Polyomavirus neutralizing antibody

Abstract

Therapies are provided that neutralize polyomavirus and can be used in immunocompromised hosts. The present invention relates to anti-VP1 antibodies, antibody fragments, and their use for the prevention and treatment of polyomavirus infection and related diseases.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present disclosure relates to anti-VP1 antibodies, antibody fragments, and methods for reducing the likelihood of polyomavirus infection. Or to their use for treatment.

[0002] 2. Background of the Invention Among the human polyomaviruses, BK virus (BKV) and JC virus (JCV These two polyomaviruses are thought to be the first to be identified in immunosuppressed patients. The two strains were isolated from the same patient and published in the same issue of the Lancet in 1971 (Gardner et al., 1971 Lancet 1:1253-1527, and Padgett et al., Lancet 1971 1:1257-1260). Maviruses are icosahedral, non-enveloped, double-stranded DNA viruses. They have a diameter of They are 40-45 nm in size and contain 88% protein and 12% DNA.

[0003] The BKV genome is a circular double-stranded DNA approximately 5 Kb in length, and is divided into three major sections: the early core and the It contains a coding region, a late coding region, and a non-coding regulatory region. The early coding region is Three regulatory proteins (large tumor antigen [TAg], small tumor antigen [tAg], and It encodes a truncated tumor antigen (truncTAg) and is expressed in newly infected cells. It is the first viral protein expressed in the human genome and facilitates viral DNA replication, favoring Responsible for establishing the cellular environment. The late coding region is composed of three genes that make up the viral capsid. The structural proteins (VP1, VP2, and VP3) and agnoproteins are encoded by However, its role during viral replication is less clear. and containing early and late promoters that induce the expression of viral gene products .

[0004] BKV has been detected in many different cell types, including epithelial cells of the kidney, bladder, and ureter (typical sites of persistence), tonsillar tissue, as well as lymphocytes (suggested sites of primary infection and seeding) (Chatterjee et al., J. Med. Virol. 2000; 60:353-362, Goudsmit et al., J. Med. Virol. 1982; 10:91-99, Heritage et al., J. Med. Virol. 1981; 8:143- 150, Shinohara et al., J. Med. Virol. 1993; 41(4):301-305). The major cellular surface receptors for BKV are gangliosides GT1b, GD1b, and GD3, all of which have terminal α2,8-linked sialic acids and are quite ubiquitous, allowing infection of a variety of cell types (see also Neu et al., PLos Patholog. 2013; 9(10):e1003714 and e1003688, O’Hara et al., Virus Res. 2014; 189:208-285). The non-enveloped icosahedral virion of BKV is composed of three different viral proteins: 360 copies of the major viral capsid protein VP1 arranged in 72 pentamers and 72 copies of the minor viral capsid proteins VP2 and VP3 combined with one molecule of VP2 or VP3 bound to each VP1 pentamer. Only VP1 is exposed on the virion surface during entry, and each pentamer has five low-affinity binding sites for ganglioside receptors. Cellular ​​​​Binding of the VP1 pentamer to ganglioside receptors on the surface initiates internalization via the caveolae-mediated endocytic pathway, followed by transport of the virus to the endoplasmic reticulum and ultimately to the nucleus (Tsai and Qian, J. Virol 2010;84(19):9840-9852).

[0005] Infection with human polyomavirus BK (BKV) is essentially ubiquitous, and it is estimated that 80 - 90% of the general population is infected (Knowles W.A., Adv. Exp. Med. Biol. 2006;577:19-45). Primary infection most often occurs during childhood (i.e., before 10 years of age) and results in a mild, non-specific, self-limiting illness or no symptoms at all. Persistent infection is established in the epithelial cells of the renal tubules, ureters, and bladder and is effectively controlled by the immune system. Transient asymptomatic viral shedding into the urine of immunocompetent adults occurs sporadically and does not result in disease or sequelae. However, immunodysfunction, particularly immunosuppression after kidney transplantation or hematopoietic stem cell transplantation, can lead to uncontrolled BKV replication and ultimately BKV-associated nephropathy (BKVAN), a painful bladder disease, or hemorrhagic cystitis (HC ). There is no effective antiviral therapy against BKV, and current standard treatment is reduction of immunosuppression, which increases the risk of acute rejection. Even with current more aggressive monitoring and prevention methods, up to 10% of kidney transplant recipients develop BKVAN, and 15 - 30% of these patients suffer graft loss due to BKVAN. Of these experienced reductions in immunosuppressive regimens at the time of BKV viremia detection, up to 30% experience an acute rejection episode as a result. ​

[0006] Although BKV was first described in 1971 (supra), until the 1990s, BK-related neph ropathy (BKVAN) was not reported in the literature as a cause of kidney transplant injury (Purigh alla et al., Am. J. Kidney Dis. 1995; 26:671-673 and Randhawa et al., Transplan tation 1999; 67:103-109). In the initial management of BKVAN, a positive BK test had serious conse quences, and more than 50% of patients had graft dysfunction and graft loss (Hirsch et al., New Engl. J. Med. 2002; 347:488-496). Reactivation of the BK virus can occur after transplant ation and is seen in approximately 30% - 50% of patients by 3 months post-transplant ( Bressollette-Bodin et al., Am J. Transplant. 2005; 5(8):1926-1933 and Brennan e t al., Am. J. Transplant. 2004;4(12):2132-2134). Reactivation of the BK virus is first detected by virus and viral DNA in urine, then in plasma, and finally in the kidney ( Brennan et al., Am. J. Transplant. 2005;5(3):582-594 and Hirsch et al., N Eng. J. Med. 2002;347(7):488-496). Approximately 80% of kidney transplant patients have BK virus in urine (BK viremia), and 5 - 10% of these patients progress to BKVAN (Bine t et al., Transplantation 1999; 67(6):918-922 and Bressollette-Bodin et al., Am J. Transplant. 2005; 5(8):1926-1933). BKV is associated with basement membrane exposure and causes necrosis and lytic destruction of tubular epithelial cells, accumulating luminal fluid in the interstitium, leading to interstitial fibrosis and tubular atrophy (Nickeleit et al., J. Am. Soc. Neprol. 1999; 10(5):1080-108 9), all of which can affect the status of the graft. Patients may present with decreased renal function, tubulo-interstitial nephritis and ureteral strictures (Garner et al., Lancet 1971; 1(7712):1253-1257 and Hirsch Am. J. Transplant 2002; 2(1)25-30).

[0007] BKV can also cause pneumonia, retinitis and meningoencephalitis in immunocompromised hosts (Repl oeg et al., Clin. Infect. Dis. 2001;33(2):191-202). BK virus disease in hematopoietic stem cell transplantation (HSCT) recipients typically presents as hemorrhagic cystitis (HC) and may vary in severity. Viruria (not viremia) and painful hematuria are associated with the clinical findings of HC. Current standard treatment is mainly supportive, including forced fluid replacement / diuresis and pain management measures. In the most severe cases, blood transfusions, blood clot evacuation are required and can lead to death in some cases. HC of any cause (e.g., drugs, radiation, virus) is relatively common among HSCT recipients, but HC associated with BKV usually occurs in about 10-12% of patients within 6 months after transplantation. There are other viral etiologies of HC, and adenovirus is more common in pediatric HS recipients compared to adult HSCT recipients. is a more common cause of HC among kidney transplant recipients. BK virus is also associated with systemic ery thematosus, other immunocompromised states such as other solid organ transplants, and in patients with HIV / AIDS (Jiang et al., Virol. 2009; 384:266-273).

[0008] In this regard, the treatment of BK nephropathy associated with organ transplantation is the reduction of immunosuppression in an attempt to prevent graft dysfunction and graft loss (Wiseman et al., Am. J. Kidney Dis. 2009; 54(1): 131-142 and Hirsch et al., Transplantation 2005; 79(1): 1277- 1286). Reduction of immunosuppression can help prevent progression from viremia to widespread damage associated with clinical nephropathy, but there is no fixed clinical regimen for reduction as this also increases the risk of acute organ rejection (Brennan et al., Am. J. Transplant 20 05; 5(3):582-594). Physicians have reported the use of therapeutic agents such as cidofovir, leflu nomide or quinolone in combination with reduction of immunosuppressive agents, but this report shows that this approach is ineffective and increases the burden of managing additional side effects (Randhawa and Brennan Am. J. Transplant 2006; 6(9):2000-2005). As such, there is an unmet useful need in the field of therapies that can neutralize polyomaviruses such as BK and be used in immunocompromised hosts.

[0009]

[0009] JC virus is also a highly prevalent (80%) polyomavirus in the population However, JC virus is generally acquired later than BK virus (Padgett et al., J. Infect. Dis. 1973;127(4):467-470 and Sabath et al., J. Infect. Dis. 20 02; 186 Suppl. 2:5180-5186). After primary infection, JC virus establishes latency in lymphoid organs and the kidney, and when reactivated, it invades the central nervous system via infected B lymphocytes. Once in the CNS, JC virus causes progressive multifocal leukoencephalopathy (PML), a progressive demyelinating central nervous system disorder. PML is most often found as an opportunistic infection in patients with HIV / AIDS and has also been reported in immunosuppressed patients (Angstrom et al., Brain 1958; 81(1):93-111 and Garcia-Suarez et al., Am. J. Hematol. 2005; 80(4):271-281). Patients with PML present with local neurological abnormalities such as confusion, changes in mental status, gait ataxia, unilateral hemiparesis, quadriparesis, and visual changes (Richardson E.P. , N. Eng. J. Med. 1961; 265:815-823). The prognosis for patients with PML is poor, especially in patients with HIV / AIDS (Antinori et al., J. Neurovirol.

[0010] SUMMARY OF THE INVENTION The present disclosure provides neutralizing antibodies against human polyomavirus Antibodies that recognize BK virus and / or JC virus, as well as their corresponding VP1 pentamers and and fragments thereof.

[0011] An antibody, wherein the antibody or an antigen-binding fragment thereof specifically binds to VP1. .

[0012] An antibody, wherein the antibody or an antigen-binding fragment thereof specifically binds to VP1 of BK virus serotypes I to IV. In one embodiment, the antibody or an antigen-binding fragment thereof binds to VP1 of BKV serotype I with a binding affinity of 5.0 pM or less, binds to VP1 of BKV serotype II with a binding affinity of 29.0 pM or less, binds to VP1 of BKV serotype III with a binding affinity of 6.0 pM or less, and / or binds to VP1 of BKV serotype IV with a binding affinity of 185.0 pM or less. In another embodiment, the antibody or an antigen-binding fragment thereof further binds to JCV VP1 and specific JCV VP1 mutants with a binding affinity in the high nanomolar concentration range. An antibody, wherein the antibody or an antigen-binding fragment thereof specifically binds to the VP1 in Table 1. In one embodiment, the antibody or an antigen-binding fragment thereof binds to two or more VP1s in Table 1. In one embodiment, the antibody or an antigen-binding fragment thereof binds to BKV VP1 serotype I and BKV VP 1 serotype II. In another embodiment, the antibody or an antigen-binding fragment thereof binds to BKV VP1 serotype I and BKV VP1 serotype III. In another embodiment, the antibody or an antigen-binding fragment thereof binds to BKV VP1 serotype I and BKV VP1 serotype I

[0013] V. In another embodiment, the antibody or an antigen-binding fragment thereof binds to BKV VP1 serotype I and BKV VP1 serotype II. In another embodiment, the antibody or an antigen-binding fragment thereof binds to BKV VP1 serotype I and BKV VP1 serotype III. In another embodiment, the antibody or an antigen-binding fragment thereof binds to BKV VP1 serotype I and BKV VP1 serotype IV. In another embodiment, the antibody or an antigen-binding fragment thereof binds to BKV VP1 serotype I and BKV VP1 serotype II. In another embodiment, the antibody or an antigen-binding fragment thereof binds to BKV VP1 serotype I and BKV VP1 serotype III. In another embodiment, the antibody or an antigen-binding fragment thereof binds to BKV VP1 serotype I and BKV VP1 serotype IV. In another embodiment, the antibody or an antigen-binding fragment thereof binds to BKV VP1 serotype I and BKV VP1 serotype V. In another embodiment, the antibody or an antigen-binding fragment thereof binds to BKV VP1 serotype I and BKV VP1 serotype V. In another embodiment, the antibody or an antigen-binding fragment thereof binds to BKV VP1 serotype Binds to ClearType II and BKV VP1 serotype III. In another embodiment, the antibody or its antigen-binding fragment binds to BKV VP1 serotype II and BKV VP1 serotype IV. In another embodiment, the antibody or its antigen-binding fragment binds to BKV VP1 serotype I and JCV VP1. In a preferred embodiment, the antibody or its antigen-binding fragment binds to BKV VP1 serotypes I, II, III, and IV. Further, the antibody or its antigen-binding fragment binds to BKV VP1 serotypes I, II, III, and IV as well as J CV VP1.

[0014] An antibody, wherein the antibody or antigen-binding fragment specifically binds to one or more amino acid residues of the VP1 epitope (SEQ ID NO: 500 or SEQ ID NO: 5 01). In one embodiment, the antibody or antigen-binding fragment specifically binds to one or more of amino acids Y169, R170, and K172, for example, as determined by the alanine scanning mutagenesis described herein, for example, binds to Y169 and R170.

[0015] An antibody, wherein the antibody or antigen-binding fragment comprises the sequence GFTFXNYWMT (SEQ ID NO: 507) (wherein X may be any amino acid (Xaa)) . In another embodiment, X may be N (Asn), S (Ser), K (Lys), or Q (Gln).

[0016] An antibody, wherein the antibody or its antigen-binding fragment (i) (a) the heavy chain variable region comprising HCDR1 (CDR - Complementary Determining Region) of SEQ ID NO: 6, (b) HCDR2 of SEQ ID NO: 7 , (c) HCDR3 of SEQ ID NO: 8, and (d) the light chain variable region comprising SEQ ID NO: 1 LCDR1 of SEQ ID NO: 6, (e) LCDR2 of SEQ ID NO: 17, and (f) LCD of SEQ ID NO: 18 A light chain variable region comprising R3; (ii) (a) HCDR1 of SEQ ID NO: 26, (b) HCDR2 of SEQ ID NO: 27, (c) A heavy chain variable region comprising HCDR3 of SEQ ID NO: 28, and (d) LCDR1 of SEQ ID NO: 36, (e) LCDR2 of SEQ ID NO: 37, and (f) a light chain variable region comprising LCDR3 of SEQ ID NO: 38; (iii) (a) HCDR1 of SEQ ID NO: 46, (b) HCDR2 of SEQ ID NO: 47, (c ) A heavy chain variable region comprising HCDR3 of SEQ ID NO: 48, and (d) LCDR1 of SEQ ID NO: 56, (e) LCDR2 of SEQ ID NO: 57, and (f) a light chain variable region comprising LCDR3 of SEQ ID NO: 58; (iv) (a) HCDR1 of SEQ ID NO: 66, (b) HCDR2 of SEQ ID NO: 67, (c) A heavy chain variable region comprising HCDR3 of SEQ ID NO: 68, and (d) LCDR1 of SEQ ID NO: 76, (e) LCDR2 of SEQ ID NO: 77, and (f) a light chain variable region comprising LCDR3 of SEQ ID NO: 78; (v) (a) HCDR1 of SEQ ID NO: 86, (b) HCDR2 of SEQ ID NO: 87, (c) a heavy chain variable region comprising HCDR3 of SEQ ID NO: 88, and (d) LCDR1 of SEQ ID NO: 96, (e ) LCDR2 of SEQ ID NO: 97, and (f) a light chain variable region comprising LCDR3 of SEQ ID NO: 98; (vi) (a) HCDR1 of SEQ ID NO: 106, (b) HCDR2 of SEQ ID NO: 107, (c) A heavy chain variable region comprising HCDR3 of SEQ ID NO: 108, and (d) LCD R1 of SEQ ID NO: 116, (e) LCDR2 of SEQ ID NO: 117, and (f) a light chain variable region comprising LCDR3 of SEQ ID NO: 118; (vii) (a) HCDR1 of SEQ ID NO: 126, (b) HCDR2 of SEQ ID NO: 127, (c) A heavy chain variable region containing the HCDR3 of SEQ ID NO: 128, and (d) the LC of SEQ ID NO: 136 DR1, (e) the LCDR2 of SEQ ID NO: 137, and (f) the LCDR3 of SEQ ID NO: 138 A light chain variable region comprising; (viii) (a) The HCDR1 of SEQ ID NO: 146, (b) the HCDR2 of SEQ ID NO: 147 , (c) a heavy chain variable region containing the HCDR3 of SEQ ID NO: 148, and (d) the L of SEQ ID NO: 156 CDR1, (e) the LCDR2 of SEQ ID NO: 157, and (f) the LCDR 3 of SEQ ID NO: 158; A light chain variable region comprising; (ix) (a) The HCDR1 of SEQ ID NO: 166, (b) the HCDR2 of SEQ ID NO: 167, ( c) a heavy chain variable region containing the HCDR3 of SEQ ID NO: 168, and (d) the LCD of SEQ ID NO: 176 R1, (e) the LCDR2 of SEQ ID NO: 177, and (f) the LCDR3 of SEQ ID NO: 178 A light chain variable region comprising; (x) (a) The HCDR1 of SEQ ID NO: 186, (b) the HCDR2 of SEQ ID NO: 187, (c ) a heavy chain variable region containing the HCDR3 of SEQ ID NO: 188, and (d) the LCDR of SEQ ID NO: 196 1, (e) the LCDR2 of SEQ ID NO: 197, and (f) the LCDR3 of SEQ ID NO: 198 A light chain variable region comprising; (xi) (a) The HCDR1 of SEQ ID NO: 206, (b) the HCDR2 of SEQ ID NO: 207, ( c) a heavy chain variable region containing the HCDR3 of SEQ ID NO: 208, and (d) the LCD of SEQ ID NO: 216 R1, (e) the LCDR2 of SEQ ID NO: 217, and (f) the LCDR3 of SEQ ID NO: 218 A light chain variable region comprising; (xii) (a) The HCDR1 of SEQ ID NO: 226, (b) the HCDR2 of SEQ ID NO: 227, (c) a heavy chain variable region containing the HCDR3 of SEQ ID NO: 228, and (d) the LC of SEQ ID NO: 236 The variable light chain region comprising DR1, (e) LCDR2 of SEQ ID NO: 237, and (f) LCDR3 of SEQ ID NO: 238 ; (xiii) The variable heavy chain region comprising (a) HCDR1 of SEQ ID NO: 246, (b) HCDR2 of SEQ ID NO: 247 , (c) HCDR3 of SEQ ID NO: 248, and the variable light chain region comprising (d) LCDR1 of SEQ ID NO: 256, (e) LCDR2 of SEQ ID NO: 257, and (f) LCDR 3 of SEQ ID NO: 258; ; (xiv) The variable heavy chain region comprising (a) HCDR1 of SEQ ID NO: 266, (b) HCDR2 of SEQ ID NO: 267 , (c) HCDR3 of SEQ ID NO: 268, and the variable light chain region comprising (d) LCDR1 of SEQ ID NO: 276, (e) LCDR2 of SEQ ID NO: 277, and (f) LCDR3 of SEQ ID NO: 278 ; ; (xv) The variable heavy chain region comprising (a) HCDR1 of SEQ ID NO: 286, (b) HCDR2 of SEQ ID NO: 287, (c) HCDR3 of SEQ ID NO: 288, and the variable light chain region comprising (d) LCDR1 of SEQ ID NO: 296, (e) LCDR2 of SEQ ID NO: 297, and (f) LCDR3 of SEQ ID NO: 298 ; ; (xvi) The variable heavy chain region comprising (a) HCDR1 of SEQ ID NO: 306, (b) HCDR2 of SEQ ID NO: 307 , (c) HCDR3 of SEQ ID NO: 308, and the variable light chain region comprising (d) LCDR1 of SEQ ID NO: 314, (e) LCDR2 of SEQ ID NO: 315, and (f) LCDR3 of SEQ ID NO: 316 ; ; (xvii) The variable heavy chain region comprising (a) HCDR1 of SEQ ID NO: 322, (b) HCDR2 of SEQ ID NO: 323 , (c) HCDR3 of SEQ ID NO: 324, and the variable light chain region comprising (d) LCDR1 of SEQ ID NO: 332, (e) LCDR2 of SEQ ID NO: 333, and (f) LCDR 3 of SEQ ID NO: 334; ; (xviii) (a) HCDR1 of SEQ ID NO: 342, (b) HCDR 2 of SEQ ID NO: 343, (c) heavy chain variable region comprising HCDR3 of SEQ ID NO: 344, and (d) LCDR1 of SEQ ID NO: 349, (e) LCDR2 of SEQ ID NO: 350, and (f) LCD R3 of SEQ ID NO: 351; (xix) (a) HCDR1 of SEQ ID NO: 356, (b) HCDR2 of SEQ ID NO: 357, (c) heavy chain variable region comprising HCDR3 of SEQ ID NO: 358, and (d) LC of SEQ ID NO: 363 DR1, (e) LCDR2 of SEQ ID NO: 364, and (f) LCDR3 of SEQ ID NO: 365; (xx) (a) HCDR1 of SEQ ID NO: 370, (b) HCDR2 of SEQ ID NO: 371, ( c) heavy chain variable region comprising HCDR3 of SEQ ID NO: 372, and (d) LCD of SEQ ID NO: 377 R1, (e) LCDR2 of SEQ ID NO: 378, and (f) LCDR3 of SEQ ID NO: 379 ; (xxi) (a) HCDR1 of SEQ ID NO: 384, (b) HCDR2 of SEQ ID NO: 385, (c) heavy chain variable region comprising HCDR3 of SEQ ID NO: 386, and (d) LC of SEQ ID NO: 391 DR1, (e) LCDR2 of SEQ ID NO: 392, and (f) LCDR3 of SEQ ID NO: 393; (xxii) (a) HCDR1 of SEQ ID NO: 398, (b) HCDR2 of SEQ ID NO: 399 , (c) heavy chain variable region comprising HCDR3 of SEQ ID NO: 400, and (d) L of SEQ ID NO: 405 CDR1, (e) LCDR2 of SEQ ID NO: 406, and (f) LCDR 3 of SEQ ID NO: 407; (xxiii) (a) HCDR1 of SEQ ID NO: 412, (b) HCDR of SEQ ID NO: 413 2, (c) heavy chain variable region comprising HCDR3 of SEQ ID NO: 414, and (d) SEQ ID NO: 419 LCDR1, (e) LCDR2 of SEQ ID NO: 420, and (f) LCD Variable light chain region containing R3; (xxiv) (a) HCDR1 of SEQ ID NO: 426, (b) HCDR2 of SEQ ID NO: 427 , (c) heavy chain variable region containing HCDR3 of SEQ ID NO: 428, and (d) L of SEQ ID NO: 433 CDR1, (e) LCDR2 of SEQ ID NO: 434, and (f) LCD Variable light chain region containing R3; (xxv) (a) HCDR1 of SEQ ID NO: 440, (b) HCDR2 of SEQ ID NO: 441, (c) heavy chain variable region containing HCDR3 of SEQ ID NO: 442, and (d) LC of SEQ ID NO: 447 DR1, (e) LCDR2 of SEQ ID NO: 448, and (f) LCDR3 of SEQ ID NO: 449 Variable light chain region containing; (xxvi) (a) HCDR1 of SEQ ID NO: 454, (b) HCDR2 of SEQ ID NO: 455 , (c) heavy chain variable region containing HCDR3 of SEQ ID NO: 456, and (d) L of SEQ ID NO: 461 CDR1, (e) LCDR2 of SEQ ID NO: 462, and (f) LCDR of SEQ ID NO: 463 Variable light chain region containing R3; (xxvii) (a) HCDR1 of SEQ ID NO: 468, (b) HCDR of SEQ ID NO: 469 2, (c) heavy chain variable region containing HCDR3 of SEQ ID NO: 470, and (d) SEQ ID NO: 475 LCDR1, (e) LCDR2 of SEQ ID NO: 476, and (f) LCD of SEQ ID NO: 477 Variable light chain region containing R3; (xxviii) (a) HCDR1 of SEQ ID NO: 482, (b) HCD of SEQ ID NO: 483 R2, (c) heavy chain variable region containing HCDR3 of SEQ ID NO: 484, and (d) SEQ ID NO: 489 LCDR1, (e) LCDR2 of SEQ ID NO: 490, and (f) LC of SEQ ID NO: 491 Variable light chain region containing DR3 An antibody comprising

[0017] an antibody, wherein said antibody or an antigen-binding fragment thereof (i) (a) an HCDR1 (CDR - Complementary Determining Region) of SEQ ID NO: 508, (b) SEQ ID NO: 509 of HCDR2, (c) a heavy chain variable region comprising HCDR3 of SEQ ID NO: 510, and (d ) an LCDR1 of SEQ ID NO: 511, (e) an LCDR2 of SEQ ID NO: 512, and (f) SEQ ID NO: 513 of LCDR3; (ii) (a) an HCDR1 of SEQ ID NO: 514, (b) an HCDR2 of SEQ ID NO: 515, ( c) a heavy chain variable region comprising HCDR3 of SEQ ID NO: 516, and (d) an LCD of SEQ ID NO: 517 R1, (e) an LCDR2 of SEQ ID NO: 518, and (f) an LCDR3 of SEQ ID NO: 519 comprising; (iii) (a) an HCDR1 of SEQ ID NO: 520, (b) an HCDR2 of SEQ ID NO: 521, (c) a heavy chain variable region comprising HCDR3 of SEQ ID NO: 522, and (d) an LC of SEQ ID NO: 523 DR1, (e) an LCDR2 of SEQ ID NO: 524, and (f) an LCDR3 of SEQ ID NO: 525 comprising; (iv) (a) an HCDR1 of SEQ ID NO: 526, (b) an HCDR2 of SEQ ID NO: 527, ( c) a heavy chain variable region comprising HCDR3 of SEQ ID NO: 528, and (d) an LCD of SEQ ID NO: 529 R1, (e) an LCDR2 of SEQ ID NO: 530, and (f) an LCDR3 of SEQ ID NO: 531 comprising; (v) (a) an HCDR1 of SEQ ID NO: 532, (b) an HCDR2 of SEQ ID NO: 533, (c ) a heavy chain variable region comprising HCDR3 of SEQ ID NO: 534, and (d) an LCDR of SEQ ID NO: 535 1, (e) an LCDR2 of SEQ ID NO: 536, and (f) an LCDR3 of SEQ ID NO: 537 comprising; (vi) (a) The HCDR1 of SEQ ID NO: 538, (b) the HCDR2 of SEQ ID NO: 539, ( c) the heavy chain variable region comprising the HCDR3 of SEQ ID NO: 540, and (d) the LCD R1 of SEQ ID NO: 541, (e) the LCDR2 of SEQ ID NO: 542, and (f) the LCDR3 of SEQ ID NO: 543 comprising the light chain variable region An antibody comprising.

[0018] At least one amino acid within the CDR is replaced by the corresponding residue of the corresponding CDR of another anti-VP1 antibody in Table 2, an antibody. An antibody.

[0019] One or two amino acids within the CDR are modified, deleted or substituted, an antibody.

[0020] An antibody that retains at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity across either the variable heavy chain region or the variable light chain region.

[0021] An antibody comprising the modifications in Table 3.

[0022] A monoclonal antibody, chimeric antibody, humanized antibody, human engineered antibody, human antibody, single-chain antibody (scFv) or an antibody fragment, an antibody.

[0023] Said antibody or its antigen-binding fragment is (i) a heavy chain variable region (vH) comprising SEQ ID NO: 12 and a light chain variable region (vL) comprising SEQ ID NO: 22; (ii) a heavy chain variable region (vH) comprising SEQ ID NO: 32 and a light chain variable region (vL) comprising SEQ ID NO: 42; (iii) a heavy chain variable region (vH) comprising SEQ ID NO: 52 and a light chain variable region (vL) comprising SEQ ID NO: 62; (iv) a heavy chain variable region (vH) comprising SEQ ID NO: 72 and a light chain variable region (vL) comprising SEQ ID NO: 82; (v) A heavy chain variable region (vH) containing SEQ ID NO: 92 and a light chain variable region (vL); (vi) A heavy chain variable region (vH) containing SEQ ID NO: 112 and a light chain variable region (vL); (vii) A heavy chain variable region (vH) containing SEQ ID NO: 132 and a light chain variable region (vL); (viii) A heavy chain variable region (vH) containing SEQ ID NO: 152 and a light chain variable region (vL); (ix) A heavy chain variable region (vH) containing SEQ ID NO: 172 and a light chain variable region (vL); (x) A heavy chain variable region (vH) containing SEQ ID NO: 192 and a light chain variable region (vL); (xi) A heavy chain variable region (vH) containing SEQ ID NO: 212 and a light chain variable region (vL); (xii) A heavy chain variable region (vH) containing SEQ ID NO: 232 and a light chain variable region (vL); (xiii) A heavy chain variable region (vH) containing SEQ ID NO: 252 and a light chain variable region (vL); (xiv) A heavy chain variable region (vH) containing SEQ ID NO: 272 and a light chain variable region (vL); (xv) A heavy chain variable region (vH) containing SEQ ID NO: 292 and a light chain variable region (vL); (xvi) A heavy chain variable region (vH) containing SEQ ID NO: 312 and a light chain variable region (vL); (xvii) A heavy chain variable region (vH) containing SEQ ID NO: 328 and a light chain variable region (vL); (xviii) A heavy chain variable region (vH) containing SEQ ID NO: 348 and a light chain variable region (vL); (xix) A heavy chain variable region (vH) containing SEQ ID NO: 362 and a light chain variable region (vL); (xx) A heavy chain variable region (vH) containing SEQ ID NO: 376 and a light chain variable region (vL); (xxi) A heavy chain variable region (vH) containing SEQ ID NO: 390 and a light chain variable region (vL); (xxii) A heavy chain variable region (vH) containing SEQ ID NO: 404 and a light chain variable region (vL); (xxiii) A heavy chain variable region (vH) containing SEQ ID NO: 418 and a light chain variable region (vL); (xxiv) A heavy chain variable region (vH) containing SEQ ID NO: 432 and a light chain variable region (vL); (xxv) A heavy chain variable region (vH) containing SEQ ID NO: 446 and a light chain variable region (vL); (xxvi) A heavy chain variable region (vH) containing SEQ ID NO: 460 and a light chain variable region (vL); (xxvii) A heavy chain variable region (vH) containing SEQ ID NO: 474 and a light chain variable region (vL); or, (xxviii) A heavy chain variable region (vH) containing SEQ ID NO: 488 and a light chain variable region (vL) comprising an antibody.

[0024] An antibody that retains at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity across either the variable light chain region or the variable heavy chain region.

[0025] One, two, three, four, or five, but less than ten, amino acids within the variable light chain region or variable heavy chain region of an antibody, which have been modified, deleted, or substituted

[0026] A monoclonal antibody, chimeric antibody, humanized antibody, human engineered antibody, human antibody, single-chain antibody (scFv) or an antibody fragment

[0027] An antibody or a fragment thereof having reduced glycosylation or having no glycosylation or being hypofucosylated The antibody according to any one of the above embodiments

[0028] A composition comprising a plurality of the antibodies or antigen-binding fragments thereof according to any one of the above embodiments wherein at least 0.05%, 0.1%, 0.5%, 1%, 2%, 3% , 5% or more of the antibodies in the composition have α2,3-linked sialic acid residues, and the antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable region comprising (a) HCDR1 (CDR - Complementary Determining Region) of SEQ ID NO: 6, (b) HCDR2 of SEQ ID NO: 7, (c) HCDR3 of SEQ ID NO: 8, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 16, (e) LCDR2 of SEQ ID NO: 17, and (f) LCDR3 of SEQ ID NO: 18; (ii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 26, (b) HCDR2 of SEQ ID NO: 27, (c) HCDR3 of SEQ ID NO: 28, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 36,( e) LCDR2 of SEQ ID NO: 37, and (f) LCDR3 of SEQ ID NO: 38; (iii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 46, (b) HCDR2 of SEQ ID NO: 47, (c ) HCDR3 of SEQ ID NO: 48, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 56, e) LCDR2 of SEQ ID NO: 57, and (f) LCDR3 of SEQ ID NO: 58; ; (iii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 46, (b) HCDR2 of SEQ ID NO: 47, (c ) HCDR3 of SEQ ID NO: 48, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 56, (e) a light chain variable region comprising LCDR2 of SEQ ID NO: 57, and (f) LCDR3 of SEQ ID NO: 58; variable region; (iv) (a) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 66, (b) HCDR2 of SEQ ID NO: 67, (c) HCDR3 of SEQ ID NO: 68, and (d) LCDR1 of SEQ ID NO: 76, ( e) LCDR2 of SEQ ID NO: 77, and (f) a light chain variable region comprising LCDR3 of SEQ ID NO: 78; region; (v) (a) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 86, (b) HCDR2 of SEQ ID NO: 87, (c) heavy chain variable region comprising HCDR3 of SEQ ID NO: 88, and (d) LCDR1 of SEQ ID NO: 96, (e ) LCDR2 of SEQ ID NO: 97, and (f) a light chain variable region comprising LCDR3 of SEQ ID NO: 98; region; (vi) (a) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 106, (b) HCDR2 of SEQ ID NO: 107, ( c) heavy chain variable region comprising HCDR3 of SEQ ID NO: 108, and (d) LCD of SEQ ID NO: 116 R1, (e) LCDR2 of SEQ ID NO: 117, and (f) a light chain variable region comprising LCDR3 of SEQ ID NO: 118; region; (vii) (a) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 126, (b) HCDR2 of SEQ ID NO: 127, (c) heavy chain variable region comprising HCDR3 of SEQ ID NO: 128, and (d) LC of SEQ ID NO: 136 DR1, (e) LCDR2 of SEQ ID NO: 137, and (f) a light chain variable region comprising LCDR3 of SEQ ID NO: 138 region; (viii) (a) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 146, (b) HCDR2 of SEQ ID NO: 147 , (c) heavy chain variable region comprising HCDR3 of SEQ ID NO: 148, and (d) L of SEQ ID NO: 156 CDR1, (e) LCDR2 of SEQ ID NO: 157, and (f) a light chain variable region comprising LCDR 3 of SEQ ID NO: 158; (ix) (a) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 166, (b) HCDR2 of SEQ ID NO: 167, ( c) A heavy chain variable region containing the HCDR3 of SEQ ID NO: 168, and (d) the LCD of SEQ ID NO: 176 R1, (e) the LCDR2 of SEQ ID NO: 177, and (f) the LCDR3 of SEQ ID NO: 178 Light chain variable region containing; (x) (a) The HCDR1 of SEQ ID NO: 186, (b) the HCDR2 of SEQ ID NO: 187, (c ) A heavy chain variable region containing the HCDR3 of SEQ ID NO: 188, and (d) the LCDR of SEQ ID NO: 196 1, (e) the LCDR2 of SEQ ID NO: 197, and (f) the LCDR3 of SEQ ID NO: 198 containing Light chain variable region; (xi) (a) The HCDR1 of SEQ ID NO: 206, (b) the HCDR2 of SEQ ID NO: 207, ( c) A heavy chain variable region containing the HCDR3 of SEQ ID NO: 208, and (d) the LCD of SEQ ID NO: 216 R1, (e) the LCDR2 of SEQ ID NO: 217, and (f) the LCDR3 of SEQ ID NO: 218 Light chain variable region containing; (xii) (a) The HCDR1 of SEQ ID NO: 226, (b) the HCDR2 of SEQ ID NO: 227, (c) A heavy chain variable region containing the HCDR3 of SEQ ID NO: 228, and (d) the LC of SEQ ID NO: 236 DR1, (e) the LCDR2 of SEQ ID NO: 237, and (f) the LCDR3 of SEQ ID NO: 238 Light chain variable region containing; (xiii) (a) The HCDR1 of SEQ ID NO: 246, (b) the HCDR2 of SEQ ID NO: 247 , (c) A heavy chain variable region containing the HCDR3 of SEQ ID NO: 248, and (d) the L of SEQ ID NO: 256 CDR1, (e) the LCDR2 of SEQ ID NO: 257, and (f) the LCDR of SEQ ID NO: 258 3 Light chain variable region containing; (xiv) (a) The HCDR1 of SEQ ID NO: 266, (b) the HCDR2 of SEQ ID NO: 267, (c) A heavy chain variable region containing the HCDR3 of SEQ ID NO: 268, and (d) the LC of SEQ ID NO: 276 The light chain variable region comprising (e) LCDR2 of SEQ ID NO: 277 and (f) LCDR3 of SEQ ID NO: 278 ; (xv) (a) HCDR1 of SEQ ID NO: 286, (b) HCDR2 of SEQ ID NO: 287, (c) the heavy chain variable region comprising HCDR3 of SEQ ID NO: 288, and (d) LCD R1 of SEQ ID NO: 296, (e) LCDR2 of SEQ ID NO: 297, and (f) LCDR3 of SEQ ID NO: 298 ; (xvi) (a) HCDR1 of SEQ ID NO: 306, (b) HCDR2 of SEQ ID NO: 307, (c) the heavy chain variable region comprising HCDR3 of SEQ ID NO: 308, and (d) LC DR1 of SEQ ID NO: 314, (e) LCDR2 of SEQ ID NO: 315, and (f) LCDR3 of SEQ ID NO: 316 ; (xvii) (a) HCDR1 of SEQ ID NO: 322, (b) HCDR2 of SEQ ID NO: 323 , (c) the heavy chain variable region comprising HCDR3 of SEQ ID NO: 324, and (d) L CDR1 of SEQ ID NO: 332, (e) LCDR2 of SEQ ID NO: 333, and (f) LCDR 3 of SEQ ID NO: 334 (xviii) (a) HCDR1 of SEQ ID NO: 342, (b) HCDR 2 of SEQ ID NO: 343, (c) the heavy chain variable region comprising HCDR3 of SEQ ID NO: 344, and (d) LCD R1 of SEQ ID NO: 349, (e) LCDR2 of SEQ ID NO: 350, and (f) LCD R3 of SEQ ID NO: 351 (xix) (a) HCDR1 of SEQ ID NO: 356, (b) HCDR2 of SEQ ID NO: 357, (c) the heavy chain variable region comprising HCDR3 of SEQ ID NO: 358, and (d) LC DR1 of SEQ ID NO: 363, (e) LCDR2 of SEQ ID NO: 364, and (f) LCDR3 of SEQ ID NO: 365 ; (xx) (a) The HCDR1 of SEQ ID NO: 370, (b) the HCDR2 of SEQ ID NO: 371, (c) a heavy chain variable region comprising the HCDR3 of SEQ ID NO: 372, and (d) the LCD R1 of SEQ ID NO: 377, (e) the LCDR2 of SEQ ID NO: 378, and (f) the LCDR3 of SEQ ID NO: 379 comprising a light chain variable region; (xxi) (a) The HCDR1 of SEQ ID NO: 384, (b) the HCDR2 of SEQ ID NO: 385, (c) a heavy chain variable region comprising the HCDR3 of SEQ ID NO: 386, and (d) the LC DR1 of SEQ ID NO: 391, (e) the LCDR2 of SEQ ID NO: 392, and (f) the LCDR3 of SEQ ID NO: 393 comprising a light chain variable region; (xxii) (a) The HCDR1 of SEQ ID NO: 398, (b) the HCDR2 of SEQ ID NO: 399 , (c) a heavy chain variable region comprising the HCDR3 of SEQ ID NO: 400, and (d) the L CDR1 of SEQ ID NO: 405, (e) the LCDR2 of SEQ ID NO: 406, and (f) the LCDR 3 of SEQ ID NO: 407 comprising a light chain variable region; (xxiii) (a) The HCDR1 of SEQ ID NO: 412, (b) the HCDR 2 of SEQ ID NO: 413, (c) a heavy chain variable region comprising the HCDR3 of SEQ ID NO: 414, and (d) the LCDR1 of SEQ ID NO: 419, (e) the LCDR2 of SEQ ID NO: 420, and (f) the LCD R3 of SEQ ID NO: 421 comprising a light chain variable region; (xxiv) (a) The HCDR1 of SEQ ID NO: 426, (b) the HCDR2 of SEQ ID NO: 427 , (c) a heavy chain variable region comprising the HCDR3 of SEQ ID NO: 428, and (d) the L CDR1 of SEQ ID NO: 433, (e) the LCDR2 of SEQ ID NO: 434, and (f) the LCDR 3 of SEQ ID NO: 435 comprising a light chain variable region; (xxv) (a) The HCDR1 of SEQ ID NO: 440, (b) the HCDR2 of SEQ ID NO: 441, (c) a heavy chain variable region comprising the HCDR3 of SEQ ID NO: 442, and (d) the LC a light chain variable region comprising DR1, (e) LCDR2 of SEQ ID NO: 448, and (f) LCDR3 of SEQ ID NO: 449 ; (xxvi) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 454, (b) HCDR2 of SEQ ID NO: 455 , (c) HCDR3 of SEQ ID NO: 456, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 461 , (e) LCDR2 of SEQ ID NO: 462, and (f) LCDR 3 of SEQ ID NO: 463; (xxvii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 468, (b) HCDR 2 of SEQ ID NO: 469, (c) HCDR3 of SEQ ID NO: 470, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 475 , (e) LCDR2 of SEQ ID NO: 476, and (f) LCD R3 of SEQ ID NO: 477; (xxviii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 482, (b) HCD R2 of SEQ ID NO: 483, (c) HCDR3 of SEQ ID NO: 484, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 489 , (e) LCDR2 of SEQ ID NO: 490, and (f) LC DR3 of SEQ ID NO: 491 A composition comprising.

[0029] A composition comprising a plurality of antibodies or antigen-binding fragments according to any of the preceding embodiments wherein none of the antibodies contain bisecting GlcNAc.

[0030] A pharmaceutical composition comprising an antibody or a fragment thereof according to any of the preceding embodiments, wherein the composition is prepared as a lyophilizate .

[0031] A pharmaceutical composition comprising an antibody or a fragment thereof according to any of the preceding embodiments and a pharmaceutically acceptable carrier. In one embodiment, the carrier is a histidine buffer. In one embodiment ​ The pharmaceutical composition contains sugar (e.g., sucrose).

[0032] Administering an effective amount of an antibody or pharmaceutical composition to a patient in need thereof by injection or infusion, A method of neutralizing BK virus or JC virus infection, comprising. The antibody or its antigen-binding fragment neutralizes BKV serotype I and BKV serotype II. In another embodiment, the antibody or its antigen-binding fragment neutralizes BKV serotype I and BKV serotype III. In another embodiment, the antibody or its antigen-binding fragment neutralizes BKV serotype I and BKV serotype IV. In another embodiment, the antibody or its antigen-binding fragment neutralizes BKV serotype II and BKV serotype III. In another embodiment, the antibody or its antigen binding fragment neutralizes BKV serotype II and BKV serotype IV. In another embodiment, the antibody or its antigen-binding fragment neutralizes BKV serotype I and JCV. In a particular embodiment, the antibody or its antigen-binding fragment neutralizes BKV serotype I, II, III, and I V. Further, the antibody or its antigen-binding fragment neutralizes BKV serotype I, II, II I, and IV as well as JCV. In a preferred embodiment, the anti-VP1 antibody neutralized infection by all four serotypes (I-IV) of BKV, and these anti-VP1 antibodies include, in particular, P8D11, modifications of P8D11, and EBB-C1975-B5.

[0033] Administering an effective amount of an antibody or pharmaceutical composition to a patient in need thereof by injection or infusion, A method of treating or reducing the likelihood of a disorder associated with BK virus or JC virus, wherein the disorder is nephropathy, BKVAN, hemorrhagic cystitis (HC) , Progressive multifocal leukoencephalopathy (PML), granulocyte neuropathy (GCN), interstitial kidney disease , ureteral stricture, vasculitis, colitis, retinitis, meningitis, and immune reconstitution syndrome (IRIS) There is a method.

[0034] A method in which an antibody or composition is reconstituted before injection or infusion.

[0035] A method in which an antibody or pharmaceutical composition is administered in combination with another therapeutic agent.

[0036] The method, wherein the therapeutic agent is an immunosuppressant.

[0037] The immunosuppressant is a monophosphate dehydrogenase inhibitor, a purine synthesis inhibitor, a calcinurin inhibitor or an mTOR inhibitor.

[0038] The immunosuppressant is mycophenolate mofetil (MMF), mycophenolate sodium, azathioprine, tacrolimus, sirolimus or cyclosporine.

[0039] The method, wherein the therapeutic agent is a further anti-VP1 antibody.

[0040] An antibody or fragment thereof according to any of the above embodiments for use as a medicament.

[0041] An antibody or fragment thereof or a pharmaceutical composition for use in the neutralization of BK virus or JC virus infection. fragment or pharmaceutical composition.

[0042] nephropathy, BKVAN, hemorrhagic cystitis (HC), progressive multifocal leukoencephalopathy (PML) , granulocyte neuropathy (GCN), interstitial kidney disease, ureteral stricture, vasculitis, colitis, retina itis, meningitis, and in the treatment of immune reconstitution syndrome (IRIS) or reduction of its likelihood An antibody or fragment thereof or a pharmaceutical composition for use thereof.

[0043] Use of an antibody or fragment thereof administered in combination with another therapeutic agent.

[0044] Use of an antibody or fragment thereof, wherein the therapeutic agent is an immunosuppressive agent.

[0045] The immunosuppressive agent is a monophosphate dehydrogenase inhibitor, a purine synthesis inhibitor, a cal Use of an antibody or fragment thereof, which is a sinurine inhibitor or an mTOR inhibitor.

[0046] The immunosuppressive agent is mycophenolate mofetil (MMF), mycophenolate sodium Use of an antibody or fragment thereof, which is mycophenolate sodium, azathioprine, tacrolimus, sirolimus or cyclosporine. Or a fragment thereof.

[0047] A nucleic acid encoding an antibody or antigen-binding fragment according to any of the above embodiments.

[0048] A vector containing the nucleic acid.

[0049] A host cell containing the vector.

[0050] A method for producing an antibody or antigen-binding fragment, which includes culturing a host cell and recovering the antibody from the culture. For use.

[0051] A diagnostic agent containing a labeled antibody or antigen-binding fragment thereof.

[0052] The label is selected from the group consisting of a radiolabel, a fluorophore, a chromophore, an imaging agent, and a metal ion. A diagnostic agent.

[0053] Definitions Unless otherwise specified, the following terms and phrases used in this specification have the following meanings. is intended to be done.

[0054] As used herein, the term "antibody" refers, without limitation, to a polypeptide of the immunoglobulin family that can bind to a corresponding antigen in a non-covalent, reversible, and specific manner. For example, a natural IgG antibody is a tetramer comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated as VL herein) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, from the amino terminus towards the carboxy terminus. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors such as various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. For example, a natural IgG antibody is a tetramer comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated as VL herein) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, from the amino terminus towards the carboxy terminus. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors such as various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors such as various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. For example, a natural IgG antibody is a tetramer comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen.

[0055] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, and anti-idiotype (anti-Id) antibodies (e.g., the present For example, a natural IgG antibody is a tetramer comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Antibodies include antibodies of any isotype / class (e.g., anti-Id antibodies against the disclosed antibodies). IgG, IgE, IgM, IgD, IgA and IgY), or subclass (e.g. For example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). This is also possible.

[0056] "Complementarity determining domains" or "complementarity determining regions ("CDRs")" refer to the VL and VH The CDRs are interchangeably referred to as the hypervariable regions of a target protein. Each human VL or VH has three CDs. CDRs (CDRs 1 to 3, numbered consecutively from the N-terminus) are present, and approximately 1 CDRs can be described by their region and order. For example, For example, "VHCDR1" or "HCDR1" both refer to the first CDR of a heavy chain variable region. The CDRs are structurally complementary to the epitope of the target protein and thus provide binding specificity. The remaining stretches of the VL or VH, the so-called framework regions, are directly responsible for the isomerism. They show little change in amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4. WH F Reeman & Co., New York, 2000).

[0057] The positions of the CDRs and framework regions may be determined according to various well-known definitions in the art, for example, abat, Chothia, and AbM (e.g., Johnson et al., Nucleic Acids Re s., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chot Hia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-8 17 (1992); Al-Lazikani et al., J.Mol.Biol., 273:927-748 (1997) (see also). It can be determined using . The definition of the antigen-binding site is also described below: Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); and Lefranc, M.P., Nucleic Aci ds Res., 29:207-209 (2001); MacCallum et al., J. Mol. Biol., 262:732-745 (1996); and Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989); Martin et al., Methods Enzymol., 203:121-153 (1991); and Rees et al., In Sternberg M.J. E. (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996). In some embodiments, in the combined Kabat and Chothia numbering schemes, the CDRs correspond to amino acid residues that are part of either the Kabat CDRs, the Chothia CDRs, or both. For example, in some embodiments, the CDRs are amino acid residues 26-35 (HC CDR 1), 50-65 (HC CDR2), and 95-102 (HC CDR3) in VH, such as mammalian VH, such as human VH; and amino acid residues 24-34 (LC in VL, such as mammalian VL, such as human VL. For example, in some embodiments, the CDRs are amino acid residues 26-35 (HC CDR 1), 50-65 (HC CDR2), and 95-102 (HC CDR3) in VH, such as mammalian VH, such as human VH; and amino acid residues 24-34 (LC correspond to 50-56 (LC CDR1), 50-56 (LC CDR2), and 89-97 (LC CDR3). respond.

[0058] Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it is understood that the variable domain portions of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light (CL) and heavy (CH1, CH2 or CH3) chains confer important biological properties such as secretion, transplacental transfer, Fc receptor binding, complement binding, etc. By convention the numbering of the constant region domains increases as they become more distant from the antigen binding site or amino terminus of the antibody. The N-terminus is the variable region and the C-terminus is the constant region ; the CH3 and CL domains actually contain the carboxy-terminal domains of the heavy and light chains, respectively. ; CH3 and CL domains actually contain the carboxy-terminal domains of the heavy and light chains, respectively. actually contain.

[0059] As used herein, the term "antigen-binding fragment" refers to one or more portions of an antibody that retain the ability to specifically interact (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution) with an epitope of an antigen. Examples of binding fragments include, but are not limited to, single-chain Fv (scFv), disulfide-bonded Fv (sdFv), Fab fragments, F (ab') fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab) 2 fragments, divalent fragments containing two Fab fragments linked by a disulfide bridge in the hinge region ; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of VL and VH domains of a single arm of an antibody; dAb fragments consisting of a VH domain (Ward et al., Nature 341: ; F(ab) 2 fragments, divalent fragments containing two Fab fragments linked by a disulfide bridge in the hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of VL and VH domains of a single arm of an antibody; dAb fragments consisting of a VH domain (Ward et al., Nature 341: (1989); and isolated complementarity determining regions (CDRs), or other epitope-binding fragments of antibodies are included.

[0060] Furthermore, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but they can be made using recombinant methods as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (single-chain Fv ( "scFv")) that can be linked by a synthetic linker; see, for example, Bird et al., Science 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. 85:5879-5883, 1988. Such single-chain antibodies are also intended to be included within the term "antigen-binding fragment." These antigen-binding fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0061] Antigen-binding fragments can also be incorporated into scaffolds based on polypeptides such as type III fibronectin (Fn3) (see, for example, U.S. Patent No. 6,703,199, which describes fibronectin polypeptide monobodies). Antigen-binding fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, tribodies, tetrabodies, v-NAR, and bis-scFv (see, for example, Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005).

[0062] An antigen-binding fragment, together with a complementary light chain polypeptide, is incorporated into a single-chain molecule comprising a pair of juxtaposed Fv segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions (Zapata et al., Protein Eng. 8:1057-1062, 1995; and U.S. Patent No. 5,64 1,870).

[0063] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to antibodies and antigen-binding fragments, such as polypeptides, that have substantially the same amino acid sequence or are derived from the same genetic origin. The term also includes preparations of antibody molecules of single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

[0064] As used herein, the term "human antibody" includes antibodies having variable regions in which both the framework regions and the CDR regions are derived from sequences of human origin. Further, when the antibody contains a constant region, the constant region is also derived from such human sequences, such as human germline sequences, or variants of human germline sequences or consensus framework sequences derived from analysis of human framework sequences, such as those described in Knappik et al., J. Mol. Biol. 296:57-86, 2000.

[0065] The human antibodies of the present disclosure may contain amino acid residues not encoded by human sequences (e.g., by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo, or by conservative substitutions to promote stability or production) mutations introduced by substitution).

[0066] As used herein, the term "recognize" means that the epitope is linear or conformational and also refers to an antibody or an antigen-binding fragment thereof that discovers and interacts (e.g., binds) with that epitope. The term "epitope" refers to the site on an antigen to which an antibody or antigen-binding fragment of the present disclosure specifically binds. An epitope can be formed from both contiguous and non-contiguous amino acids juxtaposed by the three-dimensional folding of a protein. An epitope formed from contiguous amino acids is typically retained upon exposure to a denaturing solvent, while an epitope formed by three-dimensional folding is typically lost upon treatment with a denaturing solvent. An epitope typically contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include techniques known in the art, such as x-ray crystallography and two-dimensional nuclear magnetic resonance (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)). A "paratope" is the part of an antibody that recognizes the epitope of an antigen. When used in the context of describing the interaction between an antigen (e.g., a protein) and an antibody, antibody fragment, or antibody-derived binder, the phrases "specifically binds" or "selectively binds" refer to, in a heterogeneous population of proteins and other biological agents, e.g., in a biological sample, e.g., blood An epitope formed from contiguous amino acids is typically retained upon exposure to a denaturing solvent, while an epitope formed by three-dimensional folding is typically lost upon treatment with a denaturing solvent. An epitope typically exists in a unique spatial conformation and contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids. Methods for determining the spatial conformation of an epitope include techniques known in the art, such as x-ray crystallography and two-dimensional nuclear magnetic resonance (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)). A "paratope" is the part of an antibody that recognizes the epitope of an antigen. In the context of describing the interaction between an antigen (e.g., a protein) and an antibody, antibody fragment, or antibody-derived binder, the phrases "specifically binds" or "selectively binds" refer to, in a heterogeneous population of proteins and other biological agents, e.g., in a biological sample, e.g., blood (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)). A "paratope" is the part of an antibody that recognizes the epitope of an antigen. When used in the context of describing the interaction between an antigen (e.g., a protein) and an antibody, antibody fragment, or antibody-derived binder, the phrases "specifically binds" or "selectively binds" refer to, in a heterogeneous population of proteins and other biological agents, e.g., in a biological sample, e.g., blood

[0067] In the context of describing the interaction between an antigen (e.g., a protein) and an antibody, antibody fragment, or antibody-derived binder, the phrases "specifically binds" or "selectively binds" refer to, in a heterogeneous population of proteins and other biological agents, e.g., in a biological sample, e.g., blood (e.g., in a biological sample, e.g., blood When used in the context of describing the interaction between an antigen (e.g., a protein) and an antibody, antibody fragment, or antibody-derived binder, the phrases "specifically binds" or "selectively binds" refer to, in a heterogeneous population of proteins and other biological agents, e.g., in a biological sample, e.g., blood ​Refers to a binding reaction that determines the presence of an antigen in a fluid, serum, plasma or tissue sample. Thus, Under certain specified immunoassay conditions, an antibody or binding agent having a specific binding specificity binds to at least twice the background, binds to a specific antigen, and is present in the sample Does not substantially bind to other antigens in significant amounts. In one aspect, under the specified immunoassay conditions An antibody or binding agent having a specific binding specificity is at least 10 times the background Binds to a specific antigen and does not substantially bind to other antigens present in the sample in significant amounts. Specific binding to an antibody or binding agent under such Conditions may require selection of the antibody or agent for its Specificity for a particular protein. If desired or appropriate In some cases, this selection can be achieved by removing antibodies that cross-react with molecules from other species (e.g., mouse or rat) or other subtypes. Alternatively In some embodiments, antibodies or antibody fragments that cross-react with a particular desired molecule are selected To do.

[0068] As used herein, the term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single antigenic site. Within each antigenic site, the variable regions of the antibody "arms" interact with the antigen at several sites via weak non-covalent forces; the greater the interaction, the Greater the affinity. The greater the interaction, the stronger the affinity.

[0069] The term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities. However, an isolated antibody that specifically binds to one antigen may have cross-reactivity with other antigens And. Furthermore, an isolated antibody is other cellular material and / or May have cross-reactivity with. Additionally, an isolated antibody is other cellular material and / or ​​It may not substantially contain chemical substances.

[0070] The term "corresponding human germline sequence" refers to a nucleic acid sequence that encodes a human variable region amino acid sequence or sub-sequence that has the highest determined amino acid sequence identity with the reference variable region amino acid sequence or sub-sequence compared to all other known variable region amino acid sequences encoded by the human germline immunoglobulin variable region sequences. The corresponding human germline sequence may also refer to a human variable region amino acid sequence or sub-sequence that has the highest amino acid sequence identity with the reference variable region amino acid sequence or sub-sequence compared to all other evaluated variable region amino acid sequences. The corresponding human germline sequence may be a sequence or sub-sequence that includes only the framework region, only the complementarity-determining region, the framework region and the complementarity-determining region, the variable segment (as defined above), or other combinations of sequences or sub-sequences that include the variable region. For example, sequence identity can be determined using the methods described herein, such as aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art. The corresponding human germline nucleic acid or amino acid sequence may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference variable region nucleic acid or amino acid sequence. Antibodies that specifically immunoreact with a particular protein can be selected using various immunoassay formats. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact with a protein (e.g., specific immunoreaction

[0071] ​​​​​​​​​​​​​For a description of immunoassay formats and conditions that can be used to determine properties, see Harlow & Lane, Using Antibodies, A Laboratory Manual (1998)). Typically, a specific or selective binding reaction results in a signal that is at least 2 times, more typically at least 10 - 100 times the background signal.

[0072] The term "equilibrium dissociation constant (KD, M)" refers to the dissociation rate constant (kd, time-1) divided by the association rate constant (ka, time-1, M-1). The equilibrium dissociation constant can be measured using any known method in the art. The antibodies of the present disclosure generally have an equilibrium dissociation constant of about 10 or less than 10 - 7 or 10 -8 M or less, for example, about 10 -9 M or 10 -10 M or less, and in some embodiments, about 10 M, 10 -11 M or 10 -12 M or less. -13

[0073] The term "bioavailability" refers to the systemic availability (i.e., blood / plasma level) of a given amount of a drug administered to a patient. Bioavailability is an absolute term that indicates both the time (rate) and the total amount (extent) of the drug that reaches the systemic circulation from the administered dosage form.

[0074] As used herein, the phrase "consisting essentially of" refers to the genus or species of the active agent contained in a method or composition, as well as any inert components that are inactive for the intended purpose of the method or composition. ​​​​​​Refers to an intentional excipient. In some embodiments, the phrase "consisting essentially of" clearly excludes the inclusion of one or more additional active agents other than the anti-VP 1 antibody. In some embodiments the phrase "consisting essentially of" clearly excludes the inclusion of one or more additional active agents other than the anti-VP1 antibody of the present disclosure and a second co-administered agent.

[0075] The term "amino acid" refers to natural, synthetic, and unnatural amino acids, as well as amino acid analogs and mimetics that function in a manner similar to natural amino acids. Natural amino acids are those encoded by the genetic code, as well as amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as natural amino acids, i.e., hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and α-carbon bonded to methionine methylsulfonium. Such analogs have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as natural amino acids. Amino acid mimetics are compounds that have a structure different from the general chemical structure of amino acids, but function in a manner similar to natural amino acids. pointing to the α-carbon bonded to the carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as natural amino acids. Amino acid mimetics are compounds that have a structure different from the general chemical structure of amino acids, but function in a manner similar to natural amino acids. chemical structure of amino acids, but function in a manner similar to natural amino acids. refers to compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to natural amino acids. Refers to.

[0076] The term "conservatively modified variant" applies to both amino acid sequences and nucleic acid sequences. With respect to a particular nucleic acid sequence, a conservatively modified variant is a nucleic acid that encodes the same or a substantially identical amino acid sequence, or, if the nucleic acid does not encode an amino acid sequence, refers to a substantially identical sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode the same or a substantially identical amino acid sequence. With respect to a particular nucleic acid sequence, a conservatively modified variant is a nucleic acid that encodes the same or a substantially identical amino acid sequence, or, if the nucleic acid does not encode an amino acid sequence, refers to a substantially identical sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode the same or a substantially identical amino acid sequence, or, if the nucleic acid does not encode an amino acid sequence, refers to a substantially identical sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode the same or a substantially identical amino acid sequence. A nucleic acid encodes any given protein. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at all positions specified by a codon for alanine, that codon may be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid changes are "silent mutations" and are one type of conservatively modified variant. All nucleic acid sequences that encode a polypeptide also describe all possible silent mutations of the nucleic acid. One skilled in the art will recognize that each codon in a nucleic acid (except the sole codon for methionine, AUG, and the sole codon for tryptophan, TGG) can be mutated to give functionally identical molecules. Thus, each silent mutation of a nucleic acid that encodes a polypeptide is silent in each of the described sequences.

[0077] For polypeptide sequences, "conservatively modified variants" include individual substitutions, deletions or additions to a polypeptide sequence that result in substitution of an amino acid with a chemically similar amino acid. Tables of conservative substitutions providing functionally similar amino acids are well known in the art. Such conservatively modified variants include, but are not limited to, polymorphic variants, interspecies homologs, and alleles. The following eight groups each contain amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D ), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), Valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine (C), methionine (M) (see, e.g., Creighton, Proteins (1984)). In some embodiments, the term " conservative sequence modification" is used to refer to an amino acid modification that does not significantly affect, or alternatively changes, the binding properties of an antibody containing the amino acid sequence.

[0078] As used herein, the term "optimized" refers to a nucleotide sequence that has been altered to encode an amino acid sequence using codons that are preferred in a production cell or organism, generally a eukaryotic cell, such as a yeast cell, Pichia cell, fungal cell, Trichoderma cell, Chinese hamster ovary cell (CHO), or human cell. The optimized nucleotide sequence is engineered to retain, completely or to the greatest extent possible, the amino acid sequence originally encoded by the starting nucleotide sequence, also known as the "parent" sequence.

[0079] The term "percent identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to the degree to which the two or more sequences or subsequences are the same. Two sequences are "identical" if they have the same sequence of amino acids or nucleotides over the region being compared. Two sequences are compared and aligned for maximum correspondence over a comparison window, or designated region, using one of the following sequence comparison algorithms or by manual alignment and visual inspection. ​​​​​A specified percentage of amino acid residues or nucleotides that are combined and the same (i.e., 60% identity over a specific region or, if not specified, over the entire sequence, optionally, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity), the two sequences are "substantially identical". Optionally, the identity is over a region that is at least about 30 nucleotides (or 10 amino acids) in length, and more preferably, over a region that is 100 - 500 or 1000 or more nucleotides (or 2 0, 50, 200 or more amino acids) in length. For sequence comparison, typically, one sequence acts as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are specified, and, if necessary, sequence algorithm program parameters are specified. Default program parameters can be used or alternative parameters can be

[0080] specified. Then, the sequence comparison algorithm calculates the percentage of sequence identity for the test sequence compared to the reference sequence based on the program parameters. As used herein, a "comparison window" is a reference to any one segment of a contiguous number of positions, selected from the group consisting of 20 - 600, usually about 50 - about 200, more usually about 100 - about 150, that can be compared to the reference sequence at the same number of consecutive positions after optimally aligning the two sequences. Methods of aligning sequences for comparison are well known in the art. The optimal alignment of sequences for comparison can be, for example, by specifying the subsequence coordinates and, if necessary, the sequence algorithm program parameters. Whether to use default program parameters or specify alternative parameters can be decided. Then, the sequence comparison algorithm calculates the percentage of sequence identity of the test sequence compared to the reference sequence based on the program parameters. Based on the program parameters, the sequence comparison algorithm calculates the percentage of sequence identity of the test sequence compared to the reference sequence.

[0081] As used herein, a "comparison window" refers to any one segment of a contiguous number of positions, selected from the group consisting of 20 - 600, usually about 50 - about 200, more usually about 100 - about 150, that can be compared to the reference sequence at the same number of consecutive positions after optimally aligning the two sequences. After optimally aligning the two sequences, the "comparison window" can be used to compare the sequences at the same number of consecutive positions of the reference sequence. The "comparison window" is a contiguous number of positions selected from the group consisting of 20 - 600, usually about 50 - about 200, more usually about 100 - about 150. The method of aligning sequences for comparison is well known in the art. The optimal alignment of sequences for comparison can be, for example, by using the Needleman - Wunsch algorithm (Needleman and Wunsch, J. Mol. Biol. 48:443 - 453 (1970)), which is suitable for comparing DNA and protein sequences; the Smith - Waterman algorithm (Smith and Waterman, J. Mol. Biol. 147:195 - 201 (1981)), which is suitable for local alignment; ​For example, the partial homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482c (1970) Based on the homology alignment of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970) The algorithm used was Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988) The similarity search method of Wisco nsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, W. I) or Manual alignment and visual inspection (e.g., Brent et al., Current Protocol This can be done by the method of the present invention (see, for example, "The Methods in Molecular Biology," 2003).

[0082] Two algorithms that are suitable for determining percent sequence identity and sequence similarity: Two examples are the BLAST and BLAST 2.0 algorithms, which are , Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990. Software for performing BLAST analysis. The software is supported by the National Center for Biotechnology This algorithm is publicly available at http: / / www.data.org / publications / data / information / . Identifying short word lengths W in a query sequence that match or satisfy some positive-valued threshold score T when aligned with words of the same length in the base sequence includes first identifying high scoring sequence pairs (HSPs). T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds to initiate a search to find longer HSPs that contain them . Word hits are extended in both directions along each sequence as far as possible to increase the cumulative alignment score. The cumulative score is calculated for nucleotide sequences using parameters M (reward score for pairs of matching residues; always >0) and N (penalty score for non-matching residues; always <0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. Extension of a word hit in each direction stops when the cumulative alignment score decreases by an amount X from its maximum achieved value; for the cumulative of one or more negative-score residue alignments, the cumulative score becomes zero or less; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses, by default, a word length (W) of 11, an expectation value (E) of 10, M = 5, N = -4, and comparison of both strands. For amino acid sequences, the BLASTP program uses, by default, a word length of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix . See Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915 ) using 50 alignments (B), an expectation value (E) of 10, M = 5, N = -4, and comparison of both chains.

[0083] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., , Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the minimum total probability (P (N)) that provides an indication of the probability that a match between two nucleotides or amino acid sequences occurs by chance. For example, a nucleic acid is considered similar to a reference sequence if the minimum total probability in a comparison of the test nucleic acid and the reference nucleic acid is less than about 0.2 , more preferably less than about 0.01, and most preferably less than about 0.001 .

[0084] The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4:11-17, 1988, incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 . Further, the percent identity between two amino acid sequences can be determined using the BLOSUM 62 matrix or the PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6, using GCG . ​​​​ The Needleman and Wunsch, (J. Mol. Biol. 48:444-453, 1970) algorithm incorporated in the GAP program (available from the University of South Florida) in the software package can be used to determine.

[0085] In addition to the percentage of sequence identity described above, another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with an antibody raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, for example, if two peptides differ only by conservative substitutions, the polypeptides are typically substantially identical to the second polypeptide. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that their sequences can be amplified using the same primers.

[0086] The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. The term includes nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which have similar binding properties as the reference nucleic acid, are synthetic, natural, and non-natural, and are metabolized in a manner similar to the reference nucleotides. ​​​​​​​​​​​​​​. Examples of such analogs include, but are not limited to, phosphorothioate (ph osphorothioate), phosphoramidate, methylphosphonic acid, chiral -methylphosphonic acid, 2-O-methyl ribonucleotide, peptide nucleic acid (PNA), etc. are listed.

[0087] Unless otherwise indicated, a particular nucleic acid sequence implicitly includes its conservatively modified variants (e.g., , degenerate codon substitutions) and complementary sequences, as well as explicitly shown sequences. In particular, as detailed below, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue (Batzer et al., (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al., (1985) J. Biol. Chem. 260:2605-2608; and Ro ssolini et al., (1994) Mol. Cell. Probes 8:91-98).

[0088] The term "operably linked" in the context of nucleic acids refers to the functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, when a promoter or enhancer sequence stimulates or modulates the transcription rate of a coding sequence in an appropriate host cell or other expression system, it is operably linked to the coding sequence. Generally, a promoter transcriptional regulatory sequence operably linked to a transcribed sequence is physically related to the transcribed sequence. For example, when a promoter or enhancer sequence stimulates or modulates the transcription of a coding sequence in an appropriate host cell or other expression system, it is operably linked to the coding sequence. Generally, a promoter transcriptional regulatory sequence operably linked to a transcribed sequence is physically related to the transcribed sequence. related to the transcribed sequence. ​​are contiguous, i.e., they are cis-acting. However, some transcriptional regulatory sequences such as enhancers do not need to be physically contiguous with or located in close proximity to the coding sequences they enhance transcription.

[0089] The terms "polypeptide" and "protein" are used interchangeably in this specification to refer to polymers of amino acid residues. This term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding natural amino acids, as well as to natural and non-natural amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence implicitly encompasses conservatively modified variants thereof.

[0090] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. Unless otherwise noted, the terms "patient" or "subject" are used interchangeably in this specification.

[0091] The term "BKV" or "BK virus" refers to a member of the family Polyomaviridae, genus Orthopolyomavirus. Polyomaviruses are icosahedral non-enveloped double-stranded DNA viruses with a genome of approximately 5,000 base pairs. They are approximately 40 - 45 nM in diameter (Bennett et al., Microbes and Infection. 2012:14(9):672 - 683).

[0092] "JCV" or "JC virus" refers to a member of the Polyomaviridae family, genus Orthopolyomavirus. JCV is related to BKV, which is also an icosahedral non-enveloped double-stranded DNA virus with a genome of approximately 5,000 base pairs. They are about 40-45 nM in diameter (Johne et al., Arch. Virol. 2011;156(9):1627-1634).

[0093] The term "BKV nephropathy" or "BKV-related nephropathy" or "BKVAN" refers mainly to inflammatory interstitial nephropathy resulting from lytic infection by BKV, characterized by viral cytopathic changes and viral gene expression in the renal tubular epithelium.

[0094] The term "VP1" refers to the major polyomavirus capsid subunit protein . "VP1 pentamer" is composed of 5 monomers of VP1.

[0095]

Table 1

[0096] "Virus-like particle" or "VLP" is an assembly of VP1 pentamers in the viral capsid . VLP is composed of 72 VP1 pentamers. VLP is very similar structurally to the actual virus, but lacks the minor capsid proteins (VP2 and VP3) as well as the viral DNA genome, and is therefore non-infectious. VLP is useful because the viral epitopes are presented in a conformation similar to that of the actual virus.

[0097] 「IC50」(half inhibitory concentration) refers to the concentration of a specific antibody that induces a signal midway (50%) between the baseline control and the maximum possible signal. For example, the IC50 is the concentration of an antibody at which 50% of the available binding sites on the VP1 antigen are occupied.

[0098] 「EC50」(half maximal effective concentration) refers to the concentration of a specific antibody that induces a response midway (50%) between the baseline control and the maximum possible effect after a specific exposure or treatment time. For example, the EC50 is the concentration of an antibody at which 50% of the virus infection is neutralized.

[0099] 「EC90」refers to the concentration of a specific antibody that induces a response corresponding to 90% of the maximum possible effect after a specific exposure or treatment time. For example, the EC90 is the concentration of an antibody at which 90% of the virus infection is neutralized.

[0100] 「Neutralization」refers to the inhibition of viral infection of host cells as indicated by the absence of viral gene expression. Without being bound by any theory, the mechanism of neutralization by a specific antibody can be to block the interaction between the viral capsid protein and the cell surface receptor or disrupt the entry and transport processes at any stage before the delivery of the viral genome to the nucleus of the host cell.

[0101] As used herein, the terms "treat," "treating," or "treatment" of any disease or disorder, in one aspect, refer to ameliorating the disease or disorder (i.e., slowing, stopping, or reducing the occurrence of the disease or at least one of its clinical symptoms). In another aspect, "treat," "treating," or "treatment" ​​​​​​​​​​​​​"Reducing" refers to reducing at least one physical parameter, including those not recognizable by the patient. In yet another aspect, "treating", "treatment" or "treat" refers to modulating a disease or disorder physically (e.g., stabilizing recognizable symptoms), physiologically (e.g., stabilizing physical parameters), or both. The phrase "reducing the likelihood" refers to delaying the onset, occurrence or progression of a disease, infection or disorder. The terms "therapeutically acceptable amount" or "therapeutically effective dose" interchangeably refer to an amount sufficient to obtain a desired result (i.e., reduction of tumor size, inhibition of tumor growth, prevention of metastasis, inhibition or prevention of viral, bacterial, fungal or parasitic infection). In some aspects, the therapeutically acceptable amount does not induce or cause undesirable side effects. The therapeutically acceptable amount can be determined by initially administering a low dose and then gradually increasing the dose until the desired effect is achieved. The disclosed molecules of "prophylactically effective dose" and "therapeutically effective dose" can each prevent the onset or result in a reduction in the severity of disease symptoms such as those associated with polyomavirus infection.

[0102] The phrase "administering concomitantly" refers to the concurrent presence of two active agents in an individual's blood. The active agents to be administered concomitantly can be delivered simultaneously or sequentially.

[0103]

[0104] BRIEF DESCRIPTION OF THE DRAWINGS

[0105]

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[0106] Detailed description The present disclosure relates to antibodies and antibody fragments (e.g., antigen-binding fragments) that bind to and neutralize BKV ) is provided. In particular, the present disclosure relates to antibodies and antibody fragments (e.g., antigen-binding fragments) that bind to the VP1 protein and neutralize viral infection upon such binding. Further, the present disclosure provides antibodies having desirable pharmacokinetic properties and other desirable attributes and thus capable of reducing or treating the likelihood of BK virus-related nephropathy (e.g., BKVAN). The present disclosure further provides pharmaceutical compositions containing the antibodies, as well as methods of making and using such pharmaceutical compositions for the prevention and treatment of polyomavirus infection and related disorders. Anti-VP1 Antibodies The present disclosure provides antibodies or antibody fragments (e.g., antigen-binding fragments) that specifically bind to VP1. Antibodies or antibody fragments (e.g., antigen-binding fragments) of the present disclosure include, but are not limited to, human monoclonal antibodies or fragments thereof isolated as described in the following examples. In certain embodiments, the present disclosure provides an antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to VP1 and that comprises a VH domain having an amino acid sequence set forth in SEQ ID NOs: 12, 32, 52, 72, 92, 112, 132, 152, 172, 192, 212, 232, 252, 272, 292, 312, 328, 348, 362, 376, 390, 404, 418, 432, 446, 460, 474, and 488 (Table 2). The present disclosure also provides an antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to VP1 and that is listed in Table 2.

[0107] In certain embodiments, the present disclosure provides an antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to VP1. Antibodies or antibody fragments (e.g., antigen-binding fragments) of the present disclosure include, but are not limited to, human monoclonal antibodies or fragments thereof isolated as described in the following examples. In certain embodiments, the present disclosure provides an antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to VP1 and that comprises a VH domain having an amino acid sequence set forth in SEQ ID NOs: 12, 32, 52, 72, 92, 112, 132, 152, 172, 192, 212, 232, 252, 272, 292, 312, 328, 348, 362, 376, 390, 404, 418, 432, 446, 460, 474, and 488 (Table 2). The present disclosure also provides an antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to VP1 and that is listed in Table 2.

[0108] In certain embodiments, the present disclosure provides an antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to VP1 and that comprises a VH domain having an amino acid sequence set forth in SEQ ID NOs: 12, 32, 52, 72, 92, 112, 132, 152, 172, 192, 212, 232, 252, 272, 292, 312, 328, 348, 362, 376, 390, 404, 418, 432, 446, 460, 474, and 488. The present disclosure also provides an antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to VP1 and that is listed in Table 2. In certain embodiments, the present disclosure provides an antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to VP1 and that comprises a VH domain having an amino acid sequence set forth in SEQ ID NOs: 12, 32, 52, 72, 92, 112, 132, 152, 172, 192, 212, 232, 252, 272, 292, 312, 328, 348, 362, 376, 390, 404, 418, 432, 446, 460, 474, and 488. The present disclosure also provides an antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to VP1 and that is listed in Table 2. In certain embodiments, the present disclosure provides an antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to VP1 and that The antibody also includes a VH CDR having any one of the amino acid sequences of the VH CDRs or an antibody fragment (e.g., an antigen-binding fragment). In certain embodiments, the disclosure provides an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to VP1 and has one, two, three or more VH CDRs having the amino acid sequence of any of the VH CDRs listed in Table 2 below (or alternatively, consisting of). The disclosure provides the antibody.

[0109] The disclosure provides an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to VP1 and includes a VL domain having the amino acid sequence of SEQ ID NOs: 22, 42, 62, 82, 102, 122, 142, 162, 182 202, 222, 242, 262, 282, 302, 320, 338, 355, 369 383, 397, 411, 425, 439, 453, 467, 481, and 495 (Table 2). The disclosure also provides an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to VP1 and includes a VL CDR having any one of the amino acid sequences of the VL CDRs listed in Table 2 below. In particular, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to VP1 and includes one, two, three or more VL CDRs having the amino acid sequence of any of the VL CDRs listed in Table 2 below (or alternatively, consisting of) is provided. Specifically, an antibody or antibody fragment (e.g., an antigen-binding fragment) that specifically binds to VP1 is provided. The antibody or antibody fragment (e.g., an antigen-binding fragment) includes one, two, three or more VL CDRs having the amino acid sequence of any of the VL CDRs listed in Table 2 or alternatively, consisting of.

[0110] Other antibodies or antibody fragments (e.g., antigen-binding fragments) of the disclosure are mutated but are shown in Table The CDR regions described in the sequences described in 2, and amino acids having at least 60, 70, 80 , 90 or 95 percent identity. In some embodiments, they are amino acid sequence variants in which 1, 2, 3, 4 or 5 or fewer amino acids are mutated in the CDR region, when compared to the CDR regions described in the sequences described in Table 2.

[0111] The present disclosure also provides nucleic acid sequences encoding VH, VL, full-length heavy chains, and full-length light chains of antibodies that specifically bind to VP1. Such nucleic acid sequences can be optimized for expression in mammalian cells.

[0112]

Table 2-1

[0113]

Table 2-2

[0114]

Table 2-3

[0115]

Table 2-4

[0116]

Table 2-5

[0117]

Table 2-6

[0118]

Table 2-7

[0119]

Table 2-8

[0120]

Table 2-9

[0121]

Table 2-10

[0122]

Table 2-11

[0123]

Table 2-12

[0124]

Table 2-13

[0125]

Table 2-14

[0126]

Table 2-15

[0127]

Table 2-16

[0128]

Table 2-17

[0129]

Table 2-18

[0130]

Table 2-19

[0131]

Table 2-20

[0132]

Table 2-21

[0133]

Table 2-22

[0134]

Table 2-23

[0135]

Table 2-24

[0136]

Table 2-25

[0137]

Table 2-26

[0138]

Table 2-27

[0139]

Table 2-28

[0140]

Table 2-29

[0141]

Table 2-30

[0142]

Table 2-31

[0143]

Table 2-32

[0144]

Table 2-33

[0145]

Table 2-34

[0146]

Table 2-35

[0147]

Table 2-36

[0148]

Table 2-37

[0149]

Table 2-38

[0150]

Table 2-39

[0151]

Table 2-40

[0152]

Table 2-41

[0153]

Table 2-42

[0154]

Table 2-43

[0155]

Table 2-44

[0156]

Table 2-45

[0157]

Table 2-46

[0158]

Table 2-47

[0159]

Table 2-48

[0160]

Table 2-49

[0161]

Table 2-50

[0162]

Table 2-51

[0163]

Table 2-52

[0164]

Table 2-53

[0165]

Table 2-54

[0166]

Table 2-55

[0167]

Table 2-56

[0168]

Table 2-57

[0169]

Table 2-58

[0170] Other antibodies of the present disclosure include those in which the amino acids or the nucleic acids encoding the amino acids are mutated but have at least 60, 70, 80, 90 or 95 percent identity to the sequences listed in Table 2. In some embodiments, it has 1, 2, 3, 4 or 5 or fewer amino acids in the variable region mutated compared to the variable regions described in the sequences listed in Table 2, but includes amino acid sequence variants that retain substantially the same therapeutic activity

[0171] Since these antibodies can each bind to VP1, VH, VL, full-length light chain , and full-length heavy chain sequences (amino acid sequences and nucleotide sequences encoding the amino acid sequences ) can be "mixed and matched" to generate other VP1-binding antibodies. Such "mixed and matched" VP1-binding antibodies can be tested using binding assays known in the art (e.g., ELISA, and other assays described in the Examples section ). When mixing and matching these chains, the VH sequence derived from a particular VH / VL pair should be replaced with a structurally similar VH sequence. Similarly, the full-length heavy chain sequence derived from a particular full-length heavy chain / full-length light chain pair should be replaced with a structurally similar full-length heavy chain sequence . Similarly, the VL sequence derived from a particular VH / VL pair should be replaced with a structurally similar VL sequence. Similarly, the full-length light chain sequence derived from a particular full-length heavy chain / full-length light chain pair should be replaced with a structurally similar full-length light chain sequence. Thus ​​​​Thus, in one aspect, the present disclosure provides a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 32, 52, 72, 92, 112, 132, 152, 172, 192, 212, 232, 252, 272, 292, 312, 328, 348, 362, 376, 390, 404, 418, 432, 446, 460, 474, and 488 (Table 2); and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, 302, 320, 338, 355, 369, 383, 397, 411, 425, 439, 453, 467, 481, and 495 (Table 2), wherein the antibody specifically binds to VP1; or an antigen-binding region thereof. 2, 72, 92, 112, 132, 152, 172, 192, 212, 232, 252, 272, 292, 312, 328, 348, 362, 376, 390, 404, 418, 432, 446, 460, 474, and 488 (Table 2); and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 42, 62, 82, 102, 122 , 142, 162, 182, 202, 222, 242, 262, 282, 302, 320 , 338, 355, 369, 383, 397, 411, 425, 439, 453, 467 , 481 and 495 (Table 2), or an antigen-binding region thereof.

[0172] In another aspect, the present disclosure provides a monoclonal antibody or a functional protein comprising its antigen-binding portion, wherein the monoclonal antibody has a full-length heavy chain comprising an amino acid sequence optimized for expression in mammalian cells and selected from the group consisting of SEQ ID NOs: 14, 34, 54, 74, 94, 114, 134, 154, 174, 194, 214, 234, 254, 274, 294, 313, and 330; and a full-length light chain comprising an amino acid sequence optimized for expression in mammalian cells and selected from the group consisting of SEQ ID NOs: 24, 44, 64, 84, 104, 124, 144, 164, 184, 204, 224, 244, 264, 284, 304, 321, and 340. 34, 154, 174, 194, 214, 234, 254, 274, 294, 313 and 330; and a full-length light chain comprising an amino acid sequence optimized for expression in mammalian cells and selected from the group consisting of SEQ ID NOs: 24, 44, 64, 84, 104, 124, 144, 164, 184, 204, 224, 244, 264, 284, 304, 321, 340; or (ii) its antigen binding portion.

[0173] In another aspect, the present disclosure provides a monoclonal antibody having heavy and light chain CDR1, CDR2, and CDR3 as set forth in Table 2. Provided is a VP1-binding antibody comprising R3, or a combination thereof. The amino acid sequence of the VH CDR1 of the antibody is set forth in SEQ ID NOs: 6, 26, 46, 66, 86, 106, 126, 146, 166, 186, 206, 226, 246, 266, 286, 306, 322, 342, 356, 370, 384, 398, 412, 426, 440, 454, 468, and 482. The amino acid sequence of the VH CDR2 of the antibody is set forth in SEQ ID NOs: 7, 27, 47, 67, 87, 1 07, 127, 147, 167, 187, 207, 227, 247, 267, 287, 3 07, 323, 343, 357, 371, 385, 399, 413, 427, 441, 4 55, 469, and 483. The amino acid sequence of the VH CDR3 of the antibody is set forth in SEQ ID NOs: 8, 28, 48, 68, 88, 108, 128, 148, 168, 188, 208, 22 8, 248, 268, 288, 308, 324, 344, 358, 372, 386, 40 0, 414, 428, 442, 456, 470, and 484. The amino acid sequence of the VL CD R1 of the antibody is set forth in SEQ ID NOs: 16, 36, 56, 76, 96, 116, 136, 15 6, 176, 196, 216, 236, 256, 276, 296, 314, 332, 34 9, 363, 377, 391, 405, 419, 433, 447, 461, 475 and 489. The amino acid sequence of the VL CDR2 of the antibody is set forth in SEQ ID NOs: 17, 37, 57, 7 7, 97, 117, 137, 157, 177, 197, 217, 237, 257, 277 、297, 315, 333, 350, 364, 378, 392, 406, 420, 434 、448, 462, 476 and 490. The amino acid sequence of the VL CDR3 of the antibody is 、SEQ ID NOs: 18, 38, 58, 78, 98, 118, 138, 158, 178, 198, 、SEQ ID NOs: 18, 38, 58, 78, 98, 118, 138, 158, 178, 198, 、SEQ ID NOs: 18, 38, 58, 78, 98, 118, 138, 158, 178, 198, 218, 238, 258, 278, 298, 316, 334, 351, 365, 379, as shown in 393, 407, 421, 435, 449, 463, 477 and 491.

[0174] These antibodies can each bind to VP1, and considering that the antigen-binding specificity is mainly provided by the CDR1 , 2 and 3 regions, the VH CDR1, 2 and 3 sequences and the VL CDR1, 2 and 3 sequences can be "mixed and matched" (i.e., the CDRs from different antibodies can be mixed and matched, but each antibody contains VH CDR1, 2 and 3 and VL CDR1, 2 and 3, and other VP1-binding binding molecules must be created). Such "mixed and matched" V P1-binding antibodies can be tested using binding assays known in the art and the assays described in the examples (e.g., ELISA). When mixing and matching VH CDR sequences, the CDR1, CDR2 and / or CDR3 sequences derived from a particular VH sequence should be replaced with structurally similar CDR sequence(s). Similarly, when mixing and matching VL CDR sequences, the CDR1, CDR2 and / or CDR3 sequences derived from a particular VL sequence should be replaced with structurally similar CDR sequence(s) . It will be readily apparent to those skilled in the art that new VH and VL sequences can be created by replacing one or more VH and / or VL CDR region sequences with structurally similar sequences derived from the CDR sequences shown herein for the monoclonal antibodies of the present disclosure.

[0175] ​​​Accordingly, the present disclosure provides an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 26, 46, 66, 86, 106, 126, 146, 166, 186, 206, 226, 246, 26 6, 286, 306, 322, 342, 356, 370, 384, 398, 412, 42 6, 440, 454, 468, and 482; for the heavy chain CDR1; SEQ ID NO: 7, 27, 47, 67, 87, 107, 127, 147, 1 67, 187, 207, 227, 247, 267, 287, 307, 323, 343, 3 57, 371, 385, 399, 413, 427, 441, 455, 469, and 48 3; for the heavy chain CDR2; SEQ ID NO: 8, 28, 4 8, 68, 88, 108, 128, 148, 168, 188, 208, 228, 248, 268, 288, 308, 324, 344, 358, 372, 386, 400, 414, 428, 442, 456, 470, and 484; for the heavy chain CDR3; SEQ ID NO: 16, 36, 56, 76, 96, 116, 136, 15 6, 176, 196, 216, 236, 256, 276, 296, 314, 332, 34 9, 363, 377, 391, 405, 419, 433, 447, 461, 475 and 489; for the light chain CDR1; SEQ ID NO: 17, 3 7, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237, 2 57, 277, 297, 315, 333, 350, 364, 378, 392, 406, 4 20, 434, 448, 462, 476 and 490; for the light chain CDR2; as well as SEQ ID NO: 18, 38, 58, 78, 98, 118, 57, 277, 297, 315, 333, 350, 364, 378, 392, 406, 4 20, 434, 448, 462, 476 and 490; for the light chain CDR2; as well as SEQ ID NO: 18, 38, 58, 78, 98, 118, 57, 277, 297, 315, 333, 350, 364, 378, 392, 406, 4 138, 158, 178, 198, 218, 238, 258, 278, 298, 316, 334, 351, 365, 379, 393, 407, 421, 435, 449, 463, including a light chain CDR3 comprising an amino acid sequence selected from the group consisting of 477 and 491; provided is an isolated monoclonal antibody or an antigen-binding region thereof.

[0176] In certain embodiments, an antibody that specifically binds to VP1 is an antibody or antibody fragment (e.g., an antigen-binding fragment) described in Table 2.

[0177] 1. Identification of Epitopes and Antibodies that Bind to the Same Epitopes The present disclosure provides antibodies and antibody fragments (e.g., antigen-binding fragments) that bind to epitopes of VP1. In certain embodiments, the antibodies and antibody fragments can bind to the same epitopes within all four BKV serotypes and / or JCV.

[0178] The present disclosure also provides antibodies and antibody fragments (e.g., antigen-binding fragments) that bind to the same epitopes as the anti-VP1 antibodies described in Table 2. Thus, additional antibodies and antibody fragments (e.g., antigen-binding fragments) can be identified based on their ability to cross-compete with other antibodies in a binding assay (e.g., competitively inhibit their binding in a statistically significant manner). The ability of a test antibody to inhibit the binding of the antibodies and antibody fragments (e.g., antigen-binding fragments) of the present disclosure to VP1 (e.g., human BKV or JCV VP1) indicates that the test antibody can compete with the antibody or antibody fragment (e.g., antigen-binding fragment) for binding to VP1; such an antibody, according to non-limiting theory, is the antibody with which it competes. and antibody fragments (e.g., antigen-binding fragments) of the present disclosure to VP1; such an antibody, according to non-limiting theory, is the antibody with which it competes. can compete with the antibody or antibody fragment (e.g., antigen-binding fragment) for binding to VP1; such an antibody, according to non-limiting theory, is the antibody with which it competes. or an antibody fragment (e.g., an antigen-binding fragment) that can bind to the same or a related (e.g., structurally similar or spatially proximate) epitope on VP1. In certain embodiments, an antibody that binds to the same epitope on VP1 as the antibody or antibody fragment (e.g., antigen-binding fragment) of the present disclosure is a human or humanized monoclonal antibody. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.

[0179] 2. Further Modifications of the Framework of the Fc Region The present disclosure has disclosed specific anti-VP1 antibodies. These antibodies may further include modified antibodies or antigen-binding fragments thereof that include modifications to framework residues within VH and / or VL, for example, to improve antibody properties. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "revert" one or more framework residues to their germline sequences. More specifically, an antibody that has undergone somatic mutations may contain framework residues different from the germline sequences from which the antibody was derived. By comparing the antibody framework sequence to the germline sequences from which the antibody was derived, such residues can be identified. To revert the framework region sequence to its germline configuration, somatic mutations can be "reverted" to germline sequences, for example, by site-directed mutagenesis. Such "reverted" antibodies are also intended to be included.

[0180] Another type of framework modification is within the framework region or even further, at one or ​Mutating one or more residues within multiple CDR regions to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in more detail in U.S. Patent Application Publication No. 2003 / 0153043 by Carr et al. This includes reducing the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in more detail in U.S. Patent Application Publication No. 2003 / 0153043 by Carr et al. This approach is also referred to as "deimmunization" and is described in more detail in U.S. Patent Application Publication No. 2003 / 0153043 by Carr et al. This approach is also referred to as "deimmunization" and is described in more detail in U.S. Patent Application Publication No. 2003 / 0153043 by Carr et al.

[0181] In addition to, or instead of, modifications made within the framework or CDR regions, the antibody can be engineered to change one or more functional properties of the antibody, typically including serum half-life, complement fixation, Fc receptor binding, and / or antibody-dependent cell-mediated cytotoxicity, by including modifications within the Fc region. Additionally, the antibody can be chemically modified (e.g., one or more chemical moieties can be conjugated to the antibody) or modified to change its glycosylation, again changing one or more functional properties of the antibody. These aspects are described in more detail below. In addition to, or instead of, modifications made within the framework or CDR regions, the antibody can be engineered to change one or more functional properties of the antibody, typically including serum half-life, complement fixation, Fc receptor binding, and / or antibody-dependent cell-mediated cytotoxicity, by including modifications within the Fc region. Additionally, the antibody can be chemically modified (e.g., one or more chemical moieties can be conjugated to the antibody) or modified to change its glycosylation, again changing one or more functional properties of the antibody. These aspects are described in more detail below. In addition to, or instead of, modifications made within the framework or CDR regions, the antibody can be engineered to change one or more functional properties of the antibody, typically including serum half-life, complement fixation, Fc receptor binding, and / or antibody-dependent cell-mediated cytotoxicity, by including modifications within the Fc region. Additionally, the antibody can be chemically modified (e.g., one or more chemical moieties can be conjugated to the antibody) or modified to change its glycosylation, again changing one or more functional properties of the antibody. These aspects are described in more detail below. In addition to, or instead of, modifications made within the framework or CDR regions, the antibody can be engineered to change one or more functional properties of the antibody, typically including serum half-life, complement fixation, Fc receptor binding, and / or antibody-dependent cell-mediated cytotoxicity, by including modifications within the Fc region. Additionally, the antibody can be chemically modified (e.g., one or more chemical moieties can be conjugated to the antibody) or modified to change its glycosylation, again changing one or more functional properties of the antibody. These aspects are described in more detail below. In addition to, or instead of, modifications made within the framework or CDR regions, the antibody can be engineered to change one or more functional properties of the antibody, typically including serum half-life, complement fixation, Fc receptor binding, and / or antibody-dependent cell-mediated cytotoxicity, by including modifications within the Fc region. Additionally, the antibody can be chemically modified (e.g., one or more chemical moieties can be conjugated to the antibody) or modified to change its glycosylation, again changing one or more functional properties of the antibody. These aspects are described in more detail below. In addition to, or instead of, modifications made within the framework or CDR regions, the antibody can be engineered to change one or more functional properties of the antibody, typically including serum half-life, complement fixation, Fc receptor binding, and / or antibody-dependent cell-mediated cytotoxicity, by including modifications within the Fc region. Additionally, the antibody can be chemically modified (e.g., one or more chemical moieties can be conjugated to the antibody) or modified to change its glycosylation, again changing one or more functional properties of the antibody. These aspects are described in more detail below. In addition to, or instead of, modifications made within the framework or CDR regions, the antibody can be engineered to change one or more functional properties of the antibody, typically including serum half-life, complement fixation, Fc receptor binding, and / or antibody-dependent cell-mediated cytotoxicity, by including modifications within the Fc region. Additionally, the antibody can be chemically modified (e.g., one or more chemical moieties can be conjugated to the antibody) or modified to change its glycosylation, again changing one or more functional properties of the antibody. These aspects are described in more detail below.

[0182] In one aspect, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region changes, e.g., increases or decreases. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. Changing the number of cysteine residues in the hinge region of CH1 can facilitate the assembly of the light and heavy chains, or increase or decrease the stability of the antibody. In one aspect, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region changes, e.g., increases or decreases. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. Changing the number of cysteine residues in the hinge region of CH1 can facilitate the assembly of the light and heavy chains, or increase or decrease the stability of the antibody. In one aspect, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region changes, e.g., increases or decreases. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. Changing the number of cysteine residues in the hinge region of CH1 can facilitate the assembly of the light and heavy chains, or increase or decrease the stability of the antibody. In one aspect, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region changes, e.g., increases or decreases. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. Changing the number of cysteine residues in the hinge region of CH1 can facilitate the assembly of the light and heavy chains, or increase or decrease the stability of the antibody. In one aspect, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region changes, e.g., increases or decreases. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. Changing the number of cysteine residues in the hinge region of CH1 can facilitate the assembly of the light and heavy chains, or increase or decrease the stability of the antibody.

[0183] In another aspect, the Fc hinge region of the antibody is mutated to decrease the biological half-life of the antibody. More particularly, the antibody has a weaker Staphylococcus protein A (SpA) binding compared to the native Fc-hinge domain. In another aspect, the Fc hinge region of the antibody is mutated to decrease the biological half-life of the antibody. More particularly, the antibody has a weaker Staphylococcus protein A (SpA) binding compared to the native Fc-hinge domain. One or more amino acid mutations are introduced into the CH2-CH3 domain boundary region of the Fc-hinge fragment such that it has binding to Staphylococcyl protein A (SpA). This approach is described in more detail in U.S. Patent No. 6,165,745 by Ward et al. This approach is described in more detail in U.S. Patent No. 6,165,745 by Ward et al. This approach is described in more detail in U.S. Patent No. 6,165,745 by Ward et al.

[0184] In yet another aspect, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to change the effector function of the antibody. For example, one or more amino acids can be replaced with different amino acid residues such that the antibody has an altered affinity for an effector ligand, while retaining the antigen-binding ability of the parental antibody. The effector ligand whose affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described, for example, in both U.S. Patent Nos. 5,624,821 and 5,648,260 by Winter et al. This approach is described, for example, in both U.S. Patent Nos. 5,624,821 and 5,648,260 by Winter et al. This approach is described, for example, in both U.S. Patent Nos. 5,624,821 and 5,648,260 by Winter et al. This approach is described, for example, in both U.S. Patent Nos. 5,624,821 and 5,648,260 by Winter et al. This approach is described, for example, in both U.S. Patent Nos. 5,624,821 and 5,648,260 by Winter et al.

[0185] In another aspect, one or more amino acid residues selected from amino acid residues can be replaced with different amino acid residues such that the antibody has altered C1q binding and / or reduced or abrogated complement-dependent cytotoxicity (CDC). In another aspect, one or more amino acid residues selected from amino acid residues can be replaced with different amino acid residues such that the antibody has altered C1q binding and / or reduced or abrogated complement-dependent cytotoxicity (CDC). In another aspect, one or more amino acid residues selected from amino acid residues can be replaced with different amino acid residues such that the antibody has altered C1q binding and / or reduced or abrogated complement-dependent cytotoxicity (CDC).

[0186] In yet another aspect, the ability of the antibody to fix complement is altered by changing one or more amino acid residues. This approach is described, for example, in PCT International Publication No. 94 / 29351 by Bodmer et al. In yet another aspect, the ability of the antibody to fix complement is altered by changing one or more amino acid residues. This approach is described, for example, in PCT International Publication No. 94 / 29351 by Bodmer et al. In certain aspects, the antibodies of the disclosure or replace one or more amino acids of the antigen-binding fragment thereof with one or more allotype amino acid residues to IgG1 subclass and kappa isotype. Allotype amino acid residues include, but are not limited to, the constant regions of the heavy chains of IgG1, IgG2 and IgG3 subclasses, as well as the constant regions of the light chains of kappa isotype as described by Jefferis et al., MAbs. 1:332-338 (2009).

[0187] In yet another aspect, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to modify one or more amino acids to increase the affinity of the antibody for Fcγ receptors. This approach is described, for example, in PCT International Publication No. 00 / 42072 pamphlet by Pres ta. Furthermore, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn have been mapped and variants with improved binding are described (see Shield s et al., J. Biol. Chem. 276:6591-6604, 2001).

[0188] In yet another aspect, the glycosylation of the antibody is modified. For example, an aglycosylated antibody can be produced (i.e., the antibody lacks glycosylation). Changing the glycosylation can, for example, increase the affinity of the antibody for the "antigen". Such carbohydrate modifications can be achieved, for example, by changing one or more glycosylation sites within the antibody sequence by, for example, changing one or more variable region framework glycosylation By making one or more amino acid substitutions that result in the elimination of the ligation site, Such aglycosylation can be used to enhance the binding of the nucleotides to antigens. Such techniques can be used to increase the affinity of antibodies to the target molecule. Nos. 5,714,350 and 6,350,861. It is being done.

[0189] Additionally or alternatively, hypofucosylated antibodies or bisense antibodies having a reduced amount of fucosyl residues are provided. Antibodies that have an altered type of glycosylation, such as antibodies with increased glycosylation GlcNac structures. Such altered glycosylation patterns can be used to modify the AD of antibodies. Such carbohydrate modifications have been shown to increase CC capacity. This can be achieved by expressing the antibody in a host cell with an altered cosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used to express recombinant antibodies. By this, the host cells can be used to produce antibodies with altered glycosylation. For example, European Patent No. 1,176,195 by Hang et al. The present invention describes a cell line in which the FUT8 gene encoding the transferase is functionally disrupted, Antibodies expressed in such cell lines exhibit hypoglycosylation. Publication No. 03 / 035835 discloses a method for the preparation of a cyclohexyl 1-amino-2-phenylindole (C11)-substituted cyclohexyl 1-(2-methyl-1,2-diphenyl-2-propanediol) having fucose linked to Asn(297). The ability to bind carbohydrates is reduced, resulting in hypofucosylation of antibodies expressed in the host cell. have described a variant CHO cell line, Lecl3 cells, which also produces , (see also J. Biol. Chem. 277:26733-26740 (2002)). PC by Umama et al. WO 99 / 54342 describes a cell line engineered to express an antibody showing an increase in bisecting GlcNac structures that result in an increase in the ADCC activity of the antibody such as by expressing a glycoprotein-modifying glycosyltransferase (e.g., beta(1,4)- N-acetylglucosaminyltransferase III (GnTIII)) (see also Umana et al., Nat. Biotech. 17:176-180, 1999 and J. Biol. Chem. 277:26733-26740 (2002)). ).

[0190] In another aspect, the antibody is modified to increase its biological half-life. Various methods are possible. For example, one or more of the following mutations can be introduced as described in Ward U.S. Patent No. 6,277,375: T252L, T254S, T256F . Alternatively, to increase the biological half-life, the antibody can be altered within the CH1 or CL region to contain the salvage receptor binding epitope taken from two loops of the CH2 domain of the Fc region of IgG as described in Presta et al. U.S. Patents Nos. 5,869,046 and 6,121,022. To minimize the ADCC activity of the antibody, specific mutations in the Fc region result in an "Fc silent" antibody that has minimal interaction with effector cells. Generally, the "IgG Fc region" is used to refer to immunoglobulin Fc regions such as the native sequence Fc region and variant Fc regions.

[0191] To minimize the ADCC activity of the antibody, specific mutations in the Fc region result in an "Fc silent" antibody that has minimal interaction with effector cells. Generally, the "IgG Fc region" is used to refer to immunoglobulin Fc regions such as the native sequence Fc region and variant Fc regions. Fc region" is used to refer to immunoglobulin Fc regions such as the native sequence Fc region and variant Fc regions, such as Define the C-terminal region of the heavy chain. The human IgG heavy chain Fc region is generally defined as including the amino acid residues from C226, or from position P230 to the carboxyl terminus. The numbering of the residues in the Fc region is that of the Kabat EU index. The C-terminal lysine (residue K447) of the Fc region can be removed, for example, during antibody production or purification. It is defined as including the amino acid residues from C226, or from position P230 to the carboxyl terminus. The numbering of the residues in the Fc region is that of the Kabat EU index. The C-terminal lysine (residue K447) of the Fc region can be removed, for example, during antibody production or purification.

[0192] Silenced effector functions can be obtained by mutations in the Fc region of the antibody and are described in the art: LALA and N297A (Strohl, W., 2009, Curr. Opin. Biotechnol. vol. 20(6):685-691); and D265A (Baudino et al., 2008, J. Immunol. 181: 6664-69), Heusser et al., see also WO 2012065950 pamphlet. Examples of silent Fc IgG1 antibodies are the LALA variant that includes the L234A and L235A mutations in the IgG1 Fc amino acid sequence. Another example of a silent IgG1 antibody is the DAPA (D265A, P329A) mutation (U.S. Patent No. 6,737,056). Another silent IgG1 antibody includes the N297A mutation and results in a deglycosylated / non-glycosylated antibody. It can be obtained by mutations in the Fc region of the antibody and is described in the art: LALA and N297A (Strohl, W., 2009, Curr. Opin. Biotechnol. vol. 20(6):685-691); and D265A (Baudino et al., 2008, J. Immunol. 181: 6664-69), Heusser et al., see also WO 2012065950 pamphlet. Examples of silent Fc IgG1 antibodies are the LALA variant that includes the L234A and L235A mutations in the IgG1 Fc amino acid sequence. Another example of a silent IgG1 antibody is the DAPA (D265A, P329A) mutation (U.S. Patent No. 6,737,056). Another silent IgG1 antibody includes the N297A mutation and results in a deglycosylated / non-glycosylated antibody. Another example of a silent IgG1 antibody is the DAPA (D265A, P329A) mutation (U.S. Patent No. 6,737,056). Another silent IgG1 antibody includes the N297A mutation and results in a deglycosylated / non-glycosylated antibody. See also WO 2012065950 pamphlet. Examples of silent Fc IgG1 antibodies are the LALA variant that includes the L234A and L235A mutations in the IgG1 Fc amino acid sequence. It is the LALA variant that includes the L234A and L235A mutations in the IgG1 Fc amino acid sequence. Another example of a silent IgG1 antibody is the DAPA (D265A, P329A) mutation (U.S. Patent No. 6,737,056). Another silent IgG1 antibody includes the N297A mutation and results in a deglycosylated / non-glycosylated antibody. It includes the N297A mutation and results in a deglycosylated / non-glycosylated antibody.

[0193] Fc-silent antibodies have no or low ADCC activity, which means that the Fc-silent antibody has less than 50% specific cell lysis (low ADCC activity) or less than 1% specific cell lysis (no ADCC activity). It means that the Fc-silent antibody has less than 50% specific cell lysis (low ADCC activity) or less than 1% specific cell lysis (no ADCC activity).

[0194] 3. Generation of anti-VP1 antibodies​​ Although not limited thereto, recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers and other means known in the art can be used to generate anti-VP1 antibodies and antibody fragments thereof (e.g., antigen-binding fragments), but full-length monoclonal antibodies can be obtained, for example, by hybridoma or recombinant production. Recombinant expression can be carried out in any suitable host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[0195] The present disclosure further provides a polynucleotide encoding the antibody described herein, for example, a polynucleotide encoding a segment containing a heavy or light chain variable region or a complementarity-determining region as described herein. In some embodiments, the polynucleotide encoding the heavy chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NOs: 13, 33, 53, 73, 93, 113, 133, 15 3, 173, 193, 213, 233, 253, 273, and 293. In some embodiments, the polynucleotide encoding the light chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NOs: 23, 43, 63, 83, 103, 123, 143, 163, 183, 203, 22 3, 243, 263, 283, and 303. % nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NOs: 23, 43, 63, 83, 103, 123, 143, 163, 183, 203, 22 3, 243, 263, 283, and 303. % nucleic acid sequence identity. % nucleic acid sequence identity.

[0196] In some embodiments, the polynucleotide encoding the heavy chain is at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical in nucleic acid sequence to the polynucleotides of SEQ ID NOs: 15, 35, 55, 75, 95, 115, 135, 155, 175, 195, 215, 235, 255, 275, and 295. In some embodiments, the polynucleotide encoding the light chain is at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99%, or 100% identical in nucleic acid sequence to the polynucleotides of SEQ ID NOs: 25, 45, 65, 85, 105, 125, 145, 165, 185, 205, 225, 245, 265, 285, and 305.

[0197] The polynucleotides of the disclosure may encode only the variable region sequences of the anti-VP1 antibody. They may also encode both the variable and constant regions of the antibody. Some poly nucleotide sequences encode polypeptides that include the variable regions of both the heavy and light chains of an exemplary anti-VP1 antibody. Some other polynucleotides encode two polypeptide segments that are each substantially identical to the variable regions of one heavy chain and one light chain of a murine antibody.

[0198] The polynucleotide sequences can be generated by de novo solid-phase DNA synthesis of an existing sequence encoding an anti-VP1 antibody or a binding fragment thereof (e.g., the sequences described in the Examples below), or by PCR mutagenesis. Narang et al., Meth. Enzymol. 68 : 90, Phosphotriester method of 1979; Phospho of Brown et al., Meth. Enzymol. 68:109, 1979 Phosphoester method; Diethylphosphoroamidite method of Beaucage et al., Tetra. Lett., 22:1859, 1981 Midite method; and methods known in the art such as the solid support method of U.S. Patent No. 4,458,066, etc., can achieve the direct chemical synthesis of nucleic acids. Introduction of mutations into the polynucleotide sequence by PCR can be carried out, for example, as described in PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991. It can be carried out as follows. For the introduction of mutations into the polynucleotide sequence by PCR, for example, as described in PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991. tions for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Pr ess, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and also as described in Eckert et al., PCR Methods and Applications 1:17, 1991. It can be carried out.

[0199] Also, expression vectors and host cells for generating the above anti-VP1 antibody are provided in the present disclosure. Various expression vectors can be used to express polynucleotides encoding anti-VP1 antibody chains or binding fragments. Antibodies can be generated in mammalian host cells using both virus-based expression vectors and non-viral expression vectors. Non-viral vectors and systems typically involve proteins or RNAs. It can be carried out. Using both virus-based expression vectors and non-viral expression vectors, antibodies can be generated in mammalian host cells. Using both virus-based expression vectors and non-viral expression vectors, antibodies can be generated in mammalian host cells. Non-viral vectors and systems typically involve proteins or RNAs. Plasmids, episomal vectors, and human artificial chromosomes (see, e.g., Harrington et al., Nat Genet 15:345, 1997) that contain an expression cassette for expression are exemplified. For example, non-viral vectors useful for the expression of anti-VP1 polynucleotides and polypeptides in mammalian (e.g., human) cells include pThioHis A, B, and C, pcDNA3.1 / His, pEBVHisA, B, and C (Invi trogen, San Diego, CA), MPSV vectors, and several other vectors known in the art for expressing other proteins. Useful viral vectors include retroviruses, adenoviruses, adeno-associated viruses, herpes virus-based vectors, SV40-based vectors, papillomaviruses, HBP Epstein-Barr virus, vaccinia virus vectors, and Semliki Forest virus (SFV). See Brent et al., supra; Smith, Annu. Rev. Microbiol. 49:8 07, 1995; and Rosenfeld et al., Cell 68:143, 1992.

[0200] The choice of expression vector depends on the host cell in which the vector is intended to be expressed. Typically, the expression vector contains a promoter and other regulatory sequences (e.g., enhancers) operably linked to a polynucleotide encoding an anti-VP1 antibody chain or fragment. In some embodiments, an inducible promoter is used to prevent expression of the inserted sequence except under inducing conditions. Inducible promoters include, for example, arabinose, lacZ , including a metallothionein promoter or a heat shock promoter. Culturing the transformed organism can be expanded under non-inducing conditions without biasing the population for a coding sequence that is better tolerated by the host cell. In addition to the promoter , other regulatory elements are also necessary or desired for the efficient expression of the anti-VP1 antibody chain or fragment. These elements typically include an ATG start codon and adjacent ribosome binding sites or other sequences. Furthermore, the efficiency of expression can be enhanced by the inclusion of enhancers appropriate for the cell line used (see, for example, Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., M eth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or

[0201] the CMV enhancer can be used to increase expression in mammalian host cells. The expression vector can also provide a secretion signal sequence position for forming a fusion protein with the polypeptide encoded by the inserted anti-VP1 antibody sequence. More frequently, the inserted anti-VP1 antibody sequence is ligated to the signal sequence before being included in the vector. Vectors used to receive sequences encoding the anti-VP1 antibody light and heavy chain variable domains may also encode their constant region or portion. Such vectors allow for the expression of the variable region as a fusion protein with the constant region, resulting in the production

[0202] Host cells for carrying and expressing the anti-VP1 antibody chain can be prokaryotic or eukaryotic and are acceptable. Escherichia coli (E. coli) is one prokaryotic host useful for cloning and expressing the polynucleotides of the present disclosure Another microbial host suitable for use is a bacillus such as Bacillus subtilis, as well as other enteric bacteria such as Salmonella, Serratia, and various Pseudomonas species In these prokaryotic hosts, one of ordinary skill in the art can typically create an expression vector containing an expression control sequence (e.g., origin of replication) compatible with the host cell Furthermore, there are any number of various well-known promoter systems such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system derived from lambda phage The promoter typically controls expression, optionally with an operator sequence, and has a ribosome binding site sequence etc. for initiating and completing transcription and translation Other microorganisms such as yeast can also be used to express the anti-VP1 polypeptide Insect cells combined with a baculovirus vector can also be used In other embodiments, mammalian host cells are used to express and produce the anti-VP1 polypeptides of the present disclosure For example, they can be hybridoma cell lines that express the endogenous immunoglobulin gene (e.g., the myeloma hybridoma clones described in the examples) or exogenous For example, they can be hybridoma cell lines that express the endogenous immunoglobulin gene (e.g., the myeloma hybridoma clones described in the examples) or exogenous For example, they can be hybridoma cell lines that express the endogenous immunoglobulin gene (e.g., the myeloma hybridoma clones described in the examples) or exogenous For example, they can be hybridoma cell lines that express the endogenous immunoglobulin gene (e.g., the myeloma hybridoma clones described in the examples) or exogenous

[0203] In other embodiments, mammalian host cells are used to express and produce the anti-VP1 polypeptides of the present disclosure For example, they are hybridoma cell lines that express the endogenous immunoglobulin gene (e.g., the myeloma hybridoma clones described in the examples) or exogenous -oma cell lines (e.g., the myeloma hybridoma clones described in the examples) or exogenous It may also be a mammalian cell line carrying an expression vector. These may include any normal dying, or normal or abnormal immortal animal or human cells. For example, CH O cell lines, various COS cell lines, HeLa cells, myeloma cell lines, transformed B cells and hybridomas, etc., and several suitable host cell lines capable of secreting intact immunoglobulins have been developed. The use of mammalian tissue cell culture for expressing polypeptides is generally discussed, for example, in Winnacker, From Genes to Clones, VCH Publishers, N.Y., N .Y., 1987. Expression vectors for mammalian host cells may contain expression control sequences such as replication origins, promoters, and enhancers (see, for example, Queen et al., Immunol. Rev. 89:49-68, 1986), as well as necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters may be constitutive, cell type-specific, stage-specific, and / or modifiable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polII I promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter such as the human immediate early CMV promoter, the constitutive CMV promoter, and those in the art are not limited, but include the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polII I promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter such as the human immediate early CMV promoter, the constitutive CMV promoter, and those in the art ​ Examples of known promoter-enhancer combinations are given.

[0204] Methods for introducing an expression vector containing the target polynucleotide sequence vary depending on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation may be used for other cell hosts (generally, see Sambrook et al., supra). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistics, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion with the herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), drug-enhanced uptake of DNA, and ex vivo transfection. For the long-term, high-yield production of recombinant proteins, stable expression is often desirable. For example, a cell line stably expressing an anti-VP1 antibody chain or binding fragment can be prepared using an expression vector containing a viral origin of replication or endogenous expression element and a selectable marker gene. After introduction of the vector, the cells can be grown in enriched medium for 1-2 days and then switched to selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows the growth of cells that successfully express the introduced sequence in the selective medium. Stably transfected cells resistant to the selection can be propagated using tissue culture techniques appropriate for the cell type. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation may be used for other cell hosts (generally, see Sambrook et al., supra). For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation may be used for other cell hosts (generally, see Sambrook et al., supra). For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation may be used for other cell hosts (generally, see Sambrook et al., supra). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistics, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion with the herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), drug-enhanced uptake of DNA, and ex vivo transfection. Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistics, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion with the herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), drug-enhanced uptake of DNA, and ex vivo transfection. Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistics, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion with the herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), drug-enhanced uptake of DNA, and ex vivo transfection. Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistics, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion with the herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), drug-enhanced uptake of DNA, and ex vivo transfection. Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistics, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion with the herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), drug-enhanced uptake of DNA, and ex vivo transfection. For the long-term, high-yield production of recombinant proteins, stable expression is often desirable. For the long-term, high-yield production of recombinant proteins, stable expression is often desirable. For example, a cell line stably expressing an anti-VP1 antibody chain or binding fragment can be prepared using an expression vector containing a viral origin of replication or endogenous expression element and a selectable marker gene. For example, a cell line stably expressing an anti-VP1 antibody chain or binding fragment can be prepared using an expression vector containing a viral origin of replication or endogenous expression element and a selectable marker gene. After introduction of the vector, the cells can be grown in enriched medium for 1-2 days and then switched to selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows the growth of cells that successfully express the introduced sequence in the selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows the growth of cells that successfully express the introduced sequence in the selective medium. Stably transfected cells resistant to the selection can be propagated using tissue culture techniques appropriate for the cell type.

[0205] Therapeutic and diagnostic uses The antibodies, antibody fragments (e.g., antigen-binding fragments) of the present disclosure are useful in various applications such as, but not limited to, polyomavirus infections and diseases. In certain aspects the antibodies, antibody fragments (e.g., antigen-binding fragments) are useful for neutralizing BKV or JCV infections and preventing or treating BK virus nephropathy, e.g., BKVAN. The methods of use may be in vitro, ex vivo, or in vivo methods.

[0206] In one aspect, the antibodies, antibody fragments (e.g., antigen-binding fragments) are useful for detecting the presence of BKV in a biological sample. As used herein, the term "detecting" encompasses quantitative or qualitative detection. In certain aspects, the biological sample includes cells or tissue. In certain aspects, such tissue includes normal and / or cancerous tissue that expresses BKV at higher levels compared to other tissues.

[0207] In one aspect, the present disclosure provides a method for detecting the presence of BKV in a biological sample. In certain aspects, the method includes contacting the biological sample with an anti-VP1 antibody under conditions that allow binding of the antibody to the antigen, and detecting whether a complex is formed between the antibody and the antigen. The biological sample may include, but is not limited to, urine or blood samples.

[0208] Also included is a method for diagnosing a disorder associated with the expression of BKV or JCV virus. In certain aspects, the method includes contacting test cells with an anti-VP1 antibody; the anti-VP1 ​​​​​​​​​Determining the level of BK virus expression (quantitatively or qualitatively) in test cells by detecting the binding of an antibody to BK virus; and comparing the level of infection in the test cells with the level of BK virus infection in control cells (e.g., normal cells or non-BK virus-infected cells of the same tissue origin as the test cells), wherein a higher level of the presence of BK virus in the test cells compared to the control cells indicates the presence of a disorder associated with BK virus infection. In certain embodiments, the test cells are obtained from an individual suspected of having BK virus infection. In certain embodiments, diagnostic or detection methods such as those described above include detecting the binding of an anti-VP1 antibody to BKV-infected cells. An exemplary assay for detecting the binding of an anti-VP1 antibody to BKV-infected cells is the "FACS" assay. Other methods can be used to detect the binding of the anti-VP1 antibody. Such methods include, but are not limited to, antigen-binding assays well known in the art such as Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, fluorescence immunoassay, protein A immunoassay, and immunohistochemistry (IHC). In certain embodiments, the anti-VP1 antibody is labeled. Labels include, but are not limited to, labels or moieties that are directly detectable (fluorescence, chromogenic, high electron density, chemiluminescence, and radioactive labels, etc.), and those that are indirectly detectable, for example, via an enzymatic reaction or molecular interaction.

[0209]

[0210]

[0211] ​​​​​​​​​​​​​​Examples include moieties such as enzymes or ligands.

[0212] In certain embodiments, the anti-VP1 antibody is immobilized on an insoluble matrix. Immobilization serves to separate the anti-VP1 antibody from any BKV or JCV proteins that remain free in solution. This is conveniently accomplished by adsorption to a water-insoluble matrix or surface (Bennich et al., U.S. Patent No. 3,720,760), or by covalent coupling (e.g., using glutaraldehyde cross-linking) to insolubilize the anti-VP1 antibody prior to the assay procedure, or by, for example, immunoprecipitation to insolubilize the anti-VP1 antibody after formation of a complex with BKV or JCV proteins.

[0213] Any of the above-described modes of diagnosis or detection can be carried out using the anti-VP1 antibodies of the present disclosure, instead of or in addition to another anti-VP1 antibody.

[0214] In one aspect, the present disclosure provides a method of treating, reducing the likelihood of, or ameliorating a disease, including administering to a patient an antibody, an antibody fragment (e.g., an antigen-binding fragment). In certain embodiments, the disease treated using the antibody, antibody fragment (e.g., an antigen-binding fragment) is a BK virus or JC virus infection. Examples of BKV and JCV diseases that can be treated and / or prevented include, but are not limited to, nephropathy, hemorrhagic cystitis, progressive multifocal leukoencephalopathy (PML), interstitial renal disease, ureteral stricture, granulocyte neuropathy (GCN), vasculitis, colitis, retinitis. Meningitis, myelitis, and immune reconstitution syndrome (IRIS) are included. In certain embodiments the infection is characterized by BKV or JCV expressing cells to which an anti-VP1 antibody, an antibody fragment (e.g., an antigen-binding fragment) specifically binds.

[0215] The present disclosure provides a method of treating BK virus infection and BKVAN by administering a therapeutically effective amount of an antibody, an antibody fragment (e.g., an antigen-binding fragment). In certain embodiments the subject is human.

[0216] In certain embodiments, a method of reducing BK virus infection comprises administering to a subject a therapeutically effective amount of an antibody or antibody fragment (e.g., an antigen-binding fragment). In certain embodiments the subject is human. In certain embodiments, the subject is immunosuppressed. For an immunosuppressed subject, the amount of immunosuppression may increase or decrease due to the therapeutic effect of the anti-VP1 antibody.

[0217] In certain embodiments, the transplanted tissue is infected with BK virus to which the anti-VP1 antibody binds. Since the incidence of BK infection in the general population is high, in the case of kidney transplantation, the probability that the patient receiving the kidney is BK virus positive, or the donor providing the kidney is BK virus positive, or both are BK virus positive is high. To prevent BKVAN, depending on the seroreactivity of the kidney donor or transplant recipient, an anti-VP1 antibody can be administered to the kidney transplant recipient before and / or after the kidney transplantation procedure. In another embodiment, when the virus is detected in the urine (viremia) or the virus is detected in the blood (viremia), an anti-VP1 antibody can be administered to the patient. 。

[0218] For the treatment of BK or JCV virus infection, the appropriate dosage of an antibody, or an antibody fragment (e.g., an antigen binding fragment), depends on various factors such as the type of infection to be treated, the severity and course of the infection, the responsiveness to the infection, the generation of viral resistance to the therapy, previous therapies, the patient's medical history, etc. The antibody can be administered once, or over a series of treatments lasting from several days to several months, until cure occurs, or a reduction in the infection (e.g., a reduction in viremia or a decrease in damage to the kidney caused by the virus) is achieved. The optimal dosing schedule can be calculated from measurements of drug accumulation in the patient's body and varies according to the relative potency of the individual antibody, or antibody fragment (e.g., an antigen-binding fragment). In certain embodiments, the dosage is 0.01 mg to 10 mg (e.g., 0.01 mg, 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 7 mg, 8 mg, 9 mg , or 10 mg) / kg body weight and can be administered once or more than once per day, per week, per month, or per year. In certain embodiments, the antibodies or antibody fragments of the present disclosure (e.g., antigen-binding fragments) are administered once every two weeks or once every three weeks. A treating physician can estimate the dosing frequency based on the measured half-life and antibody concentration in body fluids or tissues. In certain embodiments, the dosage is 0.01 mg to 10 mg (e.g., 0.01 mg, 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 7 mg, 8 mg, 9 mg , or 10 mg) / kg body weight and can be administered once or more than once per day, per week, per month, or per year. In certain embodiments, the antibodies or antibody fragments of the present disclosure (e.g., antigen-binding fragments) are administered once every two weeks or once every three weeks. A treating physician can estimate the dosing frequency based on the measured half-life and antibody concentration in body fluids or tissues. , or 10 mg) / kg body weight and can be administered once or more than once per day, per week, per month, or per year. In certain embodiments, the antibodies or antibody fragments of the present disclosure (e.g., antigen-binding fragments) are administered once every two weeks or once every three weeks. A treating physician can estimate the dosing frequency based on the measured half-life and antibody concentration in body fluids or tissues. In certain embodiments, the antibodies or antibody fragments of the present disclosure (e.g., antigen-binding fragments) are administered once every two weeks or once every three weeks. A treating physician can estimate the dosing frequency based on the measured half-life and antibody concentration in body fluids or tissues. In certain embodiments, the antibodies or antibody fragments of the present disclosure (e.g., antigen-binding fragments) are administered once every two weeks or once every three weeks. A treating physician can estimate the dosing frequency based on the measured half-life and antibody concentration in body fluids or tissues. A treating physician can estimate the dosing frequency based on the measured half-life and antibody concentration in body fluids or tissues. A treating physician can estimate the dosing frequency based on the measured half-life and antibody concentration in body fluids or tissues.

[0219] Combination Therapy In certain examples, the antibodies or antibody fragments of the present disclosure (e.g., antigen-binding fragments) are combined with other therapeutic agents such as other antiviral agents, anti-allergy agents, anti-emetic (or anti-nausea) agents, pain relievers, cytoprotective agents, immunosuppressive agents, and combinations thereof. In certain examples, the antibodies or antibody fragments of the present disclosure (e.g., antigen-binding fragments) are combined with other therapeutic agents such as other antiviral agents, anti-allergy agents, anti-emetic (or anti-nausea) agents, pain relievers, cytoprotective agents, immunosuppressive agents, and combinations thereof. In certain examples, the antibodies or antibody fragments of the present disclosure (e.g., antigen-binding fragments) are combined with other therapeutic agents such as other antiviral agents, anti-allergy agents, anti-emetic (or anti-nausea) agents, pain relievers, cytoprotective agents, immunosuppressive agents, and combinations thereof.

[0220] As used herein, the term "combination medicament" refers to a fixed combination in one unit dosage form, or two or more therapeutic agents administered simultaneously and independently, or particularly, a non-fixed combination that can be administered separately within a time interval in which the combination partners can cooperate, for example, exhibit a synergistic effect, or a partial kit for non-fixed combination or combination administration.

[0221] The term "combination therapy" refers to the administration of two or more therapeutic agents for treating the therapeutic conditions or infections described in this disclosure. Such administration includes the simultaneous administration of these therapeutic agents in a substantially simultaneous manner, such as a single capsule having active ingredients in a fixed ratio. Alternatively, such administration includes the simultaneous administration of each active ingredient in multiple or separate containers (e.g., capsules, powders, and liquids). The powder and / or liquid can be reconstituted or diluted to the desired dose before administration. Further, such administration also includes the use of each type of therapeutic agent in a continuous manner, either almost simultaneously or at different times. In any case, the treatment regimen provides a beneficial effect of the combination drug in treating the conditions or disorders described herein.

[0222] Combination therapy provides a "synergistic effect" and may be found to be "synergistic", that is, the effect achieved when the active ingredients are used together is greater than the sum of the effects obtained from using the compounds separately. The active ingredients are (1) co-formulated and administered simultaneously in a combined unit dosage formulation, or delivered simultaneously; (2) delivered to each other as separate formulations, or simultaneously; or (3) by some other regimen. ​​​​​​​​​​​​​ When administered in this way, a synergistic effect can be achieved. When delivered in an alternating therapy, the compound For example, when administered or delivered continuously by different injection solutions in separate syringes, a synergistic effect can be achieved. Generally, during an alternating therapy, each active ingredient at an effective dose is administered continuously, that is, sequentially, but in a combination therapy, two or more active ingredients at effective doses are administered together.

[0223] In one aspect, the present disclosure provides a method for treating BKV or JCV infection by administering an antibody together with an immunosuppressive therapy to a subject in need thereof. The anti-VP1 antibody acts prophylactically to neutralize a primary BKV or JCV infection or viral reactivation resulting from immunosuppressive therapy before or after transplantation. Examples of immunosuppressive therapies include, but are not limited to, monophosphate dehydrogenase inhibitors, purine synthesis inhibitors, calcineurin inhibitors or mTOR inhibitors. Specific examples of immunosuppressive therapy agents include, but are not limited to, mycophenolate mofetil (MMF), sodium mycophenolate, azathioprine, tacrolimus, sirolimus and cyclosporine.

[0224] Pharmaceutical Compositions To prepare a pharmaceutical composition or a sterile composition comprising an anti-VP1 antibody, the antibody of the present disclosure is mixed with a pharmaceutically acceptable carrier or excipient. The composition may further contain one or more other therapeutic agents suitable for neutralizing BKV or JCV infection.

[0225] Formulations of therapeutic and diagnostic agents can be, for example, lyophilized powders, slurries, aqueous solutions, lotions ​​​​​​​​​​... or in the form of a suspension, mixed with a physiologically acceptable carrier, excipient, or stabilizer which can be prepared by (e.g., see Hardman et al., Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y., 2001; Genna ro, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and W ilkins, New York, N.Y., 2000; Avis, et al. (eds.), Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY, 1993; Lieberman, et al. (eds.), Pharm aceutical Dosage Forms: Tablets, Marcel Dekker, NY, 1990; Lieberman, et al. (eds .) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY, 1990; Weine r and Kotkoskie, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N .Y., 2000).

[0226] In certain embodiments, the anti-VP1 antibody is a lyophilized product in a vial containing the antibody. The lyophilized product can be reconstituted with water or a pharmaceutically suitable carrier for injection. Subsequently for intravenous administration, the resulting solution is usually further diluted in a carrier solution.

[0227] ​ The antibodies disclosed herein are useful in the neutralization of BKV or JCV in immunosuppressed tissue transplant patients and thus the pharmaceutical carriers of sucrose and human albumin previously used in bone marrow transplant patients receiving CytoGam® can be used (DeRienzo et al. Pharmacotherapy 2000; 20:1175-8) . Alternatively, the anti-VP1 antibody can be introduced into transplant patients via a pharmaceutical carrier described for another antiviral antibody, Synagis® described in WO2003 / 105894. In this publication, the pharmaceutical carrier included histidine and / or lysine, saccharides (e.g., sucrose), and polyols (e.g., polysorbate ).

[0228] The selection of a dosing regimen for a therapeutic agent depends on several factors such as the severity of the infection, the level of symptoms, and the accessibility of target cells in the biological matrix. In certain instances, the dosing regimen maximizes the amount of therapeutic agent delivered to the patient consistent with an acceptable level of side effects. Thus, the amount of biologic delivered is in part dependent on the particular entity and the severity of the condition being treated. Guidelines are available for selecting appropriate doses of antibodies, cytokines, and small molecules (e.g., Wawrzynczak, Antibody Therapy, Bios Scien tific Pub. Ltd, Oxfordshire, UK, 1996; Kresina (ed.), Monoclonal Antibodies, Cy to, Humana Press, Totowa, NJ, 1993; Rosenberg (ed.), Cytokine Handbook, Academic Press, San Diego, CA, 1991). Tokines and Arthritis, Marcel Dekker, New York, N.Y., 1991; Bach (ed.), Monoclon al Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New Yor k, N.Y., 1993; Baert et al., New Engl. J. Med. 348:601-608, 2003; Milgrom et al. , New Engl. J. Med. 341:1966-1973, 1999; Slamon et al., New Engl. J. Med. 344:78 3-792, 2001; Beniaminovitz et al., New Engl. J. Med. 342:613-619, 2000; Ghosh et al., New Engl. J. Med. 348:24-32, 2003; Lipsky et al., New Engl. J. Med. 343:15 94-1602, 2000 (see, e.g.,).

[0229] The determination of an appropriate dosage is made by a physician using, for example, parameters or factors known or suspected in the art to affect or be predictive of affecting the treatment. Generally, the dosage is started at a somewhat lower amount than the optimal dosage and then increased in small increments until the desired or optimal effect is achieved as compared to any negative side effects. Important diagnostic measures include, for example, infusion reactions. The acute dosage level of the active ingredient in a pharmaceutical composition containing an anti-VP1 antibody is adjusted to achieve the desired therapeutic response for a particular patient, composition, and mode of administration such that it is not toxic to the patient. Important diagnostic measures include, for example, infusion reactions.

[0230] The acute dosage level of the active ingredient in a pharmaceutical composition containing an anti-VP1 antibody is adjusted to achieve the desired therapeutic response for a particular patient, composition, and mode of administration such that it is not toxic to the patient. can be varied to obtain the amount of the active ingredient effective therefor. The dosage levels selected will depend upon a variety of pharmacokinetic factors including the neutralizing activity of the antibody, the route of administration, the time of administration, the half-life of the antibody in the patient, the duration of treatment, other drugs, compounds and / or materials used in conjunction with the particular composition

[0231] employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. Compositions containing the antibody or fragment thereof can be provided by continuous infusion, or by dosage at intervals of for example once a day, once a week, or between 1 and 7 times a week. The dosage can be provided intravenously,

[0232] subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, or by inhalation. Specific dosage protocols include those which include the maximum dosage or dosage frequency that avoids significant undesirable side effects. For the antibodies described herein, the dosage administered to a patient can be from 0.0001 mg / kg to 100 mg / kg patient body weight. The dosage can be from 0.0001 mg / kg to 20 mg / kg, from 0.0001 mg / kg to 10 mg / kg, from 0.0001 mg / kg to 5 mg / kg, from 0.0001 to 2 mg / kg, from 0.0001 to 1 mg / kg, Use the product of the kilogram body weight of the patient (kg) and the dose administered in mg / kg to calculate it.

[0233] Next, repeat the dose of the antibody, and the administration may be spaced at least 1 day, 2 days, 3 days, 5 days, 10 days, 1 5 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months apart. .

[0234] The effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method of administration, the route and dose, and the severity of side effects (see, for example, Mayn ard et al., A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boc a Raton, Fla., 1996; Dent, Good Laboratory and Good Clinical Practice, Urch Publ ., London, UK, 2001).

[0235] The route of administration may be, for example, topical or cutaneous application, intravenous, intraperitoneal, intracerebral, intramuscular, intraocular , intraarterial, intrathecal, injection or infusion into a lesion, or by a sustained release system or implant body (see, for example, Sidman et al., Biopolymers 22:547-556, 1983; Langer et al., J. Biomed. Mater. Res. 15:167-277, 1981; Langer, Chem. Tech. 12: 98-105, 1982; Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688-3692, 1985; Hwa Ng et al., Proc. Natl. Acad. Sci. USA 77:4030-4034, 1980; U.S. Patent No. 6,350, See No. 466 and No. 6,316,024). Optionally, the composition may also contain a solubilizing agent or a local anesthetic such as lidocaine to reduce pain at the injection site, or both. Further, for example, pulmonary administration can also be used by using formulations containing an inhaler or nebulizer, and an aerosolizing agent. For example, U.S. Patent Nos. 6,019,968, 5,985,320, 5,985,3 09, 5,934,272, 5,874,064, 5, 855,913, 5,290,540, and 4,880,078 ; and PCT International Publication Nos. 92 / 19244, 97 / 325 72, 97 / 44013, 98 / 31346, and 99 / 66903 pamphlets (each of which is incorporated herein by reference in its entirety) should be referred to.

[0236] The compositions of the present disclosure can also be administered by one or more administration routes using one or more various methods known in the art. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired result. Routes of administration selected for the antibody include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral administration routes such as by injection or infusion. Parenteral administration is usually by injection and can be a mode of administration other than enteral administration and topical administration, and includes, but is not limited to, intravenous, intramuscular Intravenous, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, and intra-articular Intravenous, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions are included. Some Alternatively, the compositions of the present disclosure can be administered by parenteral routes such as topical, epidermal, or mucosal administration routes, e.g ., intranasally, orally, vaginally, rectally, sublingually, or topically. In one embodiment, the antibodies of the present disclosure are administered by infusion. In another embodiment, the antibody is subcutaneous administered.

[0237] When the antibodies of the present disclosure are administered in a controlled release system or a sustained release system, a pump can be used to achieve controlled release or sustained release (see Langer, supra; Sefton, CRC Crit. R ef Biomed. Eng. 14:20, 1987; Buchwald et al., Surgery 88:507, 1980; Saudek et al ., N. Engl. J. Med. 321:574, 1989). A polymeric material can be used to achieve controlled release or sustained release of the antibody therapy (e.g., Medical Applicatio ns of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla., 1 974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smole n and Ball (eds.), Wiley, New York, 1984; Ranger and Peppas, J. Macromol. Sci. R See, e.g., ev. Macromol. Chem. 23:61, 1983; also Levy et al., Science 228:19 0, 1985; During et al., Ann. Neurol. 25:351, 1989; Howard et al., J. Neurosurg. 7 1:105, 1989; U.S. Patent No. 5,679,377; U.S. Patent No. 5,916,59 7; U.S. Patent No. 5,912,015; U.S. Patent No. 5,989,463 ; U.S. Patent No. 5,128,326; PCT Publication No. 99 / 15154 Pam phlet; and also see PCT International Publication No. 99 / 20253 Pamphlet). Examples of polymers used in sustained release formulations include, but are not limited to, poly (2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(a crylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polygly colide (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol ), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one aspect, the polymer used in the sustained release formulation is inert, contains no leachable impurities, is stable upon storage, is sterile, and is biodegradable. A controlled release system or sustained release system can be placed near the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose (e.g., Goodson, in Medical Applications ; see also, e.g., ; see also, e.g., See Controlled Release, supra, vol. 2, pp. 115-138, 1984).

[0238] Controlled release systems are reviewed in Langer, Science 249:1527-1533, 1990. Using any technique known to those skilled in the art, sustained release formulations containing one or more antibodies of the present disclosure can be generated. For example, U.S. Patent No. 4,526,938, PCT International Publication No. 91 / 05548 pamphlet, PCT International Publication No. 96 / 20698 pamphlet, Ning et al., Radiotherapy & Oncology 39:179-189, 1996; Song et al., PDA Journal of Pharmaceutical Science & Technology 50:372-397, 1995; Cleek et al., P ro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, 1997; and Lam et al. , Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, 1997 (each of which is hereby incorporated by reference in its entirety). See also Remington's Pharmaceutical Sciences and Introd When the antibodies of the present disclosure are administered locally, they can be formulated in the form of ointments, creams, transdermal patches, lotions , gels, sprays, aerosols, solutions, emulsions, or other forms well known to those skilled in the art . See, for example, Remington's Pharmaceutical Sciences and Introd

[0239] When the antibodies of the present disclosure are administered locally, they can be formulated in the form of ointments, creams, transdermal patches, lotions , gels, sprays, aerosols, solutions, emulsions, or other forms well known to those skilled in the art . For example, Remington's Pharmaceutical Sciences and Introd Refer to Aulton's Pharmaceutics: The Design and Manufacture of Medicines, 19th ed., Mack Pub. Co., Easton, Pa. (199 5). For non-sprayable topical dosage forms, carriers or one or more excipients compatible with topical application are included and in some cases viscous or semi-solid or solid forms having a dynamic viscosity higher than water are typically used. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, plasters, etc., which are sterilized if necessary or mixed with adjuvants (e.g., preservatives, stabilizers, wetting agents, buffers, or salts ) to affect various properties such as, for example, osmotic pressure. Other suitable topical dosage forms include, in some cases, active ingredients combined with solid or liquid inert carriers filled in a mixture with a pressurized volatile substance (e.g., a gaseous propellant such as freon) or in a squeeze bottle, sprayable aerosol preparations. If necessary, a moisturizer or humectant can also be added to the pharmaceutical composition and dosage form. Examples of such

[0240] additional components are well known in the art. When administering a composition containing an antibody intranasally, it can be formulated in aerosol form, spray, mist or in the form of droplets. In particular, a prophylactic or therapeutic agent for use according to the present disclosure is conveniently delivered can be achieved. In the case of pressurized aerosols, by providing a valve for delivering a fixed amount , the dosage unit can be determined. Capsules and cartridges (e.g., composed of gelatin) for use in inhalers or nebulizers containing a powder mixture of a compound and a suitable powder base such as lactose or starch can be formulated.

[0241] Methods for co-administration or treatment with a second therapeutic agent, e.g., an immunosuppressant, cytokine, steroid, chemotherapeutic agent, antibiotic, or radiation therapy are known in the art (see, e.g., Hardman et al., (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., Pa.). The effective amount of the therapeutic agent can reduce symptoms by at

[0242] least 10%; at least 20%; at least about 30%; at least 40%, or at least 50%. ) can be administered with a time interval of less than 5 minutes, less than 30 minutes, 1 hour, about 1 hour, about 1 to about 2 hours, about 2 to about 3 hours, about 3 to about 4 hours apart, about 4 to about 5 hours apart, about 5 to about 6 hours apart, about 6 to about 7 hours apart, about 7 to about 8 hours apart, about 8 to about 9 hours apart, about 9 to about 10 hours apart, about 10 to about 11 hours apart, about 11 to about 12 hours apart, about 12 to about 18 hours apart, 18 to 24 hours apart, 24 to 36 hours apart, 36 to 4 8 hours apart, 48 to 52 hours apart, 52 to 60 hours apart, 60 to 72 hours apart, 72 to 84 hours apart, 84 to 96 hours apart, or 96 to 120 hours apart. Two or more therapies can be administered during the same patient visit.

[0243] In certain embodiments, the anti-VP1 antibody can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the anti-VP1 antibody passes through the BBB (if necessary), they can be formulated, for example, in liposomes. Methods for manufacturing liposomes are described, for example, in U.S. Patent Nos. 4,522,811; 5,374,548; and 5,399,331. Liposomes can contain one or more moieties that are selectively transported to specific cells or organs, thus enhancing targeted drug delivery. (See, for example, Ranade, (1989) J. Clin. Pharmacol. 29:685). (i). Examples of targeting moieties include folic acid or biotin (see, e.g., U.S. Patent No. 5,416,016 to Low et al.); mannoside (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibody (Bloeman et al., (1995) FEBS Lett . 357:140; Owais et al., (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al., (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al, (1994) J. Biol. Chem. 269:9090), and reference is also made to K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler (1994) Immunom ethods 4:273.

[0244] The present disclosure provides a protocol for administering a pharmaceutical composition comprising an antibody, alone or in combination with other therapies, to a subject in need thereof. Combination therapies (e.g., prophylactic or therapeutic agents) can be administered to a subject simultaneously or sequentially. The therapies of the combination therapy (e.g., prophylactic or therapeutic agents) can also be administered periodically. Periodic therapy is for reducing the development of resistance to one of the therapies (e.g., agents), avoiding or reducing one side effect of the therapies (e.g., agents), and / or improving the efficacy of the therapies, for a period of time, administration of a first therapy (e.g., a first prophylactic or therapeutic agent), then for a period of time, administration of a second therapy (e.g., a second prophylactic or therapeutic agent). Administration of a second therapy (e.g., a second prophylactic or therapeutic agent), and repetition of this continuous administration , i.e., including a cycle.

[0245] Administer the therapies (e.g., prophylactic or therapeutic agents) of the combination therapy of the present disclosure to a subject simultaneously can be. The term "simultaneously" is not limited to the administration of exactly simultaneous therapies (e.g., prophylactic or therapeutic agents), but rather, a pharmaceutical composition containing an antibody or a fragment thereof is administered to the subject continuously , and the antibody acts together with other therapy(ies) at time intervals such that it can provide an increased benefit compared to when they are administered separately . For example, each therapy can be administered simultaneously or continuously in any order at different times; however , if not administered simultaneously, they should be administered at times close enough together to provide the desired therapeutic or prophylactic effect. Each therapy can be administered to the subject separately in any suitable form and by any suitable route. In various embodiments, the therapies (e.g., prophylactic or therapeutic agents) are administered to the subject with less than 15 minutes apart, less than 30 minutes apart, less than 1 hour apart, about 1 hour apart, about 1 to about 2 hours apart, about 2 to about 3 hours apart, about 3 hours to about 4 hours apart, about 4 to about 5 hours apart, about 5 to about 6 hours apart, about 6 hours to about 7 hours apart, about 7 to about 8 hours apart, about 8 to about 9 hours apart, about 9 to about 10 hours apart, about 10 to about 11 hours apart, about 11 to about 12 hours apart, 24 hours apart, 48 hours apart, 72 hours apart, or 1 week apart. In other embodiments, two or more therapies (e.g., prophylactic or therapeutic agents) are administered during the same patient visit .

[0246] ​​A prophylactic or therapeutic agent for combination therapy can be administered to a subject in the same pharmaceutical composition. Alternatively, a prophylactic or therapeutic agent for combination therapy can be administered to a subject simultaneously in separate pharmaceutical compositions. The prophylactic or therapeutic agent may be administered to the subject by the same or different routes of administration.

Examples

[0247] Example 1: Generation of anti-VP1 antibody B cells expressing anti-VP1 antibody were lysed, and the VH (heavy) and VL (light) chains were sequenced by RT-PCR and analyzed to identify important post-translational modification (PTM) sites. Then, the plasmids of the VH and VL chains were transfected into CHO mammalian cell lines in an IgG1 backbone vector for the expression of a complete IgG1 antibody.

[0248] Methods for the generation of monoclonal antibodies using hybridoma technology are well known in the art (Antibody Methods and Protocols, Methods in Molecular Biology vol. 901, 2012, Chapter 7: 117). Briefly, female Balb / c mice were immunized with VLPs derived from BKV serotype I, serotype IV, and JCV using various prime-boost strategies, doses of immunogen, and adjuvants (including, but not limited to, Freund's adjuvant and MF 59 adjuvant) (individually or in combination). After screening the supernatants of successfully fused (growing) hybridomas for the presence of anti-VP1 antibody by ELISA, they were screened for functional activity in a neutralization assay. The CDRs derived from the selected mouse IgG were grafted into human frameworks. ​ Humanized by transplantation onto an acceptor template and cloned into a mammalian IgG1 backbone expression vector, and transfected into a CHO mammalian cell line for expression of a complete IgG1 antibody. -

[0249] Methods for generating monoclonal antibodies using phage display technology are known in the art (Antibody Methods and Protocols, Methods in Molecular Biology vol . 901, 2012, Chapter 3: 33). Briefly, a human B cell antibody library in scFv format with Vκ was subjected to three rounds of selection with increasing stringency using solution panning with streptavidin-conjugated magnetic beads complexed with biotinylated BKV serotype IV VLPs for screening for anti-VP1 antibodies. Isolates were first expressed as scFvs and screened by ELISA for binding to both BKV serotype IV VLPs and pentamers. The selected isolates were then cloned and expressed as IgG1, re-analyzed by ELISA for binding to VP1 (serotypes I and IV), and for functional activity in a neutralization assay, and transfected into a CHO mammalian cell line for expression of a complete IgG1 antibody.

[0250] An overview of the anti-VP1 antibodies is provided in Table 3.

[0251]

Table 3-1

[0252]

Table 3-2

[0253] Example 2: Affinity Maturation of Anti-VP1 Antibody The anti-VP1 antibody was affinity matured in yeast by mutagenic PCR or CDR-specific mutagenesis. The VP1 proteins (shown in Table 4) derived from each of the four serotypes of BKV were used as antigens in up to three rounds of selection by FACS analysis. Subsequently, VH (heavy) and / or VL (light) chains with enhanced binding affinity to VP1 were cloned into a mammalian IgG1 backbone expression vector and transfected into a CHO mammalian cell line for the expression of full-length IgG1 antibodies.

[0254] [Table 4]

[0255] Example 3: Production of BK Virus and Virus-Like Particles (VLPs) Genomic clones of BKV serotype I were obtained from the ATCC (pBR322-BKV MM, catalog number 45026; pBR322-BKV Dunlop, catalog number 45025). Infectious genomic clones of chimeric viruses of serotypes II, III, and IV were generated using previously described cloning strategies (Broekema et al, Virology 2010 407:368-373). Briefly, unique restriction sites (SacII, PmlI) were introduced into the BKV serotype I genome flanking the VP1-VP2-VP3 coding region using site-directed mutagenesis. Isolate SB of serotype II (GenBank accession number CAA79596.1), isolate AS of serotype III (GenBank accession number A ccession number AA46882.1) and the coding region of VP1 derived from strain ITA-4 of serotype IV (GenBank accession number BAF 75132) were synthesized such that the fragment to be synthesized included the SacII-PmlI region used for the swap combinations described by Broekema et al., supra and was synthesized in the context of the VP2 / VP 3 coding region from a serotype I isolate (Genewiz, La Jolla, CA). Subsequently, the resulting chimeric genomic clone was used to generate a high-titer infectious virus stock in primary renal proximal tubular epithelial (RPTE) cells (ATCC, catalog number PCS-400-010) as previously described (Abend et al, J. Virology 2007 81:272-279).

[0256] VLPs representing each of the four BKV serotypes were generated by expression of VP1 in Sf9 insect cells and isolated by microchip sonication (3 x 45 second pulses, 5 minute rest on ice between pulses), pelleting of the VLPs through a 20% sucrose cushion (2.5 hours at 116,000g), and extraction from frozen cell pellets derived from 1 L of culture followed by purification by anion exchange using a 5 ml GE HiTrap Q HP column (GE Healthcare, Pittsburgh, PA), purification using a size exclusion column based on 10 ml of Capto(™) Core700 resin (GE Healthcare, Pittsburgh, PA), and finally purification on a GE Sephacryl S500 26 / 60 column (GE Healthcare, Pittsburgh, PA). The prepared VLPs were used in Examples 6 and 7 Used in binding assays based on ELISA and SPR.

[0257] Example 4: Purification of BKV VP1 pentamer The VP1 proteins (sequences shown in Table 5 below) derived from each of the four serotypes of BKV were cloned using an N-terminal GST-6xHis-TEV sequence and subcloned into a pGEX destination vector (GE Healthcare, Pittsburgh, PA). The GST fusion proteins were expressed in E. coli and extracted from the cell pellet using a microfluidizer (15,000 PSI), and purified by immobilized metal ion affinity chromatography (IMAC) using a 20 ml nickel sepharose 6 Fast Flow column (GE Healthcare, Pittsburgh, PA). The GST-6xHis-TEV tag was cleaved by incubating overnight with TEV protease and the final purification was performed using a 5 ml His-Trap Fast Flow column (GE Healthcare, Pittsburgh, PA) followed by a Superdex 200 26 / 60 size exclusion column (GE Healthcare, Pittsburgh, PA).

[0258]

[0259] [Table 5]

[0259] Example 5: Affinity measurement of anti-VP1 antibodies (SET assay) The solution equilibrium titration (SET) assay was used to determine the interaction affinity (K ) between the antibody and BKV VP1 pentamers derived from all four serotypes. D The antibody was at a concentration of 1 pM (constant). Assayed, and the VP1 pentamer was serially diluted from an initial concentration of 10 nM. The antibody:VP1 pentamer solutions were incubated overnight, and then assayed for unbound antibody using MSD array plates coated with VP1 pentamer ( Meso Scale Discovery, catalog number L21XA, Rockvil le MD). The K D was determined by fitting the plot to a 1:1 binding model (Piehler et al. J. Immunol. Methods . 1997; 201(2):189-206).

[0260] In the SET assay, the K D values were in the range of 0.9 - 5.0 pM and were similar to the binding of anti-VP1 antibodies to the BKV serotype I pentamer. P8D11 and derivatives of P8D11 had comparable K values for binding to the pentamers of BKV serotypes II, III, and IV, and had at least 3.5-fold higher affinity for the serotype II pentamer and 47-fold higher affinity for the serotype IV pentamer compared to other antibodies. This is shown in FIGS. 1A D - 1D. Additionally, P8D11 and derivatives of P8D11 showed binding affinities in the range of 2.5 - 6.0 pM for the serotype III pentamer, while other antibodies did not bind detectably to the serotype III pentamer under the conditions tested. A summary of the SET affinity data for these anti-VP1 antibodies is found in FIG. 2. Example 6: Binding of anti-VP1 antibodies to VP1 pentamer and VLP (ELISA) The binding of anti-VP1 antibodies to VP1 pentamer and VLP was analyzed by ELISA.

[0261] Briefly, Immulon 2HB plates (VWR, 62402-972) were coated overnight with 100 ng / well of BKV VLP or VP1 pentamers. Antibodies were serially diluted in PBS containing 0.5% BSA and allowed to bind to the antigen-coated plates for 2 h. After washing the plates with PBS, they were incubated with a secondary antibody (HRP-conjugated rabbit anti-human IgG, Southern Bio tech catalog number 6140-05) diluted 1:6000 in 0.5% BSA in PBS. The plates were washed with PBS and the reaction was developed using tetramethylbenzidine (TMB) microwell peroxidase substrate (KPL, 52-00-03 1L).

[0262] The anti-VP1 antibodies EBB-C1975-A3, A7, E7 and B5 showed similar binding to VLP (IC50 in the range of 0.044 - 0.1 nM) or VP1 pentamers (IC50 in the range of 0.026 - 0.078 nM) derived from BKV serotype IV, but showed reduced and more variable binding activity to VLP of serotype I (IC50 in the range of 4.32 - 85.7 nM). This data is illustrated in Figures 4 - 6 and summarized in Figure 7. In contrast, the anti-VP1 antibodies derived from the 2081 and 2075 series showed enhanced binding activity to VLP of serotype I, with an IC50 in the range of 0.046 - 0.267 nM, and this data is shown in Figures 8 and 9. The JCV-specific anti-VP1 antibodies of the 2077 series showed binding activity to JCV VLP in the range of 0.034

[0263] Example 7: Binding of anti-VP1 antibodies to VP1 pentamers and VLP by SPR Binding of anti-VP1 antibodies to VP1 pentamers and VLPs was analyzed by surface plasmon resonance (SPR) analysis. Briefly, biotinylated protein A was incubated with streptavidin and analyzed by ELISA. Immobilize on the DNA-coated SPR chip surface and bind to Protein A on the resulting surface. The anti-VP1 antibody is captured. BKV VP1 pentamers or VLPs are then washed over the surface, Binding to anti-VP1 antibodies during the binding step is followed by buffer washing during the dissociation step.

[0264] Using SPR, we compared the expression of BKV to four serotypes compared to a positive control (P165E2). The binding of anti-VP1 antibodies EBB-C1975-A3, A7, E7, and B5 was evaluated. All species antibodies had very similar binding profiles to the VP1 pentamer: Atypical binding to serotype II pentamers but not to serotype I and III pentamers (large bulk shift and no return to baseline), and similar to P165E2 It binds to serotype IV pentamers but with lower affinity (Figures 3A, 3C, and 3E). For VLPs, EBB-C1975-A3, A7, and E7 have similar binding profiles. We shared files: Atypical binding to VLPs of serotype I and VLs of serotype III However, the binding profile of EBB-C1975-B5 was similar to that of serum Although there was no significant binding to types I and III VLPs, there were distinct epitopes on VP1. The binding of the ribozyme to the ribozyme was shown in Fig. 3B and 3D.

[0265] We also used SPR to convert anti-VP1 to VP1 pentamers by scanning alanine mutagenesis. 1. Characterizing the Binding of Antibodies P165E2, NEG447, P7G11A, and P8D11 ta (Figs. 13A - F and 14). All anti - VP1 antibodies showed a decrease in binding to the F66A and I145A VP1 mutants due to the overall effect of the mutation (Figs. 13B and 13F). Furthermore, K69A and E82A affected the binding of P165E2, NEG447, and P7G11A (Figs. 13D and 13E).

[0266] Example 8: Anti - VP1 antibodies bind to conformational epitopes To determine whether anti - VP1 antibodies bind to conformational epitopes, Western blots of denatured proteins by SDS - PAGE and dot blots of proteins in their native conformation were used. Briefly, VP1 pentamers of BKV serotype I or IV were electrophoresed on SDS - PAGE and transferred to nitrocellulose membranes (Western blots) or spotted directly onto nitrocellulose membranes (dot blots). Both membranes were incubated with anti - VP1 antibodies and then with an anti - human IgG secondary antibody conjugated to an infrared fluorescent dye for detection using the Licor Odyssey system.

[0267] A commercially available positive control antibody (Abcam 53977) known to recognize linear epitopes detected both denatured and non - denatured VP1. However, P165E2, P7G11, and P8D11 could not detect denatured VP1 on Western blots and recognized only native VP1 on dot blots, indicating that these antibodies bind to conformational (non - linear) epitopes of VP1 (Figs. 12A and 12B).

[0268] To further characterize the epitope of the anti-VP1 antibody, the major interaction sites for cell surface receptors within the VP1 BC loop, which is known to be mainly exposed on the virion surface, were subjected to scanning alanine mutagenesis for the residues therein. These mutant VP 1 pentamers were assayed for binding to P8D1 1 and P7G11A in the surface plasmon resonance (SPR) assay described above in Example 7. Mutations at several positions affected the binding of P7G11A (F66A, K69A, E82A, I145A) (Figure 13 A - F and Figure 14). However, mutations at only two sites resulted in a decrease in P8D11 binding (F66A, I145A) (Figure 14). Mutations at F66 and I145 resulted in the loss of binding of all the antibodies tested. While not bound by any one theory, these mutations are thought to result in the overall disruption of the VP1 pentamer structure. All other VP1 pentamers with the BC loop mutations tested retained P8D11 binding. In contrast, a protected region within the EF loop of VP1 upon binding of the P8D11 Fab fragment was identified by hydrogen - deuterium exchange studies. Follow - up scanning alanine mutagenesis studies revealed that the important contact residues for P8D11 binding within this region include Y169, R170, and K172, and D / E175, K181, N1 82, T184, and Q186 to M190 were confirmed to be important residues determined by deuterium exchange ((YRXKXX(D / E)XXXXXKNXTXQ)( SEQ ID NO: 500)). This is further described in Examples 14 - 17.

[0269] Example 9: Neutralization of BK virus by anti - VP1 antibody​​ Chimeric viruses representative of infectious BKV serotype I as well as serotypes II, III and IV were pre-incubated with purified antibodies for 1 hour to allow binding and neutralization. Next primary renal proximal tubular epithelial (RPTE) cells (ATCC, catalog number PCS-40 0-010) were exposed to the virus-antibody mixture for 4 hours, exchanged with fresh medium, and incubated for 48 hours to allow virus entry and gene expression. Cells were fixed with 4% paraformaldehyde and analyzed by immunofluorescence to detect TAg expression (Calbio chem DP02, pAb416 mouse anti-SV40 TAg antibody). High-content imaging analysis using a Cellomics ArrayScan® VTI HCS Reader was used to analyze the immunofluorescence, and the percentage of BKV-infected cells (TAg positive, DAPI positive) was quantified and the data presented as the percentage of inhibition of infection compared to untreated control wells.

[0270] As shown in Figures 15-23, anti-VP1 antibodies, including a subset of antibodies that neutralize infection by all four serotypes (I-IV) of BKV, neutralized infection by BKV. These anti-VP1 antibodies specifically included P8D11, a modification of P8D11, and EBB-C1975-B5 .

[0271] Example 10: Neutralization of JC virus by anti-VP1 viral antibodies Infectious JCV isolates Mad-1 and Mad-4 have the same VP1 sequence (Gen Bank accession number NP_043511). These JCV isolates were pre-incubated with purified antibodies for 1 hour to allow binding and neutralization. Then, COS7 cells (S African green monkey kidney fibroblast-like cell line expressing V40 TAg, ATCC catalog number CRL-1651) was exposed to the virus-antibody mixture for 4 hours and replaced with fresh medium and incubated for 72 hours to allow virus entry and gene expression. Cells were fixed with 4% paraformaldehyde and analyzed by immunofluorescence to detect JCV VP1 expression (Abcam 53977, rabbit polyclonal anti-SV40 VP1 antibody). Cellomics ArrayScan® VTI HCS Reader( Thermo Fisher, Waltham MA) was used to analyze the assay by high-content image analysis to quantify the percentage of JCV-infected cells (VP1 positive, DAPI positive) and present the data as the percent inhibition of infection compared to untreated control wells. As shown in Figures 24-26, a subset of anti-VP1 antibodies, including the P8D11 and 2077 series antibodies, neutralize infection by JCV.

[0272] Example 11: Virus resistance A resistance selection experiment using the P8D11 antibody was performed in kidney proximal tubule epithelial (RPTE) cell cultures infected with BKV serotype I or serotype IV. In the serotype I test, no virus upsurge was observed in cultures containing P8D11 over 6 passages (84 days), and thus, no resistance-associated variants (RAVs) were identified. Since no virus could be detected, no further passages were performed beyond this point. In contrast, for another antibody, a virus upsurge was detected at passage 3 (day 42). Sequencing of BKV VP1 from these cultures revealed a resistance-associated A variant (RAV) was identified, but there was no change in the cluster around specific amino acids in the VP1 sequence. Subsequent phenotypic characterization of this pooled RAV virus showed a complete loss of neutralizing activity (shift exceeding 7,692-fold of the EC50) when compared to the wild-type virus, although the change in the EC50 of P8D11 was minor (3.9-fold). Furthermore, the VP1 mutant E82K was identified as an RAV during selection with another anti-VP1 antibody (see Example 8), and characterization of the cloned E82K mutant virus showed that this variant provided a shift of 15,880-fold in the EC50 when compared to the wild-type virus, but no cross-resistance to P8D11 was shown.

[0273] Similarly, in cultures of BKV serotype IV, no resistance to P8D11 was detected after 6 passages (84 days). Again, since the virus could no longer be detected, no further passages were carried out beyond this point. However, resistance to different anti-BK antibodies was selected as early as passage 1 (14 days). Changes in the amino acids L68R and E73K were identified as mutations from the reference, providing shifts in the EC50 values of 600-fold and 227-fold, respectively, but no cross-resistance to P8D11 was shown. In summary, P8D11 has a high barrier to resistance and maintains neutralizing activity against resistant variants of both serotypes I and IV.

[0274] Example 12: Toxicity Since VP1 is an exogenous non-human target that is not expressed on the cell surface, the anti-VP1 antibodies disclosed herein have a low risk of toxicity in humans. By TCR testing, P8D1 ​​​​​​​​​​​ Staining of 42 human tissues and blood smears by 1 was shown to be absent, supporting the lack of cross-reactivity of the anti-VP1 antibody with human proteins. The anti-VP1 antibody did not show antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro, which is consistent with the fact that the VP1 protein is not expressed on the surface of host cells. itro, which is consistent with the fact that the VP1 protein is not expressed on the surface of host cells. itro did not show antibody-dependent cell-mediated cytotoxicity (ADCC), which is consistent with the fact that the VP1 protein is not expressed on the surface of host cells. P1 protein is not expressed on the surface of host cells.

[0275] Example 13: SET Affinity Assay of P8D11 for JCV VLP Progressive multifocal leukoencephalopathy (PML) is a rare but often fatal infection of the brain of immunocompromised patients caused by the JC virus. The major capsid protein (VP1) of the JC virus is involved in binding to sialic acid receptors on the surface of host cells. Certain mutations in VP1, such as amino acids L55 and S269, inactivate sialic acid recognition and play a role in the development of PML (Chen et al., mAbs 2015; 7(4), 681-692). These two mutations occur frequently in PML patients (Gorelik et al., J.Infect. Dis. 2011 204:103-114 and Reid et al., J. Infect. Dis. 2011; 204:237-244). The antibodies of the present disclosure were tested to see if they bind to mutant JCV VLPs having mutations at these positions. Binding of the anti-VP1 antibody to these VLPs indicates that JC viruses carrying these common VP1 mutations are not resistant to therapy. Progressive multifocal leukoencephalopathy (PML) is a rare but often fatal infection of the brain of immunocompromised patients caused by the JC virus. The major capsid protein (VP1) of the JC virus is involved in binding to sialic acid receptors on the surface of host cells. Certain mutations in VP1, such as amino acids L55 and S269, inactivate sialic acid recognition and play a role in the development of PML (Chen et al., mAbs 2015; 7(4), 681-692). These two mutations occur frequently in PML patients (Gorelik et al., J.Infect. Dis. 2011 204:103-114 and Reid et al., J. Infect. Dis. 2011; 204:237-244). The antibodies of the present disclosure were tested to see if they bind to mutant JCV VLPs having mutations at these positions. Binding of the anti-VP1 antibody to these VLPs indicates that JC viruses carrying these common VP1 mutations are not resistant to therapy. is involved in binding to sialic acid receptors on the surface of host cells. Certain mutations in VP1, such as amino acids L55 and S269, inactivate sialic acid recognition and play a role in the development of PML (Chen et al., mAbs 2015; 7(4), 681-692). These two mutations occur frequently in PML patients (Gorelik et al., J.Infect. Dis. 2011 204:103-114 and Reid et al., J. Infect. Dis. 2011; 204:237-244). The antibodies of the present disclosure were tested to see if they bind to mutant JCV VLPs having mutations at these positions. Binding of the anti-VP1 antibody to these VLPs indicates that JC viruses carrying these common VP1 mutations are not resistant to therapy. such as amino acids L55 and S269, inactivate sialic acid recognition and play a role in the development of PML (Chen et al., mAbs 2015; 7(4), 681-692). These two mutations occur frequently in PML patients (Gorelik et al., J.Infect. Dis. 2011 204:103-114 and Reid et al., J. Infect. Dis. 2011; 204:237-244). The antibodies of the present disclosure were tested to see if they bind to mutant JCV VLPs having mutations at these positions. Binding of the anti-VP1 antibody to these VLPs indicates that JC viruses carrying these common VP1 mutations are not resistant to therapy. and play a role in the development of PML (Chen et al., mAbs 2015; 7(4), 681-692). These two mutations occur frequently in PML patients (Gorelik et al., J.Infect. Dis. 2011 204:103-114 and Reid et al., J. Infect. Dis. 2011; 204:237-244). The antibodies of the present disclosure were tested to see if they bind to mutant JCV VLPs having mutations at these positions. Binding of the anti-VP1 antibody to these VLPs indicates that JC viruses carrying these common VP1 mutations are not resistant to therapy. frequently in PML patients (Gorelik et al., J.Infect. Dis. 2011 204:103-114 and Reid et al., J. Infect. Dis. 2011; 204:237-244). The antibodies of the present disclosure were tested to see if they bind to mutant JCV VLPs having mutations at these positions. Binding of the anti-VP1 antibody to these VLPs indicates that JC viruses carrying these common VP1 mutations are not resistant to therapy. The antibodies of the present disclosure were tested to see if they bind to mutant JCV VLPs having mutations at these positions. Binding of the anti-VP1 antibody to these VLPs indicates that JC viruses carrying these common VP1 mutations are not resistant to therapy. The antibodies of the present disclosure were tested to see if they bind to mutant JCV VLPs having mutations at these positions. Binding of the anti-VP1 antibody to these VLPs indicates that JC viruses carrying these common VP1 mutations are not resistant to therapy. Binding of the anti-VP1 antibody to these VLPs indicates that JC viruses carrying these common VP1 mutations are not resistant to therapy. Binding of the anti-VP1 antibody to these VLPs indicates that JC viruses carrying these common VP1 mutations are not resistant to therapy.

[0276] Two 22-fold serial dilutions of VLPs were prepared in sample buffer. Two fixed concentrations of the P8D11 antibody were added. The concentrations of the P8D11 antibody used were 9 nM or 1 p Two 22-fold serial dilutions of VLPs were prepared in sample buffer. Two fixed concentrations of the P8D11 antibody were added. The concentrations of the P8D11 antibody used were 9 nM or 1 p was any of M. The concentration range of the JCV consensus was 105 μg / ml to 72 pg / ml. The concentration range of the JCV L55F mutant was 300 μg / ml to 143 pg / ml. The concentration range of the JCV S269F mutant was 300 μg / ml to 143 pg / ml. Each VLP:antibody mixture with a volume of 60 μl was dispensed twice into a 384 well polypropylene microtiter plate (PP MTP). The test sample buffer served as a negative control, and the sample containing no antigen served as a positive control (Bmax). The plate was sealed and incubated overnight (o / n) at room temperature (RT). A 384-well standard MSD array plate was coated o / n with BKV- VP1 serotype I pentamer protein at 2 and 0.002 μg / ml. After washing three times with 50 μl / well of washing buffer , the plate was blocked with 50 μl / well of blocking buffer at RT for 1 hour . After washing, each VLP:antibody mixture with a volume of 30 μl / well was transferred from the PP MTP to the coated MSD plate and incubated at RT for 20 min . After further washing steps, 30 μl of the detection antibody (1 :2000 dilution) in the sample buffer was added to each well and incubated at RT for 30 min. The MSD pl ate was washed, 35 μl / well of the reading buffer was added, and incubated for 5 min . The ECL signal was measured using an MSD SECTOR Imager 6000 .

[0277] The reagents used were bovine serum albumin (BSA) (VWR catalog number 422351S ), phosphate buffered saline (PBS) 10x (Teknova catalog number P0195)​ , MSD Read Buffer T 4x (Meso Scale Discovery ry catalog number R92TC-1), Tris-buffered saline (TBS) 20x (Tek nova catalog number T1680), Tween-20 (VWR catalog number 43708 2Q). The buffers used were blocking buffer: 1xPBS + 5% ( (w / v) BSA, coating buffer: 1xPBS, sample buffer: 1xPBS + 0.5% (w / v) BSA + 0.02% (v / v) Tween-20, wash buffer: 1xTB S + 0.05% (v / v) Tween-20 and read buffer: 1xMSD Re ad Buffer.

[0278] The solution equilibrium titration (SET) assay was used to determine the interaction affinity (K ) between P8D11 and J D CV VLP as described in Example 5. The P8D11 antibody was assayed at a concentration (constant) of 9 nM or 1 pM, and the JCV VLP was serially diluted as follows: The con sensus VLP was in the range of 105 μg / ml to 72 pg / ml, and both the L55F and S2 69F mutant VLPs were in the range of 300 μg / ml to 143 pg / ml . After incubating the antibody:VP1 pentamer solution overnight, the unbound antibody was assayed using an MSD array plate (Meso Scale Discovery, catalog number L21XA , Rockville MD). K was determined by fitting the plot to a 1:1 binding model (Piehler et al. J. D ) (according to Immunol. Methods. 1997; 201(2):189-206). KinExA® Pro and n-Curve Analysis software from Sapidyne (Boise I D) were used to perform the analysis.

[0279] Figure 27 depicts the results of the SET assay in tabular form. This data is for the consensus J Determination of the affinity of the P8D11 antibody for CV VLPs and VLPs containing VP1 mutations commonly associated with PML (K ). P8D11 showed binding affinity for all JCV VLPs in the low nanomolar concentration range D . However, the binding affinity for the L55F mutant was approximately 1 / 2 of the affinity for wild-type (consensus) and S269F mutant VLPs . Thus, this indicates that the P8D11 antibody is still an effective therapy against either wild-type JC virus or JC virus with mutations commonly associated with PML .

[0280] Example 14: Deuterium exchange experiment for epitope mapping (P8D11 Fab in complex with BKV VP1 pentamer and ) Deuterium exchange mass spectrometry (HDx-MS) measures the incorporation of deuterium into the amide backbone of proteins . These measurements are sensitive to changes in amide solvent accessibility and the hydrogen bonding network of backbone amides . HDx-MS is often used to compare proteins in two different states, such as apo and ligand-bound , and is coupled with rapid digestion by pepsin . In such experiments, those skilled in the art will understand that two ​​​Regions showing different deuterium incorporations between different states, typically, 10 - 15 amino acids can be seen. The regions to be protected are either directly involved in ligand binding or allosterically affected by the binding of the antibody to the ligand.

[0281] In these experiments, the deuterium incorporation of the BKV VP1 protein (SEQ ID NO: 502) was measured in the absence and presence of the P8D11 Fab fragment. Regions in VP1 showing a decrease in deuterium incorporation upon binding of the Fab fragment are likely to be included in the epitope, but due to the nature of the measurement, it is also possible to detect changes far from the direct binding site (allosteric effect). Generally, the regions with the most protection are involved in direct binding.

[0282] Epitope mapping experiments were performed on a Waters Synapt® G2 HDx-MS platform, which includes a LEAP® robotic system, a nanoACQUITY® UPLC System, and a Synapt® G2 mass spectrometer. In this method, three sets of control experiments were performed as follows. The VP1 pentamer of BKV serotype I was diluted in 110 μl of 95% deuterated PBS buffer (pH 7.4) and incubated on a bench rotator at room temperature for 25 minutes (%D = 85.5%). Deuterium exchange was quenched by incubating on ice for 5 min and diluting 1:1 with cold quench buffer (6 M urea and 1 M TCEP pH = 2.5). After quenching, the tubes were transferred onto the LEAP system (set the thermobox to 2°C), and the quenched samples were injected onto the UPLC system by the LEAP system for analysis. ​​​​​​​​​​​​​​The PLC system incorporates an immobilized pepsin column 2.1 mm x 30 mm maintained at 12 °C (Life Technologies 2-3131-00). A 2 - 35% acetonitrile gradient and a Waters UPLC CSH C18 1.0 x 100 mm column are used for separation. Next, three sets of experiments are carried out using antibodies. The P8D11 Fab fragment is immobilized on protein G agarose beads (Thermo Scientific Cat#22851) using standard techniques. Briefly, the antibody is centrifuged to remove the storage buffer. Then, 200 μl of PBS buffer (pH 7.4) and a certain concentration of VP1 pentamer are added to the immobilized P8D11 F ab fragment and incubated at room temperature for 30 min. After incubation, the complex is centrifuged, washed with 200 μl of PBS buffer, and centrifuged again. For deuterium exchange, 200 μl of deuterated PBS is added to the antigen - antibody complex for incubation at room temperature for 25 min (%D = 85.5%). Then, the deuterium buffer is removed and immediately 125 μl of ice - cold quench buffer is added. After quenching for 5 min , the column is centrifuged and the effluent is transferred to a pre - cooled HPLC vial. The samples are analyzed using the same online pepsin digestion / LC - MS settings as in the control experiment. The results of these measurements are summarized in Figure 28. Figure 28 shows the baseline - corrected differences between the control and the samples bound to the P8D11 antibody divided by the standard error of the measurement.

[0283] In this plot, the larger the negative value, the stronger the binding of P8D to the VP1 pentamer of BKV serotype I. ​Shows that the amount of protection in a given region is large upon binding of 11 Fab fragments. P8D1 Upon binding of 1 Fab fragment, we observe the most significant amount of protection at amino acids 168 - 190 of the VP1 protein and can see in Table 1 the sequence in bold and underlined ((NYRTKYPXGTXXPKNXTXQSQVM) (SEQ ID NO: 501)). This region of the EF loop is highly conserved among all 4 serotypes of BK virus and JC virus as can be seen.

[0284] In conclusion, the deuterium mapping data indicates that the P8D11 antibody binds to an epitope within the EF loop of BKV VP1 . This region is highly conserved among all 4 BKV serotypes and JC virus, thus supporting the result that P8D11 has neutralizing activity across all 4 BKV serotypes and JC virus.

[0285] Example 15: Targeted alanine scanning and SPR for epitope mapping of P8D11 PR Binding of anti - VP1 antibodies to VP1 pentamers generated for epitope mapping by scanning alanine mutagenesis was characterized using Biacore surface plasmon resonance (SPR). The experiment was carried out at 25°C in phosphate - buffered saline (PBS) supplemented with 0.005% Tween 20 surfactant (Calbiochem catalog number 655206) and run on a Biacore T - 200 instrument (GE Healthcare Life Sciences ). Biotinylated protein A (Sigma, catalog number P2165) was immobilized at approximately 1200 response units (RU) on a Series S streptavidin sensor chip ​​​​​Fixed on the search chip, and the remaining free streptavidin sites were blocked with biotin-PEG( Pierce EZ-Link, catalog number PI21346). 3 The prepared protein A sensor chip was used to capture the antibody by injection at a flow rate of 30 μl / min for 4 seconds. The antibody was immobilized at 20 - 40 RU on flow cells 2, 3, and 4, while flow cell 1 was left as a reference cell without antibody. Then, the VP1 pentamer was injected onto the chip at 100 μl / min for 200 seconds, and then buffer was injected to monitor dissociation. Between each pentamer and pentamer concentration, the sensor chip surface was regenerated by injecting 25 mM NaOH at 30 μ l / min for 60 seconds to remove the antibody before recapturing the antibody on the protein A surface for the next cycle. Data analysis was performed in the GE BiaEvaluation software applying double reference subtraction. The evaluation of the effect of alanine mutagenesis on VP1 pentamer binding was achieved by comparing the binding RU levels, and the shape of the binding curve was compared with that of the wild-type pentamer. As previously discussed, the epitopes for antibodies P8D11 and P7G11A are steric and discontinuous (Figures 12A - B). Here, single mutations to alanine at Y169, R170, and K172 in the EF loop of BKV VP1 invalidate the binding of P8D11 (Figures 29A and 29B). Mutations at Y169 and R170 also invalidate the binding of the P7G11A antibody, but the binding of this antibody is not affected by changes at position K172 in the EF loop of BKV VP1 (Figures 29A and 29C).

[0286] As previously discussed, the epitopes for antibodies P8D11 and P7G11A are steric and discontinuous (Figures 12A - B). Here, single mutations to alanine at Y169, R170, and K172 in the EF loop of BKV VP1 invalidate the binding of P8D11 (Figures 29A and 29B). Mutations at Y169 and R170 also invalidate the binding of the P7G11A antibody, but the binding of this antibody is not affected by changes at position K172 in the EF loop of BKV VP1 (Figures 29A and 29C). invalidate the binding of the P7G11A antibody, but the binding of this antibody is not affected by changes at position K172 in the EF loop of BKV VP1 (Figures 29A and 29C). As previously discussed, the epitopes for antibodies P8D11 and P7G11A are

[0287] Example 16: Epitope mapping by x-ray crystallography Sc of antibody P8D11 bound to BKV major capsid protein VP1 in pentameric form. The crystal structure of the Fv chain was determined. As detailed below, the scFv:BKV-VP1 pentamer A 5.5:1 solution of the antibody was used to generate a fusion protein consisting of five scFv chains linked to each pentamer. Protein crystallography was then used to generate a crystallographically suitable complex of the original protein. The fragment structures were generated and the epitopes were defined.

[0288] Crystallization and structure determination The P8D11 scFv / BKV-VP1 complex was concentrated to 5.2 mg / ml and subjected to crystallization. Crystals for data collection were collected by hanging drop vapor diffusion at 18 °C. The complexes were grown in 1.0 μl of 25% (w / v) PEG3350, 0.2 M chlorine. 1.0 μl of lysine containing magnesium and 0.1 M Bis-Tris pH 7.0 Mix with the reservoir solution and equilibrate the drop against 350 μl of the same reservoir solution. Crystals were grown overnight and continued to grow for several days. Prior to storage, crystals were transferred to 75% reservoir solution + 25% glycerol and briefly cooled in liquid nitrogen. I rejected it.

[0289] Diffraction data were collected in-house on a Rigaku FRE+ copper source and R-axis X-ray diffractometer. The data was collected using Autoproc (Global Phasing, LTD). After processing and scaling, the BKV-VP1 data was processed using a cell size of a = 22 4.4 Å, b=224.4 Å, c=144.04 Å, alpha=90°, beta=90° The coordinates were processed to 2.66 Å in space group P42212 with gamma=90°. The structure of the complex was solved by molecular replacement using the BKV-VP1 pentamer as the search model with Phaser (McCoy et al. , (2007) J. Appl. Cryst. 40:658-674). The final model was built in COOT (Emsley & Cowtan (2004) Acta Cryst. D60:2126-2132) and refined using Buster (Global Phasing, LTD, Cambridge, UK) . The Rwork and Rfree values are 17.1% and 21 .4%, respectively; the root-mean-square (r.m.s) deviation values of bond lengths and bond angles are 0.010 Å and 1.18°, respectively .

[0290] The residues of the BKV-VP1 pentamer that contact P8D11 scFv, the types of interactions, and the buried surface areas were all identified by PISA (Krissinel et al., (2007) J Mol Biol. 372:774-97) and are listed in Table 6 below. Each monomer of the VP1 pentamer was found to contain a single isolated epitope for the P8D 11 antibody. Thus, the five scFv domains bind to their respective pentamers at five chemically and sterically equivalent positions . The details of the interactions at each epitope are essentially identical, and only one scFv / VP1-epitope interface is analyzed here .

[0291] The epitope of P8D11-scFv on BKV-VP1 Overall structure The overall fold of each polyomavirus VP1 pentamer structure is highly homologous at the tertiary structure level . The primary sequences are well conserved with respect to identity at 69 - 85%​ Each pentamer stacks against a β-sheet of another five chains, followed by a three-stranded β-sheet, and then is composed of five monomers, each composed of a four-stranded β-sheet. P8D11 scFv is a VH-VL fusion protein with a 20-amino acid linker between the VH and VL domains. As shown in Figure 30, the VH-VL fusion protein binds to an epitope located on the outer surface of the side of the BKV-VP1 pentamer.

[0292] Epitope of P8D11 Using the crystal structure of the BKV-VP1 / P8D11 complex, the P8D11 epitope on BKV-VP1 is identified. The interaction surface on VP1 by P8D11-scFv consists of several continuous and discontinuous (i.e., non-continuous) sequences: namely, residues 77-80, 169-186, and 191-192 detailed in Table 6. These residues form a three-dimensional conformational epitope recognized by P8D11-scFv (Figure 31A - B). This epitope defined by crystallography is in good agreement with that defined by hydrogen-deuterium exchange mass spectrometry (HDx-MS), and residues 168 - 190 are substantially protected by P8D11-Fab (Figure 28). Also, it is in good agreement with the alanine scanning performed on the important amino acids of the epitope (Figure 29A - C), and it is shown that TYR169, ARG170, and LYS172 are contact residues that are part of the epitope of the P8D11 antibody.

[0293] P8D11-scFv epitope on BKV-VP1. All residues of BKV-VP1 that contact P8D11-scFv in the crystal structure are identified by PISA, and P8D11- ​​​​​​​​​​​​​​Enumerate and sort by its buried surface area with scFv. Where applicable, enumerate the types of interactions as well.

[0294]

Table 6-1

[0295]

Table 6-2

[0296] Example 18: Formulation The anti-VP1 antibodies described herein are monoclonal antibodies having lambda light chains, IgG1 isotype, and can be lyophilized. These antibodies are soluble in histidine-sucrose formulation buffer and are stable for 4 weeks. Furthermore, the anti-VP1 antibodies were soluble at greater than 200 mg / ml as the drug substance formulated to the minimum extent (e.g., in histidine buffer in the absence of stabilizers).

[0297] For subsequent intravenous administration, the resulting solution is usually further diluted in a carrier solution to produce an immediately usable antibody solution for infusion.

[0298] Important stability indicating analytical methods for selecting the most stable formulation included, in particular, size exclusion chromatography to determine the level of aggregation, testing for particulate matter not visible to the naked eye, and potency testing.

[0299] The examples and embodiments described herein are for illustrative purposes only, and in light of them, various modifications or changes will be suggested to those skilled in the art and are understood to be included within the spirit and scope of this application and the appended claims. The following aspects may be included. [1] An antibody, wherein the antibody or its antigen-binding fragment specifically binds to VP1. [2] The antibody according to [1] above, wherein the antibody or its antigen-binding fragment specifically binds to VP1 of BK virus serotypes I to IV. [3] The antibody according to [1] above, wherein the antibody or antigen-binding fragment specifically binds to at least one VP1 in Table 1. [4] The antibody according to [1] above, wherein the antibody or its antigen-binding fragment binds to two or more VP1 serotypes in Table 1. [5] The antibody or its antigen-binding fragment is a) BKV VP1 serotype I and BKV VP1 serotype II; b) BKV VP1 serotype I and BKV VP1 serotype III; c) BKV VP1 serotype I and BKV VP1 serotype IV; d) BKV VP1 serotype II and BKV VP1 serotype III; and e) BKV VP1 serotype I and JCV VP1 The antibody according to [4] above, which binds to. [6] The antibody according to [1] above, wherein the antibody or its antigen-binding fragment binds to BKV serotype I with a binding affinity of 5.0 pM or less, or binds to BKV serotype II with a binding affinity of 29.0 pM or less, or binds to BKV serotype III with a binding affinity of 6.0 pM or less, or binds to BKV serotype IV with a binding affinity of 185.0 pM or less, or binds to JCV with a binding affinity of 436 pM or less. [7] The antibody according to [1] above, wherein the antibody or antigen-binding fragment specifically binds to the VP1 epitope (SEQ ID NO: 500 or SEQ ID NO: 501). [8] An antibody, wherein the antibody or its antigen-binding fragment is (i) A heavy chain variable region comprising (a) HCDR1 (CDR - Complementarity Determining Region) of SEQ ID NO: 6, (b) HCDR2 of SEQ ID NO: 7, (c) HCDR3 of SEQ ID NO: 8, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 16, (e) LCDR2 of SEQ ID NO: 17, and (f) LCDR3 of SEQ ID NO: 18; (ii) A heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 26, (b) HCDR2 of SEQ ID NO: 27, (c) HCDR3 of SEQ ID NO: 28, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 36, (e) LCDR2 of SEQ ID NO: 37, and (f) LCDR3 of SEQ ID NO: 38; (iii) A heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 46, (b) HCDR2 of SEQ ID NO: 47, (c) HCDR3 of SEQ ID NO: 48, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 56, (e) LCDR2 of SEQ ID NO: 57, and (f) LCDR3 of SEQ ID NO: 58; (iv) A heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 66, (b) HCDR2 of SEQ ID NO: 67, (c) HCDR3 of SEQ ID NO: 68, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 76, (e) LCDR2 of SEQ ID NO: 77, and (f) LCDR3 of SEQ ID NO: 78; (v) A heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 86, (b) HCDR2 of SEQ ID NO: 87, (c) HCDR3 of SEQ ID NO: 88, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 96, (e) LCDR2 of SEQ ID NO: 97, and (f) LCDR3 of SEQ ID NO: 98; (vi) A heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 106, (b) HCDR2 of SEQ ID NO: 107, (c) HCDR3 of SEQ ID NO: 108, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 116, (e) LCDR2 of SEQ ID NO: 117, and (f) LCDR3 of SEQ ID NO: 118; (vii) A heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 126, (b) HCDR2 of SEQ ID NO: 127, (c) HCDR3 of SEQ ID NO: 128, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 136, (e) LCDR2 of SEQ ID NO: 137, and (f) LCDR3 of SEQ ID NO: 138; (viii) A heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 146, (b) HCDR2 of SEQ ID NO: 147, and (c) HCDR3 of SEQ ID NO: 148, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 156, (e) LCDR2 of SEQ ID NO: 157, and (f) LCDR3 of SEQ ID NO: 158; (ix) A heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 166, (b) HCDR2 of SEQ ID NO: 167, and (c) HCDR3 of SEQ ID NO: 168, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 176, (e) LCDR2 of SEQ ID NO: 177, and (f) LCDR3 of SEQ ID NO: 178; (x) A heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 186, (b) HCDR2 of SEQ ID NO: 187, and (c) HCDR3 of SEQ ID NO: 188, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 196, (e) LCDR2 of SEQ ID NO: 197, and (f) LCDR3 of SEQ ID NO: 198; (xi) A heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 206, (b) HCDR2 of SEQ ID NO: 207, and (c) HCDR3 of SEQ ID NO: 208, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 216, (e) LCDR2 of SEQ ID NO: 217, and (f) LCDR3 of SEQ ID NO: 218; (xii) A heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 226, (b) HCDR2 of SEQ ID NO: 227, and (c) HCDR3 of SEQ ID NO: 228, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 236, (e) LCDR2 of SEQ ID NO: 237, and (f) LCDR3 of SEQ ID NO: 238; (xiii) A heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 246, (b) HCDR2 of SEQ ID NO: 247, and (c) HCDR3 of SEQ ID NO: 248, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 256, (e) LCDR2 of SEQ ID NO: 257, and (f) LCDR3 of SEQ ID NO: 258; (xiv) A heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 266, (b) HCDR2 of SEQ ID NO: 267, and (c) HCDR3 of SEQ ID NO: 268, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 276, (e) LCDR2 of SEQ ID NO: 277, and (f) LCDR3 of SEQ ID NO: 278; (xv)(a) The heavy chain variable region comprising HCDR1 of SEQ ID NO: 286, (b) HCDR2 of SEQ ID NO: 287, (c) HCDR3 of SEQ ID NO: 288, and (d) the light chain variable region comprising LCDR1 of SEQ ID NO: 296, (e) LCDR2 of SEQ ID NO: 297, and (f) LCDR3 of SEQ ID NO: 298; (xvi)(a) The heavy chain variable region comprising HCDR1 of SEQ ID NO: 306, (b) HCDR2 of SEQ ID NO: 307, (c) HCDR3 of SEQ ID NO: 308, and (d) the light chain variable region comprising LCDR1 of SEQ ID NO: 314, (e) LCDR2 of SEQ ID NO: 315, and (f) LCDR3 of SEQ ID NO: 316; (xvii)(a) The heavy chain variable region comprising HCDR1 of SEQ ID NO: 322, (b) HCDR2 of SEQ ID NO: 323, (c) HCDR3 of SEQ ID NO: 324, and (d) the light chain variable region comprising LCDR1 of SEQ ID NO: 332, (e) LCDR2 of SEQ ID NO: 333, and (f) LCDR3 of SEQ ID NO: 334; (xviii)(a) The heavy chain variable region comprising HCDR1 of SEQ ID NO: 342, (b) HCDR2 of SEQ ID NO: 343, (c) HCDR3 of SEQ ID NO: 344, and (d) the light chain variable region comprising LCDR1 of SEQ ID NO: 349, (e) LCDR2 of SEQ ID NO: 350, and (f) LCDR3 of SEQ ID NO: 351; (xix)(a) The heavy chain variable region comprising HCDR1 of SEQ ID NO: 356, (b) HCDR2 of SEQ ID NO: 357, (c) HCDR3 of SEQ ID NO: 358, and (d) the light chain variable region comprising LCDR1 of SEQ ID NO: 363, (e) LCDR2 of SEQ ID NO: 364, and (f) LCDR3 of SEQ ID NO: 365; (xx)(a) The heavy chain variable region comprising HCDR1 of SEQ ID NO: 370, (b) HCDR2 of SEQ ID NO: 371, (c) HCDR3 of SEQ ID NO: 372, and (d) the light chain variable region comprising LCDR1 of SEQ ID NO: 377, (e) LCDR2 of SEQ ID NO: 378, and (f) LCDR3 of SEQ ID NO: 379; (xxi)(a) The heavy chain variable region comprising HCDR1 of SEQ ID NO: 384, (b) HCDR2 of SEQ ID NO: 385, (c) HCDR3 of SEQ ID NO: 386, and (d) the light chain variable region comprising LCDR1 of SEQ ID NO: 391, (e) LCDR2 of SEQ ID NO: 392, and (f) LCDR3 of SEQ ID NO: 393; (xxii) A heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 398, (b) HCDR2 of SEQ ID NO: 399, and (c) HCDR3 of SEQ ID NO: 400, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 405, (e) LCDR2 of SEQ ID NO: 406, and (f) LCDR3 of SEQ ID NO: 407; (xxiii) A heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 412, (b) HCDR2 of SEQ ID NO: 413, and (c) HCDR3 of SEQ ID NO: 414, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 419, (e) LCDR2 of SEQ ID NO: 420, and (f) LCDR3 of SEQ ID NO: 421; (xxiv) A heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 426, (b) HCDR2 of SEQ ID NO: 427, and (c) HCDR3 of SEQ ID NO: 428, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 433, (e) LCDR2 of SEQ ID NO: 434, and (f) LCDR3 of SEQ ID NO: 435; (xxv) A heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 440, (b) HCDR2 of SEQ ID NO: 441, and (c) HCDR3 of SEQ ID NO: 442, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 447, (e) LCDR2 of SEQ ID NO: 448, and (f) LCDR3 of SEQ ID NO: 449; (xxvi) A heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 454, (b) HCDR2 of SEQ ID NO: 455, and (c) HCDR3 of SEQ ID NO: 456, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 461, (e) LCDR2 of SEQ ID NO: 462, and (f) LCDR3 of SEQ ID NO: 463; (xxvii) A heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 468, (b) HCDR2 of SEQ ID NO: 469, and (c) HCDR3 of SEQ ID NO: 470, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 475, (e) LCDR2 of SEQ ID NO: 476, and (f) LCDR3 of SEQ ID NO: 477; (xxviii) A heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 482, (b) HCDR2 of SEQ ID NO: 483, and (c) HCDR3 of SEQ ID NO: 484, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 489, (e) LCDR2 of SEQ ID NO: 490, and (f) LCDR3 of SEQ ID NO: 491 An antibody comprising the above. [9] The antibody according to [8] above, wherein at least one amino acid in the CDR is substituted by the corresponding residue of the corresponding CDR of another anti-VP1 antibody in Table 2.

[10] The antibody according to [8] above, wherein one or two amino acids in the CDR are modified, deleted or substituted.

[11] The antibody according to [8] above, containing the modifications in Table 3.

[12] The antibody according to [8] above, retaining at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity across either the variable heavy chain region or the variable light chain region.

[13] The antibody according to [8] above, which is a monoclonal antibody, chimeric antibody, humanized antibody, human engineered antibody, human antibody, single-chain antibody (scFv) or antibody fragment.

[14] The antibody or its antigen-binding fragment is (i) a variable heavy chain region (vH) containing SEQ ID NO: 12 and a variable light chain region (vL) containing SEQ ID NO: 22; (ii) a variable heavy chain region (vH) containing SEQ ID NO: 32 and a variable light chain region (vL) containing SEQ ID NO: 42; (iii) a variable heavy chain region (vH) containing SEQ ID NO: 52 and a variable light chain region (vL) containing SEQ ID NO: 62; (iv) a variable heavy chain region (vH) containing SEQ ID NO: 72 and a variable light chain region (vL) containing SEQ ID NO: 82; (v) a variable heavy chain region (vH) containing SEQ ID NO: 92 and a variable light chain region (vL) containing SEQ ID NO: 102; (vi) a variable heavy chain region (vH) containing SEQ ID NO: 112 and a variable light chain region (vL) containing SEQ ID NO: 122; (vii) a variable heavy chain region (vH) containing SEQ ID NO: 132 and a variable light chain region (vL) containing SEQ ID NO: 142; (viii) a variable heavy chain region (vH) containing SEQ ID NO: 152 and a variable light chain region (vL) containing SEQ ID NO: 162; (ix) a variable heavy chain region (vH) containing SEQ ID NO: 172 and a variable light chain region (vL) containing SEQ ID NO: 182; (x) a variable heavy chain region (vH) containing SEQ ID NO: 192 and a variable light chain region (vL) containing SEQ ID NO: 202; (xi) a variable heavy chain region (vH) containing SEQ ID NO: 212 and a variable light chain region (vL) containing SEQ ID NO: 222; (xii) a variable heavy chain region (vH) containing SEQ ID NO: 232 and a variable light chain region (vL) containing SEQ ID NO: 242; (xiii) a variable heavy chain region (vH) containing SEQ ID NO: 252 and a variable light chain region (vL) containing SEQ ID NO: 262; (xiv) a variable heavy chain region (vH) containing SEQ ID NO: 272 and a variable light chain region (vL) containing SEQ ID NO: 282; (xv) a variable heavy chain region (vH) containing SEQ ID NO: 292 and a variable light chain region (vL) containing SEQ ID NO: 302; (xvi) A heavy chain variable region (vH) comprising SEQ ID NO: 312 and a light chain variable region (vL) comprising SEQ ID NO: 320; (xvii) A heavy chain variable region (vH) comprising SEQ ID NO: 328 and a light chain variable region (vL) comprising SEQ ID NO: 338; (xviii) A heavy chain variable region (vH) comprising SEQ ID NO: 348 and a light chain variable region (vL) comprising SEQ ID NO: 355; (xix) A heavy chain variable region (vH) comprising SEQ ID NO: 362 and a light chain variable region (vL) comprising SEQ ID NO: 369; (xx) A heavy chain variable region (vH) comprising SEQ ID NO: 376 and a light chain variable region (vL) comprising SEQ ID NO: 383; (xxi) A heavy chain variable region (vH) comprising SEQ ID NO: 390 and a light chain variable region (vL) comprising SEQ ID NO: 397; (xxii) A heavy chain variable region (vH) comprising SEQ ID NO: 404 and a light chain variable region (vL) comprising SEQ ID NO: 411; (xxiii) A heavy chain variable region (vH) comprising SEQ ID NO: 418 and a light chain variable region (vL) comprising SEQ ID NO: 425; (xxiv) A heavy chain variable region (vH) comprising SEQ ID NO: 432 and a light chain variable region (vL) comprising SEQ ID NO: 439; (xxv) A heavy chain variable region (vH) comprising SEQ ID NO: 446 and a light chain variable region (vL) comprising SEQ ID NO: 453; (xxvi) A heavy chain variable region (vH) comprising SEQ ID NO: 460 and a light chain variable region (vL) comprising SEQ ID NO: 467; (xxvii) A heavy chain variable region (vH) comprising SEQ ID NO: 474 and a light chain variable region (vL) comprising SEQ ID NO: 481; or, (xxviii) A heavy chain variable region (vH) comprising SEQ ID NO: 488 and a light chain variable region (vL) comprising SEQ ID NO: 495 The antibody according to [1] above, comprising.

[15] The antibody or fragment thereof according to

[14] above, which retains at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity over either the variable light chain region or the variable heavy chain region.

[16] The antibody according to

[14] above, wherein 1, 2, 3, 4 or 5 but less than 10 amino acids within the variable light chain region or variable heavy chain region are modified, deleted or substituted.

[17] The antibody according to

[14] above, which is a monoclonal antibody, chimeric antibody, humanized antibody, human engineered antibody, human antibody, single-chain antibody (scFv) or antibody fragment.

[18] The antibody according to any one of [1] to

[17] above, wherein the antibody or fragment thereof has reduced glycosylation, or no glycosylation, or is hypofucosylated.

[19] A pharmaceutical composition comprising the antibody or fragment thereof according to any one of [1] to

[18] above, further comprising a pharmaceutically acceptable carrier.

[20] The pharmaceutical composition according to

[19] above, wherein the pharmaceutically acceptable carrier contains histidine or sugar.

[21] The pharmaceutical composition according to

[20] above, wherein the sugar is sucrose.

[22] A pharmaceutical composition comprising a plurality of the antibodies or antigen-binding fragments according to any one of the above, wherein at least 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 5% or more of the antibodies in the composition have α2,3-linked sialic acid residues.

[23] A pharmaceutical composition comprising a plurality of the antibodies or antigen-binding fragments according to any one of the above, wherein none of the antibodies contain bisecting GlcNAc.

[24] A pharmaceutical composition comprising the antibody or fragment thereof according to any one of the above, prepared as a lyophilized product.

[25] A method for neutralizing BK virus or JC virus infection, comprising administering an effective amount of the antibody or pharmaceutical composition according to [1] or

[19] above to a patient in need thereof by injection or infusion.

[26] The method according to

[25] above, wherein the patient in need is diagnosed with BK virusuria or BK viremia.

[27] A method for treating or reducing the likelihood of a disorder associated with BK virus or JC virus, comprising administering an effective amount of the antibody or pharmaceutical composition according to [1] or

[19] above to a patient in need thereof by injection or infusion, wherein the disorder is nephropathy, BKVAN, hemorrhagic cystitis (HC), progressive multifocal leukoencephalopathy (PML), granulocyte neuropathy (GCN), interstitial kidney disease, ureteral stenosis, vasculitis, colitis, retinitis, meningitis, and immune reconstitution syndrome (IRIS).

[28] The method according to

[25] or

[27] above, wherein the antibody or composition is reconstituted before injection or infusion.

[29] The method according to

[25] or

[27] above, wherein the antibody or pharmaceutical composition is administered in combination with another therapeutic agent.

[30] The method according to

[29] above, wherein the therapeutic agent is an immunosuppressive agent.

[31] The method according to

[30] above, wherein the immunosuppressive agent is a monophosphate dehydrogenase inhibitor, a purine synthesis inhibitor, a calcineurin inhibitor or an mTOR inhibitor.

[32] The method according to

[31] above, wherein the immunosuppressant is mycophenolate mofetil (MMF), sodium mycophenolate, azathioprine, tacrolimus, sirolimus or cyclosporine.

[33] The method according to

[29] above, wherein the therapeutic agent is a further anti-VP1 antibody.

[34] The antibody or fragment thereof according to any one of [1] to

[18] above for use as a medicament.

[35] The antibody or fragment thereof according to [1] above, or the pharmaceutical composition according to

[19] to

[24] above, for use in the neutralization of BK virus or JC virus infection.

[36] The antibody or fragment thereof according to [1] above, or the pharmaceutical composition according to any one of

[19] to

[24] above, for use in the treatment of nephropathy, BKVAN, hemorrhagic cystitis (HC), progressive multifocal leukoencephalopathy (PML), granulocyte neuropathy (GCN), interstitial kidney disease, ureteral stricture, vasculitis, colitis, retinitis, meningitis, and immune reconstitution syndrome (IRIS) or for reducing the likelihood thereof.

[37] Use of the antibody or fragment thereof according to

[35] above, administered in combination with another therapeutic agent.

[38] Use of the antibody or fragment thereof according to

[37] above, wherein the therapeutic agent is an immunosuppressant.

[39] Use of the antibody or fragment thereof according to

[38] above, wherein the immunosuppressant is a monophosphate dehydrogenase inhibitor, a purine synthesis inhibitor, a calcineurin inhibitor or an mTOR inhibitor.

[40] Use of the antibody or fragment thereof according to

[39] above, wherein the immunosuppressant is mycophenolate mofetil (MMF), sodium mycophenolate, azathioprine, tacrolimus, sirolimus or cyclosporine.

[41] Use of the antibody or fragment thereof according to

[37] above, wherein the therapeutic agent is a further anti-VP1 antibody.

[42] A nucleic acid encoding the antibody or antigen-binding fragment thereof according to [1] above.

[43] A vector comprising the nucleic acid according to

[42] above.

[44] A host cell comprising the vector according to

[43] above.

[45] A method for producing an antibody or antigen-binding fragment, comprising culturing a host cell and recovering the antibody from the culture.

[46] A diagnostic agent comprising the antibody or antigen-binding fragment thereof according to [1] above, which is labeled.

[47] The diagnostic agent according to

[46] above, wherein the label is selected from the group consisting of a radioactive label, a fluorophore, a chromophore, an imaging agent, and a metal ion.

Claims

**Claim 1** (i) (a) A heavy chain variable region (vH) comprising HCDR1 (CDR - Complementary Determining Region) of SEQ ID NO: 126, (b) HCDR2 of SEQ ID NO: 127, and (c) HCDR3 of SEQ ID NO: 128, and (d) LCDR1 of SEQ ID NO: 136, (e) LCDR2 of SEQ ID NO: 137, and (f) LCDR3 of SEQ ID NO: 138; a light chain variable region (vL); (ii) (a) A vH comprising HCDR1 (CDR - Complementary Determining Region) of SEQ ID NO: 146, (b) HCDR2 of SEQ ID NO: 147, and (c) HCDR3 of SEQ ID NO: 148, and (d) LCDR1 of SEQ ID NO: 156, (e) LCDR2 of SEQ ID NO: 157, and (f) LCDR3 of SEQ ID NO: 158; or (iii) (a) A vH comprising HCDR1 (CDR - Complementary Determining Region) of SEQ ID NO: 166, (b) HCDR2 of SEQ ID NO: 167, and (c) HCDR3 of SEQ ID NO: 168, and (d) LCDR1 of SEQ ID NO: 176, (e) LCDR2 of SEQ ID NO: 177, and (f) LCDR3 of SEQ ID NO: 178 An isolated antibody or an antigen - binding fragment thereof that specifically binds to VP1. **Claim 2** The antibody or an antigen - binding fragment thereof according to claim 1, wherein the antibody or an antigen - binding fragment thereof specifically binds to a VP1 epitope (SEQ ID NO: 501). **Claim 3** The antibody or an antigen - binding fragment thereof according to claim 1, wherein the antibody or an antigen - binding fragment thereof has reduced glycosylation, or no glycosylation, or is hypofucosylated. **Claim 4** A pharmaceutical composition comprising the antibody or an antigen - binding fragment thereof according to claim 1 and a pharmaceutically acceptable carrier. **Claim 5** A pharmaceutical composition comprising a plurality of the antibody or an antigen - binding fragment thereof according to claim 1, wherein at least 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 5% or more of the antibody or an antigen - binding fragment thereof in the composition has an α2,3 - linked sialic acid residue. **Claim 6** A pharmaceutical composition comprising a plurality of the antibody or an antigen - binding fragment thereof according to claim 1, wherein none of the antibody or an antigen - binding fragment thereof contains bisecting GlcNAc. **Claim 7** A pharmaceutical composition comprising the antibody or an antigen - binding fragment thereof according to claim 1, which is a lyophilized product. **Claim 8** A pharmaceutical composition for use in a method of neutralizing BK virus (BKV) or John Cunningham (JC) virus (JCV) infection, said pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to claim 1, said method comprising administering an effective amount of said antibody or antigen-binding fragment thereof to a patient in need thereof by injection or infusion.

9. The pharmaceutical composition according to claim 8, wherein the patient in need thereof is diagnosed with BK viremia, BK virusuria, JC viremia, or JC virusuria.

10. A pharmaceutical composition for use in a method of treating or reducing the likelihood of a disorder associated with BK virus or JC virus, said pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to claim 1, said method comprising administering an effective amount of said antibody or antigen-binding fragment thereof to a patient in need thereof by injection or infusion, said disorder being selected from the group consisting of nephropathy, BKV-associated nephropathy (BKAN), hemorrhagic cystitis (HC), progressive multifocal leukoencephalopathy (PML), granulocyte neuropathy (GCN), interstitial kidney disease, ureteral stricture, vasculitis, colitis, retinitis, meningitis, and immune reconstitution syndrome (IRIS).

11. The pharmaceutical composition according to claim 10, wherein said antibody or antigen-binding fragment thereof is administered in combination with another therapeutic agent.

12. The pharmaceutical composition according to claim 11, wherein said another therapeutic agent is an immunosuppressive agent.

13. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof according to claim 1.

14. A vector comprising the nucleic acid according to claim 13.

15. A host cell comprising the vector according to claim 14.

16. A method for producing an antibody or antigen-binding fragment thereof, comprising culturing the host cell according to claim 15 and recovering the antibody or antigen-binding fragment thereof from the culture.

17. A diagnostic agent comprising the antibody or antigen-binding fragment thereof according to claim 1, wherein said antibody or antigen-binding fragment thereof is labeled.

18. The diagnostic agent according to claim 17, wherein said label is selected from the group consisting of a radiolabel, a fluorophore, a chromophore, an imaging agent, and a metal ion.

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