Antibodies that specifically bind to HPV

Abstract

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

Description

Technical Field

[0001] This invention provides a set of antibodies or antigen-binding fragments thereof that specifically bind to HPV. This invention also provides polynucleotides encoding said antibodies or antigen-binding fragments thereof, vectors containing said polynucleotides, host cells containing said vectors, methods for generating said antibodies, and compositions containing said antibodies. Background Technology

[0002] Cervical cancer and precancerous lesions pose a serious threat to the health of women worldwide. Clinical, molecular biological, and epidemiological surveys have proven that human papillomavirus (HPV) is the main cause of cervical cancer and cervical dysplasia. HPV is a group of spherical, tiny, non-enveloped circular double-stranded DNA viruses belonging to the genus Papillomavirus of the family Papillovaviridae. Its capsid is an icosahedral structure composed of 72 major capsid proteins, L1 protein pentamers. Each viral particle contains 360 L1 protein monomers. It mainly causes proliferative lesions of human skin and mucous membranes. Currently, more than 200 subtypes of HPV have been identified, which are classified 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, several cervical cancer vaccines are approved for marketing in China, including: GlaxoSmithKline's (GSK) Cervarix bivalent HPV vaccine (subtypes 16 and 18), and Merck's quadrivalent HPV vaccine. and the 9-valent vaccine 9 (subtypes include HPV 6, 11, 16, 18, 31, 33, 45, 52, and 58), Wantai Biological's bivalent HPV vaccine Xinkening, and Watson Biological's bivalent HPV vaccine Wozehui. Since currently commercially available vaccines cover a maximum of nine subtypes, more and more companies are continuously developing HPV vaccines with multiple valences (such as 15-valent). In vaccine research, monoclonal antibodies are an important tool for vaccine antigen quality control, and antibody levels are a standard for evaluating vaccine efficacy. Therefore, various types of antibodies that specifically recognize HPV are needed to better assess the produced vaccines and their protective effects, and for use in the diagnosis, treatment, and prevention of HPV. Summary of the Invention

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

[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 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 of the present invention comprises:

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

[0018] (2) Light chain constant region as shown in either SEQ ID NO: 51 or SEQ ID NO: 52.

[0019] In one embodiment, the antibody or antigen-binding fragment 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 of the present invention belongs to an IgG1, IgG2, IgG3, or IgG4 subclass. In one embodiment, the antibody or antigen-binding fragment of the present invention comprises a λ light chain or a κ light chain. In one embodiment, the antibody or antigen-binding fragment of the present invention is a full-length antibody. In one embodiment, the antibody or antigen-binding fragment 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 of the present invention is a chimeric antibody or a human antibody or antigen-binding fragment thereof.

[0020] On one hand, the present invention provides a polynucleotide that encodes the antibody or antigen-binding fragment 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 carrier comprising the polynucleotide of the present invention.

[0028] On 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 another embodiment, the host cell is a CHO cell.

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

[0030] (a) The host cells of the present invention are cultured under conditions suitable for expressing the antibody or its antigen-binding fragment, and

[0031] (b) Optionally, the antibody or its antigen-binding fragment may be recovered.

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

[0033] On the one hand, the present invention provides antibodies or antigen-binding fragments or compositions thereof for use as detection reagents.

[0034] On the one hand, the present invention provides the antibody of the present invention or its antigen-binding fragment or composition for use in the identification test of HPV vaccines. Attached Figure Description

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

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

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

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

[0039] Figure 1-5 The antigen-binding curve (ELISA) of the antibody F5-127 of this invention is shown.

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

[0041] Figure 2-2 The virus neutralization curve of the antibody F5-187 of this invention is shown.

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

[0043] Figure 2-4 The virus neutralization curve of antibody F5-203 of the present invention is shown. Detailed Implementation

[0044] The term “antibody” is used in the broadest sense in this article and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0045] "Antigen-binding fragment" refers to a molecule that is distinct from the intact antibody, containing the portion of the intact antibody that binds to the antigen bound by the intact antibody. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, xFab; biantibodies; linear antibodies; single-chain antibody molecules (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 proliferating another nucleic acid linked to it. This term includes vectors that are self-replicating nucleic acid structures and vectors incorporated into the genome of the host cell to which they are introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0047] I. Composition and Method

[0048] In one aspect, the present invention provides isolated antibodies that bind to HPV.

[0049] In some implementations, antibodies that bind to HPV are 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 with the amino acid sequence of SEQ ID NO:31. In some 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 this sequence retains its ability to bind HPV. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO:31. In some embodiments, the substitutions, insertions, or deletions occur in regions other than the CDR (i.e., in the FR). Optionally, the anti-HPV antibody comprises the VH sequence of SEQ ID NO:31, including post-translational modifications of that 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:1, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) 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 with the amino acid sequence of SEQ ID NO:32. In some embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the anti-HPV antibody comprising this sequence retains its ability to bind HPV. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO:32. In some embodiments, the substitution, insertion, or deletion occurs in a region other than the CDR (i.e., in the FR). Optionally, the anti-HPV antibody comprises the VL sequence of SEQ ID NO:32, including post-translational modifications of that 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: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) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:7; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:8; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:9; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:10; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:11; and (f) a CDR-L3 comprising an 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 with the amino acid sequence of SEQ ID NO:33. In some 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 a reference sequence, but the anti-HPV antibody comprising this sequence retains its ability to bind HPV. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO:33. In some embodiments, the substitutions, insertions, or deletions occur in regions other than the CDR (i.e., in the FR). Optionally, the anti-HPV antibody comprises the VH sequence of SEQ ID NO:33, including post-translational modifications of that 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, 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 with the amino acid sequence of SEQ ID NO:34. In some 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 a reference sequence, but the anti-HPV antibody comprising this sequence retains its ability to bind HPV. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO:34. In some embodiments, the substitution, insertion, or deletion occurs in a region other than the CDR (i.e., in the FR). Optionally, the anti-HPV antibody comprises the VL sequence of SEQ ID NO:34, including post-translational modifications of that 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) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:14; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:15; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:16; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:17; and (f) a CDR-L3 comprising an 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 with the amino acid sequence of SEQ ID NO:35. In some 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 a reference sequence, but the anti-HPV antibody comprising this sequence retains its ability to bind HPV. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO:35. In some embodiments, the substitutions, insertions, or deletions occur in regions other than the CDR (i.e., in the FR). Optionally, the anti-HPV antibody comprises the VH sequence of SEQ ID NO:35, including post-translational modifications of that 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: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 with the amino acid sequence of SEQ ID NO:36. In some embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the anti-HPV antibody comprising this sequence retains its ability to bind HPV. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO:36. In some embodiments, the substitution, insertion, or deletion occurs in a region other than the CDR (i.e., in the FR). Optionally, the anti-HPV antibody comprises the VL sequence of SEQ ID NO:36, including post-translational modifications of that 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: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) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:19; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:20; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:21; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:22; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23; and (f) a CDR-L3 comprising an 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 with the amino acid sequence of SEQ ID NO:37. In some 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 a reference sequence, but the anti-HPV antibody comprising this sequence retains its ability to bind HPV. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO:37. In some embodiments, the substitutions, insertions, or deletions occur in regions other than the CDR (i.e., in the FR). Optionally, the anti-HPV antibody comprises the VH sequence of SEQ ID NO:37, including post-translational modifications of that 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: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, 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 with the amino acid sequence of SEQ ID NO:38. In some embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the anti-HPV antibody comprising this sequence retains its ability to bind HPV. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO:38. In some embodiments, the substitution, insertion, or deletion occurs in a region other than the CDR (i.e., in the FR). Optionally, the anti-HPV antibody comprises the VL sequence of SEQ ID NO:38, including post-translational modifications of that 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: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) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:25; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:26; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:27; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:28; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:29; and (f) a CDR-L3 comprising an 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 with the amino acid sequence of SEQ ID NO:39. In some 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 a reference sequence, but the anti-HPV antibody comprising this sequence retains its ability to bind HPV. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO:39. In some embodiments, the substitutions, insertions, or deletions occur in regions other than the CDR (i.e., in the FR). Optionally, the anti-HPV antibody comprises the VH sequence of SEQ ID NO:39, including post-translational modifications of that 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: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 with the amino acid sequence of SEQ ID NO:40. In some 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 a reference sequence, but the anti-HPV antibody comprising this sequence retains its ability to bind HPV. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO:40. In some embodiments, the substitution, insertion, or deletion occurs in a region other than the CDR (i.e., in the FR). Optionally, the anti-HPV antibody comprises the VL sequence of SEQ ID NO:40, including post-translational modifications of that 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: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 VH as in any of the embodiments provided above, and VL as in any of the embodiments provided above. In one embodiment, the antibody comprises VH and VL sequences respectively in SEQ ID NO:31 and SEQ ID NO:32, including post-translational modifications of those sequences. In one embodiment, the antibody comprises VH and VL sequences respectively in SEQ ID NO:33 and SEQ ID NO:34, including post-translational modifications of those sequences. In one embodiment, the antibody comprises VH and VL sequences respectively in SEQ ID NO:35 and SEQ ID NO:36, including post-translational modifications of those sequences. In one embodiment, the antibody comprises VH and VL sequences respectively in SEQ ID NO:37 and SEQ ID NO:38, including post-translational modifications of those sequences. In one embodiment, the antibody comprises VH and VL sequences respectively in SEQ ID NO:39 and SEQ ID NO:40, including post-translational modifications of those sequences.

[0072] In 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, a biantibody, or an F(ab')2 fragment.

[0073] B. Recombination methods and compositions

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

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

[0076] In the case of bispecific antibodies with heterodimeric heavy chains, four nucleic acids are required: one for the first light chain, one for the second light chain containing the polypeptide of the first heteromonomer Fc region, one for the second light chain, and one for the second heavy chain containing the polypeptide of the second heteromonomer Fc region. These four nucleic acids can be contained in one or more nucleic acid molecules or expression vectors. Such nucleic acids encode the amino acid sequence of the first VL containing the antibody and / or the amino acid sequence of the first VH containing the Fc region of the first heteromonomer and / or the amino acid sequence of the second VL containing the second VH containing the Fc region of the second heteromonomer (e.g., the first and / or second light and / or 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 heteromonad heavy chains contains a so-called “node mutation” (one of T366W and optionally 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 implementation, an isolated nucleic acid encoding the antibody used in the method reported herein is provided.

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

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

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

[0081] - In the case of antibodies composed of two identical light chains forming disulfide bonds and two identical heavy chains containing segments of VH and VL:

[0082] (1) A vector containing nucleic acids, wherein the nucleic acids encode an amino acid sequence containing antibody VL and an amino acid sequence containing antibody VH, or

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

[0084] - In the case of bispecific antibodies with heterodimeric heavy chains:

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

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

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

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

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

[0090] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be generated in bacteria, particularly where glycosylation and Fc effector function are not required. For expression of antibody fragments and peptides in bacteria, see, for example, US 5,648,237, US 5,789,199 and US 5,840,523 (also see Charlton, KA, in Methods in Molecular Biology, Vol. 248, edited by Lo, BKC, Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in *Escherichia coli*). After expression, the antibody can be separated from the bacterial cell clump in a soluble fraction and can be further purified.

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

[0092] Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used with insect cells, particularly for transfecting 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 PLATNIBODIES for generating antibodies in transgenic plants). TM technology).

[0094] Vertebrate cells can also be used as a host. For example, mammalian cell lines adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines are: monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney line (293 or 293 cells, as described in, for example, Graham, F.L. et al., J. Gen. Virol. 36 (1977) 59-74); juvenile hamster kidney cells (BHK); mouse seltoli cells (TM4 cells, as described in, for example, Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); and TRI cells, as described in, for example, Mather, JP et al., Annals. NYAcad. 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, NSO, and Sp2 / 0. For reviews of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, edited by Lo, BKC, Humana Press, Totowa, NJ (2004), pp. 255-268.

[0095] C. Determination method

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

[0097] 1. Combining the assay method with other assay methods

[0098] On the one hand, the antigen-binding activity of the antibodies of the present invention is tested, for example by known methods such as ELISA, Western blot, etc.

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

[0100] In an exemplary competitive assay, HPV or its antigen is immobilized 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 may be present in the hybridoma supernatant. As a control, HPV or its antigen is immobilized in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to HPV or its antigen, excess unbound antibody is removed, and the amount of the label conjugated with the immobilized HPV or its antigen is measured. If the amount of the label conjugated 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 Assay

[0102] On the one hand, an assay is provided for identifying biologically active anti-HPV antibodies. Biological activity may include, for example, the inhibition of HPV infection of host cells by anti-HPV antibodies. Antibodies exhibiting such biological activity in vivo and / or in vitro are also provided.

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

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

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

[0106] In some embodiments, labeled anti-HPV antibodies are provided. Labels include, but are not limited to, directly detectable labels or modules (such as fluorescence, chromogenic, electron-dense, chemiluminescence, and radioactive labels), and indirectly detectable modules, such as enzymes or ligands, for example, via enzyme reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes. 32 P, 14 C, 125 I, 3 H, and 131 I. Fluoresceins such as rare earth chelates or luciferin and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (US Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucosylamylase, 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 with enzymes such as HRP using hydrogen peroxide dye precursors), lactoperoxidase, or microperoxidase, biotin / avidin, spin-labeled, phage-labeled, stable free radicals, etc.

[0107] E. Pharmaceutical preparations

[0108] Pharmaceutical formulations of anti-HPV antibodies as described herein are prepared by mixing such antibodies of desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)) in lyophilized or aqueous solution form. Generally, pharmaceutically acceptable carriers are non-toxic to recipients at the doses and concentrations used and include, but are 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; hexanediamine chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; p-hydroxybenzoic acid hydrocarbon esters, such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); and 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; salt-forming opposite ions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). The illustrative pharmaceutically acceptable carriers described herein further comprise interstitial drug dispersants such as soluble neutral active hyaluronidase glycoproteins (sHASEGP), such as 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 Publications Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more other glycosaminoglycans such as chondroitinase.

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

[0110] The formulations described herein may also contain more than one active ingredient necessary for the specific indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitable to be present in combination in amounts effective for the desired purpose.

[0111] The active ingredient can be encapsulated in microcapsules prepared, for example, by coagulation techniques or by interfacial polymerization (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., 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. (1980).

[0112] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include semi-permeable matrices of solid hydrophobic polymers containing antibodies, which are in commercially available forms, such as membranes or microcapsules.

[0113] Formulations intended for internal administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile filter membrane.

[0114] F. Preventive, therapeutic methods and compositions

[0115] Any of the anti-HPV antibodies provided in this article can be used in preventative or therapeutic approaches.

[0116] In one aspect, an anti-HPV antibody is provided for use as a medicine. In other aspects, an anti-HPV antibody is provided for use in the prevention or treatment of HPV infection. In some embodiments, an anti-HPV antibody is provided for use in the prevention or treatment of HPV infection. In some embodiments, the present invention provides an anti-HPV antibody for use in a method of preventing an individual at risk of HPV infection, said prevention comprising administering an effective amount of the anti-HPV antibody to the individual. In some embodiments, the present invention provides an anti-HPV antibody for use in a method of treating an individual with HPV infection, said treatment comprising administering an effective amount of the anti-HPV antibody to the individual. 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 being. In another aspect, the present invention provides the use of an anti-HPV antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is used to treat HPV infection. In yet another embodiment, a method of using the medicament to treat HPV infection includes administering an effective amount of the medicament to an individual having an HPV infection. The "individual" according to any of the above embodiments can be a human being.

[0118] As used in this article, the term "HPV infection" can refer to infections of the oral cavity, skin surface, vagina, vulva, cervix, or anus, for example.

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

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

[0121] In another aspect, the present invention provides pharmaceutical formulations comprising any anti-HPV antibody provided herein, for example, used in any of the above-described 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 to a different epitope of HPV with the anti-HPV antibody described 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] The combination therapies described above encompass both combined administration (where two or more therapeutic agents are contained in the same or different formulations) and separate administration, in which case the administration of the antibody of the present invention may occur before, simultaneously with, and / or after administration of one or more other therapeutic agents. In one embodiment, the administration of the anti-HPV antibody and the administration of other therapeutic agents occur within about one month, 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 parenteral, intrapulmonary, and intranasal administration, and, if intended for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Depending in part whether the administration is transient or long-term, dosage can be administered via any suitable route, such as by injection, such as intravenous or subcutaneous injection. Various dosage schedules are covered herein, including but not limited to single administration or multiple administrations at multiple time points, bolus administration, and pulsatile infusion.

[0125] The antibodies of this invention should be formulated, and their dosage and administration determined in accordance with good medical practice. Factors considered in this regard include the specific condition being treated, the specific mammal being treated, the individual patient's clinical condition, etiology, site of drug delivery, route of administration, dosing schedule, and other factors known to the practicing physician. The antibodies may be formulated, but not necessarily, with one or more agents currently used for the prevention or treatment of the condition. The effective amount of the other agents described above depends on the amount of antibody present in the formulation, the type of condition or treatment, and other factors discussed above. These agents are generally used at the same dosage and route of administration as described herein, or at about 1-99% of the dosage described herein, or at any dosage and via any route, which is determined empirically / clinically as appropriate.

[0126] G. Products

[0127] In another aspect of the invention, an article is provided containing materials that can be used to treat, prevent, and / or diagnose the conditions described above. The article includes a container and a label or packaging insert on or in conjunction with the container. Suitable containers include, for example, bottles, tubular vials, syringes, IV solution bags, etc. The container can be formed from a variety of materials such as glass or plastic. The container contains a composition, alone or in combination with another composition, that is effective in treating, preventing, and / or diagnosing the condition, and may have a sterile access port (e.g., the container may be a tubular vial or intravenous solution bag with a stopper that can be passed through by a hypodermic needle). At least one active agent in the composition is an antibody of the present invention. The label or packaging insert indicates the use of the composition to treat a selected condition. Furthermore, the article may contain: (a) a first container containing the composition, wherein the composition contains the antibody of the present invention; and (b) a second container containing the composition, wherein the composition contains other therapeutic agents. The article in this embodiment of the invention may further include a packaging insert indicating the use of the composition to treat a specific condition. Alternatively / additionally, the article may further comprise a second (or third) container containing pharmaceutically acceptable buffers, such as antibacterial water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextran solution. It may further comprise other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0128] In this invention, peripheral blood was collected from 14 adult volunteers one month after completing the HPV 9-valent vaccine. The upper plasma layer and the middle PBMCs were obtained by density gradient centrifugation. Memory B cells specifically binding to the proteins of the nine HPV types were isolated from the PBMCs using flow cytometry with fluorescently labeled L1 proteins of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58. Nested PCR was used to obtain transfectible PCR fragments with expression activity, which were then transfected into CHO cells for expression. Cell supernatants containing secreted antibodies were obtained, and binding activity was screened by ELISA, yielding 2475 binding-positive clones. 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, and 281 clones bound to all nine L1 types. Based on the binding and pseudovirus neutralization results in cell supernatant, 25, 20, 35, 44, and 63 recombinant antibodies were constructed from the five antibody types, respectively. Considering the binding activity of each recombinant antibody to the corresponding HPV type protein and its neutralizing activity against each type of pseudovirus, one antibody from each of the five types was ultimately selected: anti-HPV6 antibody F5-77, anti-HPV11 antibody F5-187, anti-HPV16 antibody F5-196, anti-HPV18 antibody F5-203, and anti-HPV 9 type LI protein antibody F5-127. Except for F5-127, the other type-specific antibodies specifically bound to and neutralized the corresponding type protein or pseudovirus, with binding activity EC50 all below 20 ng / mL and neutralizing activity IC50 between 0.19 and 24.55 ng / mL.

[0129] Description of amino acid and nucleic acid sequences

[0130]

[0131]

[0132]

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

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

[0135] The amino acid sequence of the antibody's CDR is shown in Table 1.

[0136] Table 1: Amino acid sequence of CDR of anti-HPV antibody

[0137]

[0138]

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

[0140] Table 2: Amino acid sequences of VH, VL, and CL of anti-HPV antibodies

[0141]

[0142] The amino acid sequence of the human IgG1 constant region:

[0143] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV

[0144] HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK

[0145] SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHE

[0146] DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG

[0147] KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC

[0148] LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

[0149] Example 2: Characterization of anti-HPV antibodies—characterizing antigen binding specificity by ELISA

[0150] HPV 9 type L1 protein [HPV6 (genbank: UNG35082.1), HPV11 (genbank: AAA46935.1), HPV16 (genbank: QGC89586.1), HPV18 (genbank: ACU01871.1), HPV31 (genbank: OP900721.1), HPV33 (genbank: WAN40740.1), HPV45 (genbank: AAY86494.1), HPV52 (genbank: BBD06702.1), and HPV58 (genbank: WAN40708.1)] diluted to 2 μg / mL with PBS was added to a 96-well microplate (NEST, 504201) at 100 μL / well and incubated overnight at 4°C. After removing the solution, wash twice with PBST and block with blocking buffer (PBS + 5% BSA) at 37°C for 2 hours. After removing the solution, add 100 μL of antibody diluted with diluent (PBS + 5% BSA) to each well and incubate at 37°C for 1 hour. After removing the solution, wash three times with PBST, add 100 μL of 10,000-fold diluted mouse anti-human IgG Fc-HRP (Vazyme product) to each well, and incubate at 37°C for 1 hour. After removing the solution, wash three times with PBST, add 100 μL of chromogenic substrate TMB to each well, and incubate at 37°C in the dark for 10 minutes. After removing the solution, wash three times with PBST, and add 50 μL of 2M sulfuric acid to each well. Measure the OD value at 450 nm using 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 to only a single type of protein, exhibiting specificity; while F5-127 bound to all nine types of proteins, classifying it as a broad-spectrum binding antibody.

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

[0155] HPV type 9 L1 protein (HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, and HPV58), diluted to 2 μg / mL with PBS, was added at 100 μL / well to a 96-well microplate (NEST, 504201) and incubated overnight at 4°C. The solution was removed, and the plates were washed twice with PBST and blocked with blocking buffer (PBS + 5% BSA) at 37°C for 2 hours. The solution was then removed, and antibodies serially diluted 2-fold with dilution buffer (PBS + 5% BSA) were added at 100 μL / well to each well and incubated at 37°C for 1 hour. The solution was then removed, and the plates were washed three times with PBST. 100 μL of 10,000-fold diluted mouse anti-human IgG Fc-HRP (Vazyme product) was added to each well and incubated at 37°C for 1 hour. Remove the solution, wash three times with PBST, add 100 μL of chromogenic substrate TMB to each well, and incubate at 37°C in the dark for 10 minutes. Remove the solution, wash three times with PBST, and add 50 μL of 2M sulfuric acid to each well. Measure the OD value at 450 nm using a multi-functional microplate reader (Tecan, Spark). Plot the antibody concentration and OD value using GraphPadPrism 8.0; the results are shown below. Figure 1-1 , Figure 1-2 , Figure 1-3 , Figure 1-4 , Figure 1-5 As shown in Table 4, a four-parameter fitting was performed to calculate the EC50 value (ng / mL) of the antibody's antigen-binding activity.

[0156] Table 4: EC50 values ​​(ng / mL) of 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 five antibodies had strong binding activity to the binding type protein.

[0159] Example 4: Characterization of anti-HPV antibodies—characterizing neutralizing activity through pseudoviruses

[0160] Nine pseudoviruses of different genotypes were prepared according to the method described in the National Institutes for Food and Drug Control (NIFDC) patent (patent number: CN103333865A). The expression plasmids for the nine genotypes were P6sheLLr, P11L1w, P16sheLL, P18sheLL, P31sheLL, P33sheLL, P45sheLL, P52sheLL, and P58sheLL (all plasmids were purchased from AddGene, catalog numbers 37318, 37330, 37320, 37321, 37322, 52493, 37323, 46950, and 37324, respectively). After packaging the pseudoviruses, their TCID50 was measured. The virus solution was diluted according to the TCID50 value to obtain pseudovirus dilutions. The antibodies were serially diluted 4-fold using DMEM medium (starting with a 600-fold dilution, for a total of 7 gradients) to obtain the antibody dilutions for testing. In a 96-well plate, mix equal volumes of 60 μL virus solution and 60 μL antibody dilution buffer, and repeat the mixture in multiple wells. Incubate the 96-well plate at 4°C for 1 hour. Add 100 μL of the pseudovirus and antibody mixture to the corresponding wells of a culture plate pre-coated with 293TT cells and incubated for 6 hours. Gently tap the sides of the culture plate to mix the mixture, and incubate in a cell culture incubator (37°C, 5% CO2) for 2 hours. After incubation, transfer 15 μL of the supernatant from each well of the cell culture to a chemiluminescence detection plate. Then add 15 μL of BioLux GlucSubstrate chemiluminescence detection reagent to the detection plate and immediately place the plate in a GloMax chemiluminescence analyzer for reading. Infection inhibition rate (%) = (1 - (ratio of chemiluminescent cells in the test serum group / ratio of chemiluminescent cells in the negative control group) × 100. Serum samples with an infection inhibition rate greater than 50% are serially diluted 4-fold and subjected to a neutralization test again.

[0161] The antibody concentration and neutralization rate were plotted using GraphPad Prism 8.0, and the results are as follows: Figure 2-1 , 2-2 As shown in Figures 2-3 and 2-4, a four-parameter fitting was performed to calculate the IC50 value (ng / mL) of the antibody's virus-neutralizing activity, and the results are shown in Table 5.

[0162] Table 5: IC50 values ​​(ng / mL) of virus-neutralizing 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 showed that F5-77, F5-187, F5-196, and F5-203 neutralized only a single type of pseudovirus, demonstrating specificity; F5-127 did not neutralize any type of pseudovirus (it had no neutralizing effect even after the initial dilution was reduced to 10-fold).

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to HPV, comprising: (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; (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; (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; (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 (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 as shown in SEQ ID NO: 39 and VL as shown in SEQ ID NO:

40.

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

52.

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

5. The polynucleotide according to claim 4, comprising: (1) SEQ ID NO: 41 and 42; (2) SEQ ID NO: 43 and 44; (3) SEQ ID NO: 45 and 46; (4) SEQ ID NO: 47 and 48; or (5) SEQ ID NO: 49 and 50.

6. A vector comprising the polynucleotide according to claim 4 or 5.

7. A host cell comprising the polynucleotide of claim 4 or 5 or the vector of claim 6.

8. The host cell according to claim 7, wherein it is a CHO cell.

9. A method for generating an antibody or an antigen-binding fragment thereof, comprising: (a) Culture the host cells according to claim 7 or 8 under conditions suitable for expressing the antibody or its antigen-binding fragment, and (b) Optionally, the antibody or its antigen-binding fragment may be recovered.

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

11. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-3 or the composition according to claim 10 in the preparation of a reagent for detecting HPV in a sample.

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

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