Polyomavirus neutralizing antibody

Abstract

The present invention relates to anti-polyomavirus antibodies, antibody fragments, and their use for the prevention and treatment of BK or JC virus infection and related diseases.

Description

Technical Field

[0001] The present disclosure relates to anti-polyomavirus antibodies, antibody fragments, and their use for the treatment of polyomavirus infection or reduction of its likelihood.

Background Art

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

[0003] The BKV genome is a circular double-stranded DNA approximately 5000 base pairs in length and includes three major regions: an early coding region, a late coding region, and a non-coding control region. The early coding region encodes three regulatory proteins (large tumor antigen [TAg], small tumor antigen [tAg], and truncated tumor antigen [truncTAg]), which are the first viral proteins expressed in newly infected cells and play a role in promoting viral DNA replication and establishing a favorable cellular environment. The late coding region encodes three structural proteins (VP1, VP2, and VP3) that make up the viral capsid, as well as an agnoprotein, but its role during viral replication is not very clear. The non-coding control region includes the origin of genome replication, as well as early and late promoters that drive the expression of viral gene products.

[0004] BKV has been detected in many different cell types, such as renal epithelial cells, the bladder and ureters (typical sites of persistence), tonsillar tissue, and lymphocytes (proposed primary sites of 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 cell surface receptors for BKV are gangliosides GT1b, GD1b, and GD3, all of which have terminal α2,8 - linked sialic acid and are quite widely distributed, enabling infection of various cell types (see Neu et al., PLos Patholog. 2013;9(l0):e1103714 and e1003688, and also O’Hara et al., Virus Res. 2014;189:208 - 285). The non - enveloped icosahedral virion of BKV is composed of three different viral proteins, with 360 copies of the major viral capsid protein VP1 arranged in 72 pentamers and 72 copies of the non - major viral capsid proteins VP2 and VP3 combined, with one VP2 or VP3 molecule bound to each VP1 pentamer. During entry, only VP1 is exposed on the virion surface, and each pentamer has five low - affinity binding sites for ganglioside receptors. When the VP1 pentamer binds to the ganglioside receptor on the cell surface, internalization is initiated through the caveola - mediated endocytosis pathway, followed by transport of the virus to the endoplasmic reticulum and finally to the nucleus (Tsai and Qian, J. Virol 2010;84(19):9840 - 9852).

[0005] Infection by BK virus (BKV) is essentially ubiquitous, and the estimated range of the infected population worldwide is 80 - 90% (Knowles W.A., Adv. Exp. Med. Biol. 2006; 577: 19 - 45). Primary infection most often occurs in childhood (i.e., before 10 years of age) and leads to a mild, non - specific, self - limited disease or is asymptomatic. Persistent infection is established in the epithelial cells of the renal tubules, ureters, and bladder and is effectively controlled by the immune system. Transient and asymptomatic viral excretion in the urine of immunocompetent adults occurs sporadically, but no disease or sequelae develop. However, immune function decline, especially during immunosuppression after kidney or hematopoietic stem cell transplantation, can lead to uncontrolled BKV replication and ultimately to BK virus - associated nephropathy (BKVAN) or hemorrhagic cystitis (HC), a painful disease of the bladder. There is no effective antiviral therapy against BKV, and the current standard of care is reduction of immunosuppression, which increases the risk of acute rejection. Even with a more aggressive current approach to monitoring and prevention, up to 10% of kidney transplant patients develop BKVAN, and 15 - 30% of those patients suffer graft - function loss due to BKVAN. Up to 30% of patients who had their immunosuppressive therapy tapered at the time of detection of BK viremia ultimately experience the onset of acute rejection.

[0006] BKV was first described in 1971 (supra), but until the 1990s, BKVAN had not been reported in the literature as a cause of kidney transplant injury (Purighalla et al., Am. J. Kidney Dis. 1995;26:671-673, and Randhawa et al., Transplantation 1999;67:103-109). In the early management of BKVAN, patients who tested positive for BK faced a serious situation, and graft dysfunction and graft loss were seen in more than 50% of them (Hirsch et al., New Engl. J. Med. 2002;347:488-496). Reactivation and replication of BKV are associated with an established clinical course in kidney transplant patients, first with detection of the virus and viral DNA in urine (viremia), followed by detection of the virus in the bloodstream (viremia), and finally manifested by nephropathy and decreased renal function as a result of viral replication. Usually, within the first 3 months after transplantation, about 30-40% of all kidney transplant patients develop viremia, and 10-20% develop BK viremia (Sawinski and Goral, Nephrol Dial Transplant. 2015;30:209-217; Hirsch et al., Am J Transplant. 2013;13:136-145; Dharnidharka et al., Pediatr Nephrol. 2011;26:1763-1774; Babel et al., Transplantation. 2009;88:89-95). Usually, within 1 year after transplantation, about 1-10% of all kidney transplant patients progress to BKVAN (Bohl and Brennan, Clin J Am Soc Nephrol. 2007;2(Suppl 1):S36-46, Sawinski and Goral, Nephrol Dial Transplant. 2015;30:209-217). BKV replication in renal tubular epithelial cells causes necrosis and lytic destruction, leading to denudation of the basement membrane, accumulation of tubular fluid in the interstitium, and ultimately interstitial fibrosis and tubular atrophy (Nickeleit et al., J. Am. Soc. Neprol. 1999;10(5):1080-1089).Patients may present with worsening renal function, tubulointerstitial nephritis, and ureteral stricture (Garner et al., Lancet 1971;1(7712):1253-1257, and Hirsch Am. J. Transplant 2002;2(1)25-30).

[0007] In addition, BKV can cause interstitial pneumonia, retinitis, and meningoencephalitis in immunocompromised hosts (Reploeg et al., Clin. Infect. Dis. 2001;33(2):191-202). BKV disease in hematopoietic stem cell transplant (HSCT) patients typically manifests as hemorrhagic cystitis (HC), although the severity varies. Viruria (but not necessarily viremia) and painful hematuria are associated with the clinical symptoms of HC. Current treatment guidelines are essentially supportive and mainly include forced hydration / diuresis and pain management. In the most severe cases, blood transfusions, hematoma removal are required, and in some cases, death may result. Among HSCT patients, HC of any cause (e.g., drugs, radiation, virus) is relatively common, 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 in pediatric HSCT patients, adenovirus is a more common cause of HC compared to adult HSCT patients. Also, BK virus has been observed in other immunocompromised states such as organ transplantation and in HIV / AIDS patients (Jiang et al., Virol. 2009;384:266-273).

[0008] At present, the standard treatment for BKVAN is reduction of immunosuppression in an attempt to prevent graft dysfunction and graft injury (Wiseman et al., Am. J. Kidney Dis. 2009; 54(1): 131-142, and Hirsch et al., Transplantation 2005; 79(1): 1277-1286). Although reduction of immunosuppression helps prevent progression from viremia to significant damage associated with clinical nephropathy, there is no stable clinical treatment plan for such reduction because it increases the risk of acute organ rejection (Brennan et al., Am. J. Transplant 2005; 5(3): 582-594). Clinicians have reported using therapeutic agents such as cidofovir, leflunomide, and quinolones in combination with reduction of immunosuppressive agents, but this approach has been ineffective and has increased the burden of managing additional side effects (Randhawa and Brennan Am. J. Transplant 2006; 6(9): 2000-2005). Therefore, there are unaddressed and useful needs in the area of therapies that can neutralize polyomaviruses such as BKV and can be used in immunocompromised hosts.

[0009] JC virus (JCV) is another human polyomavirus that is widely found in the population (80%). However, JCV generally infects later than BKV (Padgett et al., J. Infect. Dis. 1973;127(4):467-470, and Sabath et al., J. Infect. Dis. 2002;186 Suppl. 2:5180-5186). After primary infection, JCV establishes a latent infection in lymphoid organs and the kidney, and upon reactivation, it invades the central nervous system (CNS) via infected B lymphocytes. Once in the CNS, JCV causes progressive multifocal leukoencephalopathy (PML), a progressive demyelinating CNS disorder. Most cases of PML are associated with immunomodulatory therapies used to treat multiple sclerosis (natalizumab, fingolimod, etc.) or rheumatoid arthritis (rituximab, etc.), and usually, the progression of the disease stops upon discontinuation of the treatment. Given the progressive nature of PML, in patients administered JCV neutralizing antibodies over several months, significant improvement may be documented over several months by clinical criteria or by MRI, which is already routinely used for monitoring multiple sclerosis. PML also manifests in patients with HIV / AIDS and has 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). In PML patients, focal neurological deficits such as confusion, changes in mental status, ataxia, hemiparesis, quadriplegia, and visual changes are observed (Richardson E.P., N. Engl. J. Med. 1961;265:815-823). The prognosis of PML patients is poor, especially in patients with HIV / AIDS (Antinori et al., J. Neurovirol. 2003;9 suppl. 1:47-53). This further emphasizes unaddressed and useful needs in the field of therapeutic methods for neutralizing polyomaviruses such as JCV. SUMMARY OF THE INVENTION

[0010] The present disclosure is directed to neutralizing antibodies against human polyomavirus, and / or fragments thereof, and antibodies that recognize BK virus and / or JC virus.

[0011] An antibody, wherein the antibody or an antigen-binding fragment thereof specifically binds to BK virus and / or JC virus.

[0012] An antibody, wherein the antibody or an antigen-binding fragment thereof specifically binds to BK virus and / or JC virus. In one embodiment, the antibody or an antigen-binding fragment thereof binds to and neutralizes BKV serotype I, BKV serotype II, BKV serotype III, or BKV serotype IV, or a combination of serotypes I-IV. In another embodiment, the antibody or an antigen-binding fragment thereof further binds to JC virus.

[0013] An antibody, wherein the antibody or antigen-binding fragment specifically binds to BK virus and / or JC virus. In one embodiment, the antibody or an antigen-binding fragment thereof binds to and neutralizes BK serotype I. In one embodiment, the antibody or an antigen-binding fragment thereof binds to and neutralizes BKV serotype I and BKV serotype II. In another embodiment, the antibody or an antigen-binding fragment thereof binds to and neutralizes BKV serotype I and BKV serotype III. In another embodiment, the antibody or an antigen-binding fragment thereof binds to and neutralizes BKV serotype I and BKV serotype IV. In another embodiment, the antibody or an antigen-binding fragment thereof binds to and neutralizes BKV serotype II and BKV serotype III. In another embodiment, the antibody or an antigen-binding fragment thereof binds to and neutralizes BKV serotype II and BKV serotype IV. In another embodiment, the antibody or an antigen-binding fragment thereof binds to and neutralizes BKV serotype I and JCV. In a preferred embodiment, the antibody or an antigen-binding fragment thereof binds to and neutralizes BKV serotypes I, II, III and IV. Further, the antibody or an antigen-binding fragment thereof binds to and neutralizes BKV serotypes I, II, III and IV, and JCV.

[0014] An isolated antibody or antigen-binding fragment thereof, comprising (i) a heavy chain region and (ii) a light chain region as defined in Table 2.

[0015] An isolated antibody, wherein the antibody or antigen-binding fragment thereof (i) a heavy chain variable region comprising (a) HCDR1 (CDR - Complementary Determining Region) of SEQ ID NO: 9, (b) HCDR2 of SEQ ID NO: 10, (c) HCDR3 of SEQ ID NO: 11, and (d) LCDR1 of SEQ ID NO: 25, (e) LCDR2 of SEQ ID NO: 26, and (f) LCDR3 of SEQ ID NO: 27, and a light chain variable region (ii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 41, (b) HCDR2 of SEQ ID NO: 42, (c) HCDR3 of SEQ ID NO: 43, and (d) LCDR1 of SEQ ID NO: 57, (e) LCDR2 of SEQ ID NO: 58, and (f) LCDR3 of SEQ ID NO: 59, and a light chain variable region (iii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 73, (b) HCDR2 of SEQ ID NO: 74, (c) HCDR3 of SEQ ID NO: 75, and (d) LCDR1 of SEQ ID NO: 89, (e) LCDR2 of SEQ ID NO: 90, and (f) LCDR3 of SEQ ID NO: 91, and a light chain variable region (iv) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 105, (b) HCDR2 of SEQ ID NO: 106, (c) HCDR3 of SEQ ID NO: 107, and (d) LCDR1 of SEQ ID NO: 121, (e) LCDR2 of SEQ ID NO: 122, and (f) LCDR3 of SEQ ID NO: 123, and a light chain variable region (v) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 137, (b) HCDR2 of SEQ ID NO: 138, (c) HCDR3 of SEQ ID NO: 139, and (d) LCDR1 of SEQ ID NO: 153, (e) LCDR2 of SEQ ID NO: 154, and (f) LCDR3 of SEQ ID NO: 155, and a light chain variable region (vi) (a) The heavy chain variable region comprising HCDR1 of SEQ ID NO: 169, (b) HCDR2 of SEQ ID NO: 170, (c) HCDR3 of SEQ ID NO: 171, and (d) the light chain variable region comprising LCDR1 of SEQ ID NO: 185, (e) LCDR2 of SEQ ID NO: 186, and (f) LCDR3 of SEQ ID NO: 187, (vii) (a) The heavy chain variable region comprising HCDR1 of SEQ ID NO: 201, (b) HCDR2 of SEQ ID NO: 202, (c) HCDR3 of SEQ ID NO: 203, and (d) the light chain variable region comprising LCDR1 of SEQ ID NO: 217, (e) LCDR2 of SEQ ID NO: 218, and (f) LCDR3 of SEQ ID NO: 219, (viii) (a) The heavy chain variable region comprising HCDR1 of SEQ ID NO: 233, (b) HCDR2 of SEQ ID NO: 234, (c) HCDR3 of SEQ ID NO: 235, and (d) the light chain variable region comprising LCDR1 of SEQ ID NO: 249, (e) LCDR2 of SEQ ID NO: 250, and (f) LCDR3 of SEQ ID NO: 251, (xi) (a) The heavy chain variable region comprising HCDR1 of SEQ ID NO: 265, (b) HCDR2 of SEQ ID NO: 266, (c) HCDR3 of SEQ ID NO: 267, and (d) the light chain variable region comprising LCDR1 of SEQ ID NO: 281, (e) LCDR2 of SEQ ID NO: 282, and (f) LCDR3 of SEQ ID NO: 283, and an isolated antibody comprising the same.

[0016] An antibody in which one or two amino acids within the CDR are modified, deleted, or substituted.

[0017] An antibody that retains at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity over either the variable heavy chain region or the variable light chain region.

[0018] An antibody, wherein the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a humanized modified antibody, a human antibody, a single-chain antibody (scFv), or an antibody fragment.

[0019] An isolated antibody or an antigen-binding fragment thereof, (i) A heavy chain variable region (vH) containing SEQ ID NO: 18 and a light chain variable region (vL) containing SEQ ID NO: 34, (ii) A heavy chain variable region (vH) containing SEQ ID NO: 50 and a light chain variable region (vL) containing SEQ ID NO: 66, (iii) A heavy chain variable region (vH) containing SEQ ID NO: 82 and a light chain variable region (vL) containing SEQ ID NO: 98, (iv) A heavy chain variable region (vH) containing SEQ ID NO: 114 and a light chain variable region (vL) containing SEQ ID NO: 130, (v) A heavy chain variable region (vH) containing SEQ ID NO: 146 and a light chain variable region (vL) containing SEQ ID NO: 162, (vi) A heavy chain variable region (vH) containing SEQ ID NO: 178 and a light chain variable region (vL) containing SEQ ID NO: 194, (vii) A heavy chain variable region (vH) containing SEQ ID NO: 210 and a light chain variable region (vL) containing SEQ ID NO: 226, (viii) A heavy chain variable region (vH) containing SEQ ID NO: 242 and a light chain variable region (vL) containing SEQ ID NO: 258, (ix) An isolated antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (vH) containing SEQ ID NO: 274 and a light chain variable region (vL) containing SEQ ID NO: 290.

[0020] An antibody or a fragment thereof 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.

[0021] An antibody in which 1, 2, 3, 4, or 5, but less than 10, amino acids within the variable light chain or variable heavy chain region are modified, deleted, or substituted.

[0022] An antibody, wherein the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human-modified antibody, a human antibody, a single-chain antibody (scFv), or an antibody fragment.

[0023] A method for isolating and producing an antibody using a natural signal / leader peptide sequence that matches an appropriate VH and / or VL gene segment.

[0024] A method for isolating and producing an antibody that uses a synthetic and / or optimized signal / leader peptide sequence to improve expression and yield.

[0025] An antibody or a fragment thereof that is an antibody with reduced glycosylation, or no glycosylation, or low fucosylation.

[0026] A pharmaceutical composition comprising an antibody or a fragment thereof and further comprising a pharmaceutically acceptable carrier.

[0027] A pharmaceutical composition in which the pharmaceutically acceptable carrier contains histidine or a saccharide.

[0028] A pharmaceutical composition in which the saccharide is sucrose

[0029] A pharmaceutical composition comprising a plurality of antibodies or antigen-binding fragments, 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.

[0030] A pharmaceutical composition comprising a plurality of antibodies or antigen-binding fragments, wherein none of the antibodies contain bisecting GlcNAc.

[0031] A pharmaceutical composition comprising an antibody or a fragment thereof, wherein the composition is prepared as a lyophilized product.

[0032] A method for neutralizing BK virus or JC virus infection, comprising administering an effective amount of an antibody to a patient in need thereof via injection or infusion.

[0033] A method in which the patient in need is diagnosed with BK virusuria or BK viremia.

[0034] A method in which the patient in need is diagnosed with JC virus uropathy or JC viremia.

[0035] A method of treating or reducing the likelihood of BK virus or JC virus-related disorders, comprising administering an effective amount of an antibody to a patient in need via injection or infusion, wherein the disorder is nephropathy, BKVAN, hemorrhagic cystitis (HC), progressive multifocal leukoencephalopathy (PML), granulocyte neuronopathy (GCN), interstitial kidney disease, ureteral stenosis, vasculitis, colitis, retinitis, meningitis, and immune reconstitution inflammatory syndrome (IRIS).

[0036] A method in which the antibody or composition is reconstituted before injection or infusion.

[0037] A method in which the antibody or pharmaceutical composition is administered in combination with another therapeutic agent.

[0038] A method in which the therapeutic agent is an immunosuppressive agent.

[0039] A method in which the immunosuppressive agent is a dehydrogenase inhibitor, a purine synthesis inhibitor, a calcineurin inhibitor or an mTOR inhibitor.

[0040] A method in which the immunosuppressive agent is mycophenolate mofetil (MMF), sodium mycophenolate, azathioprine, tacrolimus, sirolimus or cyclosporine.

[0041] A method in which the therapeutic agent is an additional anti-VP1 antibody.

[0042] A method in which PML is associated with the treatment of multiple sclerosis, rheumatoid arthritis, or psoriasis.

[0043] A method in which the treatment of multiple sclerosis is natalizumab, fingolimod, or dimethyl fumarate, dimethyl fumarate, fumaric acid, or alemtuzumab.

[0044] A method in which the treatment of rheumatoid arthritis is rituximab.

[0045] A method for treating psoriasis, which is efalizumab.

[0046] An antibody or fragment thereof for use as a medicament.

[0047] An antibody or fragment thereof for use in neutralizing BK virus or JC virus infection.

[0048] An antibody or fragment thereof for use in the treatment of nephropathy, BKVAN, hemorrhagic cystitis (HC), progressive multifocal leukoencephalopathy (PML), granulocyte neuronopathy (GCN), interstitial kidney disease, ureteral stricture, vasculitis, colitis, retinitis, meningitis, and immune reconstitution inflammatory syndrome (IRIS) or for reducing the likelihood thereof.

[0049] Use of an antibody or fragment thereof administered in combination with another therapeutic agent.

[0050] Use of an antibody or fragment thereof, wherein the therapeutic agent is an immunosuppressive agent.

[0051] Use of an antibody or fragment thereof, wherein the immunosuppressive agent is a dehydrogenase inhibitor, a purine synthesis inhibitor, a calcineurin inhibitor or an mTor inhibitor.

[0052] Use of an antibody or fragment thereof, wherein the immunosuppressive agent is mycophenolate mofetil (MMF), sodium mycophenolate, azathioprine, tacrolimus, sirolimus or cyclosporine.

[0053] Use of an antibody or fragment thereof, wherein the therapeutic agent is an additional anti-BK antibody.

[0054] Use of an antibody or fragment thereof, wherein PML is associated with the treatment of multiple sclerosis, rheumatoid arthritis, or psoriasis.

[0055] Use, wherein the treatment of multiple sclerosis is natalizumab, fingolimod, or dimethyl fumarate, fumaric acid ester, or alemtuzumab.

[0056] Use of rituximab for the treatment of rheumatoid arthritis.

[0057] Use of efalizumab for the treatment of psoriasis.

[0058] Nucleic acid encoding an antibody or antigen-binding fragment thereof.

[0059] Vector containing the nucleic acid.

[0060] Host cell containing the vector.

[0061] Diagnostic agent comprising a labeled antibody or antigen-binding fragment thereof.

[0062] Diagnostic agent, wherein the label is selected from the group consisting of a radioactive label, a fluorophore, a chromophore, an imaging agent, and a metal ion.

[0063] Definition Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings.

[0064] As used herein, the term "antibody" means a polypeptide of the immunoglobulin family that can bind to a corresponding antigen in a non-covalent, reversible, and specific manner. For example, a naturally-occurring IgG antibody is a tetramer that includes 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 herein as VH) 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 herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH region and the VL region are further divided into regions of hypervariability designated as complementarity determining regions (CDRs), which are interspersed with more conserved regions designated as framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains include 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, which include various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

[0065] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camel antibodies, chimeric antibodies, and anti-idiotype (anti-Id) antibodies (e.g., including anti-Id antibodies to the antibodies of the present disclosure). The antibody can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0066] "Complementary determining domain" or "complementary determining region" ("CDR") means, interchangeably, the hypervariable regions of VL and VH. The CDRs are the target protein-binding sites of the antibody chains that have specificity for such target proteins. Each human VL or VH, which constitutes approximately 15-20% of the variable domain, has three CDRs (CDR1-3, numbered sequentially from the N-terminus). The CDRs can be referred to by those regions and order. For example, both "VHCDR1" or "HCDR1" mean the first CDR of the heavy chain variable region. The CDRs are structurally complementary to the epitopes of the target protein and thus are directly involved in the binding specificity. The remaining segments of VL or VH, the so-called framework regions, have less variation in amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4. W.H. Freeman & Co., New York, 2000).

[0067] The positions of the CDR and framework regions can be determined using various definitions known in the art, such as Kabat, Chothia, IMGT, and AbM (see, for example, Johnson et al., Nucleic Acids Res., 29:205-206 (2001), Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987), Chothia et al., Nature, 342:877-883 (1989), Chothia et al., J. Mol. Biol., 227:799-817 (1992), Lefranc, M.P., Nucleic Acids Res., 29:207-209 (2001), Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997)). Also, the definition of the antigen-binding site is described below: Ruiz et al., Nucleic Acids Res., 28:219-221 (2000), MacCallum et al., J. Mol. Biol., 262:732-745 (1996), 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 the Kabat CDRs, the Chothia CDRs, or both.For example, in some embodiments, the CDRs correspond to amino acid residues 26 - 35 (HC CDR1), 50 - 65 (HC CDR2), and 95 - 102 (HC CDR3) in VH, e.g., mammalian VH, e.g., human VH; and to amino acid residues 24 - 34 (LC CDR1), 50 - 56 (LC CDR2), and 89 - 97 (LC CDR3) in VL, e.g., mammalian VL, e.g., human VL. Under IMGT, the CDR amino acid residues in VH are numbered approximately 26 - 35 (CDR1), 51 - 57 (CDR2), and 93 - 102 (CDR3), and the CDR amino acid residues in VL are numbered approximately 27 - 32 (CDR1), 50 - 52 (CDR2), and 89 - 97 (CDR3). Under IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0068] 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 will be understood that the variable domains of both the light chain (VL) and heavy chain (VH) portions determine antigen recognition and specificity. In contrast, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the numbering of the constant region domains increases as they become more distal 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. Indeed, the CH3 and CL domains contain the carboxy - terminal domains of the heavy and light chains, respectively.

[0069] As used herein, the term "antigen-binding fragment" refers to one or more portions of an antibody that retain the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of binding fragments include single-chain Fv (scFv), disulfide-linked Fv (sdFv), Fab fragments, F(ab’) fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments, divalent fragments comprising two Fab fragments linked by a disulfide bridge in the hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; dAb fragments consisting of a VH domain (Ward et al., Nature 341:544-546, 1989); and isolated complementarity-determining regions (CDRs), or other epitope-binding fragments of an antibody, but are not limited thereto.

[0070] Furthermore, the two domains of VL and VH of an Fv fragment are encoded by separate genes, but these can be joined by a synthetic linker using recombinant methods, enabling them to be made as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv, 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 encompassed by the term "antigen-binding fragment". These antigen-binding fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as the intact antibody.

[0071] Antigen-binding fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetra-bodies, v-NAR, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can be grafted onto a polypeptide-based scaffold such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes monobodies of fibronectin polypeptides).

[0072] Antigen-binding fragments can be incorporated into single-chain molecules, which consist of a pair of tandem Fv segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions together with a complementary light chain polypeptide (Zapata et al., Protein Eng. 8:1057-1062, 1995, and U.S. Patent No. 5,641,870).

[0073] As used herein, the terms "monoclonal antibody" or "monoclonal antibody composition" mean polypeptides that include antibodies and antigen-binding fragments having substantially the same amino acid sequence or derived from the same genetic source. Further, the term includes preparations of antibody molecules of a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

[0074] As used herein, the term "human antibody" includes antibodies having variable regions in which both the framework and CDR regions are derived from human-derived sequences. Further, when the antibody includes a constant region, the constant region is derived from such human sequences, e.g., human germline sequences or mutated versions of human germline sequences, or from antibodies that include consensus framework sequences derived from human framework sequence analysis (as described, e.g., in Knappik et al., J. Mol. Biol. 296:57-86, 2000).

[0075] The human antibodies of the present disclosure include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or somatic mutations in vivo, or conservative substitutions to promote stability or production).

[0076] As used herein, the term "recognize" means an antibody or antigen-binding fragment thereof that finds and interacts with (e.g., binds to) its epitope, and the epitope can be linear or conformational. The term "epitope" means 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 adjacent amino acids or non-adjacent amino acids juxtaposed by the tertiary folding of a protein. Epitopes formed from adjacent amino acids are typically retained even when exposed to denaturing solvents, while epitopes formed by three-dimensional folding are typically lost upon treatment with denaturing solvents. An epitope typically contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a particular spatial conformation. Methods for determining the spatial conformation of an epitope include techniques in the art, such as X-ray crystallography and two-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996)), or electron microscopy. A "paratope" is the part of an antibody that recognizes the epitope of an antigen.

[0077] The terms "specifically binds" or "selectively binds," when used in the context of describing an interaction between an antigen (e.g., a protein) and an antibody, antibody fragment, or antibody-derived binding agent, mean a binding reaction that determines the presence of the antigen in a heterogeneous collection of proteins or biological materials (e.g., in a biological sample such as blood, serum, plasma, or tissue sample). Thus, under certain specified immunoassay conditions, an antibody or binding agent having a particular binding specificity binds to a particular antigen at least 2-fold above background and does not bind significantly to other antigens present in the sample. In one aspect, under the specified immunoassay conditions, an antibody or binding agent having a particular binding specificity binds to a particular antigen at least 10-fold above background and does not bind significantly to other antigens present in the sample. Specific binding of an antibody or binding agent under such conditions may require that the antibody or agent be selected for its specificity for a particular protein. This selection can be achieved by reducing antibodies that cross-react with molecules from other species (e.g., mouse or rat) or other subtypes, as desired or appropriate. Alternatively, in some aspects, an antibody or antibody fragment is selected to cross-react with a particular desired molecule.

[0078] As used herein, the term "affinity" means 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 multiple sites via weak non-covalent binding forces, and the more interactions there are, the stronger the affinity.

[0079] The term "isolated antibody" means 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. Furthermore, an isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0080] The term "corresponding human germline sequence" means a nucleic acid sequence encoding a human variable region amino acid sequence or subsequence, which, when compared to all other known or putative variable region amino acid sequences encoded by the human germline immunoglobulin variable region sequences, shares the highest determined amino acid sequence identity with the reference variable region amino acid sequence or subsequence. Also, the corresponding human germline sequence means a human variable region amino acid sequence or subsequence that has the highest amino acid sequence identity with the reference variable region amino acid sequence or subsequence when compared to all other evaluated variable region amino acid sequences. The corresponding human germline sequence can be a sequence or subsequence that includes only framework regions, only complementarity-determining regions, framework and complementarity-determining regions, variable segments (as defined above), or other combinations of variable region-containing sequences or subsequences. Sequence identity can be determined by aligning two sequences using the methods described herein, for example, using BLAST, ALIGN, or another alignment algorithm known in the art. The corresponding human germline nucleic acid or amino acid sequence can 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 sequence or amino acid sequence.

[0081] A variety of immunoassay formats can be used to select antibodies that are specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that are specifically immunoreactive with a protein (see, for example, Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective binding reaction generates a signal that is at least 2-fold, more typically at least 10- to 100-fold, above background.

[0082] The term "equilibrium dissociation constant (KD, M)" refers to the association rate constant (Ka, time-1 , M -1 ) dissociation rate constant (Kd, time -1 ). The equilibrium dissociation constant can be measured using methods known in the art. The antibodies of the present disclosure generally have an equilibrium dissociation constant of about 10 -7 or 10 -8 M or less, for example, about 10 -9 M or 10 -10 M or less, and in some embodiments, 10 -11 M, 10 -12 M or 10 -13 M or less.

[0083] The term "bioavailability" means the systemic availability (i.e., blood / plasma level) of a given dose of a drug administered to a patient. Bioavailability is an absolute term and indicates measurements of both the time (speed) and total amount (extent) of drug reaching the general circulation from the administered dosage form.

[0084] As used in the specification, the phrase "consisting essentially of" means the genus or species of the active agent included in the method or composition, as well as any excipients that are inert for the intended purpose of the method or composition. In some embodiments, the phrase "consisting essentially of" expressly excludes the inclusion of one or more additional active agents other than the anti-BK or JC antibodies of the present disclosure. In some embodiments, the phrase "consisting essentially of" expressly excludes the inclusion of one or more additional active agents other than the anti-BK or JC antibodies of the present disclosure and a second co-administered agent.

[0085] The term "amino acid" refers to amino acids of natural origin, synthetic and non-natural amino acids, as well as amino acid analogs and mimetics that function in a manner similar to amino acids of natural origin. Amino acids of natural origin are amino acids encoded by the genetic code, as well as amino acids that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs mean compounds having the same basic chemical structure as amino acids of natural origin, i.e., an α-carbon bonded to hydrogen, a carboxyl group, an amino group, and an R group, and examples include homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Such analogs have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as amino acids of natural origin. Amino acid mimetics mean chemical compounds having a structure different from the general chemical structure of amino acids, but functioning in a manner similar to amino acids of natural origin.

[0086] The term "conservatively modified variant" applies to both amino acid 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 an essentially identical amino acid sequence, or, when the nucleic acid does not encode an amino acid sequence, a nucleic acid that encodes an essentially identical sequence. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variants are "silent variants" and are one species of conservatively modified variants. Also, all nucleic acid sequences herein that encode polypeptides describe every possible silent variant of the nucleic acids. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except for the AUG which is ordinarily the only codon for methionine and TGG which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Thus, each silent variant of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0087] For a polypeptide sequence, a "conservatively modified variant" includes individual substitutions, deletions or additions to the polypeptide sequence that result in amino acid substitutions with chemically similar amino acids. Tables of conservative substitutions that give functionally similar amino acids are known in the art. Such conservatively modified variants are polymorphic variants, interspecies homologs, and allelic additions and are not excluded. The following eight groups include amino acids that are conservative substitutes 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); 8) cysteine (C), methionine (M) (see, e.g., Creighton, Proteins (1984)). In some embodiments, the term "conservative sequence modification" is used to mean an amino acid modification that does not significantly affect or change the binding properties of an antibody comprising the amino acid sequence.

[0088] As used herein, the term "optimized" means a nucleotide sequence that has been modified to encode an amino acid sequence using codons preferred in a production cell or organism (generally a eukaryotic cell, e.g., a yeast cell, a Pichia cell, a fungal cell, a Trichoderma cell, a Chinese hamster ovary cell (CHO) or a human cell). The optimized nucleotide sequence is designed to retain, as completely as possible, the amino acid sequence originally encoded by the first nucleotide sequence, also known as the "parent sequence".

[0089] The terms "percent identity" or "percent identical" in the context of two or more nucleic acid or polypeptide sequences mean the degree to which the two or more sequences or subsequences are identical. Two sequences are "identical" when they have the same amino acid or nucleotide sequence over the region being compared. Two sequences are "substantially the same" when, using one of the following sequence comparison algorithms or by measurement by manual alignment and visual inspection, the two sequences have a specified percentage of amino acid residues or nucleotides that are identical when compared and aligned for maximum correspondence over a comparison window or specified region (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over the specified region or, if not specified, over the entire sequence). Optionally, the identity exists over a region that is at least about 30 nucleotides (or 10 amino acids) in length, more preferably over a region that is 100 - 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0090] 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 and reference sequences are input into a computer, subsequence coordinates are specified as necessary, and the program parameters of the sequence algorithm are specified. Default program parameters can be used or other parameters can be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence based on the program parameters.

[0091] As used herein, "comparison window" includes reference to any segment of a number of contiguous positions selected from the group consisting of 20 to 600, usually about 50 to about 200, more usually about 100 to about 150, wherein the sequences can be compared to a reference sequence of the same number of adjacent positions after the two sequences are optimally aligned. Methods of aligning sequences for comparison are known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482c (1970), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Brent et al., Current Protocols in Molecular Biology, 2003).

[0092] Two examples of algorithms suitable for determining sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977, and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy a positive-valued threshold score T when aligned with words of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating the search and finding longer HSPs that contain them. Word hits are extended in both directions along each sequence as long as the cumulative alignment score can increase. The cumulative score is calculated using parameters M (reward score for matching residue pairs, always >0) and N (penalty score for mismatching residues, always <0) for nucleotide sequences. For amino acid sequences, the cumulative score is calculated using a scoring matrix. Extension of the word hits in each direction stops when the cumulative alignment score drops by an amount X from its maximum gain value, when the cumulative score becomes zero or less due to the accumulation of one or more negative-score residue alignments, or when 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 a word length (W) of 11, an expectation value (E) of 10, M = 5, N = 4, and both-strand comparison as default values.For amino acid sequences, the BLASTP program uses 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). For alignments (B), it uses 50, an expectation value (E) of 10, M = 5, N = 4, and two-strand comparison as the default.

[0093] Also, the BLAST algorithm 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)), which provides an indication of the likelihood that a match between two nucleotide or amino acid sequences occurs by chance. For example, a nucleic acid is considered similar to a control sequence if the minimum total probability in the comparison of the test nucleic acid to the control nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0094] Also, the percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller incorporated into the ALIGN program (version 2.0) (Comput. Appl. Biosci. 4:11 - 17, 1988), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Further, the percent identity of two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444 - 453, 1970) incorporated into the GAP program of the GCG software package (available from the University of South Florida), using a BLOSUM 62 matrix or a 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.

[0095] Except for the percentage of sequence identity recited above, another indication that two nucleic acid sequences or polypeptides are substantially identical is that a polypeptide encoded by a first nucleic acid is immunologically cross-reactive with an antibody raised against a polypeptide encoded by a second nucleic acid, as follows. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that, as shown below, the two molecules or their complements hybridize to each other under stringent conditions. Further, another indication that two nucleic acid sequences are substantially identical is that they can be used to amplify sequences using the same primers.

[0096] The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and means deoxyribonucleotides or ribonucleotides and polymers thereof, in either single-stranded or double-stranded form. The term includes nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, have similar binding properties as the reference nucleic acid, and are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like.

[0097] Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequences explicitly indicated. Specifically, as detailed below, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed base and / or deoxyinosine residues (Batzer et al., (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al., (1985) J. Biol. Chem. 260:2605-2608; and Rossolini et al., (1994) Mol. Cell. Probes 8:91-98).

[0098] In the context of nucleic acids, the term "operably linked" refers to a functional relationship between two or more polynucleotide segments (e.g., DNA). Typically, it refers to the functional relationship between a transcriptional regulatory sequence and a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, a promoter transcriptional regulatory sequence operably linked to a transcribed sequence is physically adjacent to the transcribed sequence, i.e., cis-acting. However, some transcriptional regulatory sequences, such as enhancers, do not need to be physically adjacent or located in close proximity to the coding sequence whose transcription they enhance.

[0099] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. This term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise specified, a particular polypeptide sequence also implicitly encompasses its conservatively modified variants.

[0100] The term "subject" includes humans and non-human animals. Non-human animals include mammals and non-mammals, such as all vertebrates, for example, non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. Unless otherwise stated, the terms "patient" or "subject" are used interchangeably herein.

[0101] The term "BKV" or "BK virus" means a member of the Polyomaviridae family, Orthopolyomavirus genus. 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).

[0102] The term "JCV" or "JC virus" means a member of the Polyomaviridae family, Orthopolyomavirus genus. JCV is related to BKV and is also an icosahedral, non-enveloped, double-stranded DNA virus with a genome of approximately 5,000 base pairs. They are approximately 40 - 45 nm in diameter (Johne et al., Arch. Virol. 2011;156(9):1627 - 1634).

[0103] The term "BKV nephropathy" or "BKV-related nephropathy" or "BKVAN" means an inflammatory interstitial nephropathy resulting from lytic infection by BKV, characterized mainly by viral cytopathic transformation and viral gene expression in renal tubular epithelium.

[0104] The term "VP1" means the major polyomavirus capsid subunit protein. "VP1 pentamer" is composed of 5 monomers of VP1.

Table 1-1

Table 1-2

[0105] A "virus-like particle" or "VLP" is an assembly of VP1 pentamers into a viral capsid. The VLP is composed of 72 VP1 pentamers. The VLP is highly structurally similar to the actual virus, but lacks the minor capsid proteins (VP2, VP3) as well as the viral DNA genome, and is thus non-infectious. The VLP is useful as a viral epitope and is presented in a similar form to the actual virus.

[0106] "IC50" (half-maximal inhibitory concentration) refers to the concentration of a particular antibody that induces a signal that is intermediate (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.

[0107] "EC50" (half-maximal effective concentration) means the concentration of a particular antibody that induces a response that is intermediate (50%) between the baseline control and the maximum possible effect after a particular exposure or treatment time. For example, the EC50 is the concentration of an antibody at which 50% of the viral infection is neutralized.

[0108] "EC90" means the concentration of a particular antibody that induces a response corresponding to 90% of the maximum possible effect after a particular exposure or treatment time. For example, the EC90 is the concentration of an antibody at which 90% of the viral infection is neutralized.

[0109] "Neutralization" means the inhibition of viral infection of a host cell as indicated by the absence of viral gene expression. Without being bound by any theory, the mechanism of neutralization by a particular antibody may include preventing the interaction between the viral capsid protein and the cell surface receptor before the viral genome is delivered to the nucleus of the host cell, or preventing disruption at any stage of the entry and transport processes.

[0110] As used herein, the terms "treating," "treatment of," or "treatment" of any disease or disorder, in one aspect, means alleviating the disease or disorder (i.e., delaying, or arresting, or reducing at least one of the disease or its clinical symptoms). In another aspect, it means reducing or alleviating at least one physical parameter, including those that may not be distinguishable in a patient. In yet another aspect, "treating," "treatment of," or "treatment" means modulating the disease or disorder, either physically (e.g., stabilization of distinguishable symptoms), physiologically (e.g., stabilization of physical parameters), or both.

[0111] The phrase "reducing the likelihood of" means delaying the onset or progression of a disease, infection, or disorder.

[0112] The terms "therapeutically acceptable amount" or "therapeutically effective amount" are used interchangeably to mean an amount sufficient to produce a desired result (i.e., reduction in tumor size, inhibition of tumor growth, prevention of metastasis, inhibition or prevention of viral, bacterial, fungal, or parasitic infection). In some aspects, a therapeutically acceptable amount does not induce or cause undesirable side effects. A therapeutically acceptable amount can be administered at a low dose that is initially determined and then incrementally increased until the desired effect is achieved. The "preventively effective dosage" and "therapeutically effective dosage" of the molecules of the present disclosure can each prevent the onset of disease symptoms, including those associated with polyomavirus infection, or result in a reduction in severity.

[0113] The term "co-administration" means the concurrent presence of two active agents in the bloodstream of an individual. The active agents to be co-administered can be delivered simultaneously or sequentially. BRIEF DESCRIPTION OF THE DRAWINGS

[0114]

Figure 1

Figure 2A

Figure 2B

Figure 3A

Figure 3B

Figure 3C

Figure 3D

Figure 3E

Figure 3F

Figure 3G

Figure 3H

[0115] The present disclosure provides antibodies, antibody fragments (e.g., antigen-binding fragments) that bind to and neutralize BKV. Further, the present disclosure provides antibodies having desired pharmacokinetic properties and other desirable attributes, and thus can be used to treat or reduce the likelihood of BK virus-associated nephropathy (e.g., BKVAN) and / or JC virus-associated progressive multifocal leukoencephalopathy (PML). The present disclosure further provides pharmaceutical compositions comprising the antibodies, as well as methods of manufacturing and using such pharmaceutical compositions for the prevention and treatment of polyomavirus infections and related disorders.

[0116] Anti-polyomavirus antibody The present disclosure provides antibodies and antibody fragments (e.g., antigen-binding fragments) that specifically bind to BK or JC virus. The antibodies or antibody fragments (e.g., antigen-binding fragments) of the present disclosure include, but are not limited to, the isolated human monoclonal antibodies or fragments thereof described in the following examples.

[0117] In one aspect, the present disclosure provides an antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to BK or JC virus, wherein the antibody or antibody fragment (e.g., antigen-binding fragment) comprises a VH domain having the amino acid sequence of SEQ ID NOs: 18, 50, 82, 114, 146, 178, 210, 242, and 274 (Table 2). The present disclosure also provides an antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to BK or JC virus, wherein the antibody or antibody fragment (e.g., antigen-binding fragment) comprises a VH CDR having the amino acid sequence of any of the VH CDRs listed in Table 2. In certain aspects, the present disclosure provides an antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to BK or JC virus, wherein the antibody comprises 1, 2, 3, or more VH CDRs (or, alternatively, consists of) having the amino acid sequence of any of the VH CDRs listed in Table 2.

[0118] The present disclosure provides an antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to BK or JC virus, wherein the antibody or antibody fragment (e.g., antigen-binding fragment) comprises a VL domain having the amino acid sequence of SEQ ID NOs: 34, 66, 98, 130, 162, 194, 226, 258, and 290 (Table 2). The present disclosure also provides an antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to BK or JC virus, wherein the antibody or antibody fragment (e.g., antigen-binding fragment) comprises a VL CDR having the amino acid sequence of any of the VL CDRs listed in Table 2. In particular, the present disclosure provides an antibody or antibody fragment (e.g., antigen-binding fragment) that specifically binds to BK or JC virus, wherein the antibody or antibody fragment (e.g., antigen-binding fragment) comprises one, two, three or more VL CDRs (or, alternatively, consists of them) having the amino acid sequence of any of the VL CDRs listed in Table 2.

[0119] Other antibodies or antibody fragments (e.g., antigen-binding fragments) of the present disclosure contain mutated amino acids but have at least 60%, 70%, 80%, 90% or 95% identity in the CDR regions shown in the sequences described in Table 2. In some embodiments, when compared to the CDR regions shown in the sequences described in Table 2, the CDR regions contain an amino acid sequence in which 1, 2, 3, 4 or 5 or more amino acids have not mutated.

[0120] 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 BK or JC virus. Such nucleic acid sequences can be optimized for expression in mammalian cells.

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

Table 2-20

Table 2-21

Table 2-22

Table 2-23

Table 2-24

Table 2-25

Table 2-26

Table 2-27

Table 2-28

Table 2-29

Table 2-30

Table 2-31

Table 2-32

Table 2-33

Table 2-34

Table 2-35

Table 2-36

Table 2-37

Table 2-38

Table 2-39

Table 2-40

Table 2-41

Table 2-42

Table 2-43

Table 2-44

Table 2-45

Table 2-46

Table 2-47

Table 2-48

Table 2-49

Table 2-50

Table 2-51

Table 2-52

Table 2-53

Table 2-54

Table 2-55

Table 2-56

Table 2-57

Table 2-58

Table 2-59

Table 2-60

Table 2-61

Table 2-62

Table 2-63

Table 2-64

Table 2-65

Table 2-66

Table 2-67

Table 2-68

Table 2-69

Table 2-70

Table 2-71

Table 2-72

Table 2-73

Table 2-74

Table 2-75

Table 2-76

Table 2-77

Table 2-78

Table 2-79

Table 2-80

Table 2-81

Table 2-82

Table 2-83

Table 2-84

[0121] 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% identity to the sequences described in Table 2. In some embodiments, when compared to the variable regions shown in the sequences described in Table 2, the variable regions contain mutant amino acid sequences in which 1, 2, 3, 4 or 5 or more amino acids have not been mutated and which retain substantially the same therapeutic activity.

[0122] 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 aligned" to generate VP1-binding antibodies. Such "mixed and aligned" 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 these chains are mixed and aligned, VH sequences from a particular VH / VL pairing should be replaced with structurally similar VH sequences. Similarly, full-length heavy chain sequences from a particular full-length heavy chain / full-length light chain pairing must be replaced with structurally similar full-length heavy chain sequences. Similarly, VL sequences from a particular VH / VL pairing must be replaced with structurally similar VL sequences. Similarly, full-length light chain sequences from a particular full-length heavy chain / full-length light chain pairing must be replaced with structurally similar full-length light chain sequences. Thus, in one aspect, the disclosure provides an isolated monoclonal antibody or antigen-binding region thereof having a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 50, 82, 114, 146, 178, 210, 242, and 274 (Table 2), and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 66, 98, 130, 162, 194, 226, 258, and 290 (Table 2), wherein the antibody specifically binds to BK or JC virus.

[0123] In another aspect, the present disclosure provides an isolated monoclonal antibody having (i) a full-length heavy chain comprising an amino acid sequence selected from Table 2 and a full-length light chain comprising an amino acid sequence selected from Table 2, and having a sequence optimized for expression in mammalian cells. In a similar aspect, the present disclosure provides (i) a full-length heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 52, 84, 116, 148, 180, 212, 244, and 276 (Table 2) and optimized for expression in mammalian cells, and a full-length light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 68, 100, 132, 164, 196, 228, 260, and 292 (Table 2) and optimized for expression in mammalian cells, or (ii) a functional protein comprising an antigen-binding portion thereof.

[0124] In another aspect, the present disclosure provides a BK or JC virus-binding antibody comprising the heavy and light chain CDR1, CDR2, and CDR3 described in Table 2, or a combination thereof. The amino acid sequence of the VH CDR1 of the antibody is shown in SEQ ID NOs: 9, 41, 73, 105, 137, 169, 201, 233, and 265. The amino acid sequence of the VH CDR2 of the antibody is shown in SEQ ID NOs: 10, 42, 74, 106, 138, 170, 202, 234, and 266. The amino acid sequence of the VH CDR3 of the antibody is shown in SEQ ID NOs: 11, 43, 75, 107, 139, 171, 203, 235, and 267. The amino acid sequence of the VL CDR1 of the antibody is shown in SEQ ID NOs: 25, 57, 89, 121, 153, 185, 217, 249, and 281. The amino acid sequence of the VL CDR2 of the antibody is shown in SEQ ID NOs: 26, 58, 90, 122, 154, 186, 218, 250, and 282. The amino acid sequence of the VL CDR3 of the antibody is shown in SEQ ID NOs: 27, 59, 91, 123, 155, 187, 219, 251, and 283.

[0125] Assuming that each of these antibodies can bind to BK or JC virus and that their antigen-binding specificities are provided mainly by the CDR1, 2, and 3 regions, the VH CDR1, 2, and 3 sequences, or the VL CDR1, 2, and 3 sequences, can be "mixed and matched" (i.e., each antibody must contain VH CDR1, 2, and 3, and VL CDR1, 2, and 3 to generate other VP1-binding molecules, but the CDRs from different antibodies can be mixed and matched). Such "mixed and matched" VP1-binding antibodies can be tested using binding assays known in the art (e.g., ELISA) described in the Examples. When the VH CDR sequences are mixed and matched, the CDR1, CDR2, and / or CDR3 sequences from a particular VH sequence must be replaced with structurally similar CDR sequences. Similarly, when the VL CDR sequences are mixed and matched, the CDR1, CDR2, and / or CDR3 sequences from a particular VL sequence must be replaced with structurally similar CDR sequences. It will be readily apparent to those skilled in the art that novel VH and VL sequences can be generated for the monoclonal antibodies of the present disclosure by replacing one or more VH and / or VL CDR region sequences with structurally similar sequences from the CDR sequences shown herein.

[0126] Accordingly, the present disclosure provides an isolated monoclonal antibody or an antigen-binding region thereof, comprising a heavy-chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 41, 73, 105, 137, 169, 201, 233, and 265; a heavy-chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 42, 74, 106, 138, 170, 202, 234, and 266; a heavy-chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 43, 75, 107, 139, 171, 203, 235, and 267; a light-chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 57, 89, 121, 153, 185, 217, 249, and 281; a light-chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 58, 90, 122, 154, 186, 218, 250, and 282; and a light-chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 59, 91, 123, 155, 187, 219, 251, and 283, wherein the antibody specifically binds to BK or JC virus.

[0127] In certain embodiments, an antibody that specifically binds to BK or JC virus is an antibody or antibody fragment (e.g., antigen-binding fragment) described in Table 2.

[0128] 1. Identification of Antibodies The present disclosure provides antibodies and antibody fragments (e.g., antigen-binding fragments) that bind to BK or JC virus. In certain embodiments, the antibodies and antibody fragments can bind to the same epitope among all four BKV serotypes and / or JCV.

[0129] In addition, the present disclosure provides antibodies and antibody fragments (e.g., antigen-binding fragments) that bind to the same epitope such that they bind the anti-BK antibody or JC antibody 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 binding in a statistically significant manner). The ability of a test antibody to inhibit the binding of an antibody or antibody fragment (e.g., antibody fragment) of the present disclosure to BK or JC virus demonstrates that the test antibody can compete with the antibody or antibody fragment (e.g., antigen-binding fragment) for binding to BK or JC virus, and such an antibody, according to non-limiting theory, can bind to the same or a related (e.g., structurally similar or spatially proximal) epitope on BK or JC virus as the antibody or antibody fragment (e.g., antigen-binding fragment) with which it competes. In one aspect, an antibody that binds to the same epitope on BK or JC virus as an 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.

[0130] 2. Further modification of the framework of the Fc region The present disclosure discloses specific anti-BK or JC virus antibodies. These antibodies further include modifications to residues in the framework within VH and / or VL, and include the modified antibody or its antigen-binding fragment, for example, to improve the properties of the antibody. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "revert mutate" one or more framework residues to their corresponding germline sequences. More specifically, an antibody that has undergone somatic mutation may contain framework residues that are different from the germline sequences from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, somatic mutations can be "reverted mutated" to germline sequences, for example, by site-directed mutagenesis. Such "reverted mutated" antibodies are also intended to be encompassed.

[0131] Another type of framework modification includes mutating one or more residues within the framework region or even within one or more 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 Publication No. 2003 / 0153043 by Carr et al.

[0132] In addition to, or alternatively to, modifications made within the framework or CDR regions, the antibody can be designed to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antibody-dependent cell cytotoxicity. Further, the antibody may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter its glycosylation to change one or more functional properties of the antibody. Each of these aspects is described in more detail below.

[0133] In one embodiment, the hinge region of CH1 is modified by varying the number of cysteine residues in the hinge region, for example, increasing or decreasing. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of CH1 can be varied, for example, to facilitate the construction of the light and heavy chains, or to increase or decrease the stability of the antibody.

[0134] In another embodiment, the fc hinge region of the antibody is mutated to reduce the biological half-life of the antibody. More specifically, by introducing one or more amino acid mutations into the CH2-CH3 domain interface region of the Fc-hinge fragment, the SpA binding of the antibody is impaired relative to the binding of the native Fc-hinge domain to staphylococcal protein A (SpA). This approach is described in more detail in U.S. Patent No. 6,165,745 by Ward et al.

[0135] In yet another embodiment, the Fc region is modified by substituting 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 substituted with different amino acid residues such that the antibody has a changed affinity for an effector ligand while retaining the antigen-binding ability of the parental antibody. The effector ligand with the changed affinity can be, for example, an Fc receptor or the C1 component of complement. This approach is described, for example, in U.S. Patents Nos. 5,624,821 and 5,648,260 by Winter et al.

[0136] In another embodiment, one or more amino acids selected from amino acid residues can be substituted with different amino acid residues such that the antibody has altered C1q binding and / or reduced or abolished complement-dependent cytotoxicity (CDC). This approach is described, for example, in U.S. Patent No. 6,194,551 by Idusogie et al.

[0137] In another aspect, the ability of the antibody to fix complement is altered by modifying one or more amino acid residues. This approach is described, for example, in International Patent Application No. WO94 / 29351 by Bodmer et al. In certain aspects, one or more amino acids of the antibodies or antigen-binding fragments thereof of the present disclosure are replaced with one or more allotype amino acid residues for the IgG1 subclass and κ isotype. Also, the allotype amino acid residues include, but are not limited to, the constant regions of the heavy chains of the IgG1, IgG2, and IgG3 subclasses, and the constant region of the light chain of the κ isotype, as described in Jefferis et al., MAbs. 1:332-338 (2009).

[0138] In yet another aspect, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cell cytotoxicity (ADCC) and / or to increase the affinity of the antibody for Fcγ receptors by modifying one or more amino acids. This approach is described, for example, in International Patent Application No. WO00 / 42072 by Presta. Further, the binding sites on human IgG1 for FcγRl, FcγRII, FcγRIII, and FcRn have been mapped and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001).

[0139] In yet another aspect, the glycosylation of the antibody is modified. For example, a non-glycosylated antibody can be produced (i.e., the antibody lacks glycosylation). The glycosylation can be altered, for example, to increase the affinity of the antibody for an "antigen". Such carbohydrate modifications can be achieved, for example, by changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made, resulting in the removal of glycosylation sites in one or more variable region frameworks, thereby eliminating glycosylation at those sites. Such glycosylation can increase the affinity of the antibody for the antigen. Such an approach is described, for example, in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0140] Additionally, or alternatively, antibodies with modified forms of glycosylation can be made, such as afucosylated antibodies having a reduced amount of fucosyl residues or antibodies having an increased bisecting GlcNAc structure. Such modified glycosylation patterns have been demonstrated to increase the ADCC ability of the antibody. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in host cells having a modified glycosylation mechanism. Cells having a modified glycosylation mechanism are described in the art and can produce antibodies with modified glycosylation by expressing recombinant antibodies using them as host cells. For example, European Patent No. 1,176,195 by Hang et al. describes a cell line having the FUT8 gene encoding a functionally disrupted fucosyltransferase, and antibodies expressed in such a cell line show afucosylation. International Patent Application No. WO03 / 035835 by Presta describes a mutant CHO cell line, Lecl3 cells, with a reduced ability to attach fucose to the Asn(297)-linked carbohydrate, and as a result, the antibodies expressed in such host cells are afucosylated (see Shields et al., (2002) J. Biol. Chem. 277:26733-26740). International Patent Application No. WO99 / 54342 by Umana et al. describes a cell line designed to express a glycoprotein-modifying glycosyltransferase (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), and the antibodies expressed in the modified cell line show an increased bisecting GlcNAc structure, resulting in an increase in the ADCC activity of the antibody (see Umana et al., Nat. Biotech. 17:176-180, 1999).

[0141] In another aspect, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, as described in U.S. Patent No. 6,277,375 by Ward, one or more of the following mutations: T252L, T254S, T256F, can be introduced. Alternatively, to increase the biological half-life, as described in U.S. Patents Nos. 5,869,046 and 6,121,022 by Presta et al., the antibody can be modified within the CH1 or CL region to include the salvage receptor binding epitope derived from two loops of the CH2 domain of the Fc region of IgG.

[0142] In minimizing the ADCC activity of the antibody, specific mutations in the Fc region result in a "Fc silent" antibody with minimal interaction with effector cells. Generally, the "IgG Fc region" is used to define the C-terminal region of the immunoglobulin heavy chain, including both the native sequence Fc region and variant Fc regions. The human IgG heavy chain Fc region is generally defined as consisting of the amino acid residues from position C226 or P230 to the carboxyl terminus of the IgG antibody. The numbering of residues in the Fc region is according to the Kabat EU index. The C-terminal lysine (residue K447) of the Fc region can be removed, for example, during antibody production or purification.

[0143] The suppressed effector function is 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). See also the international patent application No. WO2012 / 065950 of Heusser et al. An example of a silent Fc IgG1 antibody is the LALA mutant containing 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. US6,737,056). Another silent IgG1 antibody contains the N297A mutation, and a non-glycosylated / unglycosylated antibody is obtained.

[0144] Fc silent antibodies have no or low ADCC activity, which means that Fc silent antibodies exhibit an ADCC activity where specific cell lysis is less than 50% (low ADCC activity) or specific cell lysis is less than 1% (no ADCC activity).

[0145] 3. Production of Antibodies Anti-BK or JC virus antibodies and antibody fragments (e.g., antigen-binding fragments) can be produced by any means known in the art, including but not limited to recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, while full-length monoclonal antibodies can be obtained, for example, by hybridoma or recombinant production. Recombinant expression is carried out from any suitable host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[0146] The present disclosure provides polynucleotides encoding the antibodies described herein, for example, polynucleotides encoding heavy or light chain variable regions or segments comprising the complementarity determining regions 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: 19, 51, 83, 115, 147, 179, 211, 243, and 275. 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: 35, 67, 99, 131, 163, 195, 227, 259, and 291.

[0147] 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: 21, 53, 85, 117, 149, 181, 213, 245, 277. 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: 37, 69, 101, 133, 165, 197, 229, 261, and 293.

[0148] The polynucleotides of the present disclosure can encode only the variable region sequences of anti-BK or JC virus antibodies. They can also encode both the variable and constant regions of an antibody. Some of the polynucleotide sequences encode polypeptides that include the variable regions of both a heavy chain and a light chain of an exemplified anti-BK or JC virus antibody. Some of the other polynucleotide sequences encode two polypeptide segments that are each substantially identical to the variable regions of a heavy chain and a light chain of a mouse antibody.

[0149] The polynucleotide sequences can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of existing sequences encoding anti-BK or JC virus antibodies or binding fragments thereof. Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art such as the phosphotriester method of Narang et al. (Meth. Enzymol. 68:90, 1979), the phosphodiester method of Brown et al. (Meth. Enzymol. 68:109, 1979), the diethylphosphoramidite method of Beaucage et al. (Tetra. Lett., 22:1859, 1981), and the solid support method of U.S. Patent No. 4,458,066. Introduction of mutations into polynucleotide sequences by PCR can be done as described, for example, 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.

[0150] In addition, the present disclosure provides expression vectors and host cells for producing the above-described anti-BK or JC virus antibodies. Various expression vectors can be used to express polynucleotides encoding anti-BK or JC virus antibody chains or binding fragments. Both virus-based expression vectors and non-virus expression vectors can be used to produce antibodies in mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors, typically expression cassettes that express proteins or RNAs, and human artificial chromosomes (see, for example, Harrington et al., Nat Genet 15:345, 1997). For example, non-viral vectors useful for the expression of anti-BK or JC virus polynucleotides and polypeptides in mammalian (e.g., human) cells include pThioHis A, B & C, pcDNA3.1 / His, pEBVHis A, B & C (Invitrogen, San Diego, CA), MPSV vectors, and numerous other vectors known in the art for expressing other proteins. Useful viral vectors include retrovirus-based vectors, adenovirus, adeno-associated virus, herpes virus, SV40-based vectors, papillomavirus, HBP Epstein-Barr virus, vaccinia virus vectors, and Semliki Forest virus (SFV). See Brent et al., supra, Smith, Annu. Rev. Microbiol. 49:807, 1995, and Rosenfeld et al., Cell 68:143, 1992.

[0151] The selection of an expression vector depends on the intended host cell in which the vector is to be expressed. Typically, an expression vector includes a promoter and other regulatory sequences (e.g., enhancers) operably linked to a polynucleotide encoding an anti-BK or JC virus antibody chain or fragment. In some embodiments, an inducible promoter is used to prevent expression of the inserted sequence under non-inducing conditions. Examples of inducible promoters include, for example, the arabinose, lacZ, metallothionein promoter or heat shock promoter. Cultures of the transformed organisms can be grown under non-inducing conditions without biasing the population of coding sequences for which the expression products are better tolerated by the host cells. In addition to the promoter, other regulatory elements may also be necessary or desirable for efficient expression of the anti-VP1 antibody chain or fragment. These elements typically include the ATG start codon and adjacent ribosome binding site or other sequences. Furthermore, the efficiency of expression can be enhanced by including an enhancer appropriate for the cell line in use (see, for example, Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., Meth Enzymol., 153:516, 1987). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

[0152] The expression vector can also provide a secretion signal sequence position to form a fusion protein with the polypeptide encoded by the inserted anti-BK antibody sequence. More often, the inserted anti-BK antibody sequence is linked to the signal sequence before being included in the vector. Vectors used to receive sequences encoding the anti-BK antibody light and heavy chain variable domains may also encode a constant region or a portion thereof. Such vectors allow for expression of the variable region as a fusion protein with the constant region, thereby resulting in the production of an intact antibody or a fragment thereof. Typically, such a constant region is human.

[0153] Host cells that possess and express an anti-BK or JC antibody chain may be either prokaryotic or eukaryotic cells. E. coli is a prokaryotic host useful for cloning and expressing the polynucleotides of the present disclosure. Other microbial hosts suitable for use include Bacilli such as Bacillus subtilis, and other Enterobacteriaceae such as Salmonella, Serratia, and Pseudomonas. In these prokaryotic hosts, expression vectors can also be prepared, typically including expression control sequences (e.g., origin of replication) compatible with the host cell. Furthermore, any number of known promoters exist, such as the lactose promoter system, the tryptophan (trp) promoter system, the β-lactamase promoter system, or the promoter system from phage λ. The promoter typically optionally controls expression with an operator sequence and has a ribosome binding site sequence, etc., for initiating and terminating 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.

[0154] In other embodiments, mammalian host cells are used to express and produce the anti-VP1 polypeptides of the disclosure. For example, they can be either a hybridoma cell line that expresses an endogenous immunoglobulin gene (e.g., the myeloma hybridoma clones described in the Examples), or a mammalian cell line having an exogenous expression vector. These include any normal cells or normal or abnormal immortalized animal or human cells. For example, many suitable host cell lines that can secrete intact immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, transformed B-cells, and hybridomas. The use of mammalian tissue cell culture to express polypeptides is generally described, for example, in Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y., 1987. Expression vectors for mammalian host cells can include expression control sequences such as origins of replication, promoters, and enhancers (see, e.g., 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 include a promoter derived from a mammalian gene or a promoter derived from a mammalian virus. Suitable promoters may be constitutive, cell-type specific, stage specific, and / or regulatable or controllable. 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 polIII promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[0155] The method of introducing an expression vector containing the target polynucleotide sequence varies depending on the type of cell host. For example, calcium chloride transfection is generally used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts (generally, Sambrook et al., supra). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection, microinjection, ballistics, virosomes, immunoliposomes, polycation nucleic acid conjugate complexes, naked DNA, artificial virions, fusion to the herpes virus structural protein VP22 (Elliot and O’Hare, Cell 88:223, 1997), drug-enhanced uptake of DNA, and ex vivo transduction. For long-term, high-yield production of recombinant proteins, stable expression is often desired. For example, cell lines that stably express anti-BK or JC virus antibody chains or binding fragments can be prepared using an expression vector containing a replication or endogenous expression element of viral origin and a selectable marker gene. After introduction of the vector, the cells can be grown in enriched medium for 1-2 days before switching 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 selective medium. Cells stably transfected with resistance can be grown using tissue culture techniques suitable for the cell type.

[0156] Therapeutic and diagnostic uses The antibodies, antibody fragments (e.g., antigen-binding fragments) of the present disclosure are useful in a variety of applications including, but not limited to, polyomavirus infection and disease. In one aspect, the antibodies, antibody fragments (e.g., antigen-binding fragments) are useful for neutralizing BKV or JCV infection and are useful for the prevention or treatment of BK virus nephropathy, e.g., BKVAN). The method of use can be an in vitro, ex vivo, or in vivo method.

[0157] In one aspect, an antibody, an antibody fragment (e.g., an antigen-binding fragment), is 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, a biological sample includes cells or tissues. In certain aspects, such tissues include normal and / or cancerous tissues that express BKV at higher levels than other tissues.

[0158] In one aspect, the present disclosure provides a method for detecting the presence of BK or JC virus in a biological sample. In certain aspects, the method includes contacting a biological sample with an anti-BK or JC virus antibody under conditions that permit binding of the antibody to the antigen, and detecting whether a complex has formed between the antibody and the antigen. The biological sample can include, but is not limited to, urine or blood samples.

[0159] Also included is a method for diagnosing a disorder associated with the expression of BK or JC virus. In certain aspects, the method includes contacting test cells with an anti-BK or JC virus antibody, detecting binding of the antibody to BK or JC virus, thereby determining the level (quantitative or qualitative) of expression of BK or JC virus in the test cells, and comparing the level of infection in the test cells to the level of infection of BK or JC virus in control cells (e.g., normal cells of the same tissue origin as the test cells or non-virus-infected cells), wherein a higher level of presence of BK or JC virus in the test cells compared to the control cells indicates the presence of a disorder associated with infection with BK or JC virus. In certain aspects, the test cells are obtained from an individual suspected of having an infection with BK or JC virus.

[0160] In certain aspects, the diagnostic or detection method as described above includes detecting binding of a BK or JC virus antibody to virus-infected cells. An exemplary assay for detecting binding of an anti-BK or JC virus antibody to virus-infected cells is the "FACS" assay.

[0161] Other methods can be used to detect the binding of anti-BK or JC virus antibodies. Such methods include, but are not limited to, antigen-binding assays 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).

[0162] In certain embodiments, the anti-BK or JC virus antibody is labeled. Labels include, but are not limited to, labels or moieties that are directly detectable (e.g., fluorescence, chromophore, high electron density, chemiluminescence, and radiolabels), as well as moieties such as enzymes or ligands that are indirectly detectable (e.g., via an enzymatic reaction or molecular interaction).

[0163] In certain embodiments, the anti-BK or JC virus antibody is immobilized on an insoluble matrix. Immobilization involves separating the anti-BK or JC virus antibody from any BKV or JCV proteins that do not remain in solution. This can be achieved by insolubilizing the anti-BK antibody or JC antibody, such as by adsorption to a water-insoluble matrix or surface, prior to the assay procedure (U.S. Patent No. 3,720,760 to Bennich et al.), by covalent bonding (e.g., using glutaraldehyde cross-linking), or by forming a complex between the anti-BK or JC antibody and the BKV or JCV protein and then insolubilizing the anti-BK or JC antibody (e.g., by immunoprecipitation).

[0164] Diagnosis or detection in any of the above embodiments can be performed using the anti-BK antibody or JC antibody of the present disclosure, instead of or in addition to, another anti-BK or JC antibody.

[0165] In one aspect, the present disclosure provides a method of administering an antibody, an antibody fragment (e.g., an antigen-binding fragment) to a patient to treat, reduce the likelihood of, or remit a disease, thereby treating the disease. In certain aspects, the disease treated with an antibody, an antibody fragment (e.g., an antigen-binding fragment) is 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 kidney disease, ureteral stenosis, granulocytic neuronopathy (GCN), vasculitis, colitis, retinitis, meningitis, and immune reconstitution inflammatory syndrome (IRIS). In certain aspects, the infection is characterized by BKV or JCV-expressing cells to which an anti-BK antibody, an antibody fragment (e.g., an antigen-binding fragment), or a JC antibody can specifically bind.

[0166] The present disclosure provides a method of treating BK virus infection, which comprises administering a therapeutically effective amount of an antibody, an antibody fragment (e.g., an antigen-binding fragment). In certain aspects, the subject is human.

[0167] In certain aspects, a method of reducing BK virus infection comprises administering to a subject a therapeutically effective amount of an antibody or an antibody fragment (e.g., an antigen-binding fragment). In certain aspects, the subject is human. In certain aspects, the subject is immunosuppressed. For an immunosuppressed subject, the amount of immunosuppression can be increased or decreased for the therapeutic effect of the anti-BK antibody.

[0168] In one aspect, the transplanted tissue is infected with BK virus to which anti-BK antibodies bind. Due to the high incidence of BK infection in the general population, in the case of kidney transplantation, there is a high probability that the patient receiving the kidney is BK virus positive, the donor providing the kidney is BK virus positive, or both are BK virus positive. To prevent BKVAN, anti-BK antibodies can be administered to the kidney transplant recipient before and / or after the kidney transplant procedure, depending on the serum positivity of the kidney donor or the transplant recipient. In another aspect, the anti-BK antibodies can be administered to the patient when the virus is detected in the urine (viremia) or when the virus is detected in the blood (viremia).

[0169] For the treatment of BK or JC virus infection, the appropriate dosage of an antibody or antibody fragment (e.g., antigen-binding fragment) depends on various factors such as the type of infection being treated, the severity and course of the infection, the responsiveness of the infection, the generation of viral resistance to the treatment, previous treatments, the patient's medical history, and the like. The antibody can be administered over a series of treatments that last for one or several days to several months, or until cure is effected or a reduction in the infection is achieved (e.g., a reduction in viremia or viral impairment of the kidney). The appropriate dosing schedule is calculated from measurements of drug accumulation in the patient's body and varies depending on the relative potency of the individual antibody or antibody fragment (e.g., antigen-binding fragment). In one aspect, the dosage is from 0.01 mg to 10 mg per kg of body weight (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) and can be administered one or more times daily, weekly, monthly, or annually. In one aspect, the antibodies or antibody fragments (e.g., antigen-binding fragments) of the present disclosure are administered once every two or three weeks. The treating physician can estimate the rate of repetition for dosing and the concentration of the antibody in body fluids or tissues based on the measured half-life.

[0170] Combination therapy In one example, an antibody or antibody fragment (e.g., antigen-binding fragment) of the present disclosure is combined with other therapeutic agents such as other antiviral agents, anti-allergy agents, anti-emetic agents (or anti-nausea agents), analgesics, cytoprotective agents, immunosuppressive agents, and combinations thereof.

[0171] As used herein, the term "pharmaceutical combination" means either a defined combination in a single dosage unit form, or an undefined combination or a kit of parts for combined administration, where two or more therapeutic agents may be administered independently simultaneously or separately within a time interval, and in particular, these time intervals are provided such that the combination partners exhibit a synergistic effect (e.g., a synergistic effect).

[0172] The term "combination therapy" means the administration of two or more therapeutic agents for treating a therapeutic condition or infection described in the present disclosure. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule where the active ingredients have a certain ratio. Alternatively, such administration includes the co-administration of each active ingredient in multiple, or separate containers (e.g., capsules, powders, and liquids). The powders and / or liquids can be reconstituted or diluted to the desired dosage before administration. Further, such administration also includes the use of each type of therapeutic agent in a sequential manner, either almost simultaneously or at different times. In any case, the treatment plan is such that the combination of agents provides a beneficial effect in treating the conditions or disorders described herein.

[0173] Combination therapy can provide a "synergistic effect", that is, when the active ingredients used together have an effect greater than the sum of the effects obtained by using the compounds separately, it can be seen that the resulting effect is "synergistic". The synergistic effect can be achieved when the active ingredients are (1) co-formulated and administered, or simultaneously delivered in a combined unit dosage formulation, (2) delivered alternately or in parallel as separate formulations, or (3) delivered by other dosing schedules. When delivered by alternating therapy, a synergistic effect can be achieved when the compounds are sequentially administered or delivered by different injections, for example, with separate syringes. Generally, during alternating therapy, the effective dosages of each active ingredient are administered sequentially, that is, continuously, while in combination therapy, the effective dosages of two or more active ingredients are administered together.

[0174] In one aspect, the present disclosure provides a method of treating BKV or JCV infection by administering to a subject in need of an antibody, together with an immunosuppressive therapy. The anti-BK antibody or JC 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, dehydrogenase inhibitor monophosphate, purine synthesis inhibitor, calcineurin inhibitor or mTOR inhibitor. Specific examples of immunosuppressive therapeutic agents include, but are not limited to, mycophenolate mofetil (MMF), sodium mycophenolate, azathioprine, tacrolimus, sirolimus and cyclosporine.

[0175] Pharmaceutical composition To prepare a pharmaceutical or sterile composition comprising an anti-BK antibody or JC antibody, the antibodies of the present disclosure are mixed with a pharmaceutically acceptable carrier or excipient. The composition may further comprise one or more other therapeutic agents suitable for neutralizing BKV or JCV infection.

[0176] Formulations of therapeutic and diagnostic agents can be prepared by mixing a physiologically acceptable carrier, excipient, or stabilizer in the form of, for example, a lyophilized powder, slurry, aqueous solution, lotion, or suspension (see, e.g., Hardman et al., Goodman and Gilman’s The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y., 2001, Gennaro, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y., 2000; Avis, et al. (eds.), Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY, 1993, Lieberman, et al. (eds.), Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY, 1990, Lieberman, et al. (eds.) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY, 1990, Weiner and Kotkoskie, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y., 2000).

[0177] In certain embodiments, the anti-BK antibody or JC antibody is a lyophilized product within a vial containing the antibody. The lyophilized product can be reconstituted with water or a pharmaceutical carrier suitable for injection. For subsequent intravenous administration, the resulting solution is usually further diluted in a carrier solution.

[0178] The antibodies disclosed herein are useful for neutralizing BKV or JCV in immunosuppressible tissue transplant patients and can use the pharmaceutical carriers of sucrose and human albumin that have been previously used in bone marrow transplant patients who receive CytoGam® (DeRienzo et al. Pharmacotherapy 2000;20:1175-8). Alternatively, the anti-BK antibody or JC antibody can be introduced into transplant patients via a pharmaceutical carrier as described for another anti-viral antibody (Synagis® described in International Patent Application No. WO2003 / 105894). In that publication, the pharmaceutical carrier is composed of histidine and / or glycine, saccharides (e.g., sucrose) and polyols (e.g., polysorbate).

[0179] Selecting a dosing regimen for treatment depends on several factors, including the severity of the infection, the level of symptoms, and the accessibility of target cells in the biological matrix. In one aspect, the dosing regimen maximizes the amount of treatment delivered to the patient that is consistent with an acceptable level of side effects. Thus, the amount of biological delivery that is delivered depends, in part, on the particular entity and the severity of the condition being treated. Guidance is available for selecting appropriate dosages of antibodies, cytokines, and small molecules (see, e.g., Wawrzynczak, Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK, 1996, Kresina (ed.), Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, N.Y., 1991, Bach (ed.), Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, 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. 344:783-792, 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. 343:1594-1602, 2000).

[0180] The determination of an appropriate dosage is made by the clinician using, for example, parameters or factors that are known or suspected in the art to affect, or are predicted to affect, the treatment. Generally, the dosage is started at an amount somewhat less than the optimal dosage and is then increased in small increments until the desired or optimal effect is obtained for any negative side effects. Important diagnostic measures include, for example, those of the symptoms of the infusion reaction.

[0181] The actual dosage level of the active ingredient in a pharmaceutical composition having an anti-BK antibody can be varied to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration without imparting toxicity to the patient. The selected dosage level depends on various 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 the treatment, the duration of the treatment, other drugs, compounds and / or substances used in combination with the particular composition being used, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and similar factors known in the medical arts.

[0182] Compositions containing an antibody or fragment thereof can be provided by continuous infusion or by doses at intervals, for example, once a day, once a week, or from 1 to 7 times a week. The doses can be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracranially, or by inhalation. Specific dosage protocols include those that include a maximum dosage or frequency of administration that avoids significant undesirable side effects.

[0183] For the antibodies described in this specification, the dosage administered to a patient may be from 0.0001 mg / kg to 100 mg / kg of the patient's body weight. The dosage may be between 0.0001 mg / kg and 20 mg / kg, 0.0001 mg / kg and 10 mg / kg, 0.0001 mg / kg and 5 mg / kg, 0.0001 and 2 mg / kg, 0.0001 and 1 mg / kg, 0.0001 mg / kg and 0.75 mg / kg, 0.0001 mg / kg and 0.5 mg / kg, 0.0001 mg / kg and 0.25 mg / kg, 0.0001 and 0.15 mg / kg, 0.0001 and 0.10 mg / kg, 0.001 and 0.5 mg / kg, 0.01 and 0.25 mg / kg, or 0.01 and 0.10 mg / kg per patient's body weight. The dosage of the antibody or its fragment can be calculated using the patient's body weight (in kilograms) multiplied by the dosage (mg / kg) administered.

[0184] Subsequently, the dosage of the antibody can be repeated, and the administrations can be spaced at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months apart.

[0185] The effective amount for a particular patient can vary depending on factors such as the condition being treated, the overall health of the patient, the method, route and dosage of administration, and the severity of side effects (see, for example, Maynard et al., A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla., 1996; Dent, Good Laboratory and Good Clinical Practice, Urch Publ., London, UK, 2001).

[0186] The route of administration can be, for example, topical or cutaneous application, intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intrathecal, intralesional injection or infusion, or by means of a sustained release system or implant (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, Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030-4034, 1980, U.S. Pat. Nos. 6,350,466 and 6,316,024). Optionally, the composition may contain a solubilizing agent or a local anesthetic (e.g., lidocaine to relieve pain at the injection site), or both. Further, pulmonary administration can be utilized using an inhaler, nebulizer, etc., and by using the composition together with an aerosolizing agent. For example, it is described in U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078, and International Patent Publications WO92 / 19244, WO97 / 32572, WO97 / 44013, WO98 / 31346, and WO99 / 66903, each of which is hereby incorporated by reference in its entirety.

[0187] In addition, the compositions of the present disclosure can be administered via one or more routes of administration using 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 antibodies include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, intraspinal, or other parenteral routes, such as administration by injection or infusion. Parenteral administration may represent a mode of administration other than enteral and topical administration and typically, but not limited to, includes intravenous, intramuscular, intraarterial, intrathecal, intra-articular, intra-orbital, intracardiac, intradermal, intraperitoneal, transtracheal, subepidermal, subclavian, intra-articular, subcapsular, intrathecal, intraspinal, epidural, and intrasternal injection and infusion. Alternatively, the compositions of the present disclosure can be administered via parenteral routes, such as topical, epithelial, or mucosal routes, for example, intranasally, orally, vaginally, rectally, sublingually, or topically. In one aspect, the antibodies of the present disclosure are administered by infusion. In another aspect, the antibodies are administered subcutaneously.

[0188] When the antibodies of the present disclosure are administered in a controlled release or sustained release system, pumps can be used to achieve controlled or sustained release (see Langer, supra, Sefton, CRC Ref Biomed. Eng. 14:20, 1987, Buchwald et al., Surgery 88:507, 1980, Saudek et al., N. Engl. J. Med. 321:574, 1989). Polymeric materials can be used to achieve controlled or sustained release of the antibody therapy (e.g., see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla., 1974, Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York, 1984, Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61, 1983, also Levy et al., Science 228:190, 1985, During et al., Ann. Neurol. 25:351, 1989, Howard et al., J. Neurosurg. 71:105, 1989, U.S. Patent Nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463, 5,128,326, International Patent Publications WO99 / 15154, and WO99 / 20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactic acid (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters.In one aspect, the polymers used in sustained release formulations are inert, contain no leachable impurities, are storage stable, sterile, and biodegradable. The controlled or sustained release system can be placed in proximity to the prophylactic or therapeutic target and thus requires only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138, 1984).

[0189] Controlled release systems are described in Langer, Science 249:1527-1533, (1990). Sustained release formulations containing one or more antibodies of the present disclosure can be manufactured using any technique known to those of skill in the art. For example, U.S. Patent No. 4,526,938, International Patent Application Nos. WO91 / 05548, WO96 / 20698, 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., Pro. 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.

[0190] 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 Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995). For non-sprayable topical dosage forms, semi-solid or solid forms that are viscous and, in some cases, have a dynamic viscosity greater than water and contain a carrier or one or more excipients compatible with topical application are typically used. Suitable formulations include solutions, suspensions, emulsions, creams, ointments, powders, liniments, plasters, etc., and may include, but are not limited to, sterilization or mixing with adjuvants (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) that affect various properties such as osmotic pressure, as required. Other suitable topical dosage forms include sprayable aerosol preparations, where, in some cases, the active ingredient is combined with a solid or liquid inert carrier and packaged as a mixture in a compressed volatile (e.g., a gaseous propellant such as freon) bottle or a squeeze bottle. Moisturizing or water-retaining agents can also be added to the pharmaceutical compositions and dosage forms, as needed. Examples of such additional components are known in the art.

[0191] When administering a composition containing an antibody intranasally, it can be formulated in the form of an aerosol, spray, mist, or droplets. In particular, the prophylactic or therapeutic agent for use according to the present disclosure can be conveniently delivered in the form of an aerosol spray presentation from a pressurized packaging pack or nebulizer using a suitable propellant (e.g., dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas). In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve that supplies a measured amount. Capsules and cartridges (e.g., made of gelatin) for use in inhalers or injectors can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.

[0192] Methods for the co - administration or treatment with a second therapeutic agent, e.g., an immunosuppressant, cytokine, steroid, chemotherapeutic agent, antibiotic or radiation, 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 the symptoms by at least 10%, at least 20%, at least about 30%, at least 40%, or at least 50%.

[0193] Additional therapies (e.g., prophylactic or therapeutic agents) that can be administered in combination with the anti-BK antibody may be administered from the anti-VP1 antibodies of the present disclosure at intervals of less than 5 minutes, less than 30 minutes, 1 hour intervals, about 1 hour intervals, about 1 hour to about 2 hours intervals, about 2 hours to about 3 hours intervals, about 3 hours to about 4 hours intervals, about 4 hours to about 5 hours intervals, about 5 hours to about 6 hours intervals, about 6 hours to about 7 hours intervals, about 7 hours to about 8 hours intervals, about 8 hours to about 9 hours intervals, about 9 hours to about 10 hours intervals, about 10 hours to about 11 hours intervals, about 11 hours to about 12 hours intervals, about 12 hours to about 18 hours intervals, 18 hours to 24 hours intervals, 24 hours to 36 hours intervals, 36 hours to 48 hours intervals, 48 hours to 52 hours intervals, 52 hours to 60 hours intervals, 60 hours to 72 hours intervals, 72 hours to 84 hours intervals, 84 hours to 96 hours intervals, 96 hours to 120 hours intervals. Two or more therapies may be administered during the same patient's hospital stay.

[0194] In one aspect, the anti-BK antibodies 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-BK antibodies cross the BBB (if necessary), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, for example, U.S. Pat. Nos. 4,522,811, 5,374,548, and 5,399,331. These liposomes can contain one or more moieties that are selectively transported to specific cells or organs and thus enhance targeted drug delivery (see, for example, Ranade, (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folic acid or biotin (see, for example, U.S. Pat. No. 5,416,016 to Low et al.), mannoside (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038), antibodies (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 also see K. Keinanen; M.L. Laukkanen (1994) FEBS Lett. 346:123, J.J. Killion; I.J. Fidler (1994) Immunomethods 4:273.

[0195] The present disclosure provides a protocol for administering a pharmaceutical composition comprising only an antibody or a combination with other therapies to a patient in need thereof. The combination therapy (e.g., prophylactic or therapeutic agent) can be administered to the subject simultaneously or sequentially. The therapy of the combination therapy (e.g., prophylactic or therapeutic agent) can also be administered periodically. Cycling therapy involves administration of a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by administration of a second therapy (e.g., a second prophylactic or therapeutic agent) for a period of time, and repeating this sequential administration, i.e., the cycle, to avoid or reduce one side effect of the therapy (e.g., the agent) and reduce the development of resistance to one of the therapies (e.g., the agent), and / or improve the effectiveness of the treatment.

[0196] The therapies of the combination therapy of the present disclosure (e.g., prophylactic or therapeutic agent) can be administered to the subject simultaneously. The term "simultaneously" is not limited to the administration of the therapies (e.g., prophylactic or therapeutic agent) exactly at the same time, but rather means that the pharmaceutical composition comprising the antibody or a fragment thereof can be administered to the subject sequentially at time intervals such that the antibody can act with the other therapy to provide an increased benefit compared to when they are administered in other ways. For example, each therapy can be administered to the subject simultaneously or sequentially in any order at different time points, but if not administered simultaneously, they must be administered at a time close enough 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 agent) are administered to the subject at intervals of less than 15 minutes, less than 30 minutes, less than 1 hour, about 1 hour interval, about 1 to about 2 hour interval, about 2 to about 3 hour interval, about 3 to about 4 hour interval, about 4 to about 5 hour interval, about 5 to about 6 hour interval, about 6 to about 7 hour interval, about 7 to about 8 hour interval, about 8 to about 9 hour interval, about 9 to about 10 hour interval, about 10 to about 11 hour interval, about 11 to about 12 hour interval, 24 hour interval, 48 hour interval, 72 hour interval, or 1 week interval. In other embodiments, two or more therapies (e.g., prophylactic or therapeutic agent) are administered during the same patient's hospital stay.

[0197] The prophylactic or therapeutic agent for combination therapy can be administered to a subject for the same pharmaceutical composition. Alternatively, the prophylactic or therapeutic agent for combination therapy can be administered simultaneously to a subject for separate pharmaceutical compositions. The prophylactic or therapeutic agent can be administered to the subject by the same or different routes of administration.

Examples

[0198] Example 1: Generation of anti-BK or JC virus antibodies B cells expressing anti-BKV and / or anti-JCV antibodies were lysed, and the VH (heavy) and VL (light) chains were amplified by RT-PCR and subsequently sequenced and analyzed to identify important post-translational modification (PTM) sites. Then, plasmids of the VH chain and VL chain were transfected into a CHO mammalian cell line as an IgG1 backbone vector for the expression of full IgG1 antibodies.

[0199] Example 2: Conjugates of anti-BKV antibodies to VLPs (ELISA) The binding of antibodies to VLPs was analyzed by ELISA. Briefly, Nunc MaxiSorp 384-well plates (Thermo scientific) were coated overnight with BKV VLPs (100 ng / well) against BKV serotype I (ST1) or serotype IV (ST4). Antibodies were serially diluted in PBS containing 0.5% BSA and allowed to bind to the antigen-coated plates for 2 hours. The plates were washed with PBS and then incubated for 1 hour with a secondary antibody (HRP-conjugated anti-human IgG goat antibody, Southern Biotech #2040-01) diluted 1:6000 in PBS containing 0.5% BSA. The plates were washed with PBS, and the reaction was developed using tetramethylbenzidine (TMB) microwell peroxidase substrate (SeramunBlau Fast, Seramun, Germany). The results of ELISA binding are shown in Figure 1. For example, antibody NOV530 binds to both BKV ST1 and BKV ST4. Antibody NOV638 binds only to BKV ST1.

[0200] Example 3: Neutralization of viral infection by anti-BKV antibodies Infectious BKV serotype I (ST1) and chimeric virus representing serotypes II (ST2), III (ST3), and IV (ST4) were pre-incubated with purified antibodies for 1 hour and subjected to binding and neutralization. Primary renal proximal tubular epithelial (RPTE) cells (ATCC, catalog number PCS-400-010) were then exposed to the virus-antibody mixture for 4 hours, replaced with fresh medium, and incubated for 48 hours to allow for virus entry and gene expression. Cells were fixed with 4% paraformaldehyde and analyzed by immunofluorescence to detect TAg expression (Calbiochem DP02, pAb416 mouse anti-SV40 TAg antibody). Immunofluorescence was analyzed by high-content image analysis using a Cellomics ArrayScan® VTI HCS Reader, and the percentage of BKV-infected cells (TAg-positive, DAPI-positive) was quantified, presenting the data as the percent inhibition of infection relative to untreated control wells. The data are presented as the concentration of antibody, EC50, at which virus infection is 50% neutralized relative to untreated control wells.

[0201] Physiologically, antibodies perform several functions that help inhibit the progressive pathogenic response, one of which is to directly block the ability of the virus to bind and / or translocate to target cells. These "neutralizing" antibodies typically represent only a subset of antigen-binding Igs. Most of the monoclonal IgG anti-BKV antibodies disclosed herein are able to neutralize at least BKV ST1 in a primary renal cell infection assay, while some are able to neutralize additional BKV subtypes and / or the related JC virus (Figure 1). For example, antibody NOV638 is able to bind and neutralize BKV ST1, while antibody NOV530 is able to bind and neutralize all four serotypes of the BK virus and also showed sub-nM EC50 for JCV (Figure 1).

[0202] Example 4: Generation of BK virus and virus-like particles (VLPs) Genomic clones of BKV ST1 were obtained from ATCC (pBR322-BKV MM, catalog number 45026, pBR322-BKV Dunlop, catalog number 45025). Infectious genomic clones of the chimeric viruses ST2, ST3, and ST4 were generated using the cloning strategy described previously (Broekema et al, Virology 2010 407:368-373). Briefly, unique restriction sites (SacII, PmlI) were introduced into the BKV serotype I genome adjacent to the VP1-VP2-VP3 coding region using site-directed mutagenesis. The coding regions of VP1 from ST2 isolate SB (GenBank accession number CAA79596.1), serotype III isolate AS (GenBank accession number AAA46882.1), and ST4 isolate ITA-4 (GenBank accession number BAF75132) were synthesized in the context of the VP2 / VP3 coding region from ST1 isolate (Genewiz, La Jolla, CA) such that synthetic fragments encompassing the SacII-PmlI region were made available for the swap combinations described by Broekema et al (supra). The resulting chimeric genomic clones were then used to generate high-titer infectious virus stocks in primary renal proximal tubular epithelial (RPTE) cells (ATCC, catalog number PCS-400-010) as described previously (Abend et al, J. Virology 2007 81:272-279).

[0203] VLPs representing each of the four BKV serotypes were generated by expression of VP1 in Sf9 insect cells, extracted from frozen cell pellets from 1 L cultures, microchip sonicated (3×45 second pulses, 5 minute rest on ice between pulses), pelleted through a 20% sucrose cushion (116,000 g, 2.5 hours), and purified by anion exchange on a 5 ml GE HiTrapQ HP column (GE Healthcare, Pittsburgh, PA), followed by purification using a 10 ml Capto™ Core700 (GE Healthcare, Pittsburgh, PA) resin-based size exclusion column, and finally purified by size exclusion chromatography on a GE Sephacryl S500 26 / 60 column (GE Healthcare, Pittsburgh, PA). The prepared VLPs were used in ELISA and SET-based binding assays.

[0204] Example 5: Affinity measurement of anti-BK antibodies (SET assay) The solution equilibrium titration (SET) assay was used to determine the interaction affinity (K d ) of antibodies against BKV VLPs from all four serotypes. Antibodies were assayed at a concentration of 1 pM (constant), and VLPs were serially diluted from an initial concentration of 10 nM. The antibody-VLP solutions were incubated overnight and then assayed for unbound antibody using an MSD array plate (Meso Scale Discovery catalog number L21XA, Rockville MD) coated with VLPs. K d was determined by fitting the plot to a 1:1 fit model (according to Piehler et al. J. Immunol. Methods. 1997;201(2):189-206).

[0205] The set of sample curves used for SET-based affinity determination of anti-BKV monoclonal IgG (clone NOV581) against BKV ST1 VLPs is shown in Figure 2A. The lower curve is K dFour-parameter fitting of the control curve (based on low concentrations of antibody NOV581), while the upper curve is a fit of the stoichiometric control curve (higher fixed antibody concentration for estimating the effective ligand concentration). Signal intensity is normalized to the initial state without BKV VLP in solution ("100% free antibody").

[0206] In Figure 2B, binding affinity was determined with cross-neutralizing monoclonal anti-BKV IgG antibodies against BKV virus-like particles (VLPs). All antibodies tested had K d values of less than 50 pM against BKV ST1. In this assay, antibody NOV581 had significant affinity for serotypes 1, 2, and 3, excluding BKV serotype 4. In contrast, antibody NOV530 had significant affinity for all four serotypes (Figure 2B).

[0207] Example 6: Cryo-Electron Microscopy To understand the mechanism by which the isolated cross-neutralizing antibody effectively inhibits infection by multiple polyomavirus strains, the inventors performed cryo-electron microscopy (cryoEM) of BKV ST1 VLPs complexed with the single-chain variable fragment (scFv) format of the cross-neutralizing IgG NOV530 and obtained a class-average density map at a resolution of 4.24 Å (Figure 3A). The inventors were able to model the capsid structure of the VLPs containing the pentameric subunit linked together via the C-terminus of each individual VP1 monomer. Surprisingly, this quaternary structure forms the basis of a composite viral epitope bound by NOV530, which has three VP1 subunits contributing amino acid residues (Figures 3B–C). A total of 20 viral residues were predicted to be within 5 Å of the antibody, and these residues are highly conserved across polyomavirus species, with 3 showing conserved homology and the remaining 17 being identical in JCV (Figures 3D–F). The interaction positions from the antibody spread throughout the heavy and light chains, with contributions from both germline-encoded (CDR1 and CDR2) and somatic recombination (CDR3) loops (Figures 3G–H). Identifying the complex binding site of NOV530 to the BKV capsid protein was not possible by other means due to its quaternary structure requirements. This binding mode raises further interesting questions about the mechanism of virus neutralization by NOV530, for example, the antibody can hold the capsid subunits together, thereby preventing the uncoating process after virus entry. Potential escape mutations can only occur at the expense of reduced virion stability. Indeed, mutations to three amino acid residues (E61, R64, and R83) within the NOV530 epitope have been previously reported and presumably dramatically reduce virus fitness by affecting receptor binding and capsid structure integrity (Dugan A.S. et al., Identification of amino acid residues in BK virus VP1 that are critical for viability and growth. J Virol 81, 11798–11808 (2007)).

[0208] CryoEM method The BKV ST1 VLPs were incubated with the scFv fragment of NOV530 (360 molecules of scFv per VLP, total protein concentration 1 mg / ml) at room temperature for 1 hour. Subsequently, the sample was concentrated 10-fold. 4.4 μL of the concentrated VLP-scFv complex was applied onto a grid (R1.2 / 1.3, Cu 300 mesh, Quantifoil Micro Tools GmbH, Grosslobichau, Germany) coated with an additional thin amorphous carbon layer. The grid was vitrified using a Leica EM GP plunger. Images were obtained using a Cs-corrected FEI Titan Krios TEM equipped with a Quantum-LS Gatan image filter (GIF) and operated at 300 kV, and were recorded on a Gatan K2-Summit direct electron detector (Gatan GmbH). Images were automatically collected in electron counting mode (nominal post-GIF magnification x105,000, calibrated pixel size 1.12 Å) (EPU, Thermo Fisher). The 7-second exposure was dose fractionated into 40 frames. The total exposure was obtained with defocus values varying from -0.8 to -2.5 μm, which was approximately 40 e- / Å2.

[0209] Low-temperature data was imaged using the following protocol. Stage drift and beam-induced motion during exposure were preprocessed, and aligned using a pipeline (StackGUI) that automates whole-image drift correction using UNBLUR (Grant, T and Grigorieff N. Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6 (eLife. 4(e06980):1-19(2015)). Contrast transfer function (CTF) parameters were estimated using the program CTFFIND4 (Mindell JA, and Grigorieff N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142:334-347(2003)). Particles were automatically picked on each micrograph using GAUTOMATCH. A total of 1,400 micrographs were acquired, and 6,000 particles were extracted for processing using the Relion software package (Scheres, S.H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519-530, doi:10.1016 / j.jsb.2012.09.006(2012)). Particle selection was by 2D classification without a reference image for 2 cycles. 5,000 particles in the best 2D classes were used for 3D refinement. A sphere was used as the initial model for 3D refinement. The inventors performed particle-based beam-induced motion correction and radiation damage weighting (known as particle polishing. See Scheres, S.H., Beam-induced motion correction for sub-megadalton cryo-EM particles. Elife 3, e03665, doi:10.7554 / eLife0.03665((2014)) on the first 20 frames (about 20e.- / Å 2 (corresponding to the total dose of). The 5,000 polished particles obtained resulted in a reconstruction with an overall resolution of 4.5 Å. Automatic refinement of the polished particles with a soft mask around the BK-VLP_scFv complex yielded a map with a resolution of 4.24 Å. The reported resolution values are based on the gold-standard Fourier shell correlation curve (FSC) with a reference of 0.143 (Scheres, S.H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519-530, doi:10.1016 / j.jsb.2012.09.006 (2012)). The cryoEM structure of the BK virion and the crystal structure of the scFv (PDB ID codes 5FUA and 4UT7 respectively) were manually fitted to the final cryoEM map using the program Coot (Emsley P. et al., Features and development of Coot Acta Crystallogr D Biol Crystallogr 66:486-501 (2010)). The resulting atomic model was subjected to multiple rounds of model rebuilding using the program Coot (Emsley P. et al., supra), and real-space refinement against the map was performed using the program Phenix (Adams P.D., et al. PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66:213-221 (2010)). This process yielded an atomic model of the pentamer and scFv complex that fits well to the cryo EM density. Structure diagrams were prepared using PyMOL (available from Schrodinger).

[0210] Example 7: Formulation The anti-BK or JC virus antibodies described herein are monoclonal antibodies, of the IgG1 isotype of the κ or λ light chain, and can be lyophilized. These antibodies are soluble and stable for 4 weeks in a histidine-sucrose formulation buffer. Furthermore, the anti-VP1 antibodies were soluble at >200 mg / ml as a minimally formulated drug substance (e.g., in histidine buffer in the absence of stabilizers).

[0211] For subsequent intravenous administration, the resulting solution is usually further diluted in a carrier solution to form an antibody solution ready for injection.

[0212] Important stability indicator assays for selecting the most stable formulation include, among others, size exclusion chromatography for determining the level of aggregation, non-visible particulate matter testing, and potency testing.

[0213] The examples and embodiments described herein are for illustrative purposes, and it is understood that various modifications or variations may be suggested to those skilled in the art in light of their content and should be included within the spirit and scope of the appended claims. The invention described in the original claims of the present application is described below. [Invention 1] An isolated antibody or antigen-binding fragment thereof, comprising (i) a heavy chain region and (ii) a light chain region as defined in Table 2. [Invention 2] An isolated antibody, wherein the antibody or antigen-binding fragment thereof (i) a heavy chain variable region comprising (a) HCDR1 (CDR - Complementary Determining Region) of SEQ ID NO: 9, (b) HCDR2 of SEQ ID NO: 10, (c) HCDR3 of SEQ ID NO: 11, and (d) LCDR1 of SEQ ID NO: 25, (e) LCDR2 of SEQ ID NO: 26, and (f) LCDR3 of SEQ ID NO: 27, and (ii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 41, (b) HCDR2 of SEQ ID NO: 42, (c) HCDR3 of SEQ ID NO: 43, and (d) LCDR1 of SEQ ID NO: 57, (e) LCDR2 of SEQ ID NO: 58, and (f) LCDR3 of SEQ ID NO: 59, and (iii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 73, (b) HCDR2 of SEQ ID NO: 74, (c) HCDR3 of SEQ ID NO: 75, and (d) LCDR1 of SEQ ID NO: 89, (e) LCDR2 of SEQ ID NO: 90, and (f) LCDR3 of SEQ ID NO: 91, and (iv) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 105, (b) HCDR2 of SEQ ID NO: 106, (c) HCDR3 of SEQ ID NO: 107, and (d) LCDR1 of SEQ ID NO: 121, (e) LCDR2 of SEQ ID NO: 122, and (f) LCDR3 of SEQ ID NO: 123, and (v) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 137, (b) HCDR2 of SEQ ID NO: 138, (c) HCDR3 of SEQ ID NO: 139, and (d) LCDR1 of SEQ ID NO: 153, (e) LCDR2 of SEQ ID NO: 154, and (f) LCDR3 of SEQ ID NO: 155, and (vi) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 169, (b) HCDR2 of SEQ ID NO: 170, (c) HCDR3 of SEQ ID NO: 171, and (d) LCDR1 of SEQ ID NO: 185, (e) LCDR2 of SEQ ID NO: 186, and (f) LCDR3 of SEQ ID NO: 187. (vii) (a) The heavy chain variable region comprising HCDR1 of SEQ ID NO: 201, (b) HCDR2 of SEQ ID NO: 202, (c) HCDR3 of SEQ ID NO: 203, and (d) the light chain variable region comprising LCDR1 of SEQ ID NO: 217, (e) LCDR2 of SEQ ID NO: 218, and (f) LCDR3 of SEQ ID NO: 219, (viii) (a) The heavy chain variable region comprising HCDR1 of SEQ ID NO: 233, (b) HCDR2 of SEQ ID NO: 234, (c) HCDR3 of SEQ ID NO: 235, and (d) the light chain variable region comprising LCDR1 of SEQ ID NO: 249, (e) LCDR2 of SEQ ID NO: 250, and (f) LCDR3 of SEQ ID NO: 251, (ix) (a) The heavy chain variable region comprising HCDR1 of SEQ ID NO: 265, (b) HCDR2 of SEQ ID NO: 266, (c) HCDR3 of SEQ ID NO: 267, and (d) the light chain variable region comprising LCDR1 of SEQ ID NO: 281, (e) LCDR2 of SEQ ID NO: 282, and (f) LCDR3 of SEQ ID NO: 283, and an isolated antibody comprising the same. [Invention 3] The antibody according to Invention 2, wherein one or two amino acids within the CDR are modified, deleted, or substituted. [Invention 4] The antibody according to Invention 2, which retains at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity over either the variable heavy chain region or the variable light chain region. [Invention 5] The antibody according to Invention 1, wherein the antibody is a monoclonal antibody, chimeric antibody, humanized antibody, humanized modified antibody, human antibody, single-chain antibody (scFv), or antibody fragment. [Invention 6] An isolated antibody or an antigen-binding fragment thereof, (i) A heavy chain variable region (vH) comprising SEQ ID NO: 18 and a light chain variable region (vL) comprising SEQ ID NO: 34, (ii) A heavy chain variable region (vH) comprising SEQ ID NO: 50 and a light chain variable region (vL) comprising SEQ ID NO: 66, (iii) A heavy chain variable region (vH) comprising SEQ ID NO: 82 and a light chain variable region (vL) comprising SEQ ID NO: 98, (iv) A heavy chain variable region (vH) comprising SEQ ID NO: 114 and a light chain variable region (vL) comprising SEQ ID NO: 130, (v) A heavy chain variable region (vH) comprising SEQ ID NO: 146 and a light chain variable region (vL) comprising SEQ ID NO: 162, (vi) A heavy chain variable region (vH) comprising SEQ ID NO: 178 and a light chain variable region (vL) comprising SEQ ID NO: 194, (vii) A heavy chain variable region (vH) containing SEQ ID NO: 210 and a light chain variable region (vL) containing SEQ ID NO: 226, and (viii) A heavy chain variable region (vH) containing SEQ ID NO: 242 and a light chain variable region (vL) containing SEQ ID NO: 258, and (ix) An isolated antibody or antigen-binding fragment thereof comprising a heavy chain variable region (vH) containing SEQ ID NO: 274 and a light chain variable region (vL) containing SEQ ID NO: 290. [Invention 7] The antibody or fragment thereof according to Invention 6, which retains at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity across any of the variable light chain regions or variable heavy chain regions. [Invention 8] The antibody according to Invention 6, 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. [Invention 9] The antibody according to Invention 6, wherein the antibody is a monoclonal antibody, chimeric antibody, humanized antibody, humanized modified antibody, human antibody, single-chain antibody (scFv), or antibody fragment. [Invention 10] The antibody or fragment thereof according to Invention 1, 2, or 6, which is hypo-glycosylated, non-glycosylated, or low-fucosylated. [Invention 11] A pharmaceutical composition comprising the antibody or fragment thereof according to Invention 1, 2, or 6, further comprising a pharmaceutically acceptable carrier. [Invention 12] The pharmaceutical composition according to Invention 11, wherein the pharmaceutically acceptable carrier comprises histidine or a saccharide. [Invention 13] The pharmaceutical composition according to Invention 12, wherein the saccharide is sucrose. [Invention 14] A pharmaceutical composition comprising a plurality of antibodies or antigen-binding fragments according to Invention 1, 2, or 6, 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. [Invention 15] A pharmaceutical composition comprising a plurality of antibodies or antigen-binding fragments according to Invention 1, 2, or 6, wherein none of the antibodies contain bisecting GlcNAc. [Invention 16] A pharmaceutical composition comprising the antibody or fragment thereof according to Invention 1, 2, or 6, wherein the composition is prepared as a lyophilized product. [Invention 17] A method of neutralizing BK virus or JC virus infection, comprising administering an effective amount of the antibody according to Invention 1, 2, or 6 to a patient in need thereof via injection or infusion. [Invention 18] The method according to invention 17, wherein the patient in need is diagnosed with BK virusuria or BK viremia. [Invention 19] The method according to invention 17, wherein the patient in need is diagnosed with JC virusuria or JC viremia. [Invention 20] A method of treating or reducing the likelihood of BK virus- or JC virus-related disorders, comprising administering to a patient in need an effective amount of the antibody according to invention 1, 2 or 6 by injection or infusion, wherein the disorder is nephropathy, BKVAN, hemorrhagic cystitis (HC), progressive multifocal leukoencephalopathy (PML), granulocyte neuronopathy (GCN), interstitial kidney disease, ureteral stricture, vasculitis, colitis, retinitis, meningitis, and immune reconstitution inflammatory syndrome (IRIS). [Invention 21] The method according to invention 20, wherein the antibody or composition is reconstituted before injection or infusion. [Invention 22] The method according to invention 20, wherein the antibody or the pharmaceutical composition is administered in combination with another therapeutic agent. [Invention 23] The method according to invention 22, wherein the therapeutic agent is an immunosuppressive agent. [Invention 24] The method according to invention 23, wherein the immunosuppressive agent is a dehydrogenase inhibitor, a purine synthesis inhibitor, a calcineurin inhibitor or an mTOR inhibitor. [Invention 25] The method according to invention 23, wherein the immunosuppressive agent is mycophenolate mofetil (MMF), sodium mycophenolate, azathioprine, tacrolimus, sirolimus or cyclosporine. [Invention 26] The method according to invention 22, wherein the therapeutic agent is an additional anti-BKV or JCV antibody. [Invention 27] The method according to invention 20, wherein the PML is associated with the treatment of multiple sclerosis, rheumatoid arthritis, or psoriasis. [Invention 28] The method according to invention 27, wherein the treatment of multiple sclerosis is natalizumab, fingolimod, dimethyl fumarate, fumaric acid ester, or alemtuzumab. [Invention 29] The method according to invention 27, wherein the treatment of rheumatoid arthritis uses rituximab. [Invention 30] The method according to invention 27, wherein the treatment of psoriasis uses efalizumab. [Invention 31] The antibody or fragment thereof according to invention 1, 2 or 6 for use as a medicament. [Invention 32] The antibody or fragment thereof according to invention 1, 2 or 6 for use in neutralizing BK virus or JC virus infection. [Invention 33] An antibody or fragment thereof according to invention 1, 2 or 6 for use in the treatment of, or reduction of the likelihood of, nephropathy, BKVAN, hemorrhagic cystitis (HC), progressive multifocal leukoencephalopathy (PML), granulocyte neuronopathy (GCN), interstitial kidney disease, ureteral stenosis, vasculitis, colitis, retinitis, meningitis, and immune reconstitution inflammatory syndrome (IRIS). [Invention 34] Use of an antibody or fragment thereof according to invention 33, administered in combination with another therapeutic agent. [Invention 35] Use of an antibody or fragment thereof according to invention 33, wherein the therapeutic agent is an immunosuppressant. [Invention 36] Use of an antibody or fragment thereof according to invention 35, wherein the immunosuppressant is a dehydrogenase inhibitor, purine synthesis inhibitor, calcineurin inhibitor or mTOR inhibitor. [Invention 37] Use of an antibody or fragment thereof according to invention 35, wherein the immunosuppressant is mycophenolate mofetil (MMF), sodium mycophenolate, azathioprine, tacrolimus, sirolimus or cyclosporine. [Invention 38] Use of an antibody or fragment thereof according to invention 34, wherein the therapeutic agent is an additional anti-BKV antibody. [Invention 39] Use of an antibody or fragment thereof according to invention 33, wherein the PML is associated with the treatment of multiple sclerosis, rheumatoid arthritis, or psoriasis. [Invention 40] Use according to invention 39, wherein the treatment of multiple sclerosis uses natalizumab, fingolimod, dimethyl fumarate, fumaric acid ester, or alemtuzumab. [Invention 41] Use according to invention 39, wherein the treatment of rheumatoid arthritis uses rituximab. [Invention 42] Use according to invention 39, wherein the treatment of psoriasis uses efalizumab. [Invention 43] A nucleic acid encoding an antibody or antigen-binding fragment according to invention 1, 2 or 6. [Invention 44] A vector comprising the nucleic acid according to invention 43. [Invention 45] A host cell comprising the vector according to invention 44. [Invention 46] A diagnostic reagent comprising a labeled antibody or antigen-binding fragment thereof according to invention 1, 2 or 6. [Invention 47] The diagnostic agent according to invention 46, wherein the label is selected from the group consisting of a radiolabel, fluorophore, chromophore, imaging agent, and metal ion.

Claims

**Claim 1** An isolated antibody or antigen-binding fragment thereof that binds to BK virus and / or JC virus, wherein the antibody or fragment (i) (a) a heavy chain variable region comprising CDR1 (complementary determining region) of SEQ ID NO: 9, (b) CDR2 of SEQ ID NO: 10, and (c) CDR3 of SEQ ID NO: 11, and (d) CDR1 of SEQ ID NO: 25, (e) CDR2 of SEQ ID NO: 26, and (f) CDR3 of SEQ ID NO: 27, of a light chain variable region; (ii) (a) a heavy chain variable region comprising CDR1 of SEQ ID NO: 169, (b) CDR2 of SEQ ID NO: 170, and (c) CDR3 of SEQ ID NO: 171, and (d) CDR1 of SEQ ID NO: 185, (e) CDR2 of SEQ ID NO: 186, and (f) CDR3 of SEQ ID NO: 187, of a light chain variable region; (iii) (a) a heavy chain variable region comprising CDR1 of SEQ ID NO: 201, (b) CDR2 of SEQ ID NO: 202, and (c) CDR3 of SEQ ID NO: 203, and (d) CDR1 of SEQ ID NO: 217, (e) CDR2 of SEQ ID NO: 218, and (f) CDR3 of SEQ ID NO: 219, of a light chain variable region; or (iv) (a) a heavy chain variable region comprising CDR1 of SEQ ID NO: 233, (b) CDR2 of SEQ ID NO: 234, and (c) CDR3 of SEQ ID NO: 235, and (d) CDR1 of SEQ ID NO: 249, (e) CDR2 of SEQ ID NO: 250, and (f) CDR3 of SEQ ID NO: 251, of a light chain variable region comprises the isolated antibody or antigen-binding fragment thereof. **Claim 2** An isolated antibody or antigen-binding fragment thereof that binds to BK virus and / or JC virus, wherein the antibody or fragment (i) a heavy chain variable region (vH) comprising SEQ ID NO: 18, and a light chain variable region (vL) comprising SEQ ID NO: 34; (ii) a heavy chain variable region (vH) comprising SEQ ID NO: 178, and a light chain variable region (vL) comprising SEQ ID NO: 194; (iii) a heavy chain variable region (vH) comprising SEQ ID NO: 210, and a light chain variable region (vL) comprising SEQ ID NO: 226; or (vi) a heavy chain variable region (vH) comprising SEQ ID NO: 242, and a light chain variable region (vL) comprising SEQ ID NO: 258 comprises the isolated antibody or antigen-binding fragment thereof. **Claim 3** The antibody or fragment thereof according to claim 1 or 2, which retains at least 90% identity over either the light chain variable region or the heavy chain variable region.

4. The antibody according to claim 2, wherein 1, 2, 3, 4, or 5, but less than 10, amino acids within the light chain variable region or the heavy chain variable region are modified, deleted, or substituted.

5. The antibody according to claim 1 or 2, wherein the antibody or fragment thereof has reduced glycosylation, is not glycosylated, or is hypofucosylated.

6. A pharmaceutical composition comprising the antibody or fragment thereof according to claim 1 or 2, further comprising a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, wherein the pharmaceutically acceptable carrier comprises histidine or a saccharide.

8. The pharmaceutical composition according to claim 7, wherein the saccharide is sucrose.

9. A pharmaceutical composition comprising a plurality of the antibodies or antigen-binding fragments thereof according to claim 1 or 2, wherein at least 0.05% of the antibodies or fragments in the composition have α2,3-linked sialic acid residues, the pharmaceutical composition according to claim 6.

10. A pharmaceutical composition comprising a plurality of the antibodies or antigen-binding fragments thereof according to claim 1 or 2, wherein none of the antibodies or fragments contain bisecting GlcNAc, the pharmaceutical composition according to claim 6.

11. The pharmaceutical composition according to claim 6, wherein the composition is prepared as a lyophilized product.

12. The pharmaceutical composition according to claim 6, for use in a method of neutralizing BK virus or JC virus infection in a patient in need thereof.

13. The pharmaceutical composition according to claim 12, wherein the patient is diagnosed with BK virusuria or BK viremia.

14. The pharmaceutical composition according to claim 12, wherein the patient is diagnosed with JC virusuria or JC viremia.

15. A pharmaceutical composition for use in a method of treating or reducing the likelihood of BK virus or JC virus-related disorders in a patient in need thereof, wherein the disorder is selected from the group consisting of nephropathy, BK virus-associated nephropathy (BKVAAN), hemorrhagic cystitis (HC), progressive multifocal leukoencephalopathy (PML), granulocyte neuronopathy (GCN), interstitial kidney disease, ureteral stenosis, vasculitis, colitis, retinitis, meningitis, and immune reconstitution inflammatory syndrome (IRIS), the pharmaceutical composition according to claim 6.

16. The pharmaceutical composition according to claim 12 or 15, wherein the composition is used to be administered by injection or infusion.

17. The pharmaceutical composition according to claim 15, wherein the composition is used to be administered in combination with another therapeutic agent.

18. The pharmaceutical composition according to claim 17, wherein the other therapeutic agent is an immunosuppressant.

19. The pharmaceutical composition according to claim 18, wherein the immunosuppressant is a dehydrogenase inhibitor, a purine synthesis inhibitor, a calcineurin inhibitor, or an mTOR inhibitor.

20. The pharmaceutical composition according to claim 18, wherein the immunosuppressant is mycophenolate mofetil (MMF), sodium mycophenolate, azathioprine, tacrolimus, sirolimus, or cyclosporine.

21. The pharmaceutical composition according to claim 17, wherein the other therapeutic agent is an additional anti-BKV or JCV antibody.

22. The pharmaceutical composition according to claim 15, wherein the PML is related to the treatment of multiple sclerosis, rheumatoid arthritis, or psoriasis.

23. The pharmaceutical composition according to claim 22, wherein the treatment of multiple sclerosis is natalizumab, fingolimod, dimethyl fumarate, fumaric acid ester, or alemtuzumab.

24. The pharmaceutical composition according to claim 22, wherein the treatment of rheumatoid arthritis uses rituximab.

25. The pharmaceutical composition according to claim 22, wherein the treatment of psoriasis uses efalizumab.

26. The antibody or fragment thereof according to claim 1 or 2 for use as a medicament.

27. The antibody or fragment thereof according to claim 1 or 2 for use in neutralizing BK virus or JC virus infection.

28. The antibody or fragment thereof according to claim 1 or 2 for use in the treatment of nephropathy, BK virus-associated nephropathy (BKVA), hemorrhagic cystitis (HC), progressive multifocal leukoencephalopathy (PML), granulocyte neuronopathy (GCN), interstitial kidney disease, ureteral stenosis, vasculitis, colitis, retinitis, meningitis, and immune reconstitution inflammatory syndrome (IRIS) or for reducing the likelihood thereof.

29. The antibody or fragment thereof according to claim 28, which is used to be administered in combination with another therapeutic agent.

30. The antibody or fragment thereof according to claim 29, wherein the other therapeutic agent is an immunosuppressive agent.

31. The antibody or fragment thereof according to claim 30, wherein the immunosuppressive agent is a dehydrogenase inhibitor, a purine synthesis inhibitor, a calcineurin inhibitor, or an mTOR inhibitor.

32. The antibody or fragment thereof according to claim 30, wherein the immunosuppressive agent is mycophenolate mofetil (MMF), sodium mycophenolate, azathioprine, tacrolimus, sirolimus, or cyclosporine.

33. The antibody or fragment thereof according to claim 29, wherein the other therapeutic agent is an additional anti-BK virus antibody.

34. The antibody or fragment thereof according to claim 28, wherein the PML is related to the treatment of multiple sclerosis, rheumatoid arthritis, or psoriasis.

35. The antibody or fragment thereof according to claim 34, wherein the treatment of multiple sclerosis uses natalizumab, fingolimod, dimethyl fumarate, fumaric acid ester, or alemtuzumab.

36. The antibody or fragment thereof according to claim 34, wherein the treatment of rheumatoid arthritis uses rituximab.

37. The antibody or fragment thereof according to claim 34, wherein the treatment of psoriasis uses efalizumab.

38. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to claim 1 or 2.

39. A vector containing the nucleic acid according to claim 38.

40. A host cell containing the vector according to claim 39.

41. A diagnostic reagent containing the labeled antibody or antigen-binding fragment thereof according to claim 1 or 2.

42. The diagnostic agent according to claim 41, wherein the label is selected from the group consisting of a radioactive label, a fluorophore, a chromophore, an imaging agent, and a metal ion.

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