibody molecule or its binding fragment does not bind to JC virus VP1. The improved specificity is achieved by not binding to closely related JC virus VP1. Increased specificity is thought to improve safety because it reduces the risk of off-target activity.

[0067] As described above, this disclosure concerns anti-BK virus antibody molecules or their binding fragments. Full-length antibodies have a constant domain and a variable domain. The constant domain does not need to be present in the antigen-binding fragment of the antibody.

[0068] Therefore, the binding fragment can include a portion of an intact full-length antibody, such as the antigen-binding region or variable region of the complete antibody. Examples of antibody fragments include Fab, F(ab')2, Id, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); multispecific antibody fragments such as bispecific, tripspecific, and multispecific antibodies (e.g., diabodies, triabodies, tetrabodies); minibodies; chelated recombinant antibodies; tribodies or vibodies; intrabodies; nanobodies; small module immunopharmaceuticals (SMIPs), binding domain immunoglobulin fusion proteins; camelized antibodies; VHH-containing antibodies; and other polypeptides formed from antibody fragments. Those skilled in the art recognize that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0069] Disclosed herein are polypeptides having a specified sequence, or a sequence that is substantially identical or similar thereto, for example, a polypeptide having at least about 85%, 90%, 95%, or 99% sequence identity with respect to the specified sequence.

[0070] Determining the percentage identity between two sequences is preferably achieved using the mathematical algorithm described in Karlin and Altschul (1993) Proc.Natl.Acad.Sci USA 90:5873-5877. Such an algorithm is incorporated into the Altschul et al. (1990) J. Mol.Biol.215:403-410 program, which is available from NCBI (https: / / blast.ncbi.nlm.nih.gov / ). Determining the percentage identity can be done using the standard parameters of the BLASTp program. Typical parameters include setting the "Max Target Sequences" box to 100, checking the "Short queries" box, setting the "Expect threshold" box to 10, the "Word Size" box to "3", and the "Max matches in a query range" box to "0". For the scoring parameters, you may set the "Matrix" box to "BLOSUM62", the "Gap Costs" box to "Existence:11 Extension:1", and the "Compositional adjustments" box to "Conditional compositional score matrix adjustment". For the filter and masking parameters, the "Low complexity regions" box may be unchecked, the "Mask for lookup table only" box may be unchecked, and the "Mask lower case letters" box may be unchecked.

[0071] According to this disclosure, “conservative amino acid substitution” is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0072] As described above, the disclosure also relates, in some embodiments, to nucleic acids encoding antibody molecules or their binding fragments, vectors comprising such nucleic acids, and host cells comprising such nucleic acids or vectors.

[0073] The antibody molecule or its bound fragment may be encoded by a single nucleic acid (for example, a single nucleic acid containing nucleotide sequences encoding the light and heavy chain polypeptides of the antibody), or by two or more separate nucleic acids, each encoding a different portion of the antibody molecule or antibody fragment. The nucleic acid may be DNA, cDNA, RNA, etc.

[0074] The nucleic acids described herein can be inserted into vectors. A “vector” is any molecule or composition that has the ability to carry a nucleic acid sequence into a suitable cell where the encoding polypeptide is synthesized.

[0075] In some aspects, this disclosure further provides host cells (e.g., isolated or purified cells) containing the nucleic acids or vectors of the present invention. The host cells can be any type of cell that can be transformed with the nucleic acids or vectors of the present invention to produce polypeptides encoded thereby.

[0076] The anti-BK virus antibody molecule or its anti-BK virus conjugated fragment of the present invention can be incorporated into compositions, particularly pharmaceutical compositions. Such compositions may contain a therapeutically effective amount of the antibody or its conjugated fragment mixed with a pharmaceutically acceptable carrier, excipient, or stabilizer.

[0077] Furthermore, the anti-BK virus antibody molecules or their anti-BK virus conjugate fragments and pharmaceutical compositions described herein may be administered in methods for treating or preventing BK virus infection and / or BK virus-related diseases.

[0078] Preferred embodiments of aspects B1 to B9 of the present invention relate to the following: 1. Anti-BK virus antibody molecules or anti-BK virus conjugate fragments, including the following: (i) A heavy chain variable region (VH) comprising the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 34 or a sequence having one or two amino acid substitutions, the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 35 or a sequence having one or two amino acid substitutions, and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 36 or a sequence having one or two amino acid substitutions, and A light chain variable region (VL) including the light chain complementarity determination region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 37 or a sequence having one or two amino acid substitutions, the light chain complementarity determination region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 38 or a sequence having one or two amino acid substitutions, and the light chain complementarity determination region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 39 or a sequence having one or two amino acid substitutions, or (ii) A heavy chain variable region (VH) including the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 34 or a sequence having one or two amino acid substitutions, the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 44 or a sequence having one or two amino acid substitutions, and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 36 or a sequence having one or two amino acid substitutions, and A light chain variable region (VL) including the light chain complementarity determination region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 37 or a sequence having one or two amino acid substitutions, the light chain complementarity determination region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 38 or a sequence having one or two amino acid substitutions, and the light chain complementarity determination region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 39 or a sequence having one or two amino acid substitutions, or (iii) A heavy chain variable region (VH) including the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 34 or a sequence having one or two amino acid substitutions, the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 44 or a sequence having one or two amino acid substitutions, and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 36 or a sequence having one or two amino acid substitutions, and A light chain variable region (VL) comprising the light chain complementarity determination region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 37 or a sequence having one or two amino acid substitutions, the light chain complementarity determination region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 38 or a sequence having one or two amino acid substitutions, and the light chain complementarity determination region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 48 or a sequence having one or two amino acid substitutions.

[0079] 2. Antibody molecules or their conjugated fragments as described in item 1, including the following: (i) Heavy chain variable region (VH) including the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 34, the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 35, and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 36; and Light chain variable region (VL) including the amino acid sequence of light chain complementarity determination region 1 (VLCDR1) of SEQ ID NO: 37, the amino acid sequence of light chain complementarity determination region 2 (VLCDR2) of SEQ ID NO: 38, and the amino acid sequence of light chain complementarity determination region 3 (VLCDR3) of SEQ ID NO: 39; or (ii) Heavy chain variable region (VH) including the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 34, the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 44, and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 36; and The light chain variable region (VL) includes the amino acid sequence of light chain complementarity determination region 1 (VLCDR1) of SEQ ID NO: 37, the amino acid sequence of light chain complementarity determination region 2 (VLCDR2) of SEQ ID NO: 38, and the amino acid sequence of light chain complementarity determination region 3 (VLCDR3) of SEQ ID NO: 39, or (iii) Heavy chain variable region (VH) including the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 34, the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 44, and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 36; and The light chain variable region (VL) includes the amino acid sequence of light chain complementarity determination region 1 (VLCDR1) of SEQ ID NO: 37, the amino acid sequence of light chain complementarity determination region 2 (VLCDR2) of SEQ ID NO: 38, and the amino acid sequence of light chain complementarity determination region 3 (VLCDR3) of SEQ ID NO: 48.

[0080] 3. Antibody molecules or their conjugated fragments as described in item 1 or 2, including the following: (i) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 40, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 41, or an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 41, or (ii) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 45, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 41, or an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 41, or (iii) A heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 49, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence having at least approximately 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 50.

[0081] 4. Antibody molecules or their conjugated fragments as described in any one of items 1 to 3, including the following: (i) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 40, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 41. (ii) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 45, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 41. (iii) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 49, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 50.

[0082] 5. An antibody molecule or its conjugated fragment described in any one of items 1 to 4, comprising one or more of the following properties (e.g., 2, 3, 4, 5, 6, 7, 8, or 9). (i) When an antibody molecule or its binding fragment binds to BK virus serotype I VP1, for example, and is tested as a bivalent molecule using ELISA as described in Example 2, for example, the EC50 is less than approximately 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.19 nM, 0.18 nM, 0.17 nM, 0.16 nM, 0.15 nM, 0.14 nM, 0.13 nM, 0.12 nM, 0.11 nM, 0.10 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, or 0.02 nM; (ii) When an antibody molecule or its binding fragment binds to BK virus serotype II VP1, for example, and is tested as a bivalent molecule using ELISA as described in Example 2, for example, the EC50 is less than approximately 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.19 nM, 0.18 nM, 0.17 nM, 0.16 nM, 0.15 nM, 0.14 nM, 0.13 nM, 0.12 nM, 0.11 nM, 0.10 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, or 0.02 nM; (iii) When an antibody molecule or its conjugate fragment binds to BK virus serotype III VP1 and is tested as a bivalent molecule using ELISA, for example as described in Example 2, the EC50 is less than approximately 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.19 nM, 0.18 nM, 0.17 nM, 0.16 nM, 0.15 nM, 0.14 nM, 0.13 nM, 0.12 nM, 0.11 nM, 0.10 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, or 0.02 nM; (iv) When an antibody molecule or its binding fragment binds to BK virus serotype IV VP1, for example, and is tested as a bivalent molecule using ELISA as described in Example 2, for example, the EC50 is less than approximately 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.19 nM, 0.18 nM, 0.17 nM, 0.16 nM, 0.15 nM, 0.14 nM, 0.13 nM, 0.12 nM, 0.11 nM, 0.10 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, or 0.02 nM; (v) Does not bind to JC virus VP1. (vi) Neutralize BK virus serotype I; (vii) Neutralize BK virus serotype II; (viii) Neutralize BK virus serotype III; (ix) Neutralizes BK virus serotype IV.

[0083] 6. An antibody molecule or its binding fragment that competes for binding to BK virus serotype I VP1, BK virus serotype II VP1, BK virus serotype III VP1, and / or BK virus serotype IV VP1 with an antibody molecule or its binding fragment described in any one of items 1 to 5.

[0084] 7. A pharmaceutical composition comprising an antibody molecule or a bound fragment thereof as described in any one of items 1 to 6, and a pharmaceutically acceptable carrier, excipient, or stabilizer.

[0085] 8. An antibody molecule or a conjugated fragment thereof as described in any one of items 1 to 6, or a pharmaceutical composition as described in item 7, for use in the treatment or prevention of BK virus infection and / or BK virus-related disease.

[0086] 9. A nucleic acid encoding the antibody heavy chain and / or light chain variable region of an antibody molecule or its binding fragment described in any one of items 1 to 6.

[0087] 10. An expression vector containing the nucleic acid described in item 9.

[0088] 11. Host cells containing nucleic acids as described in item 9 or expression vectors as described in item 10.

[0089] 12. A method for producing an antibody molecule, comprising culturing the host cells described in item 11 under conditions suitable for gene expression.

[0090] 13. A diagnostic composition comprising an antibody molecule or a bound fragment thereof as described in any one of items 1 to 6. [Examples]

[0091] Introduction Comment The amino acid sequences of the VP1 protein constructs used in the following examples are summarized in Table 1 below. Table 1 - Amino acid sequences of VP1 protein constructs [Table 1] JPEG0007840274000002.jpg240170JPEG0007840274000003.jpg129170

[0092] comparative antibody P8D11 (WO 2017 / 046676) was cloned as described for the other antibodies. The VH and VL domains of P8D11 described in WO 2017 / 046676 were fused to the same constant domain of IgG as described below. The amino acid sequence of the comparative antibody P8D11 is summarized in Table 2 below.

[0093] The negative control 24C3 is an antibody against tetanus toxoid from a healthy human. The variable fragment was fused to the same constant domain of IgG as described below.

[0094] Table 2 Amino acid sequence of comparative antibody P8D11 (WO2017 / 046676) [Table 2]

[0095] Antibody search Example 1 Using peripheral blood memory B cells from healthy human donors or kidney transplant patients, the variable regions of immunoglobulin light and heavy chains were cloned into expression cassettes combining the constant-weight region of human immunoglobulin and the transmembrane domain derived from human CD8. This prepared an antibody repertoire expression library, enabling the presentation of antibodies in mammalian cells. Screening of the antibody library was performed by introducing the library into HEK293T cells, followed by fluorescent labeling with the VP1 pentamer of the BK virus and antigen-specific sorting.

[0096] The BK-virus-specific antibody-expressing HEK cell clones obtained in this manner were further amplified, and the scale of antibody production was expanded for downstream analysis of antibody properties, such as binding assays in ELISA and BK-virus neutralization. The neutralizing ability of the antibodies was tested using BK pseudovirus (BK-PsV) and wild-type BKV, both loaded with a luciferase expression reporter plasmid. Next, BK-virus-specific antibodies with high affinity and virus neutralizing ability were subcloned into soluble antibody expression vectors and expressed after transient transfection of HEK293F cells or CHO cells. Subsequently, the antibodies were purified with protein G or protein A, and their properties were confirmed using various assays. The amino acid sequence for the identified antibody 319C07 is summarized in Table 3 below. Furthermore, variants were also created, and their corresponding amino acid sequences are summarized in Table 3 below. The amino acid sequence for the identified antibody 336F07 is summarized in Table 4 below. Furthermore, variants were also created, and their corresponding amino acid sequences are summarized in Table 4 below.

[0097] Table 3 - Amino acid sequences of anti-BK virus antibodies 319C07 and 319C07-var1. [Table 3] JPEG0007840274000006.jpg151170

[0098] Table 4 - Amino acid sequences of anti-BK virus antibodies 336F07, 336F07-var1, and 336F07-var4. [Table 4] JPEG0007840274000008.jpg243170JPEG0007840274000009.jpg198170

[0099] Assay Example 2 - Binding to VP1-pentamer in ELISA Materials and methods The binding of anti-BK virus antibodies to BKV-VP1 pentamers was analyzed by ELISA. Briefly, 1 μg / ml of BKV-VP1 pentamer was applied to 30 μl / well of Costar® 96-well Assay Plates, half-area high-binding plates (Corning Inc. #3690) overnight at 4°C. The plates were then blocked with 5% skim milk powder (Rapilait, Migros #7610200017598) diluted in PBS. The antibodies were serially diluted in PBS containing 0.5% skim milk powder and added to antigen-coated plates for 1.5 hours. Next, the plate was washed with PBS 0.05% Tween20 (AppliChem, A4974) and incubated for 45 minutes with enzyme-labeled secondary antibody (HRP-conjugated goat anti-human IgG, Jackson-Immuno #109-035-098) diluted 1 / 10,000 in PBS containing 0.5% skim milk powder. The plate was washed three times with PBS containing 0.05% Tween 20. The reaction was developed using 30 μl / well TMB liquid substrate (Sigma-Aldrich, #T0440) and stopped with 15 μl / well H2SO4. Absorbance was detected at 450 nm (Tecan, CM INFINITE MONO 200). The data were fitted to obtain the apparent EC50 ELISA value (EC50 E The parameter was determined using a three-parameter analysis with GraphPad Prism (GraphPad Software).

[0100] result Antibody 319C07 showed strong selective binding to all BKV-VP1 serotypes (Figure 1, EC50). E The values ​​are as follows: Antibody 319C07 shows higher affinity for serotypes I, II, and IV compared to the comparative antibody P8D11, and shows comparable affinity for BKV-VP1-serotype III. JPEG0007840274000010.jpg27170

[0101] Furthermore, antibody 336F07 showed strong selective binding to all BKV-VP1 serotypes (Figure 1, EC50). E The values ​​are as follows. Compared to the comparative antibody P8D11, antibody 336F07 showed high affinity in all four serotypes. JPEG0007840274000011.jpg27170

[0102] Example 3 - Neutralization of BK pseudovirus by anti-BK virus antibody Materials and methods BK pseudoviruses (BK-PsV) carrying the NanoLuc reporter gene were constructed as described (Pastrana et al, J Virol, 2013 Sep; 87(18):10105-13). Five genotypes were created: BKV-Ia (BK-D; JF894228), BKV-Ib2 (PittVR2; DQ989796), BKV-II (Q85238; CAA79596), BKV-III (Q0PDA6; BAF03017), and BKV-IVc2 (A-66H; AB369093). VP1 was purified on agarose gel beads as described (Buck et al, J Virol, 2008 Jun;82(11):5190-7 and Buck et al, Curr Protoc Cell Biol, 2007 Dec; Chapter 26; 26.21). Infectious viral titers were measured in 293TT cells. Different dilutions of BK-PsV were tested in 96-well plates with and without the inhibitor. High luminescence signals were obtained in non-inhibited cells at the dilutions selected for further assays.

[0103] 20,000 cells (293TT) were seeded in a 96-well plate using DMEM containing 9% FBS, HEPES, and penicillin / streptavidin. After incubation for 5 hours, various concentrations of antibody and fixed amounts of BK-pseudovirus were added to each well, and the plate was incubated at 37°C and 5% CO2. After 3 days of culture, the luciferase activity in the supernatant was measured using NanoGlo.TM The measurement was performed using assay reagent (Promega, #N1130).

[0104] Transfer 40 µl of cell culture medium or virus-cell-antibody co-culture medium-supernatant to a white-bottomed 96-well plate and use NanoGlo TM Luciferase activity was measured using a luminescence reader (BioTek Synergy) after adding 40 µl of substrate and incubating for 2 minutes. The data were plotted on a signal-versus-antibody concentration curve. The IC50 value was determined using GraphPad Prism with nonlinear regression (curve fitting) and the formula "log(inhibitor) vs normalized response (variable slope)" (GraphPad Software).

[0105] result 319C07 and its variant 319C07-var1 showed strong inhibitory activity against all serotypes of BK-pseudovirus. 319C07 significantly outperformed P8D11 (Figure 2; IC50 values ​​are as follows). JPEG0007840274000012.jpg27170

[0106] Furthermore, antibody 336F07 and its variants, 336F07-var1 and 336F07-var4, strongly inhibited all serotypes of BK pseudovirus. 336F07 significantly outperformed P8D11 (Figure 2; IC50 values ​​are as follows). JPEG0007840274000013.jpg27170

[0107] Example 4 - Long-term virus neutralization using anti-BK virus antibody Materials and methods To address long-term in vitro viral neutralization using anti-BK virus antibodies, human primary renal proximal tubular epithelial cells (HPRTECs) and wild-type BK virus strain I (ATCC® VR-837) were used. TMCo-culturing with anti-BK virus antibodies was repeated. At the start of the experiment, anti-BK virus antibodies were added to the cell-virus co-culture for one week at three different concentrations corresponding to their viral suppression levels (EC95, EC50, and EC5). After this, freeze-thaw cycles were repeated three times, and the virus was collected from the cells and the remaining supernatant. Next, 10 µl of this viral extract was added to newly seeded HRPTEC cells in the presence of various antibody concentrations. Two weeks after inoculation, the viral load was quantified again, and a viral extract for use in the next infection cycle was prepared. This procedure was repeated three times, resulting in a total of eight weeks of co-culturing.

[0108] The assay was performed in triple mode on a 96-well flat-bottom cell culture plate. Quantification of viral load in the culture supernatant was performed by taking 15 μl of medium, thermally inactivating it at 95°C for 10 minutes, and then applying primers 5'-GGATGGGCAGCCTATGTATG-3' (SEQ ID NO: 53) and 5'-TCATATCTGGGTCCCCTGGA-3' (SEQ ID NO: 54) along with TaqMan. TM qPCR was performed using the probe FAM-AGGGTGTTTGATGGCACAGA-TAMRA (SEQ ID NO: 55). PCR was performed using PerfeCTa qPCR ToughMix (Quantabio, #95140) under the following conditions: 8 minutes at 95°C, followed by 40 cycles of 10 seconds at 95°C and 60 seconds at 60°C.

[0109] result Antibody 319C07 exhibits more complete viral neutralization compared to P8D11. This is most evident when looking at the final infection-neutralization cycle after 8 weeks, where the virus was no longer detectable at concentrations corresponding to EC95 and EC50, whereas with P8D11, measurable viral loads were detected at both EC95 and EC50 (Figure 3, in which P8D11 is labeled "BM2").

[0110] Furthermore, antibody 336F07 shows more complete viral neutralization compared to P8D11. This is most evident when looking at the final infection-neutralization cycle after 8 weeks (Figure 3, in which P8D11 is labeled "BM2").

[0111] Example 5 - Binding of anti-BK virus antibody to a conformational epitope Materials and methods To investigate whether anti-BK virus antibodies bind to conformational epitopes, dot blots were performed using native and denatured VP1. BKV-VP1 pentamers from serotypes I, II, III, and IV were spotted onto nitrocellulose membranes either in their original form or after chemical denaturation by heating in Tris buffer containing SDS and β-mercaptoethanol at 85°C for 5 minutes. Both membranes were incubated with anti-BK virus antibodies, and binding was confirmed using a human Fc-targeting HRP-conjugated secondary antibody. Detection was performed using a colorimetric substrate (Sigma Fast DAB).

[0112] result Antibodies 319C07 and 336F07 exhibit selective binding to conformational epitopes (Figure 4). Since denatured antigens are not stained, binding to linear epitopes can be excluded. Commercially available control antibodies known to recognize linear epitopes (Ab53977, Abcam, rabbit-IgG) detected both denatured and undenatured VP1.

[0113] Example 6 - Binding of BKV-VP1 pentamer variants to JCV-VP1-pentamer in ELISA Materials and methods Mutations were introduced at various positions based on the VP1-serotype Ib wild-type sequence. Similarly, VP1 from the JC virus was also used. Binding of anti-BK virus antibodies to wild-type and variant VP1 pentamers was analyzed by ELISA. Briefly, 30 μl / well of 1 μg / ml BKV-VP1 pentamer variant was coated overnight at 4°C on a Costar® Assay Plate 96-well, half-area high-binding plate (Corning Inc. #3690). After 16 hours, nonspecific binding was blocked using 5% skim milk powder (Rapilait, Migros #7610200017598) diluted in PBS. Antibodies were serially diluted from 67 nM to 0.02 nM in PBS containing 0.5% skim milk and incubated on antigen-coated plates for 1.5 hours. Subsequently, the plate was washed three times with PBS 0.05% Tween20 (AppliChem, A4974) and incubated for 45 minutes with a secondary antibody (HRP-conjugated goat anti-human IgG, Jackson-Immuno #109-035-098) diluted to 1 / 10,000 in PBS containing 0.5% skim milk powder. The HRP activity of the conjugated secondary antibody was determined using 30 μl / well TMB liquid substrate (Sigma-Aldrich, #T0440). After 2.5 minutes, the reaction was stopped by adding 15 μl / well of 1 M H2SO4. Absorbance was detected at 450 nm (Tecan, CM INFINITE MONO 200). The data were plotted, and the EC50 value was determined by 3-parameter analysis using GraphPad Prism (GraphPad Software), and the result was named EC50ELISA (EC50E).

[0114] result The binding of antibody 319C07 to mutant VP1 of BKV compared to wild-type VP1, and to VP1 of the closely related JC polyomavirus, was examined in comparison with antibody P8D11. Antibody 319C07 did not bind to JC virus VP1 at all, while antibody P8D11 showed weak but significant binding. Mutation of the amino acids at positions N62, D175, or S275 to alanine slightly reduced the binding affinity of 319C07. Changing amino acid K172 to alanine did not reduce binding affinity. On the other hand, P8D11 showed reduced binding to the VP1-I-K172A variant, but was unaffected by binding to the N62A, D175A, or S275A variants. Compared to antibody P8D11, 319C07 showed a clearly different pattern, suggesting that it interacts with different amino acids of VP1 compared to P8D11 (Figure 5).

[0115] The binding of antibody 336F07 to mutant and wild-type VP1 of BKV, as well as to VP1 of the closely related JC polyomavirus, was also tested compared to antibody P8D11. Antibody 336F07 did not bind to JC virus VP1 at all, while antibody P8D11 showed weak but significant binding. A mutation in the amino acid at position N62 or E73 slightly reduced the binding affinity of 336F07. Changing amino acid K172 to alanine did not reduce binding affinity. On the other hand, P8D11 showed reduced binding to the VP1-I-K172A variant, but was unaffected by binding to the N62A and E73Q variants. Compared to antibody P8D11, 336F07 showed a distinctly different pattern, suggesting that it interacts with different VP1 amino acids than P8D11 (Figure 5).

[0116] Example 7 - Suppression of viral spread from infected HRPTEC cells Materials and methods Primary human renal proximal tubular epithelial cells (HRPTECs) are used in BKV (33-1 strain, ATCC® 45024) TM) was infected. Five days later, the cells were gently scratched with a cell scraper, extensively washed with PBS, and all the virus released from the cells was removed. Next, the cells were added to wells containing HRPTECs that adhered and grew and BKV-neutralizing antibodies at various dilutions. Eight days after cell seeding, 10 μl of the supernatant was taken from each well, and the virus amount was measured by quantitative PCR using the primers 5'-GGATGGGCAGCCTATGTATG-3' (SEQ ID NO: 53) and 5'-TCATATCTGGGTCCCCTGGA-3' (SEQ ID NO: 54) and the probe FAM-5'-AGGGTGTTTGATGGCACAGA-3'-TAMRA (SEQ ID NO: 55) as described (Martelli et al., Viruses, 2018 Aug 30;10(9):466).

[0117] Results Antibody 319C07 showed approximately 100-fold superiority over P8D11 in suppressing BKV spread in HRPTECs, and antibody 336F07 also showed significant superiority over P8D11 in suppressing BKV spread in HRPTECs (Figure 6, in this figure, P8D11 is denoted as "BM2").

[0118] Example 8 - Antibody-Dependent Cellular Cytotoxicity (ADCC) Materials and Methods HPRTECs seeded in flat-bottom 96-well plates that were infected or not infected with wild-type BK virus strain I (ATCC® VR-837 TM ) were used as target cells. As effector cells, artificial Jurkat cells, which are components of Promega's ADCC Reporter Bioassay G7015, were used. In this assay, as a surrogate for ADCC activity, the Fc-γRIIIa signal by effector cells was quantified by luminescence readout.

[0119] Anti-BKV antibodies or control antibodies were serially diluted and added to HPRTEC cultures together with effector cells, incubated for 6 hours, and then bioluminescence was expressed and measured according to the description of the assay kit manufacturer.

[0120] result 319C7 and 336F07 showed comparable ADCC activity against infected HPRTEC, and furthermore, comparable ADCC activity to that of P8D11. ADCC was not observed in uninfected HPRTEC for any of 319C7, 336F07, or P8D11 (Figure 7).

[0121] Example 9 - Complement-dependent toxicity Materials and methods Wild-type BK virus strain I (ATCC(registered trademark) VR-837) TM HPRTEC cells, either infected with or uninfected with BKV, seeded in flat-bottom 96-well plates, were used as target cells. Cells were incubated overnight with serial dilutions of anti-BKV antibody starting at 100 μg / ml and 20% human serum. Cell viability was quantified by determining the ratio of viable cells using an FSC / SSC criterium with a cytometer.

[0122] result 319C7 and 336F07 did not show CDC activity against infecting HPRTEC at a concentration of 100 μg / ml, and the same observation was made with antibody P8D11, which is targeted at an unrelated HRPTEC target, and the control antibody rituximab (Figure 8).

Claims

1. The following are included: anti-BK virus antibody molecules or anti-BK virus conjugate fragments: A heavy chain variable region (VH) comprising the heavy chain complementarity determination region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 21, the heavy chain complementarity determination region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 22, and the heavy chain complementarity determination region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 23; and The light chain variable region (VL) includes the amino acid sequence of light chain complementarity determination region 1 (VLCDR1) of SEQ ID NO: 24, the amino acid sequence of light chain complementarity determination region 2 (VLCDR2) of SEQ ID NO: 25, and the amino acid sequence of light chain complementarity determination region 3 (VLCDR3) of SEQ ID NO:

26.

2. The antibody molecule or its bound fragment according to claim 1, comprising the following: (i) A heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence having at least 90%, 95%, or 99% sequence identity with SEQ ID NO: 27, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence having at least 90%, 95%, or 99% sequence identity with SEQ ID NO: 28, or (ii) A heavy chain variable region (VH) consisting of the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence having at least 90%, 95%, or 99% sequence identity with SEQ ID NO: 27, and a light chain variable region (VL) consisting of the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence having at least 90%, 95%, or 99% sequence identity with SEQ ID NO:

31.

3. The antibody molecule or its conjugated fragment according to claim 1 or 2, comprising the following: (i) a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 27, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 28; or (ii) A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 27, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO:

31.

4. A pharmaceutical composition comprising an antibody molecule or a bound fragment thereof according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier, excipient, or stabilizer.

5. An antibody molecule or a conjugated fragment thereof according to any one of claims 1 to 3, or a pharmaceutical composition according to claim 4, for use in the treatment or prevention of BK virus infection and / or BK virus-related disease.

6. A nucleic acid encoding an antibody heavy chain variable region and an antibody light chain variable region of an antibody molecule or its binding fragment according to any one of claims 1 to 3.

7. An expression vector comprising the nucleic acid described in claim 6.

8. A host cell comprising the nucleic acid described in claim 6 or the expression vector described in claim 7.

9. A method for producing an antibody molecule, comprising culturing the host cell described in claim 8 under conditions suitable for gene expression.

10. A diagnostic composition comprising an antibody molecule or a bound fragment thereof as described in any one of claims 1 to 3.

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