Antibodies that specifically bind to HPV

Abstract

The present invention provides a group of antibodies or antigen binding fragments thereof that specifically bind to HPV. The invention also provides polynucleotides encoding the antibodies or antigen binding fragments thereof, vectors comprising the polynucleotides, host cells comprising the vectors, methods of generating the antibodies, and compositions comprising the antibodies.

Description

Technical Field

[0001] The present invention provides a group of antibodies specifically binding to HPV or antigen-binding fragments thereof. The present invention also provides polynucleotides encoding the antibodies or antigen-binding fragments thereof, vectors containing the polynucleotides, host cells containing the vectors, methods for generating the antibodies, and compositions containing the antibodies. Background Art

[0002] Cervical cancer and cervical precancerous lesions seriously threaten the health of women worldwide. Clinical, molecular biological, and epidemiological investigations have demonstrated that human papillomavirus (HPV) is the main cause of cervical cancer and cervical dysplasia. HPV is a group of spherical, minute, non-enveloped circular double-stranded DNA viruses belonging to the genus Papillomavirus of the family Papovaviridae. Its capsid is an icosahedral structure composed of 72 pentamers of the major capsid protein Ll protein. One virion contains 360 Ll protein monomers, which can mainly cause proliferative lesions of human skin and mucosa. Currently, more than 200 subtypes of HPV have been discovered, which are divided into high-risk types (including HPV16, 18, 31, 33, 35, 39, 45, etc.) and low-risk types (including HPV6, 11, etc.) according to their pathogenicity.

[0003] Currently, there are multiple cervical cancer vaccines approved for marketing in China, including: the HPV bivalent vaccine Cervarix (subtypes are HPV 16 and 18) of GlaxoSmithKline (GSK), the HPV quadrivalent vaccine and the 9-valent vaccine 9 (subtypes are HPV 6, 11, 16, 18, 31, 33, 45, 52, 58) of Merck & Co., the bivalent HPV vaccine Xinkening developed by Wantai Biopharm, and the bivalent HPV vaccine Wozhui developed by Watson Biologics. Since the currently marketed vaccines cover at most 9 subtypes, more and more companies are continuously researching and developing HPV vaccines with more valences (such as 15-valent). In vaccine research, monoclonal antibodies are important tools for vaccine antigen quality control, and the antibody level is the standard for evaluating vaccine efficacy. Therefore, various types of antibodies specifically recognizing HPV are needed to better judge the produced vaccines and the vaccine protection effect, and are used for the diagnosis, treatment, and prevention of HPV. Summary of the Invention

[0004] On the one hand, the present invention provides an antibody specifically binding to HPV or an antigen-binding fragment thereof, which comprises:

[0005] (1) CDR-H1 as shown in SEQ ID NO: 1, CDR-H2 as shown in SEQ ID NO: 2, CDR-H3 as shown in SEQ ID NO: 3, CDR-L1 as shown in SEQ ID NO: 4, CDR-L2 as shown in SEQ ID NO: 5, and CDR-L3 as shown in SEQ ID NO: 6;

[0006] (2) CDR-H1 as shown in SEQ ID NO: 7, CDR-H2 as shown in SEQ ID NO: 8, CDR-H3 as shown in SEQ ID NO: 9, CDR-L1 as shown in SEQ ID NO: 10, CDR-L2 as shown in SEQ ID NO: 11, and CDR-L3 as shown in SEQ ID NO: 12;

[0007] (3) CDR-H1 as shown in SEQ ID NO: 13, CDR-H2 as shown in SEQ ID NO: 14, CDR-H3 as shown in SEQ ID NO: 15, CDR-L1 as shown in SEQ ID NO: 16, CDR-L2 as shown in SEQ ID NO: 17, and CDR-L3 as shown in SEQ ID NO: 18;

[0008] (4) CDR-H1 as shown in SEQ ID NO: 19, CDR-H2 as shown in SEQ ID NO: 20, CDR-H3 as shown in SEQ ID NO: 21, CDR-L1 as shown in SEQ ID NO: 22, CDR-L2 as shown in SEQ ID NO: 23, and CDR-L3 as shown in SEQ ID NO: 24; or

[0009] (5) CDR-H1 as shown in SEQ ID NO: 25, CDR-H2 as shown in SEQ ID NO: 26, CDR-H3 as shown in SEQ ID NO: 27, CDR-L1 as shown in SEQ ID NO: 28, CDR-L2 as shown in SEQ ID NO: 29, and CDR-L3 as shown in SEQ ID NO: 30.

[0010] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises:

[0011] (1) VH as shown in SEQ ID NO: 31 and VL as shown in SEQ ID NO: 32;

[0012] (2) VH as shown in SEQ ID NO: 33 and VL as shown in SEQ ID NO: 34;

[0013] (3) VH as shown in SEQ ID NO: 35 and VL as shown in SEQ ID NO: 36;

[0014] (4) VH as shown in SEQ ID NO: 37 and VL as shown in SEQ ID NO: 38; or

[0015] (5) VH as shown in SEQ ID NO: 39 and VL as shown in SEQ ID NO: 40.

[0016] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises:

[0017] (1) A heavy chain constant region as shown in SEQ ID NO: 53, and

[0018] (2) A light chain constant region as shown in any one of SEQ ID NO: 51 and SEQ ID NO: 52.

[0019] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises an α heavy chain, a δ heavy chain, an ε heavy chain, a γ heavy chain or a μ heavy chain. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention belongs to the IgG1, IgG2, IgG3 or IgG4 subclass. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a λ light chain or a κ light chain. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is a full-length antibody. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is an antibody fragment selected from Fv, scFv, Fab, Fab', F(ab') 2 and xFab. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is a chimeric antibody or a human antibody or an antigen-binding fragment thereof.

[0020] On the one hand, the present invention provides a polynucleotide encoding the antibody or antigen-binding fragment thereof of the present invention.

[0021] In one embodiment, the polynucleotide of the present invention comprises:

[0022] (1) SEQ ID NO: 41 and 42;

[0023] (2) SEQ ID NO: 43 and 44;

[0024] (3) SEQ ID NO: 45 and 46;

[0025] (4) SEQ ID NO: 47 and 48; or

[0026] (5) SEQ ID NO: 49 and 50.

[0027] On the one hand, the present invention provides a vector comprising the polynucleotide of the present invention.

[0028] On the one hand, the present invention provides a host cell comprising the polynucleotide or vector of the present invention. In one embodiment, the host cell is a eukaryotic cell. In one embodiment, the host cell is a CHO cell.

[0029] On the one hand, the present invention provides a method for generating an antibody or an antigen-binding fragment thereof, comprising:

[0030] (a) culturing the host cell of the present invention under conditions suitable for expressing the antibody or an antigen-binding fragment thereof, and

[0031] (b) optionally, recovering the antibody or an antigen-binding fragment thereof.

[0032] On the one hand, the present invention provides a composition comprising the antibody or an antigen-binding fragment thereof of the present invention.

[0033] On the one hand, the present invention provides the antibody or an antigen-binding fragment thereof or the composition of the present invention for use as a detection reagent.

[0034] On the one hand, the present invention provides the antibody or an antigen-binding fragment thereof or the composition of the present invention for use in the identification test of HPV vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1-1 Showing the antigen-binding curve (ELISA) of the antibody F5-77 of the present invention.

[0036] Figure 1-2 Showing the antigen-binding curve (ELISA) of the antibody F5-187 of the present invention.

[0037] Figure 1-3 Showing the antigen-binding curve (ELISA) of the antibody F5-196 of the present invention.

[0038] Figure 1-4 Showing the antigen-binding curve (ELISA) of the antibody F5-203 of the present invention.

[0039] Figure 1-5 Showing the antigen-binding curve (ELISA) of the antibody F5-127 of the present invention.

[0040] Figure 2-1 Showing the virus neutralization curve of the antibody F5-77 of the present invention.

[0041] Figure 2-2 Showing the virus neutralization curve of the antibody F5-187 of the present invention.

[0042] Figure 2-3Show the virus neutralization curve of the antibody F5-196 of the present invention.

[0043] Figure 2-4 Show the virus neutralization curve of the antibody F5-203 of the present invention. Detailed implementation manners

[0044] As used herein, the term "antibody" is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided that they exhibit the desired antigen-binding activity.

[0045] "Antigen-binding fragment" refers to a molecule different from a full-length antibody that contains the portion of the full-length antibody that binds the antigen to which the full-length antibody binds. Examples of antigen-binding fragments include but are not limited to Fv, Fab, Fab’, Fab’-SH, F(ab’) 2 , xFab; diabody; linear antibody; single-chain antibody molecule (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0046] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid molecule to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors incorporated into the genome of a host cell into which it is introduced. Certain vectors are capable of directing the expression of nucleic acids operably linked thereto. Such vectors are referred to herein as "expression vectors".

[0047] I. Compositions and methods

[0048] In one aspect, the present invention provides an isolated antibody that binds to HPV.

[0049] In certain embodiments, an antibody that binds to HPV is provided.

[0050] A. Exemplary anti-HPV antibodies

[0051] In one aspect, the present invention provides an anti-HPV antibody comprising at least one, two, three, four, five, or six CDRs selected from the following: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:1; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:2; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:3; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:5; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:6.

[0052] In another aspect, the present invention provides an antibody comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:3; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:5; and (f) a CDR-L3 comprising an amino acid sequence selected from SEQ ID NO:6.

[0053] In another aspect, the anti-HPV antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:31. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-HPV antibody comprising the sequence retains the ability to bind to HPV. In certain embodiments, there are a total of 1 to 10 amino acid substitutions, insertions, and / or deletions in SEQ ID NO:31. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-HPV antibody comprises the VH sequence of SEQ ID NO:31, including post-translational modifications of the sequence. In one particular embodiment, the VH comprises one, two, or three CDRs selected from the following: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:3.

[0054] In another aspect, an anti-HPV antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 32. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-HPV antibody comprising the sequence retains the ability to bind to HPV. In certain embodiments, there are a total of 1 to 10 amino acid substitutions, insertions, and / or deletions in SEQ ID NO: 32. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the CDRs (i.e., in the FRs). Optionally, the anti-HPV antibody comprises the VL sequence of SEQ ID NO: 32, including post-translational modifications of the sequence. In a particular embodiment, the VL comprises one, two, or three CDRs selected from the following: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0055] In one aspect, the present invention provides an anti-HPV antibody comprising at least one, two, three, four, five, or six CDRs selected from the following: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 8; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 9; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12.

[0056] In another aspect, the present invention provides an antibody comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 8; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 9; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (f) CDR-L3 comprising the amino acid sequence selected from SEQ ID NO: 12.

[0057] In another aspect, the anti-HPV antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 33. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (such as conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-HPV antibody comprising the sequence retains the ability to bind to HPV. In certain embodiments, there are a total of 1 to 10 amino acid substitutions, insertions, and / or deletions in SEQ ID NO: 33. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-HPV antibody comprises the VH sequence of SEQ ID NO: 33, including post-translational modifications of the sequence. In one particular embodiment, the VH comprises one, two, or three CDRs selected from the following: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 7, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 8, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 9.

[0058] In another aspect, provided is an anti-HPV antibody, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 34. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (such as conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-HPV antibody comprising the sequence retains the ability to bind to HPV. In certain embodiments, there are a total of 1 to 10 amino acid substitutions, insertions, and / or deletions in SEQ ID NO: 34. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-HPV antibody comprises the VL sequence of SEQ ID NO: 34, including post-translational modifications of the sequence. In one particular embodiment, the VL comprises one, two, or three CDRs selected from the following: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12.

[0059] In one aspect, the present invention provides an anti-HPV antibody comprising at least one, two, three, four, five, or six CDRs selected from the following: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18.

[0060] In another aspect, the present invention provides an antibody comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17; and (f) CDR-L3 comprising the amino acid sequence selected from SEQ ID NO: 18.

[0061] In another aspect, the anti-HPV antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 35. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-HPV antibody comprising the sequence retains the ability to bind to HPV. In certain embodiments, there are a total of 1 to 10 amino acid substitutions, insertions, and / or deletions in SEQ ID NO: 35. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-HPV antibody comprises the VH sequence of SEQ ID NO: 35, including post-translational modifications of the sequence. In a particular embodiment, the VH comprises one, two, or three CDRs selected from the following: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15.

[0062] In another aspect, an anti-HPV antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 36. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-HPV antibody comprising the sequence retains the ability to bind to HPV. In certain embodiments, there are a total of 1 to 10 amino acid substitutions, insertions, and / or deletions in SEQ ID NO: 36. In certain embodiments, the substitution, insertion, or deletion occurs in a region other than the CDRs (i.e., in the FRs). Optionally, the anti-HPV antibody comprises the VL sequence of SEQ ID NO: 36, including post-translational modifications of the sequence. In a particular embodiment, the VL comprises one, two, or three CDRs selected from the following: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18.

[0063] In one aspect, the present invention provides an anti-HPV antibody comprising at least one, two, three, four, five, or six CDRs selected from the following: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24.

[0064] In another aspect, the present invention provides an antibody comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and (f) CDR-L3 comprising the amino acid sequence selected from SEQ ID NO: 24.

[0065] In another aspect, the anti-HPV antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:37. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (such as conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-HPV antibody comprising the sequence retains the ability to bind to HPV. In certain embodiments, there are a total of 1 to 10 amino acid substitutions, insertions, and / or deletions in SEQ ID NO:37. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-HPV antibody comprises the VH sequence of SEQ ID NO:37, including post-translational modifications of the sequence. In a particular embodiment, the VH comprises one, two, or three CDRs selected from the following: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:19, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:20, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:21.

[0066] In another aspect, there is provided an anti-HPV antibody, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:38. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (such as conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-HPV antibody comprising the sequence retains the ability to bind to HPV. In certain embodiments, there are a total of 1 to 10 amino acid substitutions, insertions, and / or deletions in SEQ ID NO:38. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-HPV antibody comprises the VL sequence of SEQ ID NO:38, including post-translational modifications of the sequence. In a particular embodiment, the VL comprises one, two, or three CDRs selected from the following: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:22; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:24.

[0067] In one aspect, the present invention provides an anti-HPV antibody comprising at least one, two, three, four, five, or six CDRs selected from the following: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

[0068] In another aspect, the present invention provides an antibody comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (f) CDR-L3 comprising the amino acid sequence selected from SEQ ID NO: 30.

[0069] In another aspect, the anti-HPV antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 39. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-HPV antibody comprising the sequence retains the ability to bind to HPV. In certain embodiments, there are a total of 1 to 10 amino acid substitutions, insertions, and / or deletions in SEQ ID NO: 39. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-HPV antibody comprises the VH sequence of SEQ ID NO: 39, including post-translational modifications of the sequence. In a particular embodiment, the VH comprises one, two, or three CDRs selected from the following: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27.

[0070] In another aspect, an anti-HPV antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 40. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-HPV antibody comprising the sequence retains the ability to bind to HPV. In certain embodiments, there are a total of 1 to 10 amino acid substitutions, insertions, and / or deletions in SEQ ID NO: 40. In certain embodiments, the substitutions, insertions, or deletions occur in regions other than the CDRs (i.e., in the FRs). Optionally, the anti-HPV antibody comprises the VL sequence in SEQ ID NO: 40, including post-translational modifications of the sequence. In a particular embodiment, the VL comprises one, two, or three CDRs selected from the following: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

[0071] In another aspect, an anti-HPV antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 31 and SEQ ID NO: 32, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 33 and SEQ ID NO: 34, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 35 and SEQ ID NO: 36, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 37 and SEQ ID NO: 38, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 39 and SEQ ID NO: 40, respectively, including post-translational modifications of those sequences.

[0072] In yet another aspect of the invention, the anti-HPV antibody according to any of the above embodiments is a monoclonal antibody, including chimeric, humanized or human antibodies. In one embodiment, the anti-HPV antibody is an antibody fragment, such as Fv, Fab, Fab’, xFab, scFv, diabody, or F(ab’) 2 fragment.

[0073] B. Recombinant methods and compositions

[0074] Antibodies can be generated using recombinant methods and compositions, such as those described in US 4,816,567. For these methods, one or more isolated nucleic acids encoding the antibody are provided.

[0075] In the case of a natural antibody or a natural antibody fragment, two nucleic acids are required, one for the light chain or a fragment thereof and one for the heavy chain or a fragment thereof. Such nucleic acids encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH (e.g., the light and / or heavy chains of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors.

[0076] In the case of a bispecific antibody with a heterodimeric heavy chain, four nucleic acids are required, one for the first light chain, one for the second light chain comprising the first heteromonomeric Fc region polypeptide, one for the second light chain, and one for the second heavy chain comprising the second heteromonomeric Fc region polypeptide. The four nucleic acids can be contained in one or more nucleic acid molecules or expression vectors. Such nucleic acids encode an amino acid sequence comprising the first VL of the antibody and / or an amino acid sequence comprising the first VH including the first heteromonomeric Fc region and / or an amino acid sequence comprising the second VL and / or an amino acid sequence comprising the second VH including the second heteromonomeric Fc region (e.g., the first and / or second light and / or the first and / or second heavy chains of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors. Normally, these nucleic acids are located on two or three expression vectors, i.e., one vector can contain more than one of these nucleic acids. Examples of these bispecific antibodies are CrossMab and T cell bispecific agents (see, for example, Schaefer, W. et al, PNAS, 108 (2011) 11187-1191). For example, one of the heteromonomeric heavy chains contains a so-called "knob mutation" (T366W and optionally one of S354C or Y349C) and the other contains a so-called "hole mutation" (T366S, L368A and Y407V and optionally Y349C or S354C) (see, for example, Carter, P. et al., Immunotechnol. 2 (1996) 73).

[0077] In one embodiment, an isolated nucleic acid encoding the antibody used in the methods reported herein is provided.

[0078] In yet another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided.

[0079] In yet another embodiment, a host cell comprising such nucleic acids is provided.

[0080] In one such embodiment, the host cell comprises (e.g., has been transformed with):

[0081] - In the case of an antibody consisting of two identical light chains and two identical heavy chains that form disulfide bonds and that comprise fragments of VH and VL:

[0082] (1) A vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VL and an amino acid sequence comprising the antibody VH, or

[0083] (2) A first vector and a second vector, the first vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VL, and the second vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VH.

[0084] - In the case of a bispecific antibody having heterodimeric heavy chains:

[0085] (1) A first vector comprising a first pair of nucleic acids encoding amino acid sequences, one of which comprises the first VL of the antibody and the other of which comprises the first VH, and a second vector comprising a second pair of nucleic acids encoding amino acid sequences, one of which comprises the second VL of the antibody and the other of which comprises the second VH, or

[0086] (2) A first vector comprising a first nucleic acid encoding an amino acid sequence comprising one of the variable domains (preferably the light chain variable domain), a second vector comprising a pair of nucleic acids encoding amino acid sequences, one of which comprises the light chain variable domain and the other of which comprises the first heavy chain variable domain, and a third vector comprising a pair of nucleic acids encoding amino acid sequences, one of which comprises the other light chain variable domain corresponding to the second vector and the other of which comprises the second heavy chain variable domain, or

[0087] (3) A first vector comprising a nucleic acid encoding an amino acid sequence comprising the first VL of the antibody, a second vector comprising a nucleic acid encoding an amino acid sequence comprising the first VH of the antibody, a third vector comprising a nucleic acid encoding an amino acid sequence comprising the second VL of the antibody, and a fourth vector comprising a nucleic acid encoding an amino acid sequence comprising the second VH of the antibody.

[0088] In one embodiment, the host cell is eukaryotic, such as Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells). In one embodiment, a method of generating an anti-HPV antibody is provided, the method comprising culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody, as provided above, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0089] For recombinant production of anti-HPV antibodies, the nucleic acid encoding the antibody (e.g., as described above) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the antibody), or produced by recombinant methods or obtained by chemical synthesis.

[0090] Host cells suitable for cloning or expressing antibody encoding vectors include the prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., US 5,648,237, US 5,789,199, and US 5,840,523 (see also Charlton, K.A., in Methods in Molecular Biology, Vol. 248, Lo, B.K.C. ed., Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.). After expression, the antibody can be isolated from the bacterial cell pellet in the soluble fraction and further purified.

[0091] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable as cloning or expression hosts for antibody encoding vectors, including fungal and yeast strains in which the glycosylation pathways have been "humanized" resulting in the production of antibodies with partial or fully human glycosylation patterns. See Gerngross, T.U., Nat. Biotech. 22 (2004) 1409-1414; Li et al., Nat. Biotech. 24 (2006) 210-215.

[0092] Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified which can be used with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0093] Plant cell cultures can also be used as hosts. See, for example, US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (which describe the PLANTIBODIES TM technology) for generating antibodies in transgenic plants).

[0094] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines are the simian kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (293 or 293 cells, as described, for example, in Graham, F. L. et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells, as described, for example, in Mather, J. P., Biol. Reprod. 23 (1980) 243-252); simian kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); dog kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, in Mather, J. P. et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR - CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of some mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, A. M., Methods in Molecular Biology, Vol. 248, Lo, B. K. C. ed., Humana Press, Totowa, NJ (2004), pp. 255-268.

[0095] C. Assays

[0096] The anti-HPV antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by a variety of assays known in the art.

[0097] 1. Conjugate assays and other assays

[0098] On the one hand, antibodies of the invention are tested for their antigen-binding activity, e.g., by known methods such as ELISA, Western blotting, etc.

[0099] On the other hand, competitive assays can be used to identify antibodies that compete with a reference anti-HPV antibody for binding to HPV or its antigen. In certain embodiments, such competitive antibodies bind to the same epitope (e.g., linear or conformational epitope) as the reference anti-HPV antibody. Detailed exemplary methods for localizing the epitope bound by an antibody are described in Morris (1996) “Epitope Mapping Protocols”, Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0100] In one exemplary competitive assay, immobilized HPV or its antigen is incubated in a solution containing a first labeled antibody (which binds to HPV or its antigen) (e.g., a reference anti-HPV antibody) and a second unlabeled antibody (which is to be tested for its ability to compete with the first antibody for binding to HPV or its antigen). The second antibody can be present in a hybridoma supernatant. As a control, immobilized HPV or its antigen is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that permit the first antibody to bind to HPV or its antigen, excess unbound antibody is removed, and the amount of label associated with the immobilized HPV or its antigen is measured. If the amount of label associated with the immobilized HPV or its antigen in the test sample is substantially reduced compared to the control sample, this indicates that the second antibody competes with the first antibody for binding to HPV or its antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0101] 2. Activity assays

[0102] On the one hand, assays are provided for identifying anti-HPV antibodies having biological activity. Biological activity can include, for example, an anti-HPV antibody inhibiting HPV infection of host cells. Antibodies having such biological activity in vivo and / or in vitro are also provided.

[0103] D. Methods and compositions for diagnosis and detection

[0104] In certain embodiments, any anti-HPV antibody provided herein can be used to detect the presence of HPV in a sample. As used herein, the term "detect" encompasses both quantitative and qualitative detection. In certain embodiments, the sample comprises a biological sample, such as cells or tissue, such as tumor tissue; the sample also comprises a finished vaccine or a bulk HPV-VLP antigen in vaccine production.

[0105] In one embodiment, an anti-HPV antibody for use in a diagnostic or detection method is provided. In yet another aspect, a method for detecting the presence of HPV in a sample is provided. In certain embodiments, the method comprises contacting the sample with an anti-HPV antibody under conditions that permit the anti-HPV antibody to bind to HPV or its antigen, as described herein, and detecting whether a complex is formed between the anti-HPV antibody and HPV or its antigen. Such methods can be in vitro or in vivo methods. In one embodiment, an anti-HPV antibody is used to select a subject suitable for treatment with the anti-HPV antibody, for example, where the patient is infected with HPV, or suspected of being infected with HPV, or at risk of exposure to HPV.

[0106] In certain embodiments, labeled anti-HPV antibodies are provided. Labels include, but are not limited to, labels or modules for direct detection (such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), and modules for indirect detection, such as enzymes or ligands, via, for example, an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, radioactive isotopes 32 P, 14 C, 125 I, 3 H, and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferase, such as firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), fluorescein, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases, such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase (which are coupled to enzymes such as HRP that oxidize dye precursors with hydrogen peroxide), lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, phage labels, stable free radicals, and the like.

[0107] E. Pharmaceutical Formulations

[0108] Pharmaceutical formulations of anti-HPV antibodies as described herein are prepared by mixing such antibodies having the desired purity with one or more optional pharmaceutically acceptable carriers (Remington’s Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. (1980)) in the form of a lyophilized formulation or an aqueous solution. Generally, the pharmaceutically acceptable carrier is non-toxic to the recipient at the dosages and concentrations employed and includes, but is not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; counterions for salt formation such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further comprise interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20( Baxter International, Inc.). Certain exemplary sHASEGP and methods of use, including rHuPH20 are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more other glycosaminoglycanases such as chondroitinase.

[0109] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulation containing a histidine-acetate buffer.

[0110] The formulations herein may also contain more than one active ingredient necessary for the particular indication being treated, preferably those with complementary activities and that do not have adverse effects on each other. Such active ingredients are suitably present in combination in amounts effective for the desired purpose.

[0111] The active ingredient can be encapsulated in, for example, microcapsules prepared by coacervation techniques or by interfacial polymerization (such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in coarse drop emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. (1980).

[0112] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in shaped article form, such as membranes, or microcapsules.

[0113] Formulations for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

[0114] F. Prophylactic, Therapeutic Methods and Compositions

[0115] Any anti-HPV antibody provided herein can be used in a prophylactic or therapeutic method.

[0116] In one aspect, anti-HPV antibodies for use as a medicament are provided. In further aspects, anti-HPV antibodies for use in preventing or treating HPV infection are provided. In certain embodiments, anti-HPV antibodies for use in a prophylactic or therapeutic method are provided. In certain embodiments, the present invention provides anti-HPV antibodies for use in a method of preventing an individual at risk of HPV infection, said prevention comprising administering to the individual an effective amount of the anti-HPV antibody. In certain embodiments, the present invention provides anti-HPV antibodies for use in a method of treating an individual having an HPV infection, said treatment comprising administering to the individual an effective amount of the anti-HPV antibody. In one embodiment, the antibody is used to treat HPV infection or delay its progression.

[0117] The "individual" according to any of the above embodiments is preferably a human. In a further aspect, the present invention provides the use of anti-HPV antibodies in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treating HPV infection. In a further embodiment, the medicament is for use in a method of treating HPV infection, comprising administering to an individual having an HPV infection an effective amount of the medicament. The "individual" according to any of the above embodiments can be a human.

[0118] As used herein, the term "HPV infection" can be, for example, oral, skin surface, vaginal, vulval, cervical, anal infection.

[0119] In yet another aspect, the present invention provides a method of treating an HPV infection. In one embodiment, the method comprises administering to an individual having an HPV infection an effective amount of an anti-HPV. The "individual" according to any of the above embodiments can be a human.

[0120] In yet another aspect, the present invention provides a pharmaceutical formulation comprising any anti-HPV antibody provided herein, for use, for example, in any of the above therapeutic methods. In one embodiment, the pharmaceutical formulation comprises any anti-HPV antibody provided herein and a pharmaceutically acceptable carrier.

[0121] In yet another aspect, the present invention provides a pharmaceutical formulation comprising any anti-HPV antibody provided herein, for use, for example, in any of the above therapeutic methods. In one embodiment, the pharmaceutical formulation comprises any anti-HPV antibody provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any anti-HPV antibody provided herein and at least one other therapeutic agent. In one embodiment, the at least one other therapeutic agent is, for example, another anti-HPV antibody that binds a different epitope of HPV than the anti-HPV antibody herein.

[0122] The antibodies of the present invention can be used alone or in combination with other agents in a therapy. For example, the antibodies of the present invention can be co-administered with at least one other therapeutic agent.

[0123] Such combination therapies as recorded above encompass co-administration (where two or more therapeutic agents are included in the same or different formulations), and separate administration, in which case the administration of the antibodies of the present invention can occur before, simultaneously with, and / or after the administration of one or more other therapeutic agents. In one embodiment, the administration of the anti-HPV antibody and the administration of the other therapeutic agent occur within about one month of each other, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days.

[0124] The antibodies (and any other therapeutic agents) of the present invention can be administered by any suitable means, including parenterally, intralungally, and intranasally, and, if desired for local treatment, intracutaneously. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Depending in part on whether the administration is short-term or long-term, dosing can be by any suitable route, such as by injection, such as intravenous or subcutaneous injection. A variety of dosing schedules are contemplated herein, including but not limited to single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

[0125] The antibodies of the present invention should be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular disorder being treated, the particular mammal being treated, the clinical status of the individual patient, the cause, the site to which the agent is to be delivered, the method of administration, the dosing schedule, and other factors known to the practicing physician. The antibodies need not, but may optionally, be formulated with one or more agents currently used to prevent or treat the disorder. The effective amount of the other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and the other factors discussed above. These agents are generally used at the same dosage and by the same route of administration as described herein, or at about 1-99% of the dosage described herein, or at any dosage and by any route that is empirically / clinically determined to be appropriate.

[0126] G. Articles

[0127] In another aspect of the invention, there is provided an article containing materials useful for treating, preventing, and / or diagnosing the disorders described above. The article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be formed from a variety of materials such as glass or plastic. The container contains a composition that is effective to treat, prevent, and / or diagnose a condition, either alone or in combination with another composition, and can have a sterile access port (e.g., the container can be a vial or an intravenous solution bag having a stopper through which a hypodermic needle can pass). At least one active agent in the composition is an antibody of the present invention. The label or package insert indicates the use of the composition to treat a selected condition. Additionally, the article can comprise: (a) a first container containing a composition, wherein the composition comprises an antibody of the present invention; and (b) a second container containing a composition, wherein the composition comprises another therapeutic agent. The article in this embodiment of the invention can further comprise a package insert that indicates that the composition can be used to treat a particular condition. Alternatively / additionally, the article can further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It can further comprise other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0128] In the present invention, peripheral blood was collected from 14 adult volunteers one month after completion of HPV nine-valent vaccine inoculation, and the upper-layer plasma and middle PBMC were obtained by density gradient centrifugation. Fluorescently labeled HPV type 6, 11, 16, 18, 31, 33, 45, 52, and 58 L1 proteins were used to isolate memory B cells specifically binding to the above 9 type-specific proteins from PBMC by flow sorting, and nested PCR was used to obtain transfectable PCR fragments with expression activity, which were transfected into CHO cells for expression to obtain cell supernatants containing secreted antibodies. ELISA was used for binding activity screening, and 2475 binding-positive clones were obtained. Among them, 90 clones specifically bound to HPV type 6 L1 protein, 181 clones specifically bound to HPV type 11 L1 protein, 130 clones specifically bound to HPV type 16 L1 protein, 97 clones specifically bound to HPV type 18 L1 protein; 281 clones bound to all 9 type-specific L1 proteins. According to the binding and pseudovirus neutralization results of the cell supernatants, 25, 20, 35, 44, and 63 recombinant antibodies were constructed for the above 5 types of antibodies, respectively. Considering the binding activity of each recombinant antibody to the corresponding type-specific protein and the neutralization activity against each type of pseudovirus, finally 1 clone was selected for each of the 5 types of antibodies, including anti-HPV6 antibody F5-77, anti-HPV11 antibody F5-187, anti-HPV16 antibody F5-196, anti-HPV18 antibody F5-203, and anti-HPV type 9 LI protein antibody F5-127. Except for F5-127, the remaining type-specific antibodies specifically bound and neutralized the type-specific protein or pseudovirus, with the binding activity EC50 all less than 20 ng / mL and the neutralization activity IC50 ranging from 0.19 to 24.55 ng / mL.

[0129] Description of Amino Acid Sequences and Nucleic Acid Sequences

[0130]

[0131]

[0132]

[0133] Example 1: Preparation of anti-HPV antibodies

[0134] The coding sequences of the variable regions of the light and heavy chains of the antibody were cloned into a eukaryotic expression vector carrying the coding sequence of the human IgG1 constant region, transiently transfected into CHO cells for secretory expression, and 5 anti-HPV antibody clones with a purity > 90% were obtained by affinity purification, namely F5-77, F5-187, F5-196, F5-203, and F5-127.

[0135] The amino acid sequences of the CDRs of the antibody are shown in Table 1.

[0136] Table 1: Amino Acid Sequences of CDRs of Anti-HPV Antibodies

[0137]

[0138]

[0139] The amino acid sequences of VH, VL, and CL of the antibody are shown in Table 2.

[0140] Table 2: Amino Acid Sequences of VH, VL, and CL of Anti-HPV Antibodies

[0141]

[0142] Amino acid sequence of human IgG1 constant region:

[0143] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV

[0144] HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK

[0145] SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHE

[0146] DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG

[0147] KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC

[0148] LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

[0149] Example 2: Characterization of Anti-HPV Antibody - Characterization of Antigen-Binding Specificity by ELISA

[0150] The HPV type 9 L1 proteins [HPV6 (GenBank accession number: UNG35082.1), HPV11 (GenBank accession number: AAA46935.1), HPV16 (GenBank accession number: QGC89586.1), HPV18 (GenBank accession number: ACU01871.1), HPV31 (GenBank accession number: OP900721.1), HPV33 (GenBank accession number: WAN40740.1), HPV45 (GenBank accession number: AAY86494.1), HPV52 (GenBank accession number: BBD06702.1), and HPV58 (GenBank accession number: WAN40708.1)] diluted to 2 μg / mL with PBS were added to a 96-well ELISA plate (NEST, 504201) at 100 μL / well and coated overnight at 4°C. The solution was removed, and the plate was washed twice with PBST and then blocked with a blocking solution (PBS + 5% BSA) at 37°C for 2 hours. The solution was removed, and the antibody (concentration 1 μg / mL) diluted with a dilution solution (PBS + 5% BSA) was added to the microplate at 100 μL / well and incubated at 37°C for 1 hour. The solution was removed, and the plate was washed three times with PBST. Then, 100 μL of 1W-fold diluted mouse anti-human IgG Fc-HRP (produced in-house by Vazyme) was added to each well and incubated at 37°C for 1 hour. The solution was removed, and the plate was washed three times with PBST. Then, 100 μL of the chromogenic substrate TMB was added to each well and incubated at 37°C in the dark for 10 minutes. The solution was removed, and the plate was washed three times with PBST. Then, 50 μL of 2M sulfuric acid was added to each well. The OD value at 450 nm was measured on a multi-functional microplate reader (Tecan, Spark). The results are shown in Table 3.

[0151] Table 3: Protein binding activity of anti-HPV antibodies

[0152] HPV-6 HPV-11 HPV-16 HPV-18 HPV-31 HPV-33 HPV-45 HPV-52 HPV-58 F5-77 1.21 0.07 0.05 0.05 0.04 0.05 0.05 0.05 0.05 F5-187 0.07 1.92 0.07 0.06 0.07 0.07 0.07 0.07 0.06 F5-196 0.05 0.05 2.75 0.05 0.10 0.05 0.05 0.05 0.07 F5-203 0.05 0.05 0.05 1.87 0.05 0.05 0.05 0.05 0.05 F5-127 0.99 1.37 1.90 1.80 1.64 1.27 1.38 1.40 0.42

[0153] The results showed that F5-77, F5-187, F5-196, and F5-203 bound only to single-type proteins and were specific; F5-127 bound to 9-type proteins and was a broad-spectrum binding antibody.

[0154] Example 3: Characterization of anti-HPV antibodies - Characterization of antigen-binding activity by ELISA

[0155] The HPV type 9 L1 proteins (HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, and HPV58) diluted to 2 μg / mL with PBS were added to a 96-well ELISA plate (NEST, 504201) at 100 μL / well and coated overnight at 4°C. The solution was removed, washed twice with PBST, and blocked with a blocking solution (PBS + 5% BSA) at 37°C for 2 hours. The solution was removed, and the antibody serially diluted 2-fold with a dilution solution (PBS + 5% BSA) was added to the microplate at 100 μL / well and incubated at 37°C for 1 hour. The solution was removed, washed three times with PBST, and 100 μL of a 1W-fold diluted mouse anti-human IgG Fc-HRP (produced in-house by Vazyme) was added to each well and incubated at 37°C for 1 hour. The solution was removed, washed three times with PBST, and 100 μL of the chromogenic substrate TMB was added to each well and incubated at 37°C in the dark for 10 minutes. The solution was removed, washed three times with PBST, and 50 μL of 2M sulfuric acid was added to each well. The OD value at 450 nm was measured on a multifunctional microplate reader (Tecan, Spark). The antibody concentration and OD value were plotted using GraphPad Prism 8.0, and the results are as Figure 1-1 , Figure 1-2 , Figure 1-3 , Figure 1-4 , Figure 1-5 shown. Four-parameter fitting was performed to calculate the EC50 value (ng / mL) of the antigen-binding activity of the antibody, and the results are shown in Table 4.

[0156] Table 4: EC50 values (ng / mL) of the protein-binding activity of anti-HPV antibodies

[0157] Antibody HPV-6 HPV-11 HPV-16 HPV-18 HPV-31 HPV-33 HPV-45 HPV-52 HPV-58 F5-77 11.7 / / / / / / / / F5-187 / 14.0 / / / / / / / F5-196 / / 5.4 / / / / / / F5-203 / / / 8.2 / / / / / F5-127 7.99 7.51 12.52 7.11 19.07 10.82 8.72 9.82 38.57

[0158] The results showed that all 5 antibodies had strong binding activity with the binding-type proteins.

[0159] Example 4: Characterization of anti-HPV antibodies - Characterization of neutralizing activity by pseudovirus

[0160] Prepare 9 types of pseudoviruses respectively according to the method described in the patent of the National Institutes for Food and Drug Control (Patent No.: CN103333865A). The expression plasmids of the 9 genotypes are P6sheLLr, P11L1w, P16sheLL, P18sheLL, P31sheLL, P33sheLL, P45sheLL, P52sheLL and P58sheLL (the above plasmids are all purchased from addgene, and the catalog numbers are 37318, 37330, 37320, 37321, 37322, 52493, 37323, 46950 and 37324 respectively). After packaging the pseudoviruses, determine their TCID50, and dilute the virus solution according to the TCID50 value to obtain a pseudovirus dilution. Continuously dilute the antibody 4-fold with DMEM medium (starting dilution 600-fold, a total of 7 gradients) to obtain a test antibody dilution. In a 96-well plate, mix 60 μL of the virus solution and 60 μL of the test antibody dilution in equal volume, and make duplicate wells; place the 96-well plate at 4 °C for 1 hour. Add 100 μL of the pseudovirus and test antibody mixture to the corresponding wells of a culture plate pre-coated with 293TT cells and placed for 6 hours, gently tap the periphery of the culture plate to mix them, and incubate in a cell culture incubator (37 °C, 5% CO 2 ) for 2 hours. After incubation, transfer 15 μL of the supernatant of each well of the cell culture plate to a chemiluminescence detection plate respectively, then add 15 μL of the chemiluminescence detection reagent BioLux GlucSubstrate to the detection plate, and immediately put the detection plate into a GloMax chemiluminescence detector for reading. Inhibition rate of infection (%) = (1 - ratio of chemiluminescent cells in the test serum group / ratio of chemiluminescent cells in the negative control group) × 100. For serum samples with an infection inhibition rate greater than 50%, continue to perform serial dilution at a 4-fold gradient and then conduct a neutralization test again.

[0161] Use GraphPad Prism 8.0 to plot the antibody concentration and neutralization rate, and the results are as shown in Figure 2-1 、 2-2 、2-3、2-4. Perform four-parameter fitting to calculate the IC50 value (ng / mL) of the virus neutralization activity of the antibody, and the results are shown in Table 5.

[0162] Table 5: IC50 values (ng / mL) of the virus neutralization activity of anti-HPV antibodies

[0163] HPV6 HPV11 HPV16 HPV18 HPV31 HPV33 HPV45 HPV52 HPV58 F5-77 0.19 F5-187 24.55 F5-196 2.92 F5-203 0.21 F5-127 / / / / / / / / /

[0164] The results show that F5-77, F5-187, F5-196 and F5-203 only neutralize pseudoviruses of a single type and are specific; F5-127 does not neutralize any type of pseudovirus (there is still no neutralization effect even when the initial dilution factor is reduced to 10-fold).

Claims

1. An antibody or an antigen-binding fragment thereof that specifically binds to HPV, comprising: (1) CDR-H1 as set forth in SEQ ID NO: 1, CDR-H2 as set forth in SEQ ID NO: 2, CDR-H3 as set forth in SEQ ID NO: 3, CDR-L1 as set forth in SEQ ID NO: 4, CDR-L2 as set forth in SEQ ID NO: 5, and CDR-L3 as set forth in SEQ ID NO: 6; (2) CDR-H1 as set forth in SEQ ID NO:7, CDR-H2 as set forth in SEQ ID NO:8, CDR-H3 as set forth in SEQ ID NO:9, CDR-L1 as set forth in SEQ ID NO:10, CDR-L2 as set forth in SEQ ID NO:11, and CDR-L3 as set forth in SEQ ID NO:12; (3) CDR-H1 as set forth in SEQ ID NO: 13, CDR-H2 as set forth in SEQ ID NO: 14, CDR-H3 as set forth in SEQ ID NO: 15, CDR-L1 as set forth in SEQ ID NO: 16, CDR-L2 as set forth in SEQ ID NO: 17, and CDR-L3 as set forth in SEQ ID NO: 18; (4) CDR-H1 as set forth in SEQ ID NO: 19, CDR-H2 as set forth in SEQ ID NO: 20, CDR-H3 as set forth in SEQ ID NO: 21, CDR-L1 as set forth in SEQ ID NO: 22, CDR-L2 as set forth in SEQ ID NO: 23, and CDR-L3 as set forth in SEQ ID NO: 24; or (5) CDR-H1 as shown in SEQ ID NO: 25, CDR-H2 as shown in SEQ ID NO: 26, CDR-H3 as shown in SEQ ID NO: 27, CDR-L1 as shown in SEQ ID NO: 28, CDR-L2 as shown in SEQ ID NO: 29, and CDR-L3 as shown in SEQ ID NO:

30.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising: (1) VH as shown in SEQ ID NO: 31 and VL as shown in SEQ ID NO: 32; (2) VH as shown in SEQ ID NO: 33 and VL as shown in SEQ ID NO: 34; (3) VH as shown in SEQ ID NO: 35 and VL as shown in SEQ ID NO: 36; (4) VH as shown in SEQ ID NO: 37 and VL as shown in SEQ ID NO: 38; or (5) VH shown in SEQ ID NO: 39 and VL shown in SEQ ID NO:

40.

3. The antibody or antigen-binding fragment thereof according to claim 1, comprising: (1) a heavy chain constant region as shown in SEQ ID NO: 53, and (2) The light chain constant region as shown in any one of SEQ ID NO: 51 and SEQ ID NO:

52.

4. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. The polynucleotide according to claim 4, comprising: (1) SEQ ID NOs: 41 and 42; (2) SEQ ID NOs: 43 and 44; (3) SEQ ID NOs: 45 and 46; (4) SEQ ID NOs: 47 and 48; or (5) SEQ ID NOs: 49 and 50. A vector comprising the polynucleotide according to claim 4 or 5.

7. A host cell comprising the polynucleotide according to claim 4 or 5 or the vector according to claim 6. The host cell according to claim 7 , which is a CHO cell.

9. A method for producing an antibody or an antigen-binding fragment thereof, comprising: (a) culturing the host cell according to claim 7 or 8 under conditions suitable for expressing the antibody or antigen-binding fragment thereof, and (b) optionally, recovering the antibody or antigen-binding fragment thereof.

10. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or the composition according to claim 10 for use as a medicament.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or the composition according to claim 10, for use in treating a disease.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or the composition according to claim 10, which is used as a detection reagent.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or the composition according to claim 10, for use in an identification test of an HPV vaccine.

15. The invention as herein described.

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