HIV-1 GP41 neutralizing antibodies and their uses

Abstract

The present invention discloses neutralizing antibodies that specifically bind to HIV-1 Env and antigen-binding fragments of these antibodies. Also provided are nucleic acids, vectors and host cells encoding these antibodies. Disclosed are methods for detecting HIV-1 using these antibodies. In addition, disclosed are uses of these antibodies, antigen-binding fragments, nucleic acids and vectors for preventing and / or treating HIV-1 infection.

Description

[0001] Divisional application

[0002] This application is a divisional application of the Chinese patent application with a priority date of November 3, 2015, an application date of November 3, 2016, and an application number of 201680077520.X. Field of the Invention

[0003] Concerning antibodies and antigen-binding fragments that specifically bind to human immunodeficiency virus type 1 (HIV-1) gp41 and their use, for example, in treating a subject having an HIV-1 infection. Background Art

[0004] Despite the unremitting efforts to develop anti-HIV-1 therapeutic agents, HIV-1 infection and the resulting acquired immunodeficiency syndrome (AIDS) still pose a threat to global public health. The enveloped virus HIV-1 evades humoral recognition behind a variety of protective mechanisms. The main HIV-1 envelope protein (HIV-1Env) is a glycoprotein (gp160) of approximately 160kD. During infection, the host cell's protease cuts gp160 into gp120 and gp41. Gp41 is an integral membrane protein, while gp120 protrudes from the mature virus. Gp120 and gp41 together form the HIV-1 envelope spike, which is a target for neutralizing the virus. Broadly neutralizing antibodies that bind HIV-1 Env have been identified, including the 10E8 antibody, which specifically binds to the membrane proximal external region (MPER) of gp41 and can neutralize a high percentage of HIV-1 strains. However, there is a need to develop additional HIV-1 neutralizing antibodies with different recognition and neutralization profiles for commercial production. Summary of the invention

[0005] Disclosed herein are isolated antibodies and antigen-binding fragments that specifically bind gp41 and neutralize HIV-1. In some embodiments, the isolated antibodies or antigen-binding fragments include heavy and light chain variable regions that comprise one or more amino acid substitutions (e.g., in the complementarity determining regions (CDRs) and / or framework regions) relative to the 10E8 antibody that confer a combination of improved neutralization, solubility, and autoreactivity properties relative to the 10E8 antibody.

[0006] In some embodiments, the isolated antibody or antigen-binding fragment comprises a heavy chain variable region (V H ), which includes V shown in SEQ ID NO:75 (10E8v4 S100cF)H heavy chain complementarity determining region (HCDR) 1, HCDR2 and HCDR3; and light chain variable region (V L ), which includes V shown in SEQ ID NO:6 (rL3-6mut) L light chain complementary determining region (LCDR) 1, LCDR2 and LCDR3, wherein the antibody or antigen-binding fragment specifically binds gp41 and neutralizes HIV-1. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 7, 8, 77, 10, 11 and 12, respectively (10E8v4 S100cF IMGT CDRs). In some embodiments, the V H and V L The antibody or antibody binding fragment comprises the amino acid sequences shown in SEQ ID NO: 75 and 6 (10E8v4S100cF), respectively, or comprises the amino acid sequences shown in SEQ ID NO: 76 and 6 (10E8v4 V5R S100cF), respectively, wherein the antibody or antibody binding fragment specifically binds to gp41 and neutralizes HIV-1.

[0007] In another embodiment, the isolated antibody comprises a V sequence as shown in SEQ ID NO: 5. H V of HCDR1, HCDR2 and HCDR3 H , and comprising V shown in SEQ ID NO:6 L The light chains LCDR1, LCDR2 and LCDR3 of L , where V H comprises arginine, glutamic acid, serine, lysine, aspartic acid, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105 and 110, respectively, and said V L The monoclonal antibody or antigen-binding fragment comprises alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83 and 85, respectively, and wherein the monoclonal antibody or antigen-binding fragment specifically binds to gp41 and neutralizes HIV-1.

[0008] Also disclosed are compositions comprising the antibodies and antigen-binding fragments, nucleic acids encoding the antibodies and antigen-binding fragments, expression vectors comprising the nucleic acids, and isolated host cells comprising the nucleic acids. In some embodiments, the nucleic acid molecule may be a bicistronic expression construct encoding the V or V of the antibody or antigen-binding fragment. H and V L .

[0009] The disclosed antibodies and antigen-binding fragments effectively neutralize HIV-1 in recognized in vitro models of HIV-1 infection. Thus, methods for treating or inhibiting HIV-1 infection in a subject are disclosed. The methods include administering to a subject, such as a subject at risk of HIV-1 infection or suffering from HIV-1 infection, a therapeutically effective amount of one or more of the disclosed antibodies, antigen-binding fragments, nucleic acid molecules, vectors or compositions.

[0010] The antibodies, antigen-binding fragments, nucleic acid molecules, vectors and compositions disclosed herein can be used for a variety of additional purposes, such as detecting HIV-1 infection or diagnosing HIV-1 infection in a subject, or detecting HIV-1 in a sample.

[0011] The foregoing and other features and advantages of the present disclosure will become more apparent from the following detailed description of several embodiments with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figures 1A-1F Sequence alignment of antibody heavy and light chain variable domains. Figure 1A and 1B The kabat numbering and IMGT ( Figure 1A ) or kabat( Figure 1B )CDR positioning. Figure 1C and 1D The kabat numbering and IMGT ( Figure 1C ) or kabat( Figure 1D )CDR positioning. Figure 1EV sequences for 10E8 (SEQ ID NO: 1), 10E8v4 (SEQ ID NO: 5), 10E8v4 S100cF (SEQ ID NO: 75), and 10E8v4 V5R S100cF (SEQ ID NO: 76) are shown. H Kabat numbering and IMGT CDR positioning. Figure 1F V for 10E8 (SEQ ID NO: 2) and 10E8v4 (SEQ ID NO: 6) is shown. L Kabat numbering and IMGT CDR positioning, which is also used for V in 10E8v4 S100cF and 10E8v4 V5R S100cF L .

[0013] Figure 2 For graphs and images of Coomassie blue stained gels, 10E8 antibody and HC6-S74Y V H and rL3V L Or HC6-S74Y-511 V H and rL3 V L Turbidity (measured at OD350) of 10E8 variants in phosphate buffered saline. The highly soluble VRC01 antibody was used as a control.

[0014] Figure 3 To illustrate the 10E8 antibody, and including HC6-S74Y V H and rL3 V L Or HC6-S74Y-511 V H and rL3V L Table of neutralization of a representative panel of HIV-1 pseudoviruses by 10E8 variants. Neutralization was determined using a TZM-bl cell pseudovirus neutralization assay; IC 80 value.

[0015] Figure 4A To describe the 10E8 antibody and H6 V H and L10 V L 、H8 V H and L10 V L 、H8 V H and L19 V L 、H6-511 V H and L10 V L or H8-511 V H and L10 V L Graph of the turbidity (measured at OD350) of 10E8 variants in phosphate buffered saline. The highly soluble VRC01 antibody was used as a control.

[0016] Figure 4B To describe the 10E8 antibody and H6-511 V H and L10 V L or H8-511 V H and L10 V L Table of neutralization of 10E8 variants against a representative panel of HIV-1 pseudoviruses. Neutralization was determined using a TZM-bl cell pseudovirus neutralization assay; IC 50 value.

[0017] Figure 5 Figure 1 is a ribbon diagram illustrating a portion of the structure of the 10E8 antibody bound to the MPER peptide. The "4mut" residues D28, N31, T52, and Y98 (kabat numbering) of the heavy chain variable region domain are highlighted.

[0018] Figure 6 Graph illustrating the turbidity (measured at OD350) of the 10E8 antibody and the indicated 10E8 variants in phosphate buffered saline. The highly soluble VRC01 antibody was used as a control.

[0019] Figure 7 To describe the 10E8 antibody and H6-511 V H and rL3 V L or H6-511-4mut (H6-511 with N28D, D31N, S52T and H98Y substitutions) V H and L10 V L Table of neutralization of 10E8 variants against a representative panel of HIV-1 pseudoviruses. Neutralization was determined using a TZM-bl cell pseudovirus neutralization assay; IC 50 value.

[0020] Figure 8 To describe the 10E8 antibody and HC6-S74Y-511 V H and L10 V L 、HC6-S74Y V H and L10 V L or 10E8 V H and L10 V L Graph of the turbidity (measured at OD350) of 10E8 variants in phosphate buffered saline. The highly soluble VRC01 antibody was used as a control.

[0021] Fig. 9 To illustrate the 10E8 antibody and including the V L Domain-paired 10E8 VH Graph of the turbidity (measured at OD350) of 10E8 variants in phosphate buffered saline. H Paired rL3 V with S1A, Y2S, T8P, G9A, G16K, R17Q substitutions L ("rL3-6mut") had the least turbidity of the 10E8 variant. The highly soluble VRC01 antibody was used as a control.

[0022] Fig.10 To describe the 10E8 antibody and H6-511-4mut V H and rL3 V L or H6-511-4mut V H and rL3-6mut V L Graph of the turbidity (measured at OD350) of 10E8 variants in phosphate buffered saline. The highly soluble VRC01 antibody was used as a control.

[0023] Fig.11 To describe the 10E8 antibody and H6-511 V H and rL3 V L 、H6-511-4mut V H and rL3 or H6-511-4mut V H and rL3-6mut V L Table of neutralization of a representative panel of HIV-1 pseudoviruses by 10E8 variants. Neutralization was determined using a TZM-bl cell pseudovirus neutralization assay; IC 50 and IC 80 value.

[0024] Fig.12 Figure 1 is a flow chart illustrating the development of modified 10E8 antibodies.

[0025] Figures 13A-13C The 10E8 antibody or HC6-S74Y-511 V H and rL3 V L 、HC6-S74Y-511 V H and rL3 or H6-511-4mut and rL3-6mut V L Results of kinetic concentration assays of 10E8 variants. 3 ml of the indicated antibodies (OD 0.35) were concentrated using a centrifugal concentrator ( Fig.13A ), and measure the volume of the resulting concentrate ( Fig. 13B ) and protein concentration ( Fig. 13C , OD280).

[0026] Fig.14 A series of graphs showing the presence of antibodies comprising 10E8 and HC6-S74Y-511 V H and rL3 V L 、HC6-S74Y-511 V H and rL3 or H6-511-4mut and rL3-6mut V L Results of dynamic scattering assays of solutions of 10E8 variants. The 10E8 antibody was polydisperse, indicating the presence of protein aggregates. H and rL3 V L 、HC6-S74Y-511 V H and rL3 or H6-511-4mut and rL3-6mut V L The antibodies are monodisperse.

[0027] Fig.15 A set of immunofluorescence images showing the 10E8 antibody and HC6-S74Y-511 V H and rL3 V L 、HC6-S74Y-511 V H and rL3-6mut V L or H6-511-4mut and rL3-6mut V L The 10E8 variant was not autoreactive. Reactivity to HIV-1 negative human epithelial (HEp-2) cells was determined by indirect immunofluorescence.

[0028] Figures 16A-16F A series of tables illustrating the 10E8 antibody and HC6-S74Y-511 V H and rL3 V L (Var1), HC6-S74Y-511 V H and rL3-6mut V L (Var5) or H6-511-4mut and rL3-6mut V L Neutralization of a panel of 200 HIV-1 pseudoviruses by the 10E8 variant of (Var4). IC 50 ( Figures 16A-16C ) and IC 80 ( Figures 16D-16F )value.

[0029] Fig.17 The graph shows the changes in the concentration of 10E8 antibody or 10E8 variant antibody in primate (cynomolgus monkey) serum over time after a single antibody injection.

[0030] Figures 18A-18Dis a series of graphs illustrating the size exclusion chromatography of 10E8, 10E8v4, and 10E8v5 ( Fig.18A ), in V L Size exclusion chromatography of several 10E8v4 variants with amino acid substitutions at Kabat position 32 ( Fig.18B and 18C ), and in V L Turbidity of several 10E8v4 variants with amino acid substitutions at Kabat position 32 ( Fig.18D ).

[0031] Fig.19 A series of tables are shown illustrating the IC values ​​of several broadly neutralizing HIV-1 antibodies, including the 10E8v4 S100cW antibody, against a panel of HIV-1 viruses. 50 and IC 80 value.

[0032] Fig. 20 A series of graphs and strip diagrams illustrating that changes in 10E8v4 HCDR3 improve the size exclusion chromatography profile to a single peak.

[0033] Fig.21 is a series of graphs showing the results of size exclusion chromatography for 10E8v4-DS and 10E8v4-100cW-DS antibodies.

[0034] Fig. 22 The localization of certain amino acid substitutions for 10E8v4 that improve HIV-1 neutralization within the Cryo-EM HIV-1 Env structure matched with the 10E8v4 antibody is shown. One set of mutations is in the membrane-juxtaposed region of 10E8v4 when it is bound to HIV-1 Env (for V L D7W, D26R and G68R substitutions in 10E8v4), and another group in a region not close to the cell membrane (for V H D72R, E1R D28W and P52bR replacement).

[0035] Figures 23A-23F A series of tables are shown illustrating the neutralization of a representative panel of HIV-1 pseudoviruses by several different 10E8 variant antibodies. These 10E8 variants include those in Table 10 (see Example 5). Neutralization was determined using a TZM-bl cell pseudovirus neutralization assay; IC 50 ( Fig.23A , 23C and 23D) and IC 80 ( Fig. 23B , 23E , 23F) value.

[0036] Fig.24is a series of graphs showing results from solubility assays of 10E8v4 variants comprising S100cF, S100cH, S100cL, S100cR, S100cW or S100cY substitutions, performed by measuring the volume and protein concentration of these antibody solutions after concentration using a standard centrifugal concentrator.

[0037] sequence

[0038] The nucleic acid and amino acid sequences listed in the attached sequence table are shown using standard letter abbreviations for nucleotide bases and three-letter codes for amino acids, as defined in 37 C.FR 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included in any reference to the displayed strand. The sequence table is submitted as an ASCII text file (~165 kb) in the form of a file named "Sequence.txt" generated on November 1, 2016, and is incorporated herein by reference.

[0039] In the attached sequence listing:

[0040] SEQ ID NO: 1 is 10E8 V H The amino acid sequence of

[0041] EVQLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAAPVEGRFTISRLNSINFLYLEMNNLRMEDSGLYFCARTGKYYDFWSGYPPGEEYFQDWGRGTLVTVSS

[0042] SEQ ID NO: 2 is 10E8 V L The amino acid sequence of

[0043] SYELTQETGVSVALGRTVTITCRGDSLRSHYASWYQKKPGQAPILLFYGKNNRPSGVPDRFSGSASGNRASLTISGAQAEDDAEYYCSSRDKSGSRLSVFGGGTKLTVL

[0044] SEQ ID NO: 3 is HC6-S74Y-511 V H The amino acid sequence of

[0045] EVRLAESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAAPVEGRFTISRDNYKNTLYLEMNNLRTEDSGLYFCARTGKYYDFWSGYPPGEEYFQDWGRGTLVIVSS

[0046] SEQ ID NO:4 is rL3 V L The amino acid sequence of

[0047] SYELTQDTGVSVALGRTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVL

[0048] SEQ ID NO: 5 is H6-511-4mut V H The amino acid sequence of

[0049] EVRLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWGQGTLVIVSS

[0050] SEQ ID NO:6 is rL3-6mut V L The amino acid sequence of

[0051] ASELTQDPAVSVALKQTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVL

[0052] SEQ ID NOs: 7-12 are the amino acid sequences of the IMGT CDRs of the 10E8 antibody.

[0053] SEQ ID NO: 13 is HC6-S74Y V H The amino acid sequence of

[0054] EVRLAESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAAPV

[0055] EGRFTISRLNYINFLYLEMNNLRMEDSGLYFCARTGKYYDFWSGYPPGEEYFQDWGRGTLVIVSS

[0056] SEQ ID NO:14 is the amino acid sequence of H6 V H .

[0057] EVRLVESGGGLVKPGGSLRLSCSASGFNFDDAWMTWVRQPPGKGLEWVGRISGPGEGWSVDYAESVKGRFTISRLNSINFLYLEMNNVRTEDTGYYFCARTGKHYDFWSGYPPGEEYFQDWGQGTLVIVSS

[0058] SEQ ID NO:15 is the amino acid sequence of H6-511 V H .

[0059] EVRLVESGGGLVKPGGSLRLSCSASGFNFDDAWMTWVRQPPGKGLEWVGRISGPGEGWSVDYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKHYDFWSGYPPGEEYFQDWGQGTLVIVSSSEQ ID NO:16 is the amino acid sequence of H8 V H .

[0060] EVRLVESGGRLVRPGGSLRLSCSASGFNFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAASVKGRFTISRMNSINFFYLEMNNLKIEDTGLYFCARTGKHYAFWGGYPPGEEYLEDWGQGTLVIVSSSEQ ID NO:17 is the amino acid sequence of H8-511 V H .

[0061] EVRLVESGGRLVRPGGSLRLSCSASGFNFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAASVKGRFTISRDNYKNTFYLEMNNLKTEDTGLYFCARTGKHYAFWGGYPPGEEYLEDWGQGTLVIVSSSEQ ID NO:18 is the amino acid sequence of L10 V L .

[0062] ASELTQDPAVSVALKQTVTITCRGDSLRSHYVSWYQKKPGQAPVLVFYGKNNRPSGIPDRFSGSSSGNTASLTIAGAQAEDDADYYCSSRDKSGSRLSVFGGGTKLTVL

[0063] SEQ ID NO: 19 is L19 V L The amino acid sequence of

[0064] ASELTQDPTVSVALGQTVTITCRGDSLRNYYTSWYQQKPGQAPVLLIYPKHNRPPGISDRFSASSSGNTASLTITGAQTEDEGDYYCSSRDKSGSRLVTFGRGTKLTVV

[0065] SEQ ID NO: 20 is a sequence encoding HC6-S74Y-511 V linked to an IgG1 constant region. H An exemplary nucleic acid sequence of .

[0066]

[0067] SEQ ID NO: 21 is a fragment encoding H6-511-4mut V linked to an IgG1 constant region. H An exemplary nucleic acid sequence of .

[0068]

[0069] SEQ ID NO: 22 is H6-511-4mut V linked to an IgG1 constant region H The amino acid sequence of

[0070] EVRLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEE YFQDWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO:23 is HC6-S74Y-511 V linked to IgG1 constant region H The amino acid sequence of

[0071] EVRLAESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAAPVEGRFTISRDNYKNTLYLEMNNLRTEDSGLYFCARTGKYYDFWSGYPPGEEYFQDWGRGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO:24 encodes rL3 V L Exemplary nucleic acid sequences for

[0072] tcatacgaactgactcaggacactggcgtctctgtggcactggggaggactgtgactattacttgccgaggcgactcactgcggagccactacgcttcctggtatcagaagaaacccggccaggcacctgtgctgctgttctacggaaagaacaataggccatctggcatccccgaccgcttttctggcagtgcatcagggaaccgagccagtctgaccattaccggcgcccaggctgaggacgaagccgattactattgcagctcccgggataagagcggctccagactgagcgtgttcggaggaggaactaaactgaccgtcctcagtcagcccaaggctgccccctcggtcactctgttcccgccctcgagtgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctacctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttcatag

[0073] SEQ ID NO: 25 encodes rL3-6mut V L an exemplary nucleic acid sequence.

[0074] accaccatgggatggtcatgtatcatcctttttctagtagcaactgcaaccggttctgtgaccgcatccgaactgactcaggaccctgccgtctctgtggcactgaagcagactgtgactattacttgccgaggcgactcactgcggagccactacgcttcctggtatc agaagaaacccggccaggcacctgtgctgctgttctacggaaagaacaataggccatctggcatccccgaccgcttttctggcagtgcatcagggaaccgagccagtctgaccattaccggcgcccaggctgaggacgaagccgattactattgcagctcccgggataag agcggctccagactgagcgtgttcggaggaggaactaaactgaccgtcctcagtcagcccaaggctgccccctcggtcactctgttcccgccctcgagtgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacag tggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctacctgagcctgacgcctgancagtggaangtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgt

[0075] SEQ ID NO: 26 is the amino acid sequence of HIV-1 Env from the HXB2 strain of HIV-1.

[0076] SEQ ID NOs: 27-33 are V sequences of VRC01, VRC07, VRC07-523 and VRC07-544 antibodies. H and V L Amino acid sequence of the domain.

[0077] SEQ ID NO: 34 is the amino acid sequence of the MPER peptide.

[0078] SEQ ID NO:35 is H6-511-4mut-S100cW V H The amino acid sequence of

[0079] EVRLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWWGYPPGEEYFQDWGQGTLVIVSS

[0080] SEQ ID NO:36 is the amino acid sequence of H6-511-4mut-DS V H of.

[0081] EVRLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGCPPGEEYFQDWGQGTLVIVSS

[0082] SEQ ID NO:37 is the amino acid sequence of H6-511-4mut-S100cW-DS V H of.

[0083] EVRLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWWGCPPGEEYFQDWGQGTLVIVSS

[0084] SEQ ID NO:38 is the amino acid sequence of HC6-S74Y-511-S100cW V H of.

[0085] EVRLAESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAAPVEGRFTISRDNYKNTLYLEMNNLRTEDSGLYFCARTGKYYDFWWGYPPGEEYFQDWGRGTLVIVSS

[0086] SEQ ID NO:39 is the amino acid sequence of HC6-S74Y-511-DS V H of.

[0087] EVRLAESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAAPVEGRFTISRDNYKNTLYLEMNNLRTEDSGLYFCARTGKYYDFWSGCPPGEEYFQDWGRGTLVIVSS

[0088] SEQ ID NO:40 is the amino acid sequence of HC6-S74Y-511-S100cW-DS V H .

[0089] EVRLAESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAAPVEGRFTISRDNYKNTLYLEMNNLRTEDSGLYFCARTGKYYDFWWGCPPGEEYFQDWGRGTLVIVSSSEQ ID NO:41 is the amino acid sequence of rL3-6mut-DS V L .

[0090] ASELTQDPAVSVALKQTVTITCRGDSLRCHYASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVL

[0091] SEQ ID NO:42 is the amino acid sequence of rL3-6mut-Y32W V L .

[0092] ASELTQDPAVSVALKQTVTITCRGDSLRSHWASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVL

[0093] SEQ ID NO:43 is the amino acid sequence of rL3-6mut-Y32F V L .

[0094] ASELTQDPAVSVALKQTVTITCRGDSLRSHFASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVL

[0095] SEQ ID NO:44 is an exemplary nucleic acid sequence encoding HC6-S74Y-511 V H .

[0096] gaagtgcggctggctgagagcggcggggggctggtcaaacctggcgggtcactgcggctgtcctgttctgcctccggcttcgattttgataacgcatggatgacatgggtgcgacagccacctggaaaggggctggagtgggtcggcagaatcactggacctggcgaagggtggtctgtggactacgcagctccagtcgagggacgattcaccattagtagagataactacaagaatacactgtatctggagatgaacaatctgaggactgaagacagcggcctgtatttctgcgcccgcaccgggaaatactatgatttttggtctgggtacccacccggagaggaatattttcaggactggggacggggcaccctggtgatcgtcagctcc

[0097] SEQ ID NO:45 is an exemplary nucleic acid sequence encoding H6-511-4mut V H .

[0098] gaggttagactggtggagtcaggaggggggcttgtgaagcccggtgggtctctccgcctgagctgttctgcctccggctttgatttcgataacgcctggatgacctgggtcaggcagcctccaggtaagggactggagtgggtgggaagaatcacaggtccaggcgagggctggtccgtggactacgcggaatctgttaaagggcggtttacaatctcaagggacaataccaagaataccttgtatttggagatgaacaacgtgagaactgaagacaccggatattacttctgtgccagaacaggcaaatactacgacttctggtccggctatccccctggcgaggaatattttcaagactggggtcagggaacccttgttatcgtgtcctcc

[0099] SEQ ID NO:46 is an exemplary nucleic acid sequence encoding rL3 V L as follows.

[0100] tcatacgaactgactcaggacactggcgtctctgtggcactggggaggactgtgactattacttgccgaggcgactcactgcggagccactacgcttcctggtatcagaagaaacccggccaggcacctgtgctgctgttctacggaaagaacaataggccatctggcatccccgaccgcttttctggcagtgcatcagggaaccgagccagtctgaccattaccggcgcccaggctgaggacgaagccgattactattgcagctcccgggataagagcggctccagactgagcgtgttcggaggaggaactaaactgaccgtcctc

[0101] SEQ ID NO:47 is an exemplary nucleic acid sequence encoding rL3-6mut V L as follows.

[0102] accaccatgggatggtcatgtatcatcctttttctagtagcaactgcaaccggttctgtgaccgcatccgaactgactcaggaccctgccgtctctgtggcactgaagcagactgtgactattacttgccgaggcgactcactgcggagccactacgcttcctggtatcagaagaaacccggccaggcacctgtgctgctgttctacggaaagaacaataggccatctggcatccccgaccgcttttctggcagtgcatcagggaaccgagccagtctgaccattaccggcgcccaggctgaggacgaagccgattactattgcagctcccgggataagagcggctccagactgagcgtgttcggaggaggaactaaactgaccgtcctc

[0103] SEQ ID NO:48 is an exemplary nucleic acid sequence of an expression vector encoding HC6-S74Y-511 V H as follows.

[0104] SEQ ID NO:49 is a polypeptide encoding H6-511-4mut V H Exemplary nucleic acid sequences of expression vectors.

[0105] SEQ ID NO:50 is a polypeptide encoding rL3 V L Exemplary nucleic acid sequences of expression vectors.

[0106] SEQ ID NO:51 is a polypeptide encoding rL3-6mut V L Exemplary nucleic acid sequences of expression vectors.

[0107] SEQ ID NOs: 52-57 are the amino acid sequences of IMGT CDRs.

[0108] SEQ ID NO:58 is rL3-6mut-DS-Y32W V L The amino acid sequence of

[0109] ASELTQDPAVSVALKQTVTITCRGDSLRCHWASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVL

[0110] SEQ ID NO:59 is rL3-6mut-DS-Y32F V L The amino acid sequence of

[0111] ASELTQDPAVSVALKQTVTITCRGDSLRCHFASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVL

[0112] SEQ ID NOs: 60-63 respectively encode 10E8v4-DS V H 、10E8v4-DS(or 10E8v4-100cW-DS)V L 、10E8v4-100cW V H or 10E8v4-100cW-DS V H Exemplary nucleic acid sequences of expression vectors.

[0113] SEQ ID NO:64 is a polypeptide encoding H6-511-4mut-S100cW V H (Seq_35) is an exemplary nucleic acid sequence.

[0114] atgggctggtcctgtattatcctgttcctggtcgcaactgctactggcgtccattcagaagtgaggctggtcgagagcggcggcggcctggtgaagccaggaggaagcctgcgactgagctgctccgcctctggcttcgactttgataacgcttggatgacatgggtgcgacagccccctggaaaaggcctggagtgggtcggaagaatcaccggccccggagagggatggagtgtggactacgcagaatcagtcaagggccggttcaccattagccgggataacaccaaaaatacactgtatctggagatgaacaatgtcaggactgaagacaccgggtactatttctgtgcccgcaccggaaagtactatgatttttggtggggctacccacccggagaagaatactttcaggactggggacagggaacactggtcatcgtcagcagc

[0115] SEQ ID NO: 65 encodes the exemplary nucleic acid sequence of H6-511-4mut-DS V H (Seq_36).

[0116] atgggatggtcatgtatcatcctttttctagtagcaactgcaaccggtgtacattctgaggttagactggtggagtcaggaggggggcttgtgaagcccggtgggtctctccgcctgagctgttctgcctccggctttgatttcgataacgcctggatgacctgggtcaggcagcctccaggtaagggactggagtgggtgggaagaatcacaggtccaggcgagggctggtccgtggactacgcggaatctgttaaagggcggtttacaatctcaagggacaataccaagaataccttgtatttggagatgaacaacgtgagaactgaagacaccggatattacttctgtgccagaacaggcaaatactacgacttctggtccggctgcccccctggcgaggaatattttcaagactggggtcagggaacccttgttatcgtgtcctcc

[0117] SEQ ID NO: 66 encodes the exemplary nucleic acid sequence of H6-511-4mut-S100cW-DSV H (Seq_37).

[0118] atgggatggtcatgtatcatcctttttctagtagcaactgcaaccggtgtacattctgaggttagactggtggagtcaggaggggggcttgtgaagcccggtgggtctctccgcctgagctgttctgcctccggctttgatttcgataacgcctggatgacctgggtcaggcagcctccaggtaagggactggagtgggtgggaagaatcacaggtccaggcgagggctggtccgtggactacgcggaatctgttaaagggcggtttacaatctcaagggacaataccaagaataccttgtatttggagatgaacaacgtgagaactgaagacaccggatattacttctgtgccagaacaggcaaatactacgacttctggtggggctgcccccctggcgaggaatattttcaagactggggtcagggaacccttgttatcgtgtcctcc

[0119] SEQ ID NO:67 encodes HC6-S74Y-511-S100cW V H (Seq_38) exemplary nucleic acid sequence. gaagtgcggctggctgagagcggcggggggctggtcaaacctggcgggtcactgcggctgtcctgttctgcctccggcttcgattttgataacgcatggatgacatgggtgcgacagccacctggaaaggggctggagtgggtcggcagaatcactggacctggcgaagggtggtctgtggactacgcagctccagtcgagggacgattcaccattagtagagataactacaagaatacactgtatctggagatgaacaatctgaggactgaagacagcggcctgtatttctgcgcccgcaccgggaaatactatgatttttggtgggggtacccacccggagaggaatattttcaggactggggacggggcaccctggtgatcgtcagctcc

[0120] SEQ ID NO:68 encodes HC6-S74Y-511-DS VH Exemplary nucleic acid sequence of (Seq_39).

[0121] gaagtgcggctggctgagagcggcggggggctggtcaaacctggcgggtcactgcggctgtcctgttctgcctccggcttcgattttgataacgcatggatgacatgggtgcgacagccacctggaaaggggctggagtgggtcggcagaatcactggacctggcgaagggtggtctgtggactacgcagctccagtcgagggacgattcaccattagtagagataactacaagaatacactgtatctggagatgaacaatctgaggactgaagacagcggcctgtatttctgcgcccgcaccgggaaatactatgatttttggtctgggtgcccacccggagaggaatattttcaggactggggacggggcaccctggtgatcgtcagctcc

[0122] SEQ ID NO:69 encodes HC6-S74Y-511-S100cW-DS V H Exemplary nucleic acid sequence of (Seq_40).

[0123] gaagtgcggctggctgagagcggcggggggctggtcaaacctggcgggtcactgcggctgtcctgttctgcctccggcttcgattttgataacgcatggatgacatgggtgcgacagccacctggaaaggggctggagtgggtcggcagaatcactggacctggcgaagggtggtctgtggactacgcagctccagtcgagggacgattcaccattagtagagataactacaagaatacactgtatctggagatgaacaatctgaggactgaagacagcggcctgtatttctgcgcccgcaccgggaaatactatgatttttggtgggggtgcccacccggagaggaatattttcaggactggggacggggcaccctggtgatcgtcagctcc

[0124] SEQ ID NO:70 is the exemplary nucleic acid sequence encoding rL3-6mut-DS V L (Seq_41).

[0125] atgggatggtcatgtatcatcctttttctagtagcaactgcaaccggttctgtgaccgcatccgaactgactcaggaccctgccgtctctgtggcactgaagcagactgtgactattacttgccgaggcgactcactgcggtgccactacgcttcctggtatcagaagaaacccggccaggcacctgtgctgctgttctacggaaagaacaataggccatctggcatccccgaccgcttttctggcagtgcatcagggaaccgagccagtctgaccattaccggcgcccaggctgaggacgaagccgattactattgcagctcccgggataagagcggctccagactgagcgtgttcggaggaggaactaaactgaccgtcctc

[0126] SEQ ID NO:71 is the exemplary nucleic acid sequence encoding rL3-6mut-Y32W V L (Seq_42).

[0127] atgggatggtcatgtatcatcctttttctagtagcaactgcaaccggttctgtgaccgcatccgaactgactcaggaccctgccgtctctgtggcactgaagcagactgtgactattacttgccgaggcgactcactgcggagccactgggcttcctggtatcagaagaaacccggccaggcacctgtgctgctgttctacggaaagaacaataggccatctggcatccccgaccgcttttctggcagtgcatcagggaaccgagccagtctgaccattaccggcgcccaggctgaggacgaagccgattactattgcagctcccgggataagagcggctccagactgagcgtgttcggaggaggaactaaactgaccgtcctc

[0128] SEQ ID NO:72 is an exemplary nucleic acid sequence encoding rL3-6mut-Y32F V L (Seq_43).

[0129] atgggatggtcatgtatcatcctttttctagtagcaactgcaaccggttctgtgaccgcatccgaactgactcaggaccctgccgtctctgtggcactgaagcagactgtgactattacttgccgaggcgactcactgcggagccactttgcttcctggtatcagaagaaacccggccaggcacctgtgctgctgttctacggaaagaacaataggccatctggcatccccgaccgcttttctggcagtgcatcagggaaccgagccagtctgaccattaccggcgcccaggctgaggacgaagccgattactattgcagctcccgggataagagcggctccagactgagcgtgttcggaggaggaactaaactgaccgtcctc

[0130] SEQ ID NO:73 is an exemplary nucleic acid sequence encoding rL3-6mut-DS-Y32W V L (Seq_58).

[0131] atgggatggtcatgtatcatcctttttctagtagcaactgcaaccggttctgtgaccgcatccgaactgactcaggaccctgccgtctctgtggcactgaagcagactgtgactattacttgccgaggcgactcactgcggagccactgggcttcctggtatcagaagaaacccggccaggcacctgtgctgctgttctacggaaagaacaataggccatctggcatccccgaccgcttttctggcagtgcatcagggaaccgagccagtctgaccattaccggcgcccaggctgaggacgaagccgattactattgcagctcccgggataagagcggctccagactgagcgtgttcggaggaggaactaaactgaccgtcctc

[0132] SEQ ID NO:74 is an exemplary nucleic acid sequence encoding rL3-6mut-DS-Y32F V L (Seq_59).

[0133] atgggatggtcatgtatcatcctttttctagtagcaactgcaaccggttctgtgaccgcatccgaactgactcaggaccctgccgtctctgtggcactgaagcagactgtgactattacttgccgaggcgactcactgcggagccactttgcttcctggtatcagaagaaacccggccaggcacctgtgctgctgttctacggaaagaacaataggccatctggcatccccgaccgcttttctggcagtgcatcagggaaccgagccagtctgaccattaccggcgcccaggctgaggacgaagccgattactattgcagctcccgggataagagcggctccagactgagcgtgttcggaggaggaactaaactgaccgtcctc

[0134] SEQ ID NO:75 is the amino acid sequence of H6-511-4mut-S100cF V H .

[0135] EVRLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFW F GYPPGEEYFQDWGQGTLVIVSS

[0136] SEQ ID NO:76 is the amino acid sequence of H6-511-4mut V5R S100cF V H .

[0137] EVRL R ESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFW F GYPPGEEYFQDWGQGTLVIVSS

[0138] SEQ ID NO: 77 is H6-511-4mut-S100cF V H( The amino acid sequence of HCDR3 of IMGT).

[0139] SEQ ID NO: 78 is a polypeptide encoding H6-511-4mut-S100cF V H (Seq_75) is an exemplary nucleic acid sequence.

[0140] gaggttagactggtggagtcaggaggggggcttgtgaagcccggtgggtctctccgcctgagctgttctgcctccggctttgatttcgataacgcctg gatgacctgggtcaggcagcctccaggtaagggactggagtgggtgggaagaatcacaggtccaggcgagggctggtccgtggactacgcggaatctg ttaaagggcggtttacaatctcaagggacaataccaagaataccttgtatttggagatgaacaacgtgagaactgaagacaccggatattacttctgtgccagaacaggcaaatactacgacttctggttcggctatccccctggcgaggaatattttcaagactggggtcagggaacccttgttatcgtgtcctcc

[0141] SEQ ID NO: 79 is a fragment encoding H6-511-4mut V5R S100cF V H (Seq_76) is an exemplary nucleic acid sequence.

[0142] gaggttagactgcgggagtcaggaggggggcttgtgaagcccggtgggtctctccgcctgagctgttctgcctccggctttgatttcgataacgcctg gatgacctgggtcaggcagcctccaggtaagggactggagtgggtgggaagaatcacaggtccaggcgagggctggtccgtggactacgcggaatctg ttaaagggcggtttacaatctcaagggacaataccaagaataccttgtatttggagatgaacaacgtgagaactgaagacaccggatattacttctgtgccagaacaggcaaatactacgacttctggttcggctatccccctggcgaggaatattttcaagactggggtcagggaacccttgttatcgtgtcctcc

[0143] SEQ ID NO: 80-81 encode H6-511-4mut-S100cF and H6-511-4mut V5R S100cFV, respectively. H The nucleic acid sequence of the expression vector of the region.

[0144] SEQ ID NO: 82 is H6-511-4mut-S100cF V linked to an IgG1 constant region H The amino acid sequence of

[0145] EVRLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFW FGYPPGEEYFQDWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0146] SEQ ID NO:83 is the amino acid sequence of H6-511-4mut-V5R S100cF V linked to the IgG1 constant region. H of.

[0147] EVRL R ESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFW F GYPPGEEYFQDWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0148] SEQ ID NO: 84 is rL3-6mut V linked to an Igλ constant region L The amino acid sequence of

[0149] ASELTQDPAVSVALKQTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLT VLSQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS DETAILED DESCRIPTION

[0150] I. Overview of Terminology

[0151] Unless otherwise indicated, technical terms are used in conventional usage. Definitions of commonly used terms in molecular biology can be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishers in 2009; Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in 16 volumes in 2008, and other similar reference books.

[0152] As used herein, the singular forms "a", "an", and "the" refer to the singular as well as the plural, unless the context clearly indicates otherwise. For example, the term "an antigen" includes singular or plural antigens and can be considered equivalent to the phrase "at least one antigen". As used herein, the term "comprises" means "includes". It should also be understood that any and all base sizes or amino acid sizes given for nucleic acids or polypeptides, and all molecular weight or molecular mass values, are approximate and provided for illustrative purposes, unless otherwise indicated. Although methods and materials similar to or equivalent to those described herein can be used, particularly suitable methods and materials are described herein. In the event of a conflict, the present specification including the explanation of terms shall prevail. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. In order to facilitate viewing of various embodiments, the following explanation of terms is provided.

[0153] 10E8: A neutralizing antibody that specifically binds to an epitope on the MPER of gp41. The 10E8 antibody and its binding to gp41 have been previously described, see, e.g., Huang et al. Nature, 491:406-412, 2012, which is incorporated herein by reference in its entirety. The amino acid sequences of the heavy and light chain variable regions of the 10E8 antibody are shown in SEQ ID NOs: 1 and 2.

[0154] Administration: The introduction of a composition into a subject via a chosen route. Administration can be local or systemic. For example, if the chosen route is intravenous, the composition is administered by introducing the composition into a vein of the subject. Exemplary routes of administration include, but are not limited to, oral, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (e.g., topical), intranasal, vaginal, and inhalation routes.

[0155] Amino acid substitution: An amino acid in a peptide is replaced by a different amino acid.

[0156] Antiretroviral agents: agents that specifically inhibit retroviral replication or infection of cells. Non-limiting examples of antiretroviral drugs include entry inhibitors (e.g., enfuvirtide), CCR5 receptor antagonists (e.g., apraviroc, vicriviroc, maraviroc), reverse transcriptase inhibitors (e.g., lamivudine, zidovudine, abacavir, tenofovir, emtricitabine, efavirenz), protease inhibitors (e.g., lopivar, ritonavir, raltegravir, darunavir, atazanavir), maturation inhibitors (e.g., interferon alpha, bevirimat, and vivecon).

[0157] Antiretroviral therapy (ART): It is related to the therapeutic treatment for HIV-1 infection of administering at least one antiretroviral agent (e.g., one, two, three or four antiretroviral agents) to HIV-1 infected individuals. An example of an ART regimen includes treating with a combination of tenofovir, emtricitabine and efavirenz. In some instances, ART includes highly active antiretroviral therapy (HAART). An example of a HAART regimen includes treating with a combination of tenofovir, emtricitabine and efavirenz.

[0158] Antibody: An immunoglobulin, antigen-binding fragment, or derivative thereof that specifically binds to and recognizes an analyte (antigen) such as HIV-1 gp41. The term "antibody" is used herein in its broadest sense to encompass 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.

[0159] Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof known in the art that retain antigen binding affinity. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments antigen-binding fragments, which are either produced by modification of whole antibodies or by using recombinant DNA methodology (see, e.g., Kontermann and Dubel (Ed), Antibody Engineering, Vols. 1-2, 2 nd Ed., Springer Press, 2010).

[0160] Single-chain antibodies (scFv) are genetically engineered molecules that contain one or more V chains of antibodies connected by a suitable polypeptide linker. H and V L domain, as a single-chain molecule for genetic fusion (see, e.g., Bird et al., Science, 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879-5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, ID Rugs, 13:543-549, 2010). H Domain and V LThe intramolecular orientation of the domains in the scFv is not critical for the scFv. Therefore, scRFvs with two possible arrangements can be used (V H Domain-Linker Domain-V L -Domain; V L Domain-Linker Domain-V H - domain).

[0161] In dsFv, V H and V L Mutated to introduce disulfide bonds to stabilize chain association. Also included are diabodies, which are bivalent, bispecific antibodies in which V H and V L The domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci., 90:6444-6448, 1993; Poljak et al., Structure, 2:1121-1123, 1994).

[0162] Antibodies also include genetically engineered forms, such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3 rd Ed., WH Freeman & Co., New York, 1997.

[0163] An antibody that binds to the same epitope as a reference antibody refers to an antibody that blocks 50% or more of the binding of the reference antibody to its antigen in a competition assay, whereas the reference antibody blocks 50% or more of the binding of the antibody to its antigen in a competition assay. Antibody competition assays are known, and exemplary competition assays are provided herein.

[0164] An antibody may have one or more binding sites. If there is more than one binding site, these binding sites may be identical to each other or may be different. For example, naturally occurring immunoglobulins have two identical binding sites, single-chain antibodies or Fab fragments have one binding site, and bispecific or bifunctional antibodies have two different binding sites.

[0165] Typically, naturally occurring immunoglobulins have heavy and light chains interconnected by disulfide bonds. Immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable domain genes. There are two types of light chains, lambda and kappa. There are 5 major heavy chain classes (or isotypes), which determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE.

[0166] Each heavy and light chain contains a constant region and a variable region (see, e.g., Kindt et al., Kuby Immunology, 6 th ed., WH Freeman and Co., page 91 (2007)). H " or "VH" refers to the variable region of an antibody heavy chain, including the variable region of an antigen-binding fragment (e.g., Fv, scFv, dsFv or Fab). References to "V L " or "VL" refers to the variable region of an antibody light chain, including the variable region of an Fv, scFv, dsFv or Fab. In several embodiments, the V H and V L In another embodiment, only V H . For example, naturally occurring camelid antibodies consist only of heavy chains, which are functional and stable in the absence of light chains (see, e.g., Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). Any of the disclosed antibodies includes a heterologous constant region. For example, the antibody includes a constant region that is different from the native constant region, which includes, for example, one or more modifications (e.g., "LS" mutations) to increase half-life.

[0167] The V H and V L Contains a "framework region" interrupted by three hypervariable regions also called "complementarity determining regions" or "CDRs" (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within species. The framework region of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, is used for positioning and alignment of the CDRs in three-dimensional space.

[0168] These CDRs are primarily responsible for binding to antigenic epitopes. The amino acid sequence boundaries of a given CDR can be easily determined using any of several well-known schemes, including Kabat et al. ("Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991; "Kabat" numbering scheme), Al-Lazikani et al. (JMB 273, 927-948, 1997; "Chothia" numbering scheme), and Lefranc et al. ("IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev. Comp. Immunol., 27: 55-77, 2003; "IMGT" numbering scheme). The CDRs of each chain are usually referred to as CDR1, CDR2, and CDR3 (from N-terminus to C-terminus), and are also identified by the chain in which the specific CDR is located. Therefore, V H CDR3 is a V from the antibody in which it is present. H CDR3, while V L CDR1 is a V from the antibody in which it is present. L The light chain CDR is sometimes referred to as LCDR1, LCDR2 and LCDR3. The heavy chain CDR is sometimes referred to as HCDR1, HCDR2 and HCDR3.

[0169] "Monoclonal antibody" is an antibody obtained from a substantially homogeneous antibody population (i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during the preparation of the monoclonal antibody (such variants are generally present in small amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates that the antibody is characterized as being obtained from a substantially homogeneous antibody population, and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody can be generated by a variety of techniques, including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, these methods and other exemplary methods for preparing monoclonal antibodies are described herein. In some instances, the monoclonal antibody is isolated from a subject. The monoclonal antibody may have conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. (See, e.g., Harlow & Lane, Antibodies, A Laboratory Manual, 2 nd ed. Cold Spring Harbor Publications, New York (2013). ) In some embodiments, any of the antibodies disclosed herein may be a monoclonal antibody.

[0170] "Humanized" antibodies or antigen-binding fragments include human framework regions and one or more CDRs from non-human (e.g., mouse, rat, or synthetic) antigens or antigen-binding fragments. The non-human antibody or antigen-binding fragment that provides the CDRs is called the "donor," and the human antibody or antigen-binding fragment that provides the framework regions becomes the "acceptor." In one embodiment, all CDRs in a humanized immunoglobulin come from a donor immunoglobulin. Constant regions do not have to be present, but if they are present, they may be substantially consistent with human immunoglobulin constant regions, e.g., at least about 85-90%, e.g., about 95% or more consistent. Thus, except for possible CDRs, all portions of a humanized antibody or antigen-binding fragment are substantially consistent with corresponding portions of natural human antibody sequences.

[0171] A "chimeric" antibody is an antibody that includes sequences derived from two different antibodies and usually different species. In some examples, a chimeric antibody includes one or more CDRs and / or framework regions from one human antibody and CDRs and / or framework regions from another human antibody.

[0172] A "fully human antibody" or "human antibody" is an antibody that includes sequences from (or derived from) the human genome and does not include sequences from other species. In some embodiments, a human antibody includes CDRs, framework regions, and (if present) Fc regions from the human genome. Human antibodies can be identified and isolated using techniques that generate antibodies based on sequences derived from the human genome, such as by phage display or using transgenic animals (see, e.g., Barbas et al. Phage display: A Laboratory Manuel. 1 st Ed. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20: 450-459, 2008).

[0173] Antibodies or antigen-binding fragments that neutralize HIV-1: Antibodies or antigen-binding fragments that specifically bind to HIV-1 Env (e.g., binding to gp41) in a manner that inhibits biological functions associated with HIV-1 Env (e.g., binding to its target receptor). In several embodiments, antibodies or antigen-binding fragments that neutralize HIV-1 reduce the infectious titer of HIV-1. In some embodiments, antibodies or antigen-binding fragments that specifically bind to HIV-1 Env can neutralize two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) HIV-1 strains.

[0174] Broadly neutralizing antibodies against HIV-1 differ from other antibodies against HIV-1 in that they neutralize a high percentage of many types of HIV-1 in circulation. Thus, broadly neutralizing antibodies against HIV-1 are antibodies that reduce HIV-1 infection titers by binding to and inhibiting the function of related HIV-1 antigens (e.g., antigens that share at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the antigenic surface of the antigen). In some embodiments, broadly neutralizing antibodies against HIV-1 differ from other antibodies against HIV-1 in that they neutralize a high percentage of many types of HIV-1 in circulation (e.g., at least 80%, at least 85%, at least 90% or at least 95%).

[0175] Antibody self-reactivity or autoreactivity: The property of an antibody whereby the antibody reacts with self-epitopes, i.e., epitopes of proteins and / or lipids produced by a subject. Antibodies that are not self-reactive do not substantially bind to epitopes or lipids present on the cell membranes of cells of the subject. Methods for determining whether an antibody reacts with self-epitopes are known to those of ordinary skill in the art. In one example, antibody self-reactivity is assessed using HEp-2 cell staining (cardiolipin binding assay) or an anti-nuclear antigen (ANA) assay. The anti-ANA assay includes, for example, an anti-ANA In several embodiments, the disclosed antibodies are not autoreactive (or self-reactive), or are minimally autoreactive. In a non-limiting example, the disclosed antibodies may not have substantially higher autoreactivity than the 10E8 antibody. For example, the disclosed antibodies or antigen-binding fragments may have no more than 10% higher autoreactivity than the 10E8 antibody, for example, by using HEp-2 cell staining, cardiolipin binding, anti-ANA In another non-limiting example, the disclosed antibodies do not have autoreactivity above background levels, as measured by using anti-ANA As measured by the ANA cell staining assay.

[0176] Biological sample: A sample obtained from a subject. Biological samples include all clinical samples that can be used to detect disease or infection (e.g., HIV infection) in a subject, including, but not limited to, cells, tissues, and body fluids, such as blood, blood derivatives and components (e.g., serum), cerebrospinal fluid; and biopsy or surgically removed tissues, such as unfixed, frozen, or fixed in formalin or paraffin. In a specific example, the biological sample is obtained from a subject suffering from or suspected of having HIV-1 infection.

[0177] Bispecific antibody: A recombinant molecule composed of two different antigen binding domains and thereby binding to two different antigenic epitopes. Bispecific antibodies include molecules that are chemically or genetically linked to the two domains. The antigen binding domains can be connected using a linker. The antigen binding domains can be a monoclonal antibody, an antigen binding fragment (e.g., Fab, svFv), or a combination thereof. Bispecific antibodies include one or more constant domains, but do not necessarily include a constant region.

[0178] Conditions sufficient to form an immune complex: Conditions that allow an antibody or antigen-binding fragment to bind to its cognate epitope to a detectably greater extent and / or to the exclusion of substantially all other epitopes. Conditions sufficient to form an immune complex depend on the format of the binding reaction and are generally those employed in immunoassay protocols or those encountered in vivo. For a description of immunoassay formats and conditions, see Harlow & Lane, Antibodies, A Laboratory Manual, 2 nd Ed.Cold Spring Harbor Publications, New York (2013). The conditions used in these methods are "physiological conditions", which include reference to conditions (e.g., temperature, osmotic pressure, pH) within a living mammal or mammalian cell. Although it is recognized that some organs are subjected to extreme conditions, the intracellular environment in vivo and in a cell is generally around pH 7 (e.g., pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), contains water as the main solvent, and exists at a temperature above 0°C and below 50°C. The osmotic pressure is within a range that supports cell survival and proliferation.

[0179] The formation of immune complexes can be detected by conventional methods known to the skilled person, such as immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (e.g., Western blot), magnetic resonance imaging, CT scanning, X-ray and affinity chromatography. The immunological binding properties of the selected antibodies can be quantified using methods known in the art.

[0180] Conjugate: A complex of two molecules that are linked together, for example, by a covalent bond. In one embodiment, an antibody is linked to an effector molecule; for example, an antibody that specifically binds to HIV-1 Env is covalently linked to an effector molecule. The connection can be formed by chemical or recombinant means. In one embodiment, the connection is chemical, wherein the reaction between the antibody moiety and the effector molecule produces a covalent bond formed between the two molecules to form one molecule. A peptide linker (short peptide sequence) may optionally be included between the antibody and the effector molecule. Because conjugates can be prepared from two molecules with independent functionality, such as antibodies and effector molecules, they are sometimes also referred to as "chimeric molecules".

[0181] Conservative variants: "Conservative" amino acid substitutions are those that do not substantially affect or reduce protein function (e.g., the ability of a protein to interact with a target protein). For example, an HIV-specific antibody comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 conservative substitutions compared to a reference antibody sequence and retains specific binding activity to HIV-1 antigens and / or HIV-1 neutralizing activity. The term conservative variant may also include replacing an unsubstituted parent amino acid with a substituted amino acid.

[0182] In addition, one of ordinary skill will recognize that individual substitutions, deletions or additions that change, add or delete a single amino acid or a small percentage of amino acids (e.g., less than 5%, in some embodiments less than 1%) in the encoded sequence are conservative changes, where the change results in the replacement of an amino acid with a chemically similar amino acid.

[0183] Providing conservative amino acid substitution tables for functionally similar amino acids is well known to those of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for each other:

[0184] 1) Alanine (A), serine (S), threonine (T);

[0185] 2) Aspartic acid (D), Glutamic acid (E);

[0186] 3) Asparagine (N), glutamine (Q);

[0187] 4) Arginine (R), Lysine (K);

[0188] 5) isoleucine (I), leucine (L), methionine (M), valine (V); and

[0189] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0190] Non-conservative substitutions are those that reduce the activity or function of HIV-specific antibodies (e.g., the ability to specifically bind gp41). For example, if the amino acid residue is critical to the function of the protein, even conservative substitutions may destroy the activity. Therefore, conservative substitutions do not change the basic function of the target protein.

[0191] Contacting: To place in direct physical communication; including in solid and liquid form, which can occur in vivo or in vitro. Contacting includes contact of one molecule with another molecule, for example, contact of amino acids on the surface of one polypeptide, such as an antigen, with another polypeptide, such as an antibody. Contacting can also include contacting a cell, for example, placing an antibody in direct physical communication with a cell.

[0192] Control: Reference standard. In some embodiments, the control is a negative control, such as a sample obtained from a healthy patient who has not been infected with HIV-1. In other embodiments, the control is a positive control, such as a tissue sample obtained from a patient diagnosed with HIV-1 infection. In other embodiments, the control is a historical control or a standard reference value or range value (e.g., a control sample tested previously, such as a group of HIV-1 patients with a known diagnosis or outcome, or a sample group representing a baseline or normal value).

[0193] The difference between the test sample and the control can be an increase, or conversely, a decrease. The difference can be a qualitative difference or a quantitative difference, such as a statistically significant difference. In some instances, the difference is an increase or decrease of at least about 5% relative to the control, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400% or at least about 500%.

[0194] Degenerate variants: In the context of this disclosure, "degenerate variants" refer to polynucleotides encoding proteins (e.g., antibodies that specifically bind to gp41 or their variable regions) that include sequences that are degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included as long as the amino acid sequence of the antibody that binds to gp41 encoded by the nucleotide sequence is unchanged.

[0195] Detectable marker: A detectable molecule (also referred to as a label) that is conjugated directly or indirectly to a second molecule, such as an antibody, to facilitate detection of the second molecule. For example, a detectable marker can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques (e.g., CT scans, MRI, ultrasound, fiber optics, and laparoscopy). Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). In one example, "labeled antibody" refers to the incorporation of another molecule into the antibody molecule. For example, a label is a detectable marker, such as incorporating a radiolabeled amino acid or attaching a biotinyl moiety that can be detected by a labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods) to a polypeptide. Various methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for labeling polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g.35 S or 131 I), fluorescent labels (e.g., fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a second reporter (e.g., leucine zipper pair sequences, binding sites for a second antibody, metal binding domains, epitope tags), or magnetic agents such as gadolinium chelates. In some embodiments, the labels are attached with spacer arms of varying lengths to reduce potential steric hindrance. Methods of using detectable labels and guidance on the selection of detectable labels suitable for various purposes are described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4 th ed, Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013).

[0196] Detection: Determine the existence, presence or fact of something. General methods of detection are known to the skilled artisan and can be supplemented by the procedures and reagents disclosed herein. For example, methods of detecting cells expressing gp41 in a subject are included herein.

[0197] Effector molecule: A molecule that is intended to have or produce a desired effect; for example, a desired effect on a cell to which the effector molecule is targeted. Effector molecules include, for example, polypeptides and small molecules. In one non-limiting example, the effector molecule is a toxin. The skilled artisan will appreciate that some effector molecules can have or produce more than one desired effect.

[0198] Epitope: Antigenic determinant. These are specific chemical groups or peptide sequences on a molecule that are antigenic, i.e., that elicit a specific immune response. Antibodies specifically bind to specific antigenic epitopes on polypeptides. In some examples, the disclosed antibodies specifically bind to epitopes on gp41.

[0199] Expression: Transcription or translation of a nucleic acid sequence. For example, when a DNA encoding a nucleic acid sequence (e.g., a gene) is transcribed into RNA or RNA fragments (which are processed into mRNA in some instances), it can be expressed. When an mRNA encoding a nucleic acid sequence (e.g., a gene) is translated into an amino acid sequence such as a protein or a protein fragment, it can also be expressed. In a specific example, when a heterologous gene is transcribed into RNA, it is expressed. In another example, when the RNA of a heterologous gene is translated into an amino acid sequence, it is expressed. Regulation of expression includes control of transcription, translation, RNA transport and processing, degradation of intermediate molecules such as mRNA, or by activation, inactivation, compartmentalization, or degradation of protein molecules after production.

[0200] Expression control sequence: a nucleic acid sequence that regulates the expression of a heterologous nucleic acid sequence that is operably connected thereto. When the expression control sequence controls and regulates the transcription and translation (optionally) of the nucleic acid sequence, the expression control sequence is operably connected to the nucleic acid sequence. Therefore, the expression control sequence includes a start codon (ATG) before a suitable promoter, enhancer, transcription terminator, protein coding gene, intron splicing signal, the correct reading frame of the gene is maintained to allow the correct translation of mRNA, and a stop codon. The term "control sequence" is intended to include at least the components whose presence can affect expression, and may also include additional components, whose presence is advantageous, such as leader sequences and fusion partner sequences. The expression control sequence includes a promoter.

[0201] A promoter is a minimal sequence sufficient to direct transcription. Also included are promoter elements that are sufficient to confer promoter-dependent gene expression that is controllable in terms of cell-specific, tissue-specific, or inducible by external signals or agents; these elements may be located in the 5' or 3' region of a gene. Constitutive and inducible promoters are included (see, e.g., Bitter et al., Methods in Enzymology 153:516-544, 1987). For example, when cloning in a bacterial system, inducible promoters such as pL, plac, ptrp, ptac (ptrp-lac hybrid promoter) of bacteriophage λ may be used. In one embodiment, when cloning in a mammalian cell system, promoters derived from mammalian cell genomes (e.g., metallothionein promoters) or derived from mammalian viruses (e.g., retroviral long terminal repeats; adenovirus late promoters; vaccinia virus 7.5K promoters) may be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide transcription of nucleic acid sequences.

[0202] The polynucleotide can be inserted into an expression vector containing a promoter sequence that promotes efficient transcription of the inserted gene sequence in the host. The expression vector usually contains a replication origin, a promoter, and a specific nucleic acid sequence that allows phenotypic screening of transformed cells.

[0203] Expression vector: A vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleic acid sequence to be expressed. The expression vector includes sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or located in an in vitro expression system. Expression vectors include all expression vectors known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0204] Fc region: A polypeptide comprising the constant region of an antibody except the first constant region immunoglobulin domain. The Fc region generally refers to the last two constant region immunoglobulin domains of IgA, IgD and IgG, and the last three constant region immunoglobulin domains of IgE and IgM. The Fc region may also include part or all of the flexible hinge at the N-terminus of these domains. For IgA and IgM, the Fc region may include or may not include a tailpiece, and may or may not be bound by a J chain. For IgG, the Fc region includes the immunoglobulin domains Cγ2 and Cγ3, and the lower part of the hinge between Cγ2 and Cγ3. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is generally defined as including residues C226 or P230 at its carboxyl terminus, where the numbering follows the EU index as in Kabat. For IgA, the Fc region includes the immunoglobulin domains Cα2 and Cα3, and the lower part of the hinge between Cα1 and Cα2. Any of the disclosed antibodies includes a heterologous Fc region or a heterologous constant domain. For example, the antibody includes an Fc region or a constant domain that is different from a native Fc region or a constant domain, such as an Fc region or a constant domain that includes one or more modifications (e.g., an "LS" mutation) to increase half-life.

[0205] HIV-1 Envelope Protein (Env): The HIV-1 Envelope protein is initially synthesized as a precursor protein of 845 to 870 amino acids in size, called gp160. Individual gp160 polypeptides form homotrimers and undergo glycosylation and processing in the Golgi to remove the signal peptide, as well as cleavage by cellular proteases between approximately positions 511 / 512 to produce separate gp120 and gp41 polypeptide chains, which remain associated as gp120 / gp41 protomers within the homotrimer. The extracellular domain (i.e., the extracellular portion) of the HIV-1 Env trimer undergoes several structural rearrangements, from a pre-fusion mature (cleaved) closed conformation that escapes antibody recognition, through an intermediate conformation that binds to the receptor CD4 and co-receptors (CCR5 or CXCR4), to a post-fusion conformation.

[0206] The numbering used in the disclosed HIV-1 Env protein and fragments thereof is related to the HXB2 numbering scheme described in Numbering Positions in HIV Relative to HXB2CG Bette Korber et al., Human Retroviruses and AIDS 1998: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Korber et al., Eds Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM, which is incorporated herein by reference in its entirety.

[0207] HIV-1 gp120: A polypeptide that is part of the HIV-1 Env protein. Mature gp120 includes approximately HIV-1 Env residues 31 to 511, comprising the majority of the external surface-exposed domains of the HIV-1 Env trimer, and it is gp120 that binds to the cellular CD4 receptor and cellular chemokine receptors (e.g., CCR5). The mature gp120 polypeptide is a heavily N-glycosylated extracellular polypeptide that interacts with the gp41 extracellular domain to form HIV-1 Env protomers, which trimerize to form HIV-1 Env trimers.

[0208] HIV-1 gp41: A polypeptide that is part of the HIV-1 Env protein. Mature gp41 includes approximately HIV-1 Env residues 512 to 860, and includes cytoplasmic, transmembrane, and extracellular domains. The gp41 extracellular domain (including approximately HIV-1 Env residues 512 to 644) can interact with gp120 to form HIV-1 Env protomers, which trimerize to form HIV-1 Env trimers.

[0209] Gp41 includes a membrane-proximal external region (MPER) that is adjacent to the N-terminus of the transmembrane region of gp41. The MPER is highly hydrophobic (50% of the residues are hydrophobic) and is highly conserved among many HIV-1 clades (Zwick, MB et al., J Virol, 75(22): p.10892-905, 2001). The MPER of HIV-1 gp41 includes the target epitope of the 10E8 monoclonal antibody.

[0210] Human immunodeficiency virus type 1 (HIV-1): retrovirus, causes human immunosuppression (HIV-1 disease), and leads to the disease complex (disease complex) called acquired immunodeficiency syndrome (AIDS). "HIV-1 disease" refers to a series of signs and symptoms (including the appearance of opportunistic infections) recognized in people infected by HIV-1 (as determined by antibodies or western blot studies). Laboratory results associated with this disease include progressive decline in T cells. Related viruses used as animal models include simian immunodeficiency virus (SIV) and feline immunodeficiency virus (FIV). Treating HIV-1 with HAART is effective in reducing viral load and has alleviated HIV-1 infection effects in infected individuals.

[0211] HXB2 numbering system: A reference numbering system for HIV-1 protein and nucleic acid sequences, using the HIV-1 HXB2 strain sequence as a reference for all other HIV-1 strain sequences. Those of ordinary skill in the art are familiar with the HXB2 numbering system, which is described in "Numbering Positions in HIV Relative to HXB2CG," Bette Korber et al., Human Retroviruses and AIDS 1998: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Korber B, Kuiken CL, Foley B, Hahn B, McCutchan F, Mellors JW, and Sodroski J, Eds. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM, which is incorporated herein by reference in its entirety. HXB2 is also known as: HXBc2, which refers to HXB clone 2; HXB2R, in the Los Alamos HIV database, where the R stands for revised, because it is slightly revised from the original HXB2 sequence; and HXB2CG, in the GENBANK TM In the present invention, the numbering scheme used in the gp120 and gp41 polypeptides disclosed herein is relative to the HXB2 numbering scheme. For reference, the HIV-1 Env amino acid sequence of HXB2 is shown below:

[0212] (SEQ ID NO: 26; Accession number K03455, the sequence present in the database on May 4, 2015 is incorporated herein by reference).

[0213] IgA: A polypeptide belonging to the class of antibodies substantially encoded by the recognized immunoglobulin alpha genes. In humans, this class or isotype includes IgA1 and IgA 2 IgA antibodies can exist as monomers, multimers (called pIgA) that are mainly dimers, and secretory IgA. The invariant chain of wild-type IgA contains an 18-amino acid extension at its C-terminus, called the tail piece (tp). Polymeric IgA is secreted by plasma cells and consists of a 15 kDa peptide called the J chain connecting two IgA monomers through a conserved cysteine ​​residue in the tail piece.

[0214] IgG: A polypeptide belonging to the antibody class or isotype that is essentially encoded by the recognized immunoglobulin gamma genes. In humans, this class includes IgG 1 IgG 2 IgG 3 and IgG 4 In mice, this class includes IgG 1 IgG 2a IgG 2b IgG 3 .

[0215] Immune complex: The combination of an antibody or antigen binding fragment (e.g., scFv) and a soluble antigen forms an immune complex. The formation of immune complexes can be detected by conventional methods known to the skilled artisan, such as immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (e.g., Western blot), magnetic resonance imaging, CT scanning, X-rays, and affinity chromatography. The immunological binding properties of the selected antibody can be quantified using methods known in the art.

[0216] Isolated: A biological component (e.g., a nucleic acid, peptide, protein, or protein complex, such as an antibody) that has been substantially separated, produced separately, or purified away from other biological components (i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins) in the cells of an organism in which the component naturally occurs. Thus, isolated nucleic acids, peptides, and proteins include proteins and proteins purified by standard purification methods. The term also encompasses nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids. An isolated nucleic acid, peptide, or protein, such as an antibody, may be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

[0217] Linker: A bifunctional molecule that can be used to connect two molecules into one continuous molecule, such as an effector molecule to an antibody. In some embodiments, the conjugates provided include a linker between the effector molecule or detectable marker and the antibody. In some cases, the linker is a peptide within an antigen binding fragment (e.g., an Fv fragment) that acts as an indirect bond to the V Hand V L Non-limiting examples of peptide linkers include (G 4 S) 1 Connector, (G 4 S) 2 Connector or (G 4 S) 3 Connector.

[0218] The terms "conjugating", "joining", "bonding" or "linking" may refer to making two molecules into one continuous molecule; for example, joining two polypeptides into one continuous polypeptide, or covalently attaching an effector molecule or a detectable marker radionuclide or other molecule to a polypeptide such as a scFv. In certain contexts, these terms include reference to linking a ligand such as an antibody moiety to an effector molecule. The connection may be formed by chemical or recombinant means. "Chemical means" refers to a reaction between an antibody moiety and an effector molecule such that a covalent bond is formed between the two molecules to form one molecule.

[0219] Nucleic acid molecule: A polymeric form of nucleotides, which may include the sense and antisense strands of RNA, cDNA, genomic DNA, and their synthetic forms and mixed polymers. Nucleotide refers to a ribonucleotide, a deoxynucleotide, or a modified form of either type of nucleotide. The term "nucleic acid molecule" is used herein synonymously with "nucleic acid" and "polynucleotide". Nucleic acid molecules are generally at least 10 bases long, unless otherwise specified. The term includes single-stranded and double-stranded forms of DNA. Polynucleotides may include one or both of naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide bonds. "cDNA" refers to DNA that is complementary or identical to mRNA, whether in single-stranded or double-stranded form. "Encoding" refers to the inherent properties of a specific nucleotide sequence in a polynucleotide such as a gene, cDNA or mRNA, which is used as a template for the synthesis of other polymers and macromolecules in biological processes, which have a defined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a defined amino acid sequence and the resulting biological properties.

[0220] Operably linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter such as the CMV promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, an operably linked DNA sequence is continuous and, if necessary, connects two protein coding sequences in the same reading frame.

[0221] Pharmaceutically acceptable carriers: The use of pharmaceutically acceptable carriers is conventional. Remington's Pharmaceutical Science, 22nd ed., Pharmaceutical Press, London, UK (2012) describes compositions and formulations suitable for drug delivery of the disclosed agents.

[0222] Generally, the property of the carrier depends on the specific mode of administration adopted. For example, parenteral preparations generally include injectable fluids, which include pharmaceutically and physiologically acceptable fluids as vehicles, such as water, saline, balanced salt solutions, aqueous glucose solutions, glycerol, etc. For solid compositions (e.g., powders, pills, tablets or capsule forms), conventional non-toxic solid carriers include, for example, pharmaceutical grade mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may also contain a small amount of non-toxic auxiliary substances, such as wetting agents or emulsifiers, added preservatives (e.g., natural preservatives (on-natural preservatives)), and pH buffers, etc., such as sodium acetate or sorbitan monolaurate. In a particular example, the pharmaceutically acceptable carrier is sterile and is suitable for, for example, parenteral administration to a subject by injection. In some embodiments, the activating agent and the pharmaceutically acceptable carrier are provided in a unit dosage form such as a pill, or are provided in an optional amount in a vial. Unit dosage form includes single or multiple doses (eg, in vials from which metered doses of the agent can be selectively dispensed).

[0223] Polypeptide: A polymer in which the monomers are amino acid residues linked together by amide bonds. When the amino acids are alpha amino acids, either the L optical isomer or the D optical isomer can be used, with the L isomer being preferred. The terms "polypeptide" or "protein" as used herein are intended to encompass any amino acid sequence, including modified sequences, such as glycoproteins. Polypeptides include naturally occurring proteins, as well as those produced recombinantly or synthetically. Polypeptides have an amino terminus (N terminus) and a carboxyl terminus. In some embodiments, the polypeptide is a disclosed antibody or fragment thereof.

[0224] Polypeptide modification: Polypeptides can be modified by various chemical techniques to produce derivatives that have substantially the same activity and conformation as the unmodified peptide and optionally have other desired properties. For example, the carboxylic acid group of the protein, either at the carboxyl terminus or on the side chain, can be provided in the form of a salt of a pharmaceutically acceptable cation or esterified to form a C 1 To C 16 Ester, or converted to formula NR 1 R 2 An amide, wherein R 1 and R2 Each is independently H or C 1 -C 16 The amino group of the peptide, whether the amino terminal or the side chain, can be in the form of a pharmaceutically acceptable acid addition salt, such as HCl, HBr, acetic acid, benzoic acid, toluenesulfonic acid, maleic acid, tartaric acid or other organic salt, or can be modified to C 1 -C 16 The alkyl or dialkylamino group can be further converted into an amide.

[0225] The hydroxyl groups on the side chains of the polypeptide can be converted to C 1 -C 16 Alkoxy or C 1 -C 16 The phenyl and phenol rings of the peptide side chains can be substituted with one or more halogen atoms such as F, Cl, Br or I, or with C 1 -C 16 Alkyl, C 1 -C 16 Alkoxy, carboxylic acid and its ester or amide of these carboxylic acids. The methylene group of the peptide side chain can be extended to the corresponding C 2 -C 4 Alkylene. Mercapto groups may be protected with any of several well-recognized protecting groups such as acetamide.

[0226] Recombination: A recombinant nucleic acid is a nucleic acid having a sequence that is not naturally occurring, or having a sequence formed by artificially combining two originally separate sequence segments. This artificial combination can be accomplished by chemical synthesis, or more often, by artificially manipulating separated nucleic acid segments, such as by genetic engineering techniques. A recombinant protein is a protein having a sequence that is not naturally occurring, or having a sequence formed by artificially combining two originally separate sequence segments. In several embodiments, a recombinant protein is encoded by a heterologous (e.g., recombinant) nucleic acid that has been introduced into a host cell such as a bacterium or eukaryotic cell. The nucleic acid can be introduced, for example, on an expression vector having a signal capable of expressing a protein encoded by the introduced nucleic acid, or the nucleic acid can be integrated into a host chromosome.

[0227] Sequence identity: The similarity between two amino acid sequences is expressed by the similarity between the sequences, also known as sequence identity. Sequence identity is often measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologues or variants of a polypeptide will have a relatively high degree of sequence identity when aligned using standard methods.

[0228] Methods of sequence alignment for comparison are well known in the art. Various programs and alignment algorithms are described in Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations.

[0229] NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and the Internet for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. A description of how to use this program to determine sequence identity is available at the NCBI website on the Internet.

[0230] V of the antibody that specifically binds to the peptide L or V HHomologues and variants are typically characterized by having at least about 75%, such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity when compared to the target amino acid sequence over its entire length. When evaluated by this method, proteins with even higher similarities to a reference sequence will show an increased percentage identity, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity. When comparing sequence identities less than the full-length sequence, homologues and variants typically have at least 80% sequence identity within a short window of 10 to 20 amino acids, and may have at least 85% or at least 90% or 95% sequence identity, depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the Internet. Those skilled in the art will recognize that these sequence identity ranges are provided for guidance only; it is entirely possible to obtain very significant homologs that fall outside the ranges provided.

[0231] Terms used to describe sequence relationships between two or more nucleotide sequences or amino acid sequences include "reference sequence," "selected from," "comparison window," "identical," "percentage of sequence identity," "substantially identical," "complementary," and "substantially complementary."

[0232] For sequence comparison of nucleic acid sequences, one sequence is usually used as a reference sequence and the test sequence is compared thereto. When a sequence alignment algorithm is used, the test and reference sequences are input into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Default program parameters are used. Sequence alignment methods for comparison are well known in the art. Optimal alignment of sequences for comparison can be accomplished, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970, by the search for similarity algorithm of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4 th ed, Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013). An example of a useful algorithm is PILEUP. PILEUP uses a simplified progressive alignment algorithm of Feng & Doolittle, J. Mol. Evol. 35:351-360, 1987. The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151-153, 1989. Using PILEUP, the reference sequence is compared to other test sequences to determine the percent sequence identity relationship, using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP is available from the GCG sequence analysis software package, such as version 7.0 (Devereaux et al., Nuc. Acids Res. 12:387-395, 1984).

[0233] Another example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., J. Mol. Biol. 215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402, 1977. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands as defaults. The BLASTP program (for amino acid sequences) uses a wordlength (W) of 3 and an expectation (E) of 10, and the BLOSUM62 scoring matrix as defaults (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915, 1989). Oligonucleotides are linear polynucleotide sequences of up to about 100 nucleotides in length.

[0234] Specific binding: When referring to an antibody or antigen binding fragment, it refers to a binding reaction that determines the presence of a target protein, peptide or polysaccharide in the presence of a variety of protein populations and other biomolecules. Thus, under specified conditions, the antibody preferentially binds to a specific target protein, peptide or polysaccharide (e.g., an antigen present on the surface of a pathogen, such as HIV-1 Env), and does not bind to other proteins or polysaccharides present in a sample or subject in significant amounts. Specific binding can be determined by methods known in the art. With respect to antibody-antigen complexes, the specific binding of the antigen and the antibody has a specific affinity of less than about 10 -7 Moore's K D , for example less than about 10 -8 Moore, 10 -9 or even less than about 10 -10 Moore.

[0235] K D Refers to the dissociation constant for a given interaction, such as a polypeptide-ligand interaction or an antigen-antigen interaction. For example, for a bimolecular interaction of an antibody or antigen-binding fragment with an antigen, it is the concentration of the individual components of the bimolecular interaction divided by the concentration of the complex.

[0236] The antibodies disclosed herein specifically bind to a defined target (or multiple targets, in the case of bispecific antibodies). Thus, an antibody that specifically binds to an epitope on gp41 is an antibody that substantially binds to gp41, including cells or tissues expressing gp41 (to which gp41 is attached as a substrate), or gp41 in a biological sample. Of course, it will be appreciated that non-specific interactions may occur between an antibody or a conjugate comprising an antibody (e.g., an antibody that specifically binds to gp41 or a conjugate comprising the antibody) and a non-target (e.g., a cell that does not express gp41). Typically, specific binding results in a stronger association between the antibody and a protein or cell loaded with the antigen than between the antibody and a protein or cell lacking the antigen. Specific binding typically results in an increase in the amount of antibody bound to a protein comprising the epitope or a cell or tissue expressing the target epitope by greater than 2-fold, such as greater than 5-fold, greater than 10-fold, or greater than 100-fold when compared to a protein or cell or tissue lacking the epitope. Specific binding to a protein under these conditions requires that the antibody be selected for its specificity for a particular protein. Various immunoassay formats are suitable for selecting antibodies or other ligands that are specifically immunoreactive with a particular protein. For example, solid phase ELISA immunoassays are routinely used to select monoclonal antibodies that are specifically immunoreactive with a protein. For a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see Harlow & Lane, Antibodies, A Laboratory Manual, 2 nd ed., Cold Spring Harbor Publications, New York (2013).

[0237] Subject: A living multicellular vertebrate organism, a class that includes humans and non-human mammals. In examples, the subject is a human. In specific examples, the subject is a newborn infant. In other examples, a subject is selected in need of inhibiting HIV-1 infection. For example, the subject is either uninfected and at risk for HIV-1 infection, or is infected and in need of treatment.

[0238] Therapeutically effective amount: An amount of an agent, such as a disclosed gp41-specific antibody or antigen-binding fragment, that is sufficient to prevent, treat (including prophylaxis), reduce and / or alleviate the symptoms and / or underlying causes of a condition or disease, such as preventing, inhibiting and / or treating HIV-1 infection. In some embodiments, a therapeutically effective amount is sufficient to reduce or eliminate the symptoms of a disease, such as AIDS. For example, this may be the amount necessary to inhibit or prevent HIV-1 replication or to measurably alter the outward symptoms of HIV-1 infection. Typically, such an amount will be sufficient to measurably inhibit HIV-1 replication or infectivity. Ideally, a therapeutically effective amount provides a therapeutic effect without causing significant cytotoxic effects in a subject.

[0239] In some embodiments, administration of a therapeutically effective amount of a disclosed antibody or antigen-binding fragment that binds gp41 can reduce or inhibit HIV-1 infection by a desired amount (e.g., as measured by infected cells, or by the number or percentage of HIV-1 infected subjects, or by prolonged survival of infected subjects), such as by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (eliminating or preventing detectable HIV-1 infection) compared to an appropriate control.

[0240] Several preparations disclosed herein are administered in a therapeutically effective amount. The therapeutically effective amount of an antibody or antigen-binding fragment that specifically binds to gp41 administered to a subject will vary with a number of factors associated with the subject, such as overall health and / or subject weight. The therapeutically effective amount can be determined by varying the dose and measuring the resulting therapeutic response, such as a reduction in viral titer. The therapeutically effective amount can also be determined by various in vitro, in vivo, or in situ immunoassays.

[0241] The therapeutically effective amount encompasses divided doses, which contribute to obtaining a therapeutic response in combination with previous or subsequent administration. For example, a therapeutically effective amount of an agent can be administered, for example, daily, in a single dose or several doses during a course of treatment that lasts for days or weeks. However, the therapeutically effective amount can depend on the subject being treated, the severity of the condition being treated, and the tumor, as well as the mode of administration. The unit dosage form of an agent can be packaged in, for example, a vial or a syringe with sterile components in a therapeutic amount or in multiples of a therapeutic amount.

[0242] Transformed: A transformed cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule can be introduced into the cell, including transfection with viral vectors, transformation with plasmid vectors, and by electroporation, lipofection, and particle gun acceleration.

[0243] Treat or prevent disease: Preventing disease refers to, for example, inhibiting the complete appearance of a disease or condition in a subject at risk of HIV-1 infection or with HIV-1 infection. Treating a disease refers to a therapeutic intervention that alleviates the signs or symptoms of a disease or pathological condition after the disease or pathological condition has begun to appear. When referring to a disease or pathological condition, the term "alleviate" refers to any observable beneficial effect of the treatment. The beneficial effect can be confirmed, for example, by the delayed onset of clinical symptoms of the disease in susceptible subjects, a reduction in the severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in viral load, an improvement in the overall health or well-being of the subject, or other parameters known in the art that are specific to a particular disease. "Preventive" treatment is a treatment that is applied to a subject who does not show signs of the disease for the purpose of reducing the risk of developing pathology.

[0244] The term "prevent" does not necessarily mean that an agent completely eliminates a disease or symptom, as long as at least one characteristic of the disease or condition is eliminated. Thus, an antibody that inhibits or prevents an infection may, but need not, completely eliminate such infection, as long as the infection is measurably reduced, such as by at least about 50%, such as at least about 70% or about 80%, or even about 90% of the infection in the absence of the agent, or compared to a reference agent.

[0245] Vector: The vector includes a nucleic acid sequence (e.g., mRNA or DNA) that allows it to replicate in a host cell, such as an origin of replication. The vector may also include one or more selectable markers and other genetic elements known in the art. A viral vector is a recombinant nucleic acid vector having at least some nucleic acid sequences derived from one or more viruses. In some embodiments, a viral vector is provided, comprising one or more nucleic acid molecules encoding antibodies or antigen-binding fragments that specifically bind to HIV-1gp41 and neutralize HIV-1. In some embodiments, the viral vector may be an adeno-associated virus (AAV) vector. A replication-deficient viral vector is a vector that requires one or more regions required for replication of the viral genome to be supplemented due to a defect in at least one replication-critical gene function. For example, so that the viral vector does not replicate in typical host cells, especially those cells in human patients that may be infected by viral vectors during therapeutic methods.

[0246] VRC01 class antibodies, heavy chain, or light chain: a class of antibodies that bind to the CD4 binding site on gp120 and can neutralize HIV-1 infection, and their heavy and light chains. VRC01, the prototype member of the VRC01 class of antibodies, can neutralize more than 90% of circulating HIV-1 isolates with an average 50% inhibitory concentration (IC 50) is ~0.3 μg / ml. Despite the overall sequence differences between VRC01 class antibodies, the antibody-gp120 co-crystal structure revealed that VRC01 class recognition of gp120 is consistent within the class. In fact, three-dimensional structural analysis of HIV-1 gp120 from different HIV-1 clades in complex with different VRC01 class antibodies from multiple donors showed that VRC01 class antibodies share striking similarities in physical structure and revealed several antibody features that contribute to gp120 binding and HIV-1 neutralization. The major structural and ontogenetic features of VRC01 class antibodies allow the identification of members of this class by antibody sequence interrogation.

[0247] For example, the V H With VH1-2 germline origin, VRC01 class V H The coding sequence differs by 20-35% (e.g., 25-30%) from the corresponding germline gene sequence. H The Kabat position 50 includes a tryptophan residue (V H Trp 50 ), including an asparagine residue at Kabat position 58 (V H Asn 58 ), and including an arginine residue at Kabat position 71 (V H Arg 71 ). These residues form specific interactions with amino acids on gp120, which contributes to the specificity and neutralizing properties of VRC01. When VRC01 antibodies bind to gp120, V H Trp 50 gp120 Asn 280 Form hydrogen bonds, V H Asp 58 gp120Arg 456 and Gly 458 Form hydrogen bonds, V H Arg 71 gp120 Asp 368 Forming a salt bridge, and V H Trp100B and gp120 Asn 279 Form hydrogen bonds.

[0248] In addition, the V L Has germline origin of IGKV1-33, IGKV3-11, IGKV3-15, IGKV3-20 or IGLV2-14, of which VRC01 class V LThe coding sequence differs by 15-35% (e.g., 25-30%) from the corresponding germline gene sequence. L Or include LCDR1 with 2 to 6 amino acid deletions (kabat positioning), or include LCDR1 with glycine residues at kabat positions 28 and 30. The deletion or the presence of glycine residues provides flexibility, which allows the LCDR1 to avoid structural clashes with the D ring of gp120 when the antibody binds to the CD4 binding site. In addition, the VRC01 class V L The LCDR3 comprises a 5 amino acid long LCDR3 (according to the kabat positioning) and includes a hydrophobic residue (e.g., leucine or tyrosine) at kabat position 91, deletions at kabat positions 92-95, and a glutamic acid or glutamine residue at kabat position 96. The hydrophobic residues at position 91 cluster against gp120 Loop D, and the glutamic acid or glutamine residue at kabat position 96 interacts with a conserved positively charged region at the base of the gp120 V5 domain.

[0249] Non-limiting examples of antibodies that fall into the VRCOl class include VRCOl, VRC03, VRC07, VRC07-523, VRC13, 3BCN117, 12A12, 12A21, VRC-PG04, NIH45-46, VRC23, VRC-CH30, VRC-CH31, and VRC-PG20. The description, characterization and generation of these antibodies, as well as VRC01 class antibodies, are available and familiar to those of ordinary skill in the art (see, e.g., Diskin et al., Science, 334(6060):1289-93, 2011; Kwong and Mascola, Immunity, 37, 412-425, 2012; Li et al., J. Virol., 85, 8954-8967, 2011; Rudicell et al., J. Virol., 88, 12669-12682, 2012; Scheid et al., Science, 333(6049):16 33-1637, 2011; West et al., PNAS, 109: E2083-2090, 2012; Wu et al., Science, 329(5993): 856-861, 2010; Wu et al., Science, 333(6049): 1593-1602, 2011; Zhou et al., Immunity, 39: 245-258, 2013; Georgiev et al., Science, 340: 751-756, 2013; Zhu et al., PNAS, 110, E4088-E4097, 2013; and WIPO Publication Nos. WO 2012 / 158948, WO2011038290, WO2012154312, WO2013142324 and WO2013016468, the entire contents of each of which are incorporated herein by reference).

[0250] VRC01, VRC07, VRC07-523 (VRC07H-G54H heavy chain and VRC01L-E1I2del-V3S light chain) and VRC07-544 (VRC07H-I37V / G54H / T93A heavy chain and VRC01-EIdel-V3S light chain) antibodies are described in U.S. Patent Publications 8337036 and 2014 / 0322163 and Rudicell et al., J. Virol., 88:12669-12682, 2014, each of which is incorporated by reference in its entirety. V H and V L The sequence is provided as follows:

[0251] VRC01 V H

[0252] QVQLVQSGGQMKKPGESMRISCRASGYEFIDCTLNWIRLAPGKRPEWMGWLKPRGGAVNYARPLQGRVTMTRDVYSDTAFLELRSLTVDDTAVYFCTRGKNCDYNWDFEHWGRGTPVIVSS(SEQ ID NO:27)

[0253] VRC01 V L

[0254] EIVLTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFSGSRWGPDYNLTISNLESGDFGVYYCQQYEFFGQGTKVQVDIK(SEQ ID NO:28)

[0255] VRC07 V H

[0256] QVRLSQSGGQMKKPGDSMRISCRASGYEFINCPINWIRLAPGKRPEWMGWMKPRGGAVSYARQLQGRVTMTRDMYSETAFLELRSLTSDDTAVYFCTRGKYCTARDYYNWDFEHWGQGTPVTVSS(SEQ ID NO:29)

[0257] VRC07 V L

[0258] EIVLTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFSGSRWGPDYNLTISNLESGDFGVYYCQQYEFFGQGTKVQVDIK(SEQ ID NO:28)

[0259] VRC07-523 V H

[0260] QVRLSQSGGQMKKPGDSMRISCRASGYEFINCPINWIRLAPGKRPEWMGWMKPRHGAVSYARQLQGRVTMTRDMYSETAFLELRSLTSDDTAVYFCTRGKYCTARDYYNWDFEHWGQGTPVTVSS(SEQ ID NO:30)

[0261] VRC07-523 V L

[0262] SLTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFSGSRWGPDYNLTISNLESGDFGVYYCQQYEFFGQGTKVQVDIK(SEQ ID NO:31)

[0263] VRC07-544 V H

[0264] QVRLSQSGGQMKKPGDSMRISCRASGYEFINCPINWVRLAPGKRPEWMGWMKPRHGAVSYARQLQGRVTMTRDMYSETAFLELRSLTSDDTAVYFCARGKYCTARDYYNWDFEHWGQGTPVTVSS(SEQ ID NO:32)

[0265] VRC07-544 V L

[0266] SLTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFSGSRWGPDYNLTISNLESGDFGVYYCQQYEFFGQGTKVQVDIK(SEQ ID NO:33)

[0267] II. Description of Several Embodiments

[0268] Provided are isolated monoclonal antibodies and antigen-binding fragments that specifically bind to epitopes on gp41. The antibodies and antigen-binding fragments may be fully human. In several embodiments, the antibodies and antigen-binding fragments may be used to neutralize HIV-1 infection. Also disclosed herein are compositions comprising the antibodies and antigen-binding fragments and pharmaceutically acceptable carriers. Also provided are nucleic acids encoding the antibodies or antigen-binding fragments, and expression vectors (e.g., adeno-associated virus (AAV) viral vectors) comprising these nucleic acids.

[0269] These antibodies, Fabs, nucleic acid molecules, expression vectors, and compositions can be used for research, diagnostic and therapeutic purposes. For example, monoclonal antibodies and Fabs can be used to diagnose or treat subjects with HIV-1 infection, or can be administered prophylactically to prevent HIV-1 infection in subjects. In some embodiments, these antibodies can be used to determine HIV-1 titers in subjects.

[0270] A. Neutralizing Monoclonal Antibodies and Antigen-Binding Fragments

[0271] The parental 10E8 antibody neutralized 98% of a panel of 200 HIV-1 pseudotyped viruses, IC 50 Less than 50 μg / ml (see Example 1). However, as disclosed in Example 1, the solubility of the 10E8 antibody may not be optimal for commercial production purposes. Earlier attempts to modify the 10E8 antibody successfully improved solubility and the efficacy of the antibody, but resulted in increased autoreactivity. In some embodiments, disclosed herein are modified 10E8 antibodies and antigen-binding fragments having CDRs of the 10E8 antibody and framework region amino acid substitutions compared to the parent 10E8 antibody sequence, which impart a combination of improved neutralization, solubility and autoreactivity properties relative to 10E8. In additional embodiments, the modified 10E8 antibody includes amino acid substitutions in the CDRs that impart a combination of improved neutralization, solubility and autoreactivity properties relative to 10E8.

[0272] The following discussion of monoclonal antibodies refers to the IMGT numbering scheme including the V sequences including the CDRs. H and V L The monoclonal antibody of 10E8 (unless the context indicates otherwise). One of ordinary skill in the art will appreciate that various CDR numbering schemes (e.g., Kabat, Chothia, or IMGT numbering schemes) can be used to determine CDR positions. The amino acid sequences and CDR positions of the heavy and light chains of 10E8 and several variants thereof are shown in Table 1 according to the IMGT numbering scheme.

[0273] Table 1. IMGT CDR sequences of 10E8 and 10E8 variants.

[0274]

[0275]

[0276]

[0277] 10E8 variant 1 (10E8v1)

[0278] As disclosed in the Examples, the "10E8 variant 1" comprises HC6-S74Y-511 V H (SEQ ID NO:3) and rL3V L (SEQ ID NO: 4), specifically binds gp41 and has an unexpectedly excellent combination of HIV-1 neutralization breadth and potency, solubility, and autoreactivity. H Includes 10E8 heavy chain CDRs and relative to 10E8 V H(SEQ ID NO: 1) with Q3R, V5A, L72D, S74Y, I75K, F77T, M84T and T110I framework region amino acid substitutions. L Includes 10E8 light chain CDRs and relative to 10E8 V L (SEQ ID NO: 2) with amino acid substitutions in the E7D, I45V, V58I, S76T, D83E and E85D framework regions.

[0279] Thus, in some embodiments, the disclosed antibodies or antigen-binding fragments include a 10E8 V H CDRs (e.g., SEQ ID NOs: 7, 8, and 9) and may further comprise arginine, alanine, aspartic acid, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively. H , and contains 10E8 V L CDRs (e.g., SEQ ID NOs: 10, 11, 12) and V residues of aspartic acid, valine, isoleucine, threonine, glutamic acid, and aspartic acid at kabat positions 7, 45, 58, 76, 83, and 85, respectively. L .

[0280] In other embodiments, the antibody or antigen-binding fragment comprises V H , which includes 10E8 V H CDRs (e.g., SEQ ID NOs: 7, 8, and 9), arginine, alanine, aspartic acid, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO: 3 (HC6-S74Y-511); and V L , which includes 10E8 V L CDRs (e.g., SEQ ID NOs: 10, 11, and 12), aspartic acid, valine, isoleucine, threonine, glutamic acid, and aspartic acid residues at kabat positions 7, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO: 4 (rL3).

[0281] In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 3 (HC6-S74Y-511). Hand a V comprising the amino acid sequence shown in SEQ ID NO:4 (rL3) L .

[0282] 10E8 variant 3 (10E8v3)

[0283] As disclosed in the Examples, the "10E8 variant 3" includes H6-511-4mut V H (SEQ ID NO: 5) and rL3V L (SEQ ID NO: 4), specifically binds gp41 and has an unexpectedly excellent combination of HIV-1 neutralization breadth and potency, solubility, and autoreactivity. H Includes 10E8 heavy chain CDRs and relative to 10E8 V H (SEQ ID NO: 1) Q3R, A61E, P62S, E64K, L72D, S74T, I75K, F77T, L82cV, M84T, S87T, L89Y, R105Q, T110I framework amino acid substitutions. rL3 V L As described above.

[0284] Thus, in some embodiments, the disclosed antibodies or antigen-binding fragments include V H , which includes 10E8 V H CDRs (e.g., SEQ ID NOs: 7, 8, and 9) and arginine, glutamic acid, serine, lysine, aspartic acid, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively; and V L , which includes 10E8 V L CDRs (e.g., SEQ ID NOs: 10, 11, 12) and aspartic acid, valine, isoleucine, threonine, glutamic acid and aspartic acid residues at kabat positions 7, 45, 58, 76, 83 and 85, respectively.

[0285] In other embodiments, the antibody or antigen-binding fragment comprises V H , which includes 10E8 V HCDRs (e.g., SEQ ID NOs:7, 8, and 9), arginine, glutamic acid, serine, lysine, aspartic acid, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO:5 (H6-511-4mut); and V L , which includes 10E8 V L CDRs (e.g., SEQ ID NOs: 10, 11, and 12), aspartic acid, valine, isoleucine, threonine, glutamic acid, and aspartic acid residues at kabat positions 7, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO: 4 (rL3).

[0286] In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 5 (H6-511-4mut). H and a V comprising the amino acid sequence shown in SEQ ID NO:4 (rL3) L .

[0287] 10E8 variant 4 (10E8v4)

[0288] As disclosed in the Examples, the "10E8 variant 4" includes H6-511-4mut V H (SEQ ID NO: 5) and rL3-6mut V L (SEQ ID NO: 6), specifically binds gp41 and has an unexpectedly superior combination of HIV-1 neutralization breadth and potency, solubility, and autoreactivity. H6-511-4mut V H As described above. L Includes 10E8 light chain CDRs and relative to 10E8 V L (SEQ ID NO: 2), S1A, Y2S, E7D, T8P, G9A, G16K, R17Q, I45V, V58I, S76T, D83E, E85D framework amino acid substitutions.

[0289] Thus, in some embodiments, the disclosed antibodies or antigen-binding fragments include V H , which includes 10E8 V HCDRs (e.g., SEQ ID NOs: 7, 8, and 9) and arginine, glutamic acid, serine, lysine, aspartic acid, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively; and V L , which includes 10E8 V L CDRs (e.g., SEQ ID NOs: 10, 11, 12) and alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83 and 85, respectively.

[0290] In other embodiments, the antibody or antigen-binding fragment comprises V H , which includes 10E8 V H CDRs (e.g., SEQ ID NOs:7, 8, and 9), arginine, glutamic acid, serine, lysine, aspartic acid, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO:5 (H6-511-4mut); and V L , which includes 10E8 V L CDRs (e.g., SEQ ID NOs: 10, 11, and 12), alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid, and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO: 6 (rL3-6mut).

[0291] In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 5 (H6-511-4mut). H and a V comprising the amino acid sequence shown in SEQ ID NO:6 (rL3-6mut) L .

[0292] 10E8 variant 5 (10E8v5)

[0293] As disclosed in the Examples, the "10E8 variant 5" includes HC6-S74Y-511 V H (SEQ ID NO: 3) and rL3-6mut V L (SEQ ID NO:6), specifically binds gp41 and has an unexpectedly superior combination of HIV-1 neutralization breadth and potency, solubility, and autoreactivity. HC6-S74Y-511 V H and rL3-6mut V L As described above.

[0294] Thus, in some embodiments, the disclosed antibodies or antigen-binding fragments include a 10E8 V H CDRs (e.g., SEQ ID NOs: 7, 8, and 9) and may further comprise arginine, alanine, aspartic acid, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively. H , and contains 10E8 V L CDRs (e.g., SEQ ID NOs: 10, 11, 12) and V residues of alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid and aspartic acid at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83 and 85, respectively. L .

[0295] In other embodiments, the antibody or antigen-binding fragment comprises V H , which includes 10E8 V H CDRs (e.g., SEQ ID NOs: 7, 8, and 9), arginine, alanine, aspartic acid, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO: 3 (HC6-S74Y-511); and V L , which includes 10E8 V LCDRs (e.g., SEQ ID NOs: 10, 11, and 12), alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid, and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO: 6 (rL3-6mut).

[0296] In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 3 (HC6-S74Y-511). H and a V comprising the amino acid sequence shown in SEQ ID NO:6 (rL3-6mut) L .

[0297] S100cW, S100cF, and V5R

[0298] As disclosed in the Examples, incorporation of hydrophobic amino acid substitutions (e.g., S100cW or S100cF substitutions) into certain 10E8 antibodies (e.g., 10E8 variant 4 antibodies) at VH kabat position S100c produces antibodies that specifically bind gp41 and have an unexpectedly superior combination of HIV-1 neutralization breadth and potency, solubility, and autoreactivity. Accordingly, in some embodiments, any antibody described herein (e.g., 10E8, or 10E8 variants, including variants 1, 3, 4, or 5) may also include an S100cW substitution. In some embodiments, any antibody described herein (e.g., 10E8, or 10E8 variants, including variants 1, 3, 4, or 5) may also include an S100cF substitution.

[0299] 10E8 variant 4 S100cF (10E8v4 S100cF)

[0300] In some embodiments, the VH:H6-511-4mut, SEQ ID NO:5; VL:rL3-6mut, SEQ ID NO:6) of the combination 10E8v4 antibody that binds gp41 and has unexpectedly superior HIV-1 neutralization breadth and potency, solubility, and autoreactivity is H and V L , in V H S100cF is replaced in the H and V L The antibody is called 10E8v4S100cF, and the corresponding V H and V LThe amino acid sequences are provided herein as SEQ ID NOs: 75 and 6, respectively. The 10E8v4S100cF antibody specifically binds gp41 and neutralizes HIV-1, and also has an unexpectedly superior combination of HIV-1 neutralization breadth and potency, solubility, and autoreactivity.

[0301] In some embodiments, the antibody or antigen-binding fragment comprises 10E8 V H CDR with S100cF replaced V H (e.g., SEQ ID NOs: 7, 8, and 77), comprising 10E8 V L CDR (e.g., SEQ ID NO: 10, 11, 12) V L , where the antibody specifically binds to gp41 and neutralizes HIV-1.

[0302] In some embodiments, the antibody or antigen-binding fragment comprises V H , which includes 10E8V with S100cF replacement H CDRs (e.g., SEQ ID NOs: 7, 8, and 77) and arginine, glutamic acid, serine, lysine, aspartic acid, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively; and V L , which includes 10E8 V L CDRs (e.g., SEQ ID NOs: 10, 11, 12) and alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83 and 85, respectively, wherein the monoclonal antibody specifically binds to gp41 and neutralizes HIV-1.

[0303] In other embodiments, the antibody or antigen-binding fragment comprises V H , which includes 10E8 V with S100cF substitution HCDRs (e.g., SEQ ID NOs:7, 8, and 77), arginine, glutamic acid, serine, lysine, aspartic acid, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO:75 (H6-511-4mut-S100cF); and V L , which includes 10E8 V L CDRs (e.g., SEQ ID NOs: 10, 11, and 12), alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid, and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO: 6 (rL3-6mut), wherein the monoclonal antibody specifically binds to gp41 and neutralizes HIV-1.

[0304] In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 75 (H6-511-4mut-S100cF). H and a V comprising the amino acid sequence shown in SEQ ID NO:6 (rL3-6mut) L .

[0305] 10E8 variant 4V5R S100cF (10E8v4 V5R S100cF)

[0306] In some embodiments, the antibody or antigen-binding fragment comprises V of the 10E8v4 antibody. H and V L (VH: H6-511-4mut, SEQ ID NO: 5; VL: rL3-6mut, SEQ ID NO: 6), in V H V5R and S100cF are available. H and V L The antibody is called 10E8v4 V5R S100cF, and the corresponding V H and V LThe amino acid sequences are provided herein as SEQ ID NOs: 76 and 6, respectively. The 10E8v4 V5RS100cF antibody specifically binds gp41 and neutralizes HIV-1, and also has an unexpectedly excellent combination of HIV-1 neutralization breadth and potency, solubility, and autoreactivity.

[0307] In some embodiments, the antibody or antigen-binding fragment comprises V H , which includes 10E8 V with S100cF substitution H CDRs (e.g., SEQ ID NOs: 7, 8, and 77) and arginine, arginine, glutamic acid, serine, lysine, aspartic acid, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 5, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively; and V L , which includes 10E8 V L CDRs (e.g., SEQ ID NOs: 10, 11, 12) and alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83 and 85, respectively, wherein the monoclonal antibody specifically binds to gp41 and neutralizes HIV-1.

[0308] In other embodiments, the antibody or antigen-binding fragment comprises V H , which includes 10E8 V with S100cF substitution H CDRs (e.g., SEQ ID NOs:7, 8, and 77), arginine, arginine, glutamic acid, serine, lysine, aspartic acid, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 5, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO:76 (H6-511-4mut-V5R S100cF); and V L , which includes 10E8 V LCDRs (e.g., SEQ ID NOs: 10, 11, and 12), alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid, and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO: 6 (rL3-6mut), wherein the monoclonal antibody specifically binds to gp41 and neutralizes HIV-1.

[0309] In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 76 (H6-511-4mut-V5R S100cW). H and a V comprising the amino acid sequence shown in SEQ ID NO:6 (rL3-6mut) L .

[0310] Additional modifications of 10E8 variant 4V5R S100cF

[0311] In some embodiments, the antibody or antigen-binding fragment comprises V of the 10E8v4 V5R S100cF antibody. H and V L , also includes one or more amino acid substitutions that improve the HIV-1 neutralization breadth and potency, solubility and / or autoreactivity of the modified antibody relative to 10E8v4 V5R S100cF. The modified 10E8v4V5R S100cF antibody specifically binds gp41 and neutralizes HIV-1, and also has an unexpectedly excellent combination of HIV-1 neutralization breadth and potency, solubility, and autoreactivity.

[0312] In some embodiments, the antibody or antigen-binding fragment comprises V H , which includes the amino acid sequence shown in SEQ ID NO:76 (H6-511-4mut-V5R S100cW), and further comprises one of E1R, E1W, G15R, S25W, D28W, D30W, D30R, N31W, N31R, P52bR, D72R or E81R substitutions relative to SEQ ID NO:76; and V L , which includes the amino acid sequence shown in SEQ ID NO:6 (rL3-6mut), wherein the antibody specifically binds to gp41 and neutralizes HIV-1.

[0313] In some embodiments, the antibody or antigen-binding fragment comprises V H, which includes the amino acid sequence shown in SEQ ID NO:76 (H6-511-4mut-V5R S100cW); and V L , which includes the amino acid sequence shown in SEQ ID NO:6 (rL3-6mut), and further includes one of the substitutions (kabat numbering) of D7W, A14R, Q17R, T18R, T20R, D26R, K51R, N53R, S65R, G68R, G77R or G100R relative to SEQ ID NO:6, wherein the antibody specifically binds gp41 and neutralizes HIV-1. In a preferred embodiment, the antibody or antigen-binding fragment includes V H , which includes the amino acid sequence shown in SEQ ID NO:76 (H6-511-4mut-V5R S100cW); and V L , which includes the amino acid sequence shown in SEQ ID NO:6 (rL3-6mut), and also includes a G68R substitution relative to SEQ ID NO:6 (kabat numbering), wherein the antibody specifically binds to gp41 and neutralizes HIV-1.

[0314] In some embodiments, the antibody or antigen-binding fragment comprises V H , which includes the amino acid sequence shown in SEQ ID NO:76 (H6-511-4mut-V5R S100cW); and V L , which includes the amino acid sequence shown in SEQ ID NO:6 (rL3-6mut), wherein the V H and V L Also included are one of the paired substitutions relative to SEQ ID NOs: 76 and 6, respectively: E1R and D7W; E1R and D26R; E1R and G68R; D28W and D7W; D28W and D26R; D28W and G68R; D28W and Q17R; D28W and T18R; D28W and T20R; D28W and K51R; D28W and G77R; P52bR and D7W ; P52bR and D26R; P52bR and G68R; P52bR and Q17R; P52bR and T18R; P52bR and T20R; P52bR and K51R; P52bR and G77R; D72R and D7W; D72R and D26R; or D72R and G68R, wherein the antibody specifically binds to gp41 and neutralizes HIV-1.

[0315] In some embodiments, the antibody or antigen-binding fragment comprises V H , which includes the amino acid sequence shown in SEQ ID NO:76 (H6-511-4mut-V5R S100cW); and V L, which includes the amino acid sequence shown in SEQ ID NO:6 (rL3-6mut), wherein the V H Also included are D72R, E1R D28W and P52bR substitutions, and wherein the antibody specifically binds gp41 and neutralizes HIV-1. In some embodiments, the antibody or antigen-binding fragment includes V H , which includes the amino acid sequence shown in SEQ ID NO:76 (H6-511-4mut-V5R S100cW); and V L , which includes the amino acid sequence shown in SEQ ID NO:6 (rL3-6mut), wherein the V L Also included are D7W, D26R and G68R substitutions, and wherein the antibody specifically binds gp41 and neutralizes HIV-1. In some embodiments, the antibody or antigen-binding fragment comprises V H , which includes the amino acid sequence shown in SEQ ID NO:76 (H6-511-4mut-V5R S100cW); and V L , which includes the amino acid sequence shown in SEQ ID NO:6 (rL3-6mut), wherein the V H Also includes D72R, E1R D28W and P52bR replacements, the V L Also included are D7W, D26R and G68R substitutions, and wherein the antibody specifically binds gp41 and neutralizes HIV-1.

[0316] In some embodiments, the antibody or antigen-binding fragment comprises V H , which includes the amino acid sequence shown in SEQ ID NO:76 (H6-511-4mut-V5R S100cW); and V L , which includes the amino acid sequence shown in SEQ ID NO:6 (rL3-6mut), wherein the V H and / or V L Also included are amino acid substitutions of any combination of substitutions listed in Table 10, and wherein the antibody specifically binds gp41 and neutralizes HIV-1. In some embodiments, the antibody or antigen-binding fragment comprises V H , which includes the amino acid sequence shown in SEQ ID NO:76 (H6-511-4mut-V5R S100cW); and V L , which includes the amino acid sequence shown in SEQ ID NO:6 (rL3-6mut), wherein the V H and / or V LAlso included are the compounds listed in Table 10 for 10E8-1053, 10E8-1054, 10E8-1055, 10E8-1056, 10E8-1057, 10E8-1058, 10E8-1059, 10E8-1060, 10E8-1061, 10E8-1062, 10E8-1063, 10E8-1064, 10E8-1065, 10E8-1066, 10E8-1067, 10E8-1068, 10E8-1069, 10E8-1070, 10E8-1071, 10E8-1072, 10E8-1073, 10E8-1074, 82, 10E8-1083, 10E8-1084, 10E8-1085, 10E8-1086, 10E8-1087, 10E8-1088, 10E8-1089, 10E8-1090, 10E8-1091, 10E8-1092, 10E8-1093, 10E8-1094 or 10E8-1095 antibodies, and wherein the antibody specifically binds gp41 and neutralizes HIV-1.

[0317] In some embodiments, the antibody or antigen-binding fragment comprises V H , which includes the amino acid sequence shown in SEQ ID NO:76 (H6-511-4mut-V5R S100cW); and V L , which includes the amino acid sequence shown in SEQ ID NO:6 (rL3-6mut) (corresponding to the 10E8-1073 antibody listed in Table 10), wherein the V H Also included is a D28Y substitution, and wherein the antibody specifically binds gp41 and neutralizes HIV-1. In some embodiments, the antibody or antigen-binding fragment includes V H , which includes the amino acid sequence shown in SEQ ID NO: 76 (H6-511-4mut-V5R S100cW); and V L , which includes the amino acid sequence shown in SEQ ID NO:6 (rL3-6mut), wherein the V H Also includes P52bR replacement, the V L Also included are D7W and G68R substitutions (corresponding to the 10E8-1081 antibody listed in Table 10), and wherein the antibody specifically binds gp41 and neutralizes HIV-1.

[0318] 10E8 Variant 4 S100cW

[0319] In some embodiments, the disclosed antibodies or antigen-binding fragments include V H , which includes the 10E8 V with S100cW replacement H CDRs (e.g., SEQ ID NOs: 7, 8, and 52) and arginine, glutamic acid, serine, lysine, aspartic acid, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively; and V L , which includes 10E8 V L CDRs (e.g., SEQ ID NOs: 10, 11, 12) and alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83 and 85, respectively.

[0320] In other embodiments, the antibody or antigen-binding fragment comprises V H , which includes the 10E8 V with S100cW replacement H CDRs (e.g., SEQ ID NOs: 7, 8, and 52), arginine, glutamic acid, serine, lysine, aspartic acid, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO: 35 (H6-511-4mut-S100cW); and V L , which includes 10E8 V L CDRs (e.g., SEQ ID NOs: 10, 11, and 12), alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid, and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO: 6 (rL3-6mut).

[0321] In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 35 (H6-511-4mut-S100cF).H and a V comprising the amino acid sequence shown in SEQ ID NO:6 (rL3-6mut) L .

[0322] 10E8 Variant 5 S100cW

[0323] In some embodiments, the disclosed antibodies or antigen-binding fragments include a 10E8 V having an S100cW substitution. H CDRs (e.g., SEQ ID NOs: 7, 8, and 52) and may further comprise arginine, alanine, aspartic acid, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively. H , and contains 10E8 V L CDRs (e.g., SEQ ID NOs: 10, 11, 12) and V residues of alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid and aspartic acid at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83 and 85, respectively. L .

[0324] In other embodiments, the antibody or antigen-binding fragment comprises V H , which includes the 10E8 V with S100cW replacement H CDRs (e.g., SEQ ID NOs: 7, 8, and 52), arginine, alanine, aspartic acid, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO: 38 (HC6-S74Y-511-S100cW); and V L , which includes 10E8 V L CDRs (e.g., SEQ ID NOs: 10, 11, and 12), alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid, and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO: 6 (rL3-6mut).

[0325] In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 38 (HC6-S74Y-511-S100cW). H and a V comprising the amino acid sequence shown in SEQ ID NO:6 (rL3-6mut) L .

[0326] DS

[0327] As disclosed in the Examples, incorporation of a "DS" modification, i.e., a non-native disulfide bond, between LCDR1 kabat position 30 and HCDR3 kabat position 100e into certain 10E8 antibodies (e.g., 10E8 variant 4 antibody) resulted in antibodies that specifically bind gp41 and have an unexpectedly superior combination of HIV-1 neutralization breadth and potency, solubility, and autoreactivity. Accordingly, any antibody described herein (e.g., 10E8, or 10E8 variants, including variants 1, 3, 4, or 5) may also include a DS replacement.

[0328] 10E8 Variant 4 DS

[0329] In some embodiments, the disclosed antibodies or antigen-binding fragments include V H , which includes 10E8 V with DS replacement H CDRs (e.g., SEQ ID NOs: 7, 8, and 53) and arginine, glutamic acid, serine, lysine, aspartic acid, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively; and V L , which includes 10E8 V with DS replacement L CDRs (e.g., SEQ ID NOs: 55, 11, 12) and alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83 and 85, respectively.

[0330] In other embodiments, the antibody or antigen-binding fragment comprises V H , which includes 10E8V with DS replacement HCDRs (e.g., SEQ ID NOs: 7, 8, and 53), arginine, glutamic acid, serine, lysine, aspartic acid, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO: 36 (H6-511-4mut-DS); and V L , which includes 10E8 V with DS replacement L CDRs (e.g., SEQ ID NOs:55, 11, and 12), alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid, and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO:41 (rL3-6mut-DS).

[0331] In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 36 (H6-511-4mut-DS). H and a V comprising the amino acid sequence shown in SEQ ID NO:41 (rL3-6mut-DS) L .

[0332] 10E8 Variant 5 DS

[0333] In some embodiments, the disclosed antibodies or antigen-binding fragments include 10E8V with a DS substitution. H CDRs (e.g., SEQ ID NOs: 7, 8, and 53) and may also contain arginine, alanine, aspartic acid, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively. H , and contains 10E8 V with DS replacement L CDRs (e.g., SEQ ID NOs: 55, 11, 12) and V residues of alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid and aspartic acid at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83 and 85, respectively. L .

[0334] In other embodiments, the antibody or antigen-binding fragment comprises V H , which includes 10E8V with DS replacement H CDRs (e.g., SEQ ID NOs: 7, 8, and 53), arginine, alanine, aspartic acid, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO: 39 (HC6-S74Y-511-DS); and V L , which includes 10E8 V with DS replacement L CDRs (e.g., SEQ ID NOs:55, 11, and 12), alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid, and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO:41 (rL3-6mut-DS).

[0335] In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 39 (HC6-S74Y-511-DS). H and a V comprising the amino acid sequence shown in SEQ ID NO:41 (rL3-6mut-DS) L .

[0336] S100cW-DS

[0337] As disclosed in the Examples, incorporation of a "DS" modification, i.e., a non-native disulfide bond and an S100cW substitution, between LCDR1 kabat position 30 and HCDR3 kabat position 100e into certain 10E8 antibodies (e.g., 10E8 variant 4 antibody) produces antibodies that specifically bind gp41 and have an unexpectedly superior combination of HIV-1 neutralization breadth and potency, solubility, and autoreactivity. Accordingly, any antibody described herein (e.g., 10E8, or 10E8 variants, including variants 1, 3, 4, or 5) may also include an S100cW and DS substitution.

[0338] 10E8 Variant 4 S100cW-DS

[0339] In some embodiments, the disclosed antibodies or antigen-binding fragments include V H , which includes 10E8 V with s100cW and DS replacementH CDRs (e.g., SEQ ID NOs: 7, 8, and 54) and arginine, glutamic acid, serine, lysine, aspartic acid, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively; and V L , which includes 10E8 V with DS replacement L CDRs (e.g., SEQ ID NOs: 55, 11, 12) and alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83 and 85, respectively.

[0340] In other embodiments, the antibody or antigen-binding fragment comprises V H , which includes 10E8 V with S-100cW and DS replacement H CDRs (e.g., SEQ ID NOs: 7, 8, and 54), arginine, glutamic acid, serine, lysine, aspartic acid, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO: 37 (H6-511-4mut-S100cW-DS); and V L , which includes 10E8 V with DS replacement L CDRs (e.g., SEQ ID NOs:55, 11, and 12), alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid, and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO:41 (rL3-6mut-DS).

[0341] In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 37 (H6-511-4mut-S100cW-DS). Hand a V comprising the amino acid sequence shown in SEQ ID NO:41 (rL3-6mut-DS) L .

[0342] 10E8 Variant 5 S100cW-DS

[0343] In some embodiments, the disclosed antibodies or antigen-binding fragments include 10E8 V with S100cW and DS substitutions. H CDRs (e.g., SEQ ID NOs: 7, 8, and 54) and may further comprise arginine, alanine, aspartic acid, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively. H , and contains 10E8 V with DS replacement L CDRs (e.g., SEQ ID NOs: 55, 11, 12) and V residues of alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid and aspartic acid at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83 and 85, respectively. L .

[0344] In other embodiments, the antibody or antigen-binding fragment comprises V H , which includes 10E8 V with S100cW and DS replacement H CDRs (e.g., SEQ ID NOs: 7, 8, and 54), arginine, alanine, aspartic acid, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO: 40 (HC6-S74Y-511-S100cW-DS); and V L , which includes 10E8 V with DS replacement L CDRs (e.g., SEQ ID NOs:55, 11, and 12), alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid, and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO:41 (rL3-6mut-DS).

[0345] In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence shown in SEQ ID NO: 40 (HC6-S74Y-511-S100cW-DS); H and a V comprising the amino acid sequence shown in SEQ ID NO:41 (rL3-6mut-DS) L .

[0346] V L Substitution at position 32 of Kabat

[0347] As disclosed in the Examples, in certain 10E8 antibodies (e.g., 10E8 variant 4 antibodies) V L Incorporation of amino acid substitutions at kabat position 32 of the invention generates antibodies that specifically bind gp41 and have an unexpectedly superior combination of HIV-1 neutralization breadth and potency, solubility, and autoreactivity. Accordingly, any of the antibodies described herein (e.g., 10E8, or 10E8 variants, including 1, 3, 4, or 5; or, 10E8 or 10E8 variants, including variants 1, 3, 4, or 5 with S100cW substitutions; or, 10E8 or 10E8 variants, including variants 1, 3, 4, or 5 with DS substitutions; or, 10E8 or 10E8 variants, including variants 1, 3, 4, or 5 with S100cW-DS substitutions) may also be present at V L The kabat position 32 of the present invention comprises an amino acid substitution. In some embodiments, the substitution may be a Y32F substitution. In other embodiments, the substitution may be a Y32W substitution.

[0348] In some embodiments, V of any of the 10E8 variants disclosed in the "10E8 variant 4" section above is L An amino acid substitution (e.g., a Y32F or Y32W substitution) may also be included at kabat position 32. In some embodiments, V of any of the 10E8 variants disclosed in the "10E8 variant 5" section above is L An amino acid substitution may also be included at kabat position 32 (e.g., a Y32F or Y32W substitution). In some embodiments, V of any of the 10E8 variants disclosed above in the "S100cW and S100cF" section is L An amino acid substitution may also be included at kabat position 32 (e.g., a Y32F or Y32W substitution). In some embodiments, V of any of the 10E8 variants disclosed in the "DS" section above is L An amino acid substitution may also be included at kabat position 32 (e.g., a Y32F or Y32W substitution). In some embodiments, V of any of the 10E8 variants disclosed in the "S100cW-DS" section above is LAmino acid substitutions may also be included at kabat position 32 (eg, Y32F or Y32W substitutions).

[0349] In some embodiments, the antibody or antigen-binding fragment comprises a V sequence comprising SEQ ID NO: 3 (HC6-S74Y-511) and SEQ ID NO: 42 (rL3-6mut-Y32W), respectively. H and V L In some embodiments, a V sequence comprising SEQ ID NO: 3 (HC6-S74Y-511) and SEQ ID NO: 43 (rL3-6mut-Y32F) is provided. H and V L Antibodies or antigen-binding fragments thereof.

[0350] In some embodiments, the antibody or antigen-binding fragment comprises a V sequence comprising SEQ ID NO: 5 (H6-511-4mut) and SEQ ID NO: 42 (rL3-6mut-Y32W), respectively. H and V L In some embodiments, a V sequence comprising SEQ ID NO: 5 (H6-511-4mut) and SEQ ID NO: 43 (rL3-6mut-Y32F) is provided. H and V L Antibodies or antigen-binding fragments thereof.

[0351] In some embodiments, the antibody or antigen-binding fragment comprises a V sequence comprising SEQ ID NO: 35 (H6-511-4mut-S100cW) and SEQ ID NO: 42 (rL3-6mut-Y32W), respectively. H and V L In some embodiments, a V sequence comprising SEQ ID NO: 35 (H6-511-4mut-S100cW) and SEQ ID NO: 43 (rL3-6mut-Y32F) is provided. H and V L Antibodies or antigen-binding fragments thereof.

[0352] In some embodiments, the antibody or antigen-binding fragment comprises a V sequence comprising SEQ ID NO: 38 (HC6-S74Y-511-S100cW) and SEQ ID NO: 42 (rL3-6mut-Y32W), respectively. H and V LIn some embodiments, a V sequence comprising SEQ ID NO: 38 (HC6-S74Y-511-S100cW) and SEQ ID NO: 43 (rL3-6mut-Y32F) is provided. H and V L Antibodies or antigen-binding fragments thereof.

[0353] In some embodiments, the antibody or antigen-binding fragment comprises a V sequence comprising SEQ ID NO: 36 (H6-511-4mut-DS) and SEQ ID NO: 58 (rL3-6mut-DS-Y32W), respectively. H and V L In some embodiments, a V sequence comprising SEQ ID NO: 36 (H6-511-4mut-DS) and SEQ ID NO: 59 (rL3-6mut-DS-Y32F) is provided. H and V L Antibodies or antigen-binding fragments thereof.

[0354] In some embodiments, the antibody or antigen-binding fragment comprises a V sequence comprising SEQ ID NO: 39 (HC6-S74Y-511-DS) and SEQ ID NO: 58 (rL3-6mut-DS-Y32W), respectively. H and V L In some embodiments, a V sequence comprising SEQ ID NO: 39 (HC6-S74Y-511-DS) and SEQ ID NO: 59 (rL3-6mut-DS-Y32F) is provided. H and V L Antibodies or antigen-binding fragments thereof.

[0355] In some embodiments, the antibody or antigen-binding fragment comprises a V sequence comprising SEQ ID NO: 37 (H6-511-4mut-S100cW-DS) and SEQ ID NO: 58 (rL3-6mut-DS-Y32W), respectively. H and V L In some embodiments, a V sequence comprising SEQ ID NO: 37 (H6-511-4mut-S100cW-DS) and SEQ ID NO: 59 (rL3-6mut-DS-Y32F) is provided. H and V L Antibodies or antigen-binding fragments thereof.

[0356] In some embodiments, the antibody or antigen-binding fragment comprises a VH and a VL comprising SEQ ID NO: 40 (HC6-S74Y-511-S100cW-DS) and SEQ ID NO: 58 (rL3-6mut-DS-Y32W), respectively. In some embodiments, an antibody or antigen-binding fragment comprising a VH and a VL comprising SEQ ID NO: 40 (HC6-S74Y-511-S100cW-DS) and SEQ ID NO: 59 (rL3-6mut-DS-Y32F), respectively, is provided.

[0357] Separate heavy and light chain variable regions

[0358] Any of the following heavy chain variable regions can be "mixed and matched" with any of the 10E8 light chain (or variants thereof) variable regions disclosed herein to generate antibodies that specifically bind to gp41 and neutralize HIV-1. In addition, any of the following light variable regions can be "mixed and matched" with any of the 10E8 heavy chain (or variants thereof) variable regions disclosed herein to generate antibodies that specifically bind to gp41 and neutralize HIV-1.

[0359] HC6-S74Y-511 V H

[0360] In some embodiments, the antibody or antigen-binding fragment comprises V H , which includes 10E8 V H CDRs (e.g., SEQ ID NOs: 7, 8, and 9), and arginine, alanine, aspartic acid, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively. In another embodiment, the antibody or antigen-binding fragment comprises V H , which includes 10E8 V H CDRs (e.g., SEQ ID NOs: 7, 8, and 9), and arginine, alanine, aspartic acid, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO: 3 (HC6-S74Y-511). In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 3 (HC6-S74Y-511). H .

[0361] H6-511-4mut V H

[0362] In some embodiments, the antibody or antigen-binding fragment comprises V H , which includes 10E8 VH CDRs (e.g., SEQ ID NOs: 7, 8, and 9), and arginine, glutamic acid, serine, lysine, aspartic acid, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively. In additional embodiments, the antibody or antigen-binding fragment includes V H , which includes 10E8 V H CDRs (e.g., SEQ ID NOs: 7, 8, and 9), and arginine, glutamic acid, serine, lysine, aspartic acid, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO: 5 (H6-511-4mut). In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 5 (H6-511-4mut). H .

[0363] HC6-S74Y-511-S100cW V H

[0364] In some embodiments, the antibody or antigen-binding fragment comprises V H , which includes the 10E8 V with S100cW replacement H CDRs (e.g., SEQ ID NOs: 7, 8, and 62), and arginine, alanine, aspartic acid, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively. In another embodiment, the antibody or antigen-binding fragment comprises V H , which includes the 10E8 V with S100cW replacement HCDRs (e.g., SEQ ID NOs: 7, 8, and 62), and arginine, alanine, aspartic acid, tyrosine, lysine, threonine, threonine, and isoleucine residues at kabat positions 3, 5, 72, 74, 75, 77, 84, and 110, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO: 38 (HC6-S74Y-511-S100cW). In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 38 (HC6-S74Y-511-S100cW). H .

[0365] H6-511-4mut-S100cW V H

[0366] In some embodiments, the antibody or antigen-binding fragment comprises V H , which includes the 10E8 V with S100cW replacement H CDRs (e.g., SEQ ID NOs: 7, 8, and 62), and arginine, glutamic acid, serine, lysine, aspartic acid, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively. In additional embodiments, the antibody or antigen-binding fragment comprises V H , which includes 10E8 VH CDRs with S100cW substitutions (e.g., SEQ ID NOs: 7, 8, and 62), and arginine, glutamic acid, serine, lysine, aspartic acid, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO: 35 (H6-511-4mut-S100cW). In further embodiments, the antibody or antigen-binding fragment includes a VH CDR comprising the amino acid sequence set forth in SEQ ID NO: 35 (H6-511-4mut-S100cW). H .

[0367] LV L

[0368] In some embodiments, the antibody or antigen-binding fragment comprises V L , which includes 10E8 VL CDRs (e.g., SEQ ID NOs: 10, 11, and 12), and may further include aspartic acid, valine, isoleucine, threonine, glutamic acid, and aspartic acid residues at kabat positions 7, 45, 58, 76, 83, and 85, respectively. In another embodiment, the antibody or antigen-binding fragment includes V L , which includes 10E8 V L CDRs (e.g., SEQ ID NOs: 10, 11, and 12), and aspartic acid, valine, isoleucine, threonine, glutamic acid, and aspartic acid residues at kabat positions 7, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO: 4 (rL3). In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 4 (rL3). L .

[0369] rL3-6mut V L

[0370] In some embodiments, the antibody or antigen-binding fragment comprises V L , which includes 10E8 V L CDRs (e.g., SEQ ID NOs: 10, 11, and 12), and alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid, and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively. In another embodiment, the antibody or antigen-binding fragment comprises V L , which includes 10E8 V L CDRs (e.g., SEQ ID NOs: 10, 11, and 12), and alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid, and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO: 6 (rL3-6mut). In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 6 (rL3-6mut). L .

[0371] rL3-6mut V with Y32W substitution L

[0372] In some embodiments, the antibody or antigen-binding fragment comprises V L , which includes 10E8V with Y32W replacement L CDRs (e.g., SEQ ID NOs: 10, 11, and 66), and alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid, and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively. In another embodiment, the antibody or antigen-binding fragment comprises V L , which includes 10E8 V with Y32W replacement L CDRs (e.g., SEQ ID NOs: 10, 11, and 66), and alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid, and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO: 42 (rL3-6mut-Y32W). In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 42 (rL3-6mut-Y32W). L .

[0373] rL3-6mut V with Y32F substitution L

[0374] In some embodiments, the antibody or antigen-binding fragment comprises V L , which includes 10E8V with Y32F replacement L CDRs (e.g., SEQ ID NOs: 10, 11, and 67), and alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid, and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively. In another embodiment, the antibody or antigen-binding fragment comprises V L , which includes 10E8 V with Y32F substitution LCDRs (e.g., SEQ ID NOs: 10, 11, and 67), and alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid, and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively, and an amino acid sequence that is at least 80% (e.g., at least 90% or at least 95%) identical to SEQ ID NO: 43 (rL3-6mut-Y32F). In further embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 43 (rL3-6mut-Y32F). L .

[0375] 1. Additional Description of the Disclosed Antibodies and Antigen-Binding Fragments

[0376] In certain embodiments, substitutions, insertions or deletions may occur in one or more CDRs, as long as these changes do not significantly reduce the ability of the antibody to bind antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not significantly reduce binding affinity may be made in the CDRs. H and V L In certain embodiments of the sequences, each CDR is either unchanged or contains no more than one, two or three amino acid substitutions. H and V L In some embodiments, the V region of the antibody or antigen-binding fragment is substituted with amino acids (e.g., conservative amino acids) to enhance production or solubility. H Compared with the publicly available 10E8 variant V H The antibody or antigen-binding fragment comprises up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8 or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the antibody or antigen-binding fragment, and / or the V L comprising up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the disclosed 10E8 variant VL.

[0377] The disclosed modified 10E8 antibodies specifically bind to the MPER of gp41 at an epitope referred to as the 10E8 epitope, which is included in the peptide sequence provided as NEQELLELDKWASLWNWFDITNWLWYIR (SEQ ID NO: 34). The 10E8 epitope has been elucidated by solving the structure of the 10E8 antibody in complex with a gp41 peptide (see Huang et al., Nature, 491: 406-412, 2012, which is incorporated herein by reference in its entirety). As described in Huang et al. (Nature, 491: 406-412, 2012) and PCT Pub. US Pub. 2014 / 0348785A1, the V H Kabat positions 28, 31, 33, 50, 52, 52B, 52C, 53, 56, 58, and 97-100J, and V L The amino acids at Kabat positions 91 and 95B contribute to the buried surface area of ​​the antibody interface with gp41. Accordingly, in some embodiments, any modified 10E8 antibody or antigen-binding fragment disclosed herein has a V H Kabat positions 28, 31, 33, 50, 52, 52B, 52C, 53, 56, 58, and 97-100J, and V L Kabat positions 91 and 95B include the corresponding amino acids of the parent 10E8 antibody. These residues are D, N, W, R, T, P, G, E, S, D, K, Y, Y, D, F, W, S, G, Y, P, P, G, E, and E, respectively. In some embodiments, V of any of the modified 10E8 antibodies or antigen-binding fragments provided herein is H An arginine residue is included at kabat position 50 and an aspartic acid residue is included at kabat position 58.

[0378] In some embodiments, any of the disclosed modified 10E8 antibodies or antigen-binding fragments that specifically bind gp41 and neutralize HIV-1 may also include one or more gain of function amino acid substitutions listed in one of Tables 3, 4, 9, or 10.

[0379] In some embodiments, the antibodies or antigen-binding fragments can also be distinguished by neutralization breadth. For example, in some embodiments, the antibodies or antigen-binding fragments can be distinguished by neutralization breadth with an IC value of less than 50 μg / ml. 50Neutralizes at least 80% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the Fig.16A HIV-1 isolates in. Those of ordinary skill in the art are familiar with methods for measuring the neutralization breadth and potency of monoclonal antibodies specific for HIV-1 Env, such as methods including a single round of HIV-1 Env pseudovirus infection of TZM-bl cells. Exemplary pseudovirus neutralization assays and HIV-1 pseudovirus panels are described, for example, in Li et al., J Virol 79, 10108-10125, 2005, Seaman et al., J. Virol., 84: 1439-1452, 2010; Sarzotti-Kelsoe et al., J. Immunol. Methods, 409: 131-46, 2014; and WO 2011 / 038290, each of which is incorporated herein by reference.

[0380] In some embodiments, the antibody or antigen binding fragment can also be distinguished by its solubility in aqueous solution. For example, in some embodiments, the antibody or antigen binding fragment (e.g., including 10E8v4 S100cF or 10E8v4 V5R S100cF variable region) can be dissolved in phosphate buffered saline (pH 7.4) at room temperature (e.g., 20 to 22 degrees Celsius) to a concentration of at least 1.0 mg / ml (e.g., at least 1.0 mg / ml, at least 2.0 mg / ml, at least 3.0 mg / ml, at least 4.0 mg / ml, at least 5.0 mg / ml, at least 6.0 mg / ml, at least 7.0 mg / ml, at least 8.0 mg / ml, at least 9.0 mg / ml, or at least 10.0 mg / ml) and remain dissolved for at least 12 hours (at least 24 hours, at least 48 hours, at least one week, at least two weeks, or longer). In some embodiments, the antibody or antigen-binding fragment (e.g., comprising the 10E8v4 S100cF or 10E8v4 V5R S100cF variable region) can be dissolved in phosphate-buffered saline (pH 7.4) at room temperature (e.g., 20 to 22 degrees Celsius) to a concentration of at least 10 mg / ml (e.g., at least 15 mg / ml, at least 20 mg / ml, at least 25 mg / ml, at least 30 mg / ml, at least 35 mg / ml, at least 40 mg / ml, at least 45 mg / ml, or at least 50 mg / ml) and remain dissolved for at least 12 hours (at least 24 hours, at least 48 hours, at least one week, at least two weeks, or longer). In some embodiments, the antibody or antigen-binding fragment (e.g., comprising the 10E8v4 S100cF or 10E8v4 V5R S100cF variable region) can be dissolved in phosphate-buffered saline (pH 7.4) at room temperature (e.g., 20 to 22 degrees Celsius) to a concentration of at least 10 mg / ml (e.g., at least 15 mg / ml, at least 20 mg / ml, at least 25 mg / ml, at least 30 mg / ml, at least 35 mg / ml, at least 40 mg / ml, at least 45 mg / ml, or at least 50 mg / ml) and remain dissolved for at least 12 hours (at least 24 hours, at least 48 hours, at least one week, at least two weeks, or longer). In some embodiments, the antibody or antigen-binding fragment (e.g., comprising the 10E8v4S100cF or 10E8v4 V5R S100cF variable region) can be dissolved in phosphate-buffered saline (pH 7.4) at room temperature (e.g., 20 to 22 degrees Celsius) to a concentration of at least 20 mg / ml and remain dissolved for at least 12 hours (at least 24 hours, at least 48 hours, at least one week, at least two weeks, or longer).In some embodiments, the antibody or antigen-binding fragment (e.g., including the variable region of 10E8v4 S100cF or 10E8v4 V5R S100cF) can be dissolved in phosphate-buffered saline (pH 7.4) at room temperature (e.g., 20 to 22 degrees Celsius) to a concentration of at least 40 mg / ml and remain dissolved for at least 12 hours (at least 24 hours, at least 48 hours, at least one week, at least two weeks, or longer). In some embodiments, the antibody or antigen-binding fragment (e.g., including the variable region of 10E8v4 S100cF or 10E8v4 V5R S100cF) can be dissolved in phosphate-buffered saline (pH 7.4) at room temperature (e.g., 20 to 22 degrees Celsius) to a concentration of at least 30 mg / ml and remain dissolved for at least 12 hours (at least 24 hours, at least 48 hours, at least one week, at least two weeks, or longer). In one embodiment, the phosphate buffered saline comprises NaCl (137 mM), KCl (2.7 mM), Na. 2 HPO 4 (10 mM), KH 2 PO 4 (1.8 mM), pH 7.4. In some embodiments, the phosphate buffered saline further comprises CaCl 2 (1 mM) and MgCl 2 (0.5 mM). Those skilled in the art are familiar with methods for determining whether a protein remains dissolved over time. For example, standard methods can be used to test the concentration of a protein dissolved in an aqueous solution. In some embodiments, the antibodies or antigen-binding fragments can also be distinguished by their autoreactivity (i.e., binding to self-antigens). For example, in some embodiments, the disclosed antibodies or antigen-binding fragments include autoreactivity comparable to that of the 10E8 antibody. For example, the antibodies or antigen-binding fragments can have no more than 10% higher autoreactivity than the 10E8 antibody, as measured using standard assays, such as a HEp-2 staining assay or a cardiolipin binding assay.

[0381] The antibody or antigen-binding fragment may be a human antibody or fragment thereof. Chimeric antibodies are also provided. The antibody or antigen-binding fragment includes any suitable framework region, such as (but not limited to) a human framework region. Human framework regions and mutations that can be generated in human antibody framework regions are known in the art (see, e.g., U.S. Patent No. 5,585,089, which is incorporated herein by reference). Alternatively, a heterologous framework region, such as, but not limited to, a mouse or monkey framework region, may be included in the heavy chain or light chain of the antibody. (See, e.g., Jones et al., Nature 321:522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Carter et al., Proc. Natl. Acad. Sci. USA 89:4285, 1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; and Singer et al., J. Immunol. 150:2844, 1993).

[0382] The antibody may be of any isotype. The antibody may, for example, be an IgM or IgG antibody, such as IgG 1 IgG 2 IgG 3 or IgG 4 The class of antibodies that specifically bind to gp41 can be switched to another class. In one aspect, the encoding V L or V H The nucleic acid molecule is then modified so that it does not include any nucleic acid sequence encoding the constant region of the light chain or heavy chain, respectively. L or V H The nucleic acid molecule is operably linked to a C-terminal fragment encoding an immunoglobulin molecule from a different class. L or C H This can be done by including C L or C H This can be accomplished by vectors or nucleic acid molecules that specifically bind to gp41, which are known in the art. For example, an antibody that is initially IgG and specifically binds to gp41 can be class-switched to IgM. Class switching can be used to convert one IgG subtype to another, such as from IgG 1 To IgG 2 IgG 3 or IgG 4 .

[0383] In some examples, the disclosed antibodies are antibody oligomers, such as dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, etc.

[0384] (a) Binding affinity

[0385] In some embodiments, the antibody or antigen-binding fragment may be present at a rate of no more than 1.0×10 -8 M, not more than 5.0×10 -8 M, not more than 1.0×10 -9 M, not more than 5.0×10 -9 M, not more than 1.0×10 -10 M, not more than 5.0×10 -10 M or not more than 1.0×10 -11 The affinity of M (e.g., through K D Measured) specific binding to gp41. K D It can be measured, for example, by a radiolabeled antigen binding assay (RIA) using known methods with the Fab form of the antibody of interest and its antigen. In one assay, the solution binding affinity of the Fab for the antigen is measured by mixing the Fab with a minimal concentration of ( 125 I) The labeled antigen was equilibrated in the presence of a titration series of unlabeled antigen, and then the unbound antigen was captured by an anti-Fab antibody-coated plate for measurement (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the assay conditions, 5 μg / ml of the capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) was used to coat the plate. Multiwell plates (Thermo Scientific) were incubated overnight and then blocked with 2% (w / v) bovine serum albumin in PBS for 2 to 5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 μM or 26 pM [ 125 I]-antigen is mixed with a serial dilution of the target Fab (e.g., consistent with the evaluation of anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The target Fab is then incubated overnight; however, the incubation can be continued for a longer period of time (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to the capture plate and incubated at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed with 0.1% polysorbate 20 in PBS. Wash the plates 8 times. When the plates are dry, add scintillant (MICROSCINT-20 TM ; Packard), 150 μl / well, and the plate was placed on TOPCOUNT TM Counting was performed on a gamma counter (Packard) for 10 min. Each Fab concentration that was less than or equal to 20% of maximal binding was selected for competitive binding assays.

[0386] In another assay, or K was measured at ∼10 response units (RU) using surface plasmon resonance at 25°C with an immobilized antigen CM5 chip (BIAcore, Inc., Piscataway, NJ). D Briefly, carboxymethyl dextran biosensor chips (CM5, Inc.) was activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The antigen was diluted to 5 μg / ml (~0.2 μM) with 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 l / min to obtain approximately 10 response units (RU) of coupled protein. After the injection of the antigen, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were injected at a flow rate of approximately 25 l / min at 25 ° C into a 4% PBS containing 0.05% polysorbate 20 (TWEEN-20 TM ) surfactant in PBS (PBST). The binding rate (k on ) and dissociation rate (k off ) using a simple one-to-one Langmuir binding model ( EvaluationSoftware version 3.2) by simultaneously fitting the binding and dissociation sensorgrams. The equilibrium dissociation constant (K D ) is calculated as the ratio k off / k on See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 106 M by the surface plasmon resonance determination above, -1 s -1 , the association rate can be determined by using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 at 25°C in the presence of increasing concentrations of antigen as measured in a spectrophotometer, such as a spectrophotometer equipped with stopped flow (Aviv Instruments) or an 8000-series SLM-AMINCO with a stirred cuvette. TM Spectrophotometer.

[0387] (b) Multispecific Antibodies

[0388] In some embodiments, the antibody or antigen binding fragment is included in a multispecific antibody, such as a bispecific antibody or a trispecific antibody. These multispecific antibodies can be produced by known methods, such as cross-linking two or more antibodies or antigen binding fragments (such as scFv) of the same or different types. Exemplary methods for preparing multispecific antibodies include those described in PCT Publication No. WO2013 / 163427, which are incorporated herein by reference in their entirety. Suitable cross-linking agents include heterobifunctional, i.e., having two different reactive groups separated by a suitable separator (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), and homobifunctional (e.g., suberate disuccinimide ester). These linkers can be obtained from Pierce Chemical Company, Rockford, Illinois (Pierce Chemical Company, Rockford, Ill).

[0389] In some embodiments, the antibody or antigen-binding fragment is included on a bifunctional antibody that specifically binds gp41 and also specifically binds CD3. Examples of CD3 binding domains that can be included on a bispecific antibody or antigen-binding fragment are known, including those disclosed in PCT Publication No. WO2013 / 163427, which is incorporated herein by reference in its entirety.

[0390] Various types of multispecific antibodies are known. Bispecific single chain antibodies can be encoded by a single nucleic acid molecule. Examples of bispecific single chain antibodies, and methods for constructing such antibodies are known in the art (see, e.g., U.S. Patent Nos. 8,076,459, 8,017,748, 8,007,796, 7,919,089, 7,820,166, 7,635,472, 7,575,923, 7,435,549, 7,332,168, 7,323,440, 7,235,641, 7,229,760, 7,112,324, 6,723,538, incorporated herein by reference). Additional examples of bispecific single chain antibodies can be found in PCT Application No. WO 99 / 54440; Mack, J. Immunol., 158:3965-3970, 1997; Mack, PNAS, 92:7021-7025, 1995; Kufer, Cancer Immunol. Immunother., 45:193-197, 1997; Loffler, Blood, 95:2098-2103, 2000; and Bruhl, J. Immunol., 166:2420-2426, 2001. The production of bispecific Fab-scFv ("bibody") molecules is described, for example, in Schoonjans et al. (J. Immunol. 165: 7050-57, 2000) and Willems et al. (J Chromatogr B Analyt Technol Biomed Life Sci. 786: 161-76, 2003). For diabodies, the scFv can be fused to one of the VL-CL(L) or VH-CH1 chains, for example to produce a diabody with one scFv fused to the C-terminus of a Fab chain.

[0391] (c) Fragment

[0392] The present disclosure encompasses antigen-binding fragments, such as Fab, F(ab') 2 and Fv, which includes a heavy chain and a V L In some embodiments, the antigen-binding fragment comprises the heavy chain and light chain variable regions from the 10E8v4 S100cF or 10E8v4 V5R S100cF antibody.

[0393] These antibody fragments retain the ability to selectively bind to antigen and are "antigen-binding" fragments. Non-limiting examples of these fragments include:

[0394] (1) Fab, a monovalent antigen-binding fragment of an antibody molecule, which can be produced by digesting an intact antibody with papain to obtain a complete light chain and a portion of one heavy chain;

[0395] (2) Fab', which can be obtained by treating the whole antibody with pepsin and then reducing it to obtain the complete light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;

[0396] (3)(Fab') 2 , which antibody fragment can be obtained by treating intact antibodies with pepsin without subsequent reduction; F(ab') 2 It is a dimer of two Fab' fragments held together by two disulfide bonds;

[0397] (4) Fv, a genetically engineered fragment containing the V expressed as two chains L and V L ;as well as

[0398] (5) Single-chain antibodies (e.g., scFv), which are defined as single-chain molecules containing V and V linked by a suitable polypeptide linker. H and V L Genetically engineered molecules (see, for example, Ahmad et al., Clin. Dev. Immunol., 2012, doi: 10.1155 / 2012 / 980250; Marbry, ID Rugs, 13: 543-549, 2010). H Domain and V L The intramolecular orientation of the domains in the scFv is not critical for the antibodies provided (e.g., for the multispecific antibodies provided). Thus, scRFvs with two possible arrangements (V H Domain-Linker Domain-V L -Domain; V L Domain-Linker Domain-V H - domain).

[0399] (6) Single-chain antibody dimer (scFv 2 ), defined as a dimer of scFV. This is also called a miniantibody.

[0400] Methods for preparing these fragments are known in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, 2 nd, Cold Spring Harbor Laboratory, New York, 2013).

[0401] In some embodiments, the antigen-binding fragment may be a Fv antibody, which is generally about 25 kDa, contains a complete antigen-binding site, and has three CDRs for each heavy chain and each light chain. H and V L If the Fv antibody is not expressed continuously, the chains of the Fv antibody are generally held together by non-covalent interactions. However, these chains tend to dissociate after dilution, so some methods have been developed to cross-link these chains through glutaraldehyde, intramolecular disulfide bonds or peptide linkers. Therefore, in one example, the Fv can be a disulfide-stabilized Fv (dsFv), in which V H and V L In another example, the Fv fragment comprises a V and a V connected by a peptide linker. H and V L These single-chain antigen-binding proteins (scFv) can be constructed by constructing a V-encoding H and V L The nucleic acid molecule of the V domain is prepared. The nucleic acid molecule is inserted into an expression vector, and the expression vector is subsequently introduced into a host cell such as a mammalian cell. The recombinant host cell synthesizes a single polypeptide chain having a linker peptide bridging two V domains. The method of producing scFv is known in the art (see Whitlow et al., Methods: a Companion to Methods in Enzymology, Vol. 2, p. 97, 1991; Bird et al., Science 242: 423, 1988; U.S. Patent No. 4,946,778; Pack et al., Bio / Technology 11: 1271, 1993; Ahmad et al., Clin. Dev. Immunol., 2012, doi: 10.1155 / 2012 / 980250; Marbry, ID Rugs, 13: 543-549, 2010. It is also considered that dimers of single-chain antibodies (scFV 2 ).

[0402] Antigen-binding fragments can be prepared by proteolysis of antibodies or by expressing DNA encoding the fragments in host cells. Antigen-binding fragments can also be obtained by conventional methods of digesting intact antibodies with pepsin or papain. For example, antibodies can be enzymatically cleaved by pepsin to provide a fragment known as F(ab'). 2The antigen-binding fragment is produced by cleaving the 5S fragment of the Fab' fragment. The fragment can also be further cleaved using a sulfhydryl reducing agent, and optionally a blocking group for the sulfhydryl groups generated by cleaving the disulfide bonds, to produce a 3.5S Fab' monovalent fragment. Alternatively, enzymatic cleavage using pepsin directly produces two monovalent Fab' fragments and an Fc fragment (see U.S. Pat. No. 4,036,945 and U.S. Pat. No. 4,331,647, and references therein; Nisonhoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press, 1967; and Coligan et al., Sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

[0403] Other methods of cleaving antibodies may also be used, for example, separating the heavy chains to form monovalent light-heavy chain fragments, further cleaving the fragments, or other enzymatic, chemical or genetic techniques, as long as the fragments bind the antigen recognized by the intact antibody.

[0404] Mouse V amplified from genomic DNA of immunized mice H Gene library identified a single V binding antigen H Domains are referred to as domain antibodies (dAbs) (Ward et al. Nature 341: 544-546, 1989). Human single immunoglobulin variable domain polypeptides capable of binding antigen with high affinity have also been described (see, e.g., PCT Publication Nos. WO 2005 / 035572 and WO 2003 / 002609). The CDRs disclosed herein may also be included in dAbs.

[0405] In some embodiments, one or more heavy chain and / or light chain complementary determining regions (CDRs) from a disclosed antibody (e.g., HC6-S74Y-511 / rL3-6mut or H6-511-4mut / rL3-6mut antibody) are expressed on the surface of another protein, such as a scaffold protein. The expression of antibody domains on the surface of scaffold proteins is known in the art (see, e.g., Liu et al., J. Virology 85(17):8467-8476, 2011). Such expression generates a chimeric protein that retains binding to gp41. In some specific embodiments, one or more heavy chain CDRs are transplanted onto a scaffold protein, such as one or more of heavy chain CDR1, CDR2, and / or CDR3. One or more CDRs may also be included in a diabody or another type of single-chain antibody molecule.

[0406] (d) Variant

[0407] In certain embodiments, the amino acid sequence variants of the antibodies provided herein are of interest. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. The amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. These modifications include, for example, deletions and / or insertions and / or residue substitutions in the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be performed to obtain the final construct, provided that the final construct has desired characteristics, such as antigen binding.

[0408] In certain embodiments, antibody variants with one or more amino acid replacements are provided. The target site for replacement mutations includes CDR and framework regions. Amino acid replacements can be introduced into the target antibody, and the product can be screened for desired activity, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0409] These variants generally retain the H and V L The amino acid residues necessary for correct folding and stabilization between the regions are retained, and the charge characteristics of the residues are retained to maintain the low pI and low toxicity of the molecule. H and V L Region-forming amino acid substitutions (eg, conservative amino acid substitutions) may be made, for example, to enhance production or solubility.

[0410] In certain embodiments, substitutions, insertions or deletions may occur in one or more CDRs, as long as these changes do not significantly reduce the ability of the antibody to bind antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not significantly reduce binding affinity may be made in the CDRs. H and V L In certain embodiments of the sequences, each CDR is either unaltered or contains no more than one, two or three amino acid substitutions.

[0411] To increase the binding affinity of the antibody, V H and V L The segments can be randomly mutated, for example, in the HCDR3 region or the LCDR3 region, in a process similar to the in vivo somatic mutation process responsible for affinity maturation of antibodies during a natural immune response. Such in vitro affinity maturation can be performed by amplifying V sequences using PCR primers complementary to HCDR3 or LCDR3, respectively. H and V LIn this process, the primers have been "spiked" with a random mixture of four nucleotide bases at certain positions, so that the resulting PCR product encodes the V region in which the primers have been spiked. H and / or V L Random mutations were introduced into the CDR3 region. H and V L These random mutations can be tested H and V L Segments are used to determine binding affinity for gp41. Methods for in vitro affinity maturation are known (see, for example, Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, (2001)).

[0412] A useful method for identifying residues or regions of antibodies that can be used for mutation is called "alanine scanning mutation", as described in Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, residues or groups of target residues (e.g., charged residues, such as arg, asp, his, lys and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Replacements can be further introduced to the initial replacement at the amino acid position that has demonstrated functional sensitivity. Alternatively, or additionally, the crystal structure of the antigen-antibody complex can be used to identify the contact points between the antigen and the antigen. These contact residues and adjacent residues can be targeted or eliminated as candidates for replacement. Variants can be screened to determine whether they contain desired properties.

[0413] In certain embodiments, the antibody or antigen-binding fragment is altered to increase or decrease the degree of glycosylation of the antibody or antigen-binding fragment. The addition or deletion of glycosylation sites can be conveniently achieved by altering the amino acid sequence to form or remove one or more glycosylation sites.

[0414] Where the antibody includes an Fc region, the carbohydrate attached to the Fc region may be altered. Native antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are typically bound by the same CH 2The oligosaccharide may be attached to Asn297 of the oligonucleotide domain. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "trunk" of the diagonal oligosaccharide structure. In some embodiments, modification of the oligosaccharides in the antibody may be performed to generate antibody variants with certain improved properties.

[0415] In one embodiment, antibody variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies may be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all sugar structures (e.g., complex, hybrid, and high mannose structures) attached to Asn 297 as measured by MALDI-TOF mass spectrometry, as described, for example, in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 within the Fc region; however, due to small sequence variations in antibodies, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. These fucosylated variants may have improved ADCC function. See, for example, U.S. Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of disclosures concerning "defucosylated" or "fucose-deficient" antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec 13 CHO cells deficient in protein glycosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); U.S. Patent Application No US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94 (4): 680-688 (2006); and WO 2003 / 085107).

[0416] Also provided are antibodies with bisected oligosaccharides, for example, wherein the bisected oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of these antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Also provided are antibody variants having at least one galactose residue in the oligosaccharides attached to the Fc region. These antibody variants may have improved CDC function. These antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0417] In several embodiments, the constant region of the antibody comprises one or more amino acid substitutions to optimize the in vivo half-life of the antibody. The serum half-life of IgG Abs is regulated by the neonatal Fc receptor (FcRn). Thus, in several embodiments, the antibody comprises amino acid substitutions that increase binding to the FcRn. Several such substitutions are known to those of ordinary skill in the art, such as the substitutions T250Q and M428L in the IgG constant region (see, e.g., Hinton et al., J. Immunol., 176:346-356, 2006); M428L and N434S ("LS" mutations, see, e.g., Zalevsky et al., Nature Biotechnology, 28:157-159, 2010); N434A (see, e.g., Petkova et al., Int. Immunol., 18:1759-1769, 2006); T307A, E380A and N434A (see, e.g., Petkova et al., Int. Immunol., 18:1759-1769, 2006); and M252Y, S254T and T256E (see, e.g., Dall'Acqua et al., J. Biol. Chem., 281:23514-23524, 2006). The disclosed antibodies and antigen-binding fragments can be linked to an Fc polypeptide comprising any of the substitutions listed above, for example, the Fc polypeptide comprises M428L and N434S substitutions.

[0418] In several embodiments, the constant region of the antibody includes one or more amino acid replacements to optimize antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC is mainly mediated by a group of closely related Fcγ receptors. In some embodiments, the antibody includes one or more amino acid replacements that increase binding to FcγRIIIa. Several such replacements are known to those of ordinary skill in the art, such as IgG constant region replacements S239D and I332E (see, e.g., Lazar et al., Proc. Natl., Acad. Sci. USA, 103: 4005-4010, 2006); and S239D, A330L and I332E (see, e.g., Lazar et al., Proc. Natl., Acad. Sci. USA, 103: 4005-4010, 2006).

[0419] Combinations of the above substitutions are also included to generate IgG constant regions with increased binding to FcRn and FcγRIIIa. This combination increases antibody half-life and ADCC. For example, such combinations include antibodies with the following amino acid substitutions in the Fc region: (1) S239D / I332E and T250Q / M428L; (2) S239D / I332E and M428L / N434S; (3) S239D / I332E and N434A; (4) S239D / I332E and T307A / E380A / N434A; (5) S239D / I332E and M252Y / S254T / T256E; (6) S239D / I332E and M252Y / S254T / T256E; (7) S239D / I332E and M252Y / S254T / T256E; (8) S239D / I332E and M252Y / S254T / T256E; (9) S239D / I332E and M252Y / S254T / T256E; (10) S239D / I332E and M252Y / S254T / T256E; (11) S239D / I332E and T250Q / M428L; (12) S239D / I332E and M428L / N434S; (13) S239D / I332E and N434A; (14) S239D / I332E and T307A / E380A / N434A; (15) S239D / I332E and M252Y / S254T / 6) S239D / A330L / I332E and 250Q / M428L; (7) S239D / A330L / I332E and M428L / N434S; (8) S239D / A330L / I332E and N434A; (9) S239D / A330L / I332E and T307A / E380A / N434A; or (10) S239D / A330L / I332E and M252Y / S254T / T256E. In some embodiments, the antibody or antigen-binding fragment thereof is modified to be directly toxic to infected cells or to utilize natural defenses, such as complement, antibody-dependent cellular toxicity, or macrophage phagocytosis.

[0420] In certain embodiments, the antibodies provided herein can be further modified to contain additional non-proteinaceous modules known in the art and readily available. Modules suitable for derivatized antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, hydroxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer may be of any molecular weight, branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under a defined condition, etc.

[0421] The antibody or antigen binding fragment may be derivatized or linked to another molecule (e.g., another peptide or protein). Typically, the antigen or antigen binding fragment is derivatized so that binding to gp41 is not adversely affected by the derivatization or labeling. For example, the antibody or antigen binding fragment may be functionally linked (by chemical coupling, genetic fusion, non-covalent association or other means) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detectable marker, an effector molecule, or a protein or peptide that can mediate association of the antibody or antigen binding fragment with another molecule (e.g., a streptavidin core region or a polyhistidine tag).

[0422] B. Conjugates

[0423] The antibodies and antigen-binding fragments that specifically bind to an epitope on gp41 can be conjugated to an agent, such as an effector molecule or a detectable marker, using any number of means known to those skilled in the art. Covalent and non-covalent attachment means can be used. It will be appreciated by those skilled in the art that a variety of effector molecules and detectable markers can be used, including but not limited to toxins and radioactive agents such as 125 I. 32 P. 14 C. 3 H and 35 S and other tags, targeting modules and ligands, etc. The choice of a specific effector molecule or detectable marker depends on the specific target molecule or cell, and the desired biological effect.

[0424] The choice of a specific effector molecule or detectable marker depends on the specific target molecule or cell, and the desired biological effect. Thus, for example, the effector molecule may be a cytotoxin used to cause death of a specific target cell (e.g., HIV-1 infected cell). In other embodiments, the effector molecule may be a cytokine, such as IL-15; conjugates comprising cytokines may be used, for example, to locally stimulate immune cells.

[0425] The procedure for attaching an effector molecule or detectable label to an antibody or antigen-binding fragment varies with the chemical structure of the effector. Polypeptides typically contain various functional groups; for example, carboxylic acids, free amines (-NH 2) or sulfhydryl (-SH), which can be used to react with appropriate functional groups on the polypeptide, resulting in the binding of the effector molecule or detectable marker. Alternatively, the antibody or antigen binding fragment is derivatized to expose or attach additional reactive functional groups. The derivatization can involve the attachment of any number of known linker molecules (such as those available from Pierce Chemical Company, Rockford, IL). The linker can be any molecule used to link the antibody or antigen binding fragment to the effector molecule or detectable marker. The linker is capable of forming a covalent bond with the antibody or antigen binding fragment and with the effector molecule or detectable marker. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody or antigen binding fragment and the effector molecule or detectable marker are polypeptides, the linker can be linked to the constituent amino acids through the side chains of the constituent amino acids (e.g., through a disulfide bond to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.

[0426] In view of the large number of methods reported for attaching various radiodiagnostic compounds, radiotherapeutic compounds, labels (e.g., enzymes or fluorescent molecules), toxins, and other agents to antibodies, one skilled in the art will be able to determine the appropriate method for attaching a given agent to an antibody or antigen-binding fragment or other polypeptide. For example, the antibody or antigen-binding fragment can be conjugated to an effector molecule, such as a small molecular weight drug, such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansine, maytansine derivatives, including maytansine derivatives known as DM1 (also known as mertansine), or other agents to prepare an antibody drug conjugate (ADC). In several embodiments, conjugates of an antibody or antigen-binding fragment with one or more small molecule toxins are provided, such as calicheamicin, maytansines, dolastatins, auristatins, trichothecenes, and CC1065, as well as derivatives of these toxins having toxin activity.

[0427] The antibody or antigen-binding fragment can be conjugated to a detectable label; for example, a detectable label that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques (e.g., computed tomography (CT), computed axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiberoptic examination, and laparoscopy). Specific non-limiting examples of detectable labels include fluorophores, chemiluminescent agents, enzymatic linkages, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable labels include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phospholipids, and the like. Bioluminescent labels, such as luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), can also be used. The antibody or antigen binding fragment can also be conjugated with an enzyme that can be used for detection, such as horseradish peroxidase, beta galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc. When the antibody or antigen binding fragment is conjugated with a detectable enzyme, it can be used to produce a reagent that can detect the reaction product by adding another enzyme. For example, when the reagent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine produces a colored reaction product that is visually detectable. The antibody or antigen binding fragment can also be conjugated with biotin and detected by indirectly measuring the binding of avidin or streptavidin. It should be noted that avidin itself can be conjugated with an enzyme-active fluorescent label.

[0428] The antibody or antigen-binding fragment can be conjugated with a paramagnetic agent such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide are also used as labels. Antibodies can also be conjugated with lanthanides (europium and dysprosium) and manganese. Antibodies or antigen-binding fragments can also be labeled with a predetermined polypeptide epitope recognized by a second reporter (e.g., a leucine zipper pair sequence, a binding site for a second antibody, a metal binding domain, an epitope tag).

[0429] The antibody or antigen-binding fragment may also be conjugated to a radiolabeled amino acid. The radiolabel may be used for diagnostic and therapeutic purposes. For example, the radiolabel may be used to detect gp41 and cells expressing gp41 by x-ray, emission spectroscopy or other diagnostic techniques. Examples of labels for polynucleotides include, but are not limited to, the following radioisotopes or radionuclides: 3 H. 14 C. 15 N. 35 S. 90 Y. 99 Tc, 111 In, 125 I. 131 I.

[0430] Means for detecting these detectable labels are well known to those skilled in the art. Thus, for example, radioactive labels can be detected using film or a scintillation counter, and fluorescent labels can be detected using a photodetector to detect emitted light. Enzymatic labels are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply observing the colored label.

[0431] The average number of effector molecules or detectable labels per antibody or antigen binding fragment in the conjugate can range, for example, from 1 to 20 modules per antibody or antigen binding fragment. In certain embodiments, the average number of effector molecules or detectable labels per antibody or antigen binding fragment in the conjugate ranges from about 1 to about 2, about 1 to about 3, about 1 to about 8; about 2 to about 6; about 3 to about 5; or about 3 to about 4. The loading (e.g., effector molecule / antibody ratio) of the conjugate can be controlled in various ways, for example by: (i) controlling the molar excess of effector molecule-linker intermediate or linker reagent relative to the antibody, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limited reducing conditions for modification of cysteine ​​thiol groups, (iv) engineering the amino acid sequence of the antibody by recombinant technology so that the number and position of cysteine ​​residues are modified to control the number or position of linker-effector molecule attachments.

[0432] C. Polynucleotides and Expression

[0433] Nucleic acid molecules (e.g., cDNA molecules) encoding amino acid sequences of antibodies, antigen-binding fragments, and conjugates that specifically bind gp41 are provided. Nucleic acids encoding these molecules can be easily generated by one skilled in the art using the amino acid sequences (e.g., CDR sequences and V H and V L sequences), sequences available in the art (e.g., framework or constant region sequences), and genetic code generation. In several embodiments, the nucleic acid molecule may encode the V region of the disclosed antibody or antigen-binding fragment. H 、V L or V H and V L (eg, in a bicistronic expression vector.) In several embodiments, the nucleic acid molecules can be expressed in a host cell (eg, a mammalian cell) to produce the disclosed antibodies or antigen-binding fragments.

[0434] A person skilled in the art can easily use the genetic code to construct various functionally equivalent nucleic acids, for example, nucleic acids that differ in sequence but encode the same antibody sequence or encode a conjugate or fusion protein and include V L and / or V H Nucleic acid sequence.

[0435] In a non-limiting example, an isolated nucleic acid molecule encodes a V or V region of an antibody or antigen-binding fragment disclosed herein. H , and includes a nucleic acid sequence as shown in any one of SEQ ID NO: 20, 21, 44, 45, 78 or 79. In a non-limiting example, the isolated nucleic acid molecule encodes a V or V sequence of an antibody or antigen-binding fragment disclosed herein. L , and includes a nucleic acid sequence as shown in any one of SEQ ID NO: 24, 46 or 47. In a non-limiting example, the isolated nucleic acid molecule encodes a V or V sequence of an antibody or antigen-binding fragment disclosed herein. H and V L , and include the nucleic acid sequence shown in any one of SEQ ID NOs:44 and 46, SEQ ID NOs:44 and 47, SEQ ID NOs:45 and 46, SEQ ID NOs:45 and 47 (10E8v4), SEQ ID NOs:78 and 47 (10E8v4 S100cF) or SEQ ID NOs:79 and 47 (10E8v4V5R, S100cF), respectively.

[0436] Nucleic acid sequences encoding antibodies, antigen-binding fragments, and conjugates that specifically bind to gp41 can be prepared by any suitable method, such as cloning appropriate sequences or by direct chemical synthesis using methods such as the phosphotriester method, Narang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method, Brown et al., Meth. Enzymol. 68:109-151, 1979; the dimethylphosphoramidite method, Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862, 1981 described solid phase phosphoramidite triester method, for example, using, for example, Needham-Van Devanter et al., Nucl. Acids Res. 12:6159-6168, 1984; and, solid support method, U.S. Pat. No. 4,458,066 Chemical synthesis produces a single stranded oligonucleotide. This can be converted to double-stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.

[0437] Exemplary nucleic acids can be prepared by cloning techniques. Examples of suitable cloning and sequencing techniques, as well as instructions sufficient to guide the skilled artisan in performing many cloning operations, are known (see, e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4 th ed., Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013). Product information from manufacturers of biological reagents and experimental equipment also provides useful information. These manufacturers include SIGMA Chemical Company (Saint Louis, MO), R&D Systems (Minneapolis, MN), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (Carlsbad, CA), and Applied Biosystems (Foster City, CA), as well as many other commercial sources known to those of skill.

[0438] Nucleic acids can be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification system (TAS), and self-maintained sequence amplification system (3SR). Various cloning methods, host cells, and in vitro amplification methodologies are well known to technicians.

[0439] These nucleic acid molecules can be expressed in recombinant engineered cells, such as bacteria, plants, yeast, insects and mammalian cells. Antibodies, antigen-binding fragments and conjugates can be used as single V H and / or V LThe V chains can be expressed (linked to an effector molecule or detectable marker if desired), or can be expressed as a fusion protein. Methods for expressing and purifying antibodies and antigen-binding fragments are known and further described herein (see, e.g., Al-Rubeai (ed), Antibody Expression and Production, Springer Press, 2011). Immunoadhesins can also be expressed. Thus, in some examples, a V encoding a H and V L , and a nucleic acid of an immunoadhesin. The nucleic acid sequence may optionally encode a leader sequence.

[0440] To generate scFv, the V H and V L The DNA fragment is operably linked to a flexible linker encoding an amino acid sequence (Gly 4 -Ser) 3 Another fragment of V H and V L The sequence can be expressed as a continuous single-chain protein, and V H and V L The domains are linked by this flexible linker (see, e.g., Bird et al., Science 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; McCafferty et al., Nature 348:552-554, 1990; Kontermann and Dubel (Ed), Antibody Engineering, Vols. 1-2, 2 nd Ed., Springer Press, 2010; Harlow and Lane, Antibodies: A Laboratory Manual, 2 nd , Cold Spring Harbor Laboratory, New York, 2013). Optionally, a cleavage site, such as a furin cleavage site, can be included in the linker.

[0441] Code V H and / or V L The nucleic acid may optionally encode an Fc region (immunoadhesin). The Fc region may be IgA, IgM or IgGFc. The Fc region may be an optimized Fc region, as described in U.S. Published Patent Application No. 20100 / 093979, which is incorporated herein by reference. In one example, the immunoadhesin is IgG 1 Fc.

[0442] The single-chain antibody may be monovalent if only a single V H and V L ; Bivalent, if two V H and V L ; Multivalent, if more than two V H and V L Bispecific or multivalent antibodies can be generated that specifically bind gp41 and another antigen such as, but not limited to, CD3. H and V L Optionally at V H and V L A furin cleavage site is included between the domains.

[0443] Those skilled in the art are aware of several expression systems that can be used to express proteins, including Escherichia coli (E. coli), other bacterial hosts, yeast, and various higher eukaryotic cells, such as COS, CHO, HeLa, and myeloma cell lines.

[0444] One or more DNA sequences encoding antibodies, Fabs or conjugates can be expressed in vitro by DNA transfer into suitable host cells. The cell can be prokaryotic or eukaryotic. The term can also include any offspring of the host cell. It should be understood that all offspring may not be identical to the parental cell because mutations may occur during replication. Methods for stable transfer (meaning that foreign DNA is continuously maintained in the host) are known in the art. Hybridomas expressing target antibodies are also encompassed in the present disclosure.

[0445] The expression of nucleic acids encoding antibodies and antigen-binding fragments described herein can be accomplished by operably linking the DNA or cDNA to a promoter (which is constitutive or inducible) and then incorporating it into an expression cassette. The promoter can be any target promoter, including a cytomegalovirus promoter and a human T-cell-tropic virus promoter (HTLV)-1. Optionally, an enhancer, such as a cytomegalovirus enhancer, is included in the construct. The expression cassette can be suitable for replication and integration in prokaryotic or eukaryotic cells. A typical expression cassette contains specific sequences that can be used to regulate the expression of the DNA encoding the protein. For example, the expression cassette includes an appropriate promoter, an enhancer, transcription and translation terminators, a start sequence, an initial codon (i.e., ATG) before a protein-coding gene, an intron splicing signal, a sequence that maintains the correct reading frame of the gene to allow the correct translation of mRNA, and a stop codon. The vector can encode a selectable marker, such as a marker encoding drug resistance (e.g., ampicillin or tetracycline resistance).

[0446] In order to obtain the high level expression of the cloned gene, it is desirable to construct an expression cassette containing a strong promoter to guide transcription, a ribosome bind site for translation initiation (internal ribosome bind sequence), and transcription / translation terminator. For Escherichia coli, this includes promoters such as T7, trp, lac or lambda promoters, ribosome bind site, and preferably transcription termination signal. For eukaryotic cells, control sequences include promoters / enhancers and polyadenylation sequences derived from, for example, immunoglobulin genes, HTLV, SV40 or cytomegalovirus, and may also include indirect donor and / or acceptor sequences (for example, CMV and / or HTLV indirect acceptor and donor sequences). The expression cassette may be transferred into a selected host cell by known methods such as for Escherichia coli conversion or electroporation and for mammalian cell calcium phosphate treatment, electroporation or lipofection. The cell transformed through the expression cassette may be selected by the antibiotic resistance given by the gene contained in the expression cassette, for example amp, gpt, neo and hyg genes.

[0447] When the host is a eukaryotic cell, such a DNA transfection method can be used, such as calcium phosphate precipitation, conventional mechanical operations such as microinjection, electroporation, insertion of plasmids encapsulated in liposomes, or viral vectors. Eukaryotic cells can also be co-transfected with polynucleotide sequences encoding antibodies, labeled antibodies or antigen-binding fragments and a second foreign DNA molecule encoding a selectable phenotype such as herpes simplex virus thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express proteins (see, for example, Viral Expression Vectors, Springer press, Muzyczka ed., 2011). Those skilled in the art can easily use expression systems such as plasmids and vectors for producing proteins in cells including advanced eukaryotic cells such as COS, CHO, HeLa and myeloma cell lines.

[0448] Also provided is a cell group including at least one host cell described herein. The cell group may be a heterogeneous group, including a host cell comprising any described recombinant expression vector, plus at least one other cell, such as a host cell (e.g., T cell) that does not comprise any recombinant expression vector, or a cell different from a T cell, such as a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc. Alternatively, the cell group may be a substantially uniform group, wherein the cell group mainly includes a host cell (e.g., mainly consisting of them) comprising a recombinant expression vector. The cell group may also be a clonal group of cells, wherein all cells of the group are clones of a single host cell comprising a recombinant expression vector, so that all cells of the group include a recombinant expression vector. In one embodiment of the invention, the cell group is a clonal group including a host cell, and the host cell includes an expression vector described herein.

[0449] The nucleic acids encoding the polypeptides described herein can be modified without reducing their biological activity. Some modifications can be made to facilitate clonal expression or to incorporate targeting molecules into fusion proteins. These modifications are well known to those skilled in the art and include, for example, stop codons, methionine added at the amino terminus to provide a start site, additional amino acids placed at either end to generate restriction sites for convenient localization, or additional amino acids for their auxiliary effects (e.g., poly-His) in purification steps. In addition to recombinant methods, the immunoconjugates, effector modules, and antibodies of the present disclosure can also be constructed in whole or in part using standard peptide synthesis known in the art.

[0450] Once expressed, the antibodies, antigen-binding fragments, and conjugates can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, etc. (see generally, Simpson ed., Basic methods in Protein Purification and Analysis: A laboratory Manual, Cold Harbor Press, 2008). The antibodies, antigen-binding fragments, and conjugates do not have to be 100% pure. Once purified (partially or to homogeneity as desired), if it is to be used therapeutically, the polypeptide should be substantially free of endotoxins.

[0451] Methods for expressing antibodies, antigen-binding fragments and conjugates from mammalian cells and bacterial cells such as E. coli and / or refolding them into suitable active forms have been described, are well known, and can be applied to the antibodies disclosed herein. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, 2 nd, Cold Spring Harbor Laboratory, New York, 2013, Simpson ed., Basic methods in ProteinPurification and Analysis: A laboratory Manual, Cold Harbor Press, 2008, and Ward et al., Nature 341:544, 1989.

[0452] In addition to recombinant methods, these antibodies, antigen-binding fragments and / or conjugates can also be constructed in whole or in part using standard peptide synthesis. Solid phase synthesis of polypeptides can be accomplished by attaching the C-terminal amino acid sequence to an insoluble support, followed by sequential addition of the remaining amino acids in the sequence. The technique for solid phase synthesis is described by Barany & Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85: 2149-2156, 1963, and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill., 1984. Longer proteins can be synthesized by condensation of the amino and carboxyl termini of shorter fragments. Methods for forming peptide bonds by activating the carboxyl terminus (eg, using the coupling agent N,N'-dicyclohexylcarbodiimide) are well known in the art.

[0453] D. Methods and Compositions

[0454] 1. Treatment Methods

[0455] The methods disclosed herein are used to prevent or treat HIV-1 infection. Prevention includes inhibition of HIV-1 infection. The method includes contacting a cell with a therapeutically effective amount of a disclosed antibody, antigen binding fragment, or conjugate that specifically binds to gp41, or with a nucleic acid encoding such an antibody, antigen binding fragment, or conjugate. The method may also include contacting a subject with a therapeutically effective amount of a disclosed antibody, antigen binding fragment, or conjugate that specifically binds to gp41, or administering to a subject a nucleic acid encoding such an antibody, antigen binding fragment, or conjugate. In some instances, the antibody, antigen binding fragment, conjugate, or nucleic acid molecule may be used before exposure (e.g., to prevent or inhibit HIV-1 infection). In some instances, the antibody, antigen binding fragment, conjugate, or nucleic acid molecule may be used in post-exposure prevention. In some instances, the antibody, antigen binding fragment, conjugate, or nucleic acid molecule may be used to eliminate or reduce the viral reservoir of HIV-1 in a subject. For example, a therapeutically effective amount of an antibody, Fab, conjugate, or nucleic acid molecule can be administered to a subject with HIV-1, such as a subject being treated with antiviral therapy. In some instances, the antibody, Fab, conjugate, or nucleic acid molecule is modified so that it is directly cytotoxic to infected cells (e.g., by conjugation to a toxin), or so that natural defenses such as complement, antibody-dependent cellular cytotoxicity (ADCC), or macrophage phagocytosis are used.

[0456] For the method to be effective, HIV-1 infection need not be completely eliminated or prevented. For example, the method can reduce the HIV-1 infection of the desired amount, for example, compared with the HIV-1 infection lacking the treatment, reducing at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or even at least 100% (elimination of detectable HIV-1 infected cells). In some embodiments, the cell can also be contacted with other reagents such as antiviral agents of therapeutically effective amount. The cell can be in vivo or in vitro. The method includes the use of one or more other reagents known in the art. In other embodiments, HIV-1 replication can be reduced or suppressed by similar methods. For the method to be effective, HIV-1 replication need not be completely eliminated. For example, the method can reduce a desired amount of HIV-1 replication, such as by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or even at least 100% (elimination of detectable HIV-1) compared to HIV-1 replication in the absence of the treatment.

[0457] Methods for determining neutralizing activity include, but are not limited to, a single-cycle infection assay as described by Martin et al. (2003) Nature Biotechnology 21:71-76. In this assay, the level of viral activity is measured by a selectable marker whose activity reflects the amount of live virus in the sample and determines the IC 50 In other assays, acute infection can be monitored in the PM1 cell line or in primary cells (normal PBMCs). In this assay, the level of viral activity can be monitored by determining the p24 concentration using ELISA. See, e.g., Martin et al. (2003) Nature Biotechnology 21:71-76.

[0458] In one embodiment, the administration of the disclosed antibody, Fab, conjugate or nucleic acid molecule results in a reduction in the establishment of HIV-1 infection in the subject and / or a weakening of the subsequent HIV-1 disease progression. The reduction in the establishment of HIV-1 infection and / or the weakening of the subsequent HIV-1 disease progression encompass any statistically significant reduction in HIV-1 activity. In some embodiments, methods for treating a subject with HIV-1 infection are disclosed. These methods include administering a therapeutically effective amount of the disclosed antibody, Fab, conjugate or nucleic acid molecule to the subject, thereby preventing or treating the HIV-1 infection.

[0459] Studies have shown that when zidovudine is applied to HIV-infected women during pregnancy and childbirth and to children after birth, the mother-to-child transmission rate of HIV-1 can be significantly reduced (Connor et al., 1994 Pediatr Infect Dis J 14: 536-541). Several studies on mother-to-child transmission of HIV-1 have confirmed the correlation between maternal viral load and the risk of HIV-1 transmission to children during childbirth. The present disclosure provides antibodies, antigen-binding fragments, conjugates and nucleic acid molecules that can be used to reduce mother-to-child transmission of HIV. Therefore, in some instances, a therapeutically effective amount of gp41-specific antibodies or their antigen-binding fragments or nucleic acids encoding such antibodies or antibody antigen-binding fragments are administered to prevent HIV-1 from spreading from mother to baby or reduce the risk of HIV-1 spreading from mother to baby. In some instances, a therapeutically effective amount of antibodies, antigen-binding fragments or nucleic acids encoding such antibodies or antigen-binding fragments are administered to mothers and / or children during childbirth. In other examples, a therapeutically effective amount of an antibody, an antigen binding fragment, or a nucleic acid encoding the antibody or antigen binding fragment is administered to the mother and / or infant prior to breastfeeding to prevent or reduce the risk of viral transmission to the infant. In some embodiments, a therapeutically effective amount of an antibody, an antigen binding fragment, or a nucleic acid encoding the antibody or antigen binding fragment and a therapeutically effective amount of another agent, such as zidovudine, are administered to the mother and / or infant.

[0460] For any application, the antibody, antigen-binding fragment, conjugate or nucleic acid molecule can be combined with antiretroviral therapy. Antiretroviral drugs are broadly classified by the stage of the retroviral life cycle that the drug inhibits. The disclosed antibodies can be administered in combination with nucleoside analog reverse transcriptase inhibitors (e.g., zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine, entecavir and apricitabine), nucleoside reverse transcriptase inhibitors (e.g., tenofovir and adefovir), non-nucleoside reverse transcriptase inhibitors (e.g., efavirenz, nevirapine, delavirdine, etravirine ) and rilpivirine), protease inhibitors (e.g., saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir, fosamprenavir, atazanavir, tipranavir, and darunavir), entry or fusion inhibitors (e.g., maraviroc and enfuvirtide), maturation inhibitors (e.g., bevirimat and vivecon), or broad-spectrum inhibitors, such as natural antivirals. In some examples, the disclosed antibodies or active fragments thereof, or nucleic acids encoding them, are administered in combination with IL-15 or conjugated to IL-15.

[0461] Studies have shown that a mixture of HIV-1 neutralizing antibodies targeting different HIV-1 Env epitopes can treat SHIV chronically infected macaques (Shingai et al., Nature, 503, 277-280, 2013; and Barouch et al., Nature, 503, 224-228, 2013). Therefore, in some examples, one or more additional antibodies that bind to HIV-1 Env (e.g., it binds gp120 and / or gp41) and neutralize HIV-1 infection are also administered to the subject. The additional antibodies can be administered before, during, or after the administration of the novel antibodies disclosed herein (e.g., 10E8v4, 10E8v5, 10E8v4 S100cF or 10E8v4 V5R S100cF). In some embodiments, the additional antibody may be an antibody that specifically binds to an epitope on HIV-1 Env, such as the CD4 binding site (e.g., b12, 3BNC117, VRC01, VRC07, VRC07-523 antibodies), the V1 / V2 domain (e.g., PG9 antibodies, CAP256-VRC26), the V3 loop (e.g., 10-1074, PGT 121 or PGT128 antibodies), or those that bind to gp120 and gp41 subunits (e.g., 35022, PGT151 or 8ANC195). In these combined antibody treatment methods, these antibodies may be in IgG form or in another form. Antibodies that specifically bind to these regions and neutralize HIV-1 infection are known to those of ordinary skill in the art. Non-limiting examples can be found, for example, in PCT Publication Nos. WO 2011 / 038290, WO / 2013 / 086533, WO / 2013 / 090644, WO / 2012 / 158948, each of which is incorporated herein by reference in its entirety.

[0462] In some examples, DNA encoding an antibody or antigen-binding fragment thereof is administered to a subject, for example, to provide in vivo antibody production using the subject's cellular machinery. Immunization by nucleic acid constructs is well known in the art and is taught, for example, in U.S. Pat. No. 5,643,578, U.S. Pat. No. 5,593,972, and U.S. Pat. No. 5,817,637. U.S. Pat. No. 5,880,103 describes several methods for delivering encoding nucleic acids to an organism. One method of administering nucleic acids is direct administration of plasmid DNA, such as administration of a mammalian expression plasmid. The nucleotide sequence encoding the disclosed antibody or antigen-binding fragment thereof may be placed under the control of a promoter to increase expression. The method includes liposome delivery of nucleic acids. Such methods may be applied by a person of ordinary skill in the art to the production of antibodies or antigen-binding fragments thereof. In some embodiments, the disclosed antibodies or antigen-binding fragments are expressed in a subject using a pVRC8400 vector (described in Barouch et al., J. Virol, 79, 8828-8834, 2005, which is incorporated herein by reference).

[0463] Nucleic acid molecules encoding the disclosed antibodies (e.g., 10E8v4 S100cW-DS) or antigen-binding fragments can be included in a viral vector, for example, for expression of the antigen or antigen-binding fragment in a host cell or subject (e.g., a subject suffering from HIV-1 infection or at risk of HIV-1 infection). A variety of viral vectors that can be used to express the disclosed antibodies or antigen-binding fragments have been constructed, such as retroviral vectors, adenoviral vectors, or adeno-associated virus (AAV) vectors. In several examples, the viral vector can be replicable. For example, the viral vector can have a mutation in the viral genome that does not inhibit viral replication in the host cell. The viral vector can also be conditionally replicable. In other examples, the viral vector is replication-defective in the host cell.

[0464] In several embodiments, a therapeutically effective amount of an adeno-associated virus (AAV) viral vector comprising one or more nucleic acid molecules encoding a disclosed antibody or antigen-binding fragment (e.g., 10E8v4 S100cF antibody) can be administered to a subject (e.g., a human subject having HIV-1 infection or at risk of HIV-1 infection). The AAV viral vector is designed to express a nucleic acid molecule encoding a disclosed antibody or antigen-binding fragment, and administration of a therapeutically effective amount of the AAV viral vector to a subject results in expression of a therapeutically effective amount of the antibody or antigen-binding fragment in the subject. Non-limiting examples of AAV viral vectors that can be used to express the disclosed antibodies or antigen-binding fragments in a subject include those provided in Johnson et al. (“Vector-mediated gene transfer engenders long-lived neutralizing activity and protection against SIV infection in monkeys,” Nat. Med., 15(8):901-906, 2009) and Gardner et al. (“AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges,” Nature, 519(7541):87-91, 2015).

[0465] In one embodiment, nucleic acid encoding the disclosed antibodies or antigen-binding fragments thereof is introduced directly into cells. For example, the nucleic acid can be loaded into gold microspheres by standard methods and expressed by, for example, Bio-Rad's HELIOS TM The gene gun device is introduced into the skin. The nucleic acid can be "naked", consisting of a therapeutic under the control of a strong promoter.

[0466] Typically, the DNA is injected into muscle, although other sites may be injected directly. Dosages for injection are generally about 0.5 μg / kg to about 50 mg / kg, and typically about 0.005 mg / kg to about 5 mg / kg (see, e.g., U.S. Pat. No. 5,589,466).

[0467] In another embodiment, mRNA-based administration schemes can be used for delivering nucleic acids encoding disclosed antibodies into cells. In some embodiments, mRNA-based therapeutic agents based on nucleic acids can provide powerful alternatives for the methods mentioned above. mRNA therapeutic agents eliminate safety concerns about DNA integration into the host genome and can be directly translated in the host cell cytoplasm. In addition, this simple cell-free, in vitro synthetic RNA avoids the manufacturing complexity associated with the cell generation and purification of viral vectors or complex biomolecules. Two exemplary forms of delivery of RNA-based therapeutics that can be used to deliver nucleic acids encoding the disclosed antibodies include conventional non-amplified specific mRNA therapeutic delivery (see, e.g., Petsch et al., “Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection,” Nature biotechnology, 30(12):1210–6, 2012) and self-amplifying mRNA therapeutic delivery (see, e.g., Geall et al., “Nonviral delivery of self-amplifying RNA vaccines,” PNAS, 109(36):14604-14609, 2012; Magini et al., “Self-Amplifying mRNA Vaccines Expressing Multiple Conserved Influenza Antigens Confer Protection against Homologous and Heterosubtypic Viral Challenge,” PLoS One, 11(8):e0161193, 2016; and Brito et al., “Self-amplifying mRNA vaccines,” Adv Genet., 89:179-233, 2015).

[0468] 2. Dosage

[0469] The therapeutically effective amount of the gp41-specific antibody, antigen-binding fragment, conjugate, or nucleic acid molecule encoding these molecules will depend on the severity of the disease and / or infection and the overall condition of the patient's health. The therapeutically effective amount provides a subjective alleviation of symptoms or an objectively identifiable improvement noted by a physician or other qualified observer. The gp41-specific antibody, antigen-binding fragment, conjugate, or nucleic acid molecule encoding these molecules can be administered in combination with another therapeutic agent, either simultaneously or sequentially.

[0470] Single or multiple administrations of compositions comprising the disclosed gp41-specific antibodies, antigen-binding fragments, conjugates, or nucleic acid molecules encoding these molecules can be administered according to the dosage and frequency required and tolerated by the patient. Compositions comprising gp41-specific antibodies, antigen-binding fragments, conjugates, or nucleic acid molecules encoding these molecules should provide a sufficient amount of at least one of the gp41-specific antibodies, antigen-binding fragments, conjugates, or nucleic acid molecules encoding these molecules to effectively treat the patient. The dose can be administered once, but can be applied regularly until a therapeutic result is achieved or until side effects make it necessary to stop treatment. In one example, a dose of the antibody or antigen-binding fragment is infused for 30 minutes every other day. In this example, about 1 dose to about 10 doses can be administered, for example, 3 doses or 6 doses can be administered every other day. In another example, a continuous infusion of about 5 to about 10 days is administered. The subject can be treated regularly, for example, monthly, until the desired therapeutic result is achieved. Generally, the dose is sufficient to treat or alleviate the symptoms or signs of the disease without causing unacceptable toxicity to the patient.

[0471] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. This dosage is normally within the range that includes the ED 50 The dosage may vary within this range depending on the dosage form employed and the route of administration used. The therapeutically effective dose may be determined from cell culture assays and animal studies.

[0472] In certain embodiments, an antibody or antigen-binding fragment that specifically binds to gp41, or a conjugate thereof, or a nucleic acid molecule or vector encoding such a molecule, or a composition comprising such a molecule is administered at a dose ranging from about 5 or 10 nmol / kg to about 300 nmol / kg, or from about 20 nmol / kg to about 200 nmol / kg, or at a dose of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 125, 130, 140, 150, 160, 170, 175, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 750, 1000, 1250, 1500, 1750 or 2000 nmol / kg, or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 5 00, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 μg / kg, or at a dose of about 1, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg / kg, or at other doses deemed appropriate by the treating physician. In some embodiments, the antigen or antigen binding fragment can be administered to a subject at a dose of about 0.5 to about 40 mg / kg, such as about 1 to about 30, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 1 to about 5, about 1 to about 3, about 0.5 to about 40 mg / kg, such as about 0.5 to about 30, about 0.5 to about 20, about 0.5 to about 15, about 0.5 to about 10, about 0.5 to about 5, about 0.5 to about 3, about 3 to about 7, about 8 to about 12, about 15 to about 25, about 18 to about 22, about 28 to about 32, about 10 to about 20, about 5 to about 15, about 20 to about 40 mg / kg. The dosages described herein can be administered according to the dosing frequency / administration frequency described herein, including but not limited to daily, 2 or 3 times per week, weekly, every 2 weeks, every 3 weeks, monthly, every 1 month, etc.

[0473] In some embodiments, the disclosed therapeutic agent can be administered intravenously, subcutaneously or by other modes for a period of time daily or multiple times per week, followed by a period of no treatment, and then the cycle is repeated. In some embodiments only, the initial treatment time (e.g., daily or weekly multiple times) is 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks. In related embodiments, the time without treatment lasts for 3 days, 1 week, 2 weeks, 3 weeks or 4 weeks. In certain embodiments, the dosing regimen of the therapeutic agent is once a day for 3 days, followed by 3 days of no treatment; daily or weekly multiple times for 1 week, followed by 3 days or 1 week of no treatment; daily or weekly multiple times for 2 weeks, followed by 1 or 2 weeks of no treatment; daily or weekly multiple times for 3 weeks, followed by 1, 2 or 3 weeks of no treatment; or daily or weekly multiple times for 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks, followed by 1, 2, 3 or 4 weeks of no treatment.

[0474] 3. Mode of administration

[0475] The gp41-specific antibodies, antigen-binding fragments, conjugates, or nucleic acid molecules encoding these molecules or compositions comprising these molecules, and additional agents can be administered in various ways, including local and systemic administration, for example, by subcutaneous, intravenous, intraarterial, intraperitoneal, intramuscular, intradermal or intraspinal injection. In one embodiment, the therapeutic agent is administered by subcutaneous, intravenous, intraarterial, intraperitoneal, intramuscular, intradermal or intraspinal injection once a day. The therapeutic agent can also be administered by injection directly at or near the disease site.

[0476] The gp41-specific antibodies, antigen-binding fragments, conjugates, or nucleic acid molecules encoding these molecules, or compositions comprising these molecules can also be orally administered in the form of microspheres, microcapsules, liposomes (uncharged or charged (e.g., cationic)), polymer microparticles (e.g., polyamide, polylactic acid, polyglycolide, poly(lactide-glycolide)), microemulsions, etc.

[0477] Other methods of administration are by osmotic pumps (e.g., Alzet pumps) or miniature pumps (e.g., Alzet miniature osmotic pumps), which allow controlled, continuous and / or slow release delivery of the therapeutic agent or pharmaceutical composition at a predetermined time. The osmotic pump or miniature pump can be implanted subcutaneously or near the target site.

[0478] It will be apparent to those skilled in the art that the gp41-specific antibodies, antigen binding fragments, conjugates, or nucleic acid molecules encoding these molecules, or compositions comprising these molecules, may also be administered by other modes. It is within the skill of the skilled artisan to determine the most effective mode of administration. The gp41-specific antibodies, antigen binding fragments, conjugates, or nucleic acid molecules encoding these molecules, or compositions comprising these molecules, may be administered as pharmaceutical formulations suitable for, for example, oral (including oral and sublingual), rectal, nasal, topical, pulmonary, vaginal, or parenteral (including intramuscular, intraarterial, intraspinal, subcutaneous, or intravenous) administration, or in a form suitable for administration by inhalation or insufflation. Depending on the desired mode of administration, the pharmaceutical formulation may be a solid, liquid, semisolid, or liquid dosage form, such as a tablet, suppository, pill, capsule, powder, liquid, suspension, emulsion, ointment, cream, cream, lotion, etc. The formulation may be provided in a unit dosage form suitable for single administration of a precise dose. The formulation includes an effective amount of a therapeutic agent and one or more pharmaceutically acceptable excipients, carriers and / or diluents, and optionally one or more other biologically active agents.

[0479] 4. Composition

[0480] Compositions are provided, which include one or more of the gp41-specific antibodies, antigen binding fragments, conjugates, or nucleic acid molecules encoding these molecules disclosed herein in a carrier. The composition can be used, for example, to treat or detect HIV-1 infection. The composition can be prepared in a unit dosage form for administration to a subject. The amount and timing of administration are determined by the treating physician to achieve the desired purpose. The gp41-specific antibodies, antigen binding fragments, conjugates, or nucleic acid molecules encoding these molecules can be formulated for systemic or local administration. In one example, the gp41-specific antibodies, antigen binding fragments, conjugates, or nucleic acid molecules encoding these molecules are formulated for parenteral administration, such as intravenous administration.

[0481] In some embodiments, the composition comprises at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) pure antibodies, antigen-binding fragments, or conjugates thereof. In certain embodiments, the composition contains less than 10% (e.g., less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or even less) of macromolecular contaminants, such as other mammalian (e.g., human) proteins.

[0482] The composition for administration comprises a solution of the gp41-specific antibody, antigen binding fragment, conjugate or nucleic acid molecule encoding these molecules dissolved in a pharmaceutically acceptable carrier such as an aqueous carrier. Various aqueous carriers can be used, for example, buffered saline, etc. These solutions are sterile and generally free of undesirable substances. These compositions can be sterilized by conventional, well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjustment and buffering agents, toxicity regulators, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the antibody in these formulations can vary widely and will be selected based primarily on fluid volume, viscosity, body weight, etc., according to the specific mode of administration selected and the needs of the subject.

[0483] Typical compositions for intravenous administration include about 0.01 to about 50 mg / kg of the antibody or antigen-binding fragment or conjugate / subject / day (or a corresponding dose of a conjugate including the antibody or antigen-binding fragment). Actual methods for preparing the administrable compositions will be known or apparent to those skilled in the art and are described in more detail in publications such as Remington's Pharmaceutical Science, 22nd ed., Pharmaceutical Press, London, UK (2012). In some embodiments, the composition may be a liquid formulation comprising one or more antibodies, antigen-binding fragments (e.g., antibodies or antigen-binding fragments that specifically bind gp41) in a concentration range of about 0.1 mg / ml to about 40 mg / ml or about 0.5 mg / ml to about 20 mg / ml or about 1 mg / ml to about 20 mg / ml or about 0.1 mg / ml to about 10 mg / ml or about 0.5 mg / ml to about 10 mg / ml or about 1 mg / ml to about 10 mg / ml.

[0484] The antibodies or antigen-binding fragments or conjugates thereof or nucleic acid molecules encoding these molecules can be provided in lyophilized form and rehydrated using aseptic techniques prior to administration, although they can also be provided as sterile solutions of known concentrations. The antibody solution or antigen-binding fragment or nucleic acid encoding these antibodies or antigen-binding fragments can then be added to an infusion bag containing 0.9% sodium chloride, USP, and typically administered at a dose of 0.5 to 15 mg / kg. There is considerable experience available in the art in the administration of antibody drugs, with antibody drugs approved since 1997. It has been on the market in the United States since 2001. Antibodies, antigen binding fragments, conjugates, or nucleic acids encoding these molecules can be administered by slow infusion, rather than by intravenous push or bolus administration. In one example, a higher loading dose is administered, followed by a maintenance dose administered at a lower level. For example, an initial 4 mg / kg loading dose can be infused over approximately 90 minutes, followed by a 2 mg / kg maintenance dose infused over 30 minutes per week for 4 to 8 weeks (if the previous dose is well tolerated).

[0485] The parenteral formulations of controlled release can be prepared as implants, oily injections or as particle systems. For an overview of protein delivery systems, see Banga, AJ, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particle systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres and nanoparticles. Microcapsules contain therapeutic proteins as central cores, such as cytotoxins or drugs. In microspheres, the therapeutic agent is dispersed in particles. Particles, microspheres and microcapsules less than about 1 μm are generally referred to as nanoparticles, nanospheres and nanocapsules, respectively. Capillaries have a diameter of about 5 μm, so that only nanoparticles are administered intravenously. Microparticles are generally about 100 μm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatment on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).

[0486] Polymers can be used for the ion controlled release of antibody compositions disclosed herein. Various degradable and non-degradable polymer matrices for controllable drug delivery are known in the art (Langer, Accounts Chem. Res. 26: 537-542, 1993). For example, block copolymer poloxamer 407 is a viscous and flowing liquid at low temperatures, but forms a semisolid gel at body temperature. It has been shown to be an effective carrier for the formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9: 425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44 (2): 58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm. 112: 215-224, 1994). On the other hand, liposomes are used for controlled release and drug targeting of lipid-encapsulated drugs (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Many additional systems for the controlled delivery of therapeutic proteins are known (see U.S. Pat. No. 5,055,303; U.S. Pat. No. 5,188,837; U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; U.S. Pat. No. 4,957,735; U.S. Pat. No. 5,019,369; U.S. Pat. No. 5,055,303; U.S. Pat. No. 5,514,670; U.S. Pat. No. 5,413,797; U.S. Pat. No. 5,268,164; U.S. Pat. No. 5,004,697; U.S. Pat. No. 4,902,505; U.S. Pat. No. 5,506,206; U.S. Pat. No. 5,271,961; U.S. Pat. No. 5,254,342 and U.S. Pat. No. 5,534,496).

[0487] 5. Detection and diagnostic methods

[0488] Also provided is a method for in vitro or in vivo detection of the expression of gp41. In one example, the expression of gp41 is detected in a biological sample and can be used to detect HIV-1 infection as the presence of HIV-1 in the sample. The sample can be any sample, including but not limited to tissues from biopsies, autopsies, and case specimens. Biological samples also include tissue sections, for example, frozen sections obtained for histological purposes. Biological samples also include body fluids, such as blood, serum, plasma, sputum, cerebrospinal fluid, or urine. The detection method includes contacting an antibody or antigen-binding fragment or a conjugate thereof (e.g., a conjugate including a detectable marker) that specifically binds to gp41 with a cell or sample or being administered to a subject under conditions sufficient to form an immune complex, and detecting the immune complex (e.g., by detecting a detectable marker conjugated to the antibody or antigen-binding fragment).

[0489] In several embodiments, methods for detecting HIV-1 infection in a subject are provided. The present disclosure provides methods for detecting HIV-1 in a biological sample, wherein the method comprises contacting a biological sample from a subject with a disclosed antibody or antigen-binding fragment under conditions sufficient to form an immune complex, and detecting the immune complex to detect the gp41 in the biological sample. In one example, detection of gp41 in a sample indicates that the subject has HIV-1 infection. In another example, detection of gp41 in a sample verifies the diagnosis of HIV-1 infection in a subject.

[0490] In some embodiments, the disclosed antibodies or antigen-binding fragments thereof are used to test vaccines. For example, a vaccine composition comprising gp41 is tested to see if it presents a conformation comprising the 10E8 epitope. Therefore, provided herein is a method for testing a vaccine, wherein the method comprises contacting a sample containing a vaccine, such as an HIV-1 Env immunogen, with the disclosed antibody or antigen-binding fragment under conditions sufficient to form an immune complex, and detecting the immune complex to detect a vaccine having an HIV-1 immunogen comprising the 10E8 epitope in the sample. In one example, detection of immune complexes in a sample indicates that a vaccine component, such as an HIV-1 Env immunogen, presents a conformation capable of binding to the antibody or antigen-binding fragment.

[0491] In one embodiment, the antibody or Fab is directly labeled with a detectable label. In another embodiment, the antibody (first antibody) in conjunction with HIV-1Env is unlabeled, and a second antibody or other molecules that can bind to the antibody in conjunction with the first antibody are used for detection. As known to those skilled in the art, a second antibody that can specifically bind to the first antibody of a specific species and class is selected. For example, if the first antibody or human IgG, the second antibody can be anti-human IgG. Other molecules that can bind to antibodies include, but are not limited to, protein A and protein G, which are all commercially available.

[0492] Suitable labels for the antibody, antigen-binding fragment, or secondary antibody are as described above, and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. A non-limiting example of a luminescent material is luminol; a non-limiting exemplary magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include 125 I. 131 I. 35 S or 3 H.

[0493] E. Kit

[0494] Kits are also provided. For example, a kit for treating a subject with HIV-1 infection, or for detecting gp41 in a sample or in a subject. The kit will typically include a disclosed gp41-specific antibody, antigen-binding fragment, conjugate, or nucleic acid molecule encoding these molecules, or a composition comprising these molecules. More than one disclosed gp41-specific antibody, antigen-binding fragment, conjugate, or nucleic acid molecule encoding these molecules, or a composition comprising these molecules may be included in the kit.

[0495] In one embodiment, the kit is a diagnostic kit and includes an immunoassay. Although the details of the immunoassay may vary depending on the specific format used, the method of detecting gp41 in a biological sample generally includes the step of contacting the biological sample with a specifically reactive antibody under conditions sufficient to form an immune complex with gp41. The antibody allows specific binding under immunologically reactive conditions to form an immune complex, and directly or indirectly detects the presence of the immune complex (bound antibody).

[0496] The kit includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed of various materials such as glass or plastic. The container typically contains a composition including one or more disclosed antibodies, Fabs, conjugates, nucleic acid molecules or compositions. In several embodiments, the container can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle). The label or package insert indicates that the composition is used to treat a specific condition.

[0497] The label or package insert will typically also include instructions for use of the antibody, Fab, conjugate, nucleic acid molecule or composition included in the kit. Package inserts typically include instructions typically included in the commercial packaging of therapeutic products, which include information about the indications, usage, dosage, application, contraindications and / or warnings for using such therapeutic products. The illustrative material can be written in electronic form (e.g., computer disk or compressed disk) or can be imaged (e.g., video file). The kit can also include additional components to facilitate the specific application targeted by the kit. Therefore, for example, the kit can additionally contain means for detecting labels (e.g., enzyme substrates for enzyme labels, filter sets for detecting fluorescent labels, suitable second labels such as second antibodies, etc.). The kit can also include conventional buffers and other reagents for implementing ad hoc methods. Such a kit and suitable contents are well known to those skilled in the art.

[0498] F. Alternative Embodiments

[0499] Item 1. An isolated monoclonal antibody or antigen-binding fragment comprising:

[0500] (A) Heavy chain variable region (V H ), which includes V shown in SEQ ID NO:35 (10E8v4 S100cW) H heavy chain complementarity determining region (HCDR) 1, HCDR2 and HCDR3; and light chain variable region (V L ), which includes V shown in SEQ ID NO:6 (rL3-6mut) L light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3;

[0501] (B) V comprising HCDR1, HCDR2 and HCDR3 of the variable region shown in SEQ ID NO: 36 (10E8v4 DS) H , and a V region comprising LCDR1, LCDR2 and LCDR3 of the variable region shown in SEQ ID NO:41 (rL3-6mut-DS) L ;or

[0502] (C) a V sequence comprising HCDR1, HCDR2 and HCDR3 of the variable region shown in SEQ ID NO: 37 (10E8v4 S100cW-DS) H , and a V region comprising LCDR1, LCDR2 and LCDR3 of the variable region shown in SEQ ID NO:41 (rL3-6mut-DS) L ;as well as

[0503] The monoclonal antibody or antigen-binding fragment specifically binds to gp41 and neutralizes HIV-1.

[0504] Item 2. The isolated monoclonal antibody or antigen-binding fragment of Item 1, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3

[0505] comprising the amino acid sequences shown in SEQ ID NOs: 7, 8, 52, 10, 11 and 12 (10E8v4 / v5 S100cW IMGT CDRs), respectively;

[0506] comprising the amino acid sequences shown in SEQ ID NOs: 7, 8, 53, 55, 11 and 12 (10E8v4 / v5 DS IMGT CDRs), respectively;

[0507] Or include the amino acid sequences shown in SEQ ID NOs: 7, 8, 54, 55, 5 and 6 (10E8v4 / v5 S100cW-DS IMGT CDRs), respectively.

[0508] Item 3. The isolated monoclonal antibody or antigen-binding fragment of Item 1, wherein V H and V L

[0509] Respectively include the amino acid sequences shown in SEQ ID NO: 35 and 6 (10E8v4 S100cW);

[0510] comprising the amino acid sequences shown in SEQ ID NOs: 36 and 41 (10E8v4 DS), respectively;

[0511] Respectively include the amino acid sequences shown in SEQ ID NO: 37 and 41 (10E8v4 S100cW-DS);

[0512] comprising the amino acid sequences shown in SEQ ID NOs: 38 and 6 (10E8v5 S100cW), respectively;

[0513] comprising the amino acid sequences shown in SEQ ID NOs: 39 and 41 (10E8v5 DS), respectively; or

[0514] They respectively include the amino acid sequences shown in SEQ ID NOs: 40 and 41 (10E8v5 S100cW-DS).

[0515] Item 4. An isolated monoclonal antibody or antigen-binding fragment comprising:

[0516] (A) heavy chain and light chain variable regions comprising the amino acid sequences shown in SEQ ID NOs: 5 and 6 (10E8v4), respectively;

[0517] (B) heavy chain and light chain variable regions comprising the amino acid sequences shown in SEQ ID NOs: 3 and 6 (10E8v5), respectively;

[0518] The monoclonal antibody or antigen-binding fragment specifically binds to gp41 and neutralizes HIV-1.

[0519] Item 5. The isolated antibody or antigen-binding fragment of any of the preceding items, wherein LCDR1 further comprises an amino acid substitution of a serine residue at kabat position 32.

[0520] Item 6. The isolated antibody or antigen-binding fragment of Item 5, wherein the amino acid substitution is a Y32F substitution.

[0521] Item 7. The isolated antibody or antigen-binding fragment of Item 5, wherein the amino acid substitution is a Y32W substitution.

[0522] Item 8. The isolated monoclonal antibody or antigen-binding fragment of Item 5, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3

[0523] comprising the amino acid sequences shown in SEQ ID NOs: 7, 8, 9, 56, 11 and 12 (10E8v4 / v5 Y32W IMGT CDRs), respectively;

[0524] comprising the amino acid sequences shown in SEQ ID NOs: 7, 8, 52, 56, 11 and 12 (10E8v4 / v5 S100cW Y32W IMGT CDRs), respectively;

[0525] comprising the amino acid sequences shown in SEQ ID NOs: 7, 8, 53, 85, 11 and 12 (10E8v4 / v5 DS Y32W IMGT CDRs), respectively;

[0526] comprising the amino acid sequences shown in SEQ ID NOs: 7, 8, 54, 85, 5 and 6 (10E8v4 / v5 S100cW-DS Y32W IMGT CDRs), respectively;

[0527] comprising the amino acid sequences shown in SEQ ID NOs: 7, 8, 52, 57, 11 and 12 (10E8v4 / v5 S100cW Y32F IMGT CDRs), respectively;

[0528] comprising the amino acid sequences shown in SEQ ID NOs: 7, 8, 53, 86, 11 and 12 (10E8v4 / v5 DS Y32F IMGT CDRs), respectively; or

[0529] They respectively include the amino acid sequences shown in SEQ ID NOs: 7, 8, 54, 86, 5 and 6 (10E8v4 / v5 S100cW-DS Y32F IMGT CDRs).

[0530] Item 9. The isolated monoclonal antibody or antigen-binding fragment of Item 5, wherein V H and V L

[0531] Respectively include the amino acid sequences shown in SEQ ID NO: 5 and 41 (10E8v4 Y32W);

[0532] Respectively include the amino acid sequences shown in SEQ ID NO: 35 and 6 (10E8v4 S100cW);

[0533] comprising the amino acid sequences shown in SEQ ID NOs: 36 and 41 (10E8v4 DS), respectively;

[0534] Respectively include the amino acid sequences shown in SEQ ID NO: 37 and 41 (10E8v4 S100cW-DS);

[0535] Respectively include the amino acid sequences shown in SEQ ID NOs: 3 and 41 (10E8v5 Y32W);

[0536] Respectively include the amino acid sequences shown in SEQ ID NO: 38 and 6 (10E8v5 S100cW Y32W);

[0537] comprising the amino acid sequences shown in SEQ ID NOs: 39 and 41 (10E8v5 DS Y32W), respectively; or

[0538] They respectively include the amino acid sequences shown in SEQ ID NOs: 40 and 41 (10E8v5 S100cW-DS Y32W).

[0539] Item 10. The antibody or antigen-binding fragment of any of the preceding items, further comprising one or more gain-of-function amino acid substitutions listed in Table 3 or Table 4.

[0540] Item 11. The isolated antibody or antigen-binding fragment of any of the preceding items, wherein the antibody or antigen-binding fragment is soluble in phosphate-buffered saline, pH 7.4 at 20°C to a concentration of at least 5 mg / ml.

[0541] Item 12. The isolated antibody or antigen-binding fragment of any of the preceding items, wherein the antibody or antigen-binding fragment has an IC of 50 μg / ml or less. 50 Neutralize at least 95% of the Fig.16A The virus strains in .

[0542] Item 13. The antibody or antigen-binding fragment of any of the preceding items, comprising a human framework region.

[0543] Item 14. The antibody of any of the preceding items, comprising a human constant region.

[0544] Item 15. The antibody of any of the preceding items, wherein the antibody is IgG, IgM or IgA.

[0545] Item 16. The antibody of any of the preceding items, comprising a recombinant constant domain comprising a modification that increases binding to a neonatal Fc receptor.

[0546] Item 17. The antibody of Item 16, wherein the antibody is IgG1, and the modifications that increase binding to the neonatal Fc receptor include M428L and N434S amino acid substitutions.

[0547] Item 18. An antigen-binding fragment of any one of Items 1 to 13.

[0548] Item 19. The antigen-binding fragment of Item 18, wherein the antigen-binding fragment is Fv, Fab, F(ab') 2 , scFV or scFV 2 Fragment.

[0549] Item 20. A bispecific antibody comprising the isolated human monoclonal antibody or antigen-binding fragment of any of the preceding items.

[0550] Item 21. The bispecific antibody of Item 20, wherein the antibody specifically binds gp41 and CD3.

[0551] Item 22. The antibody or antigen-binding fragment of any of the preceding items, linked to an effector molecule or a detectable label.

[0552] Item 23. The antibody or antigen-binding fragment of Item 22, wherein the detectable label is a fluorescent, enzymatic or radioactive label.

[0553] Item 24. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment of any one of Items 1 to 21.

[0554] Item 25. V encoding the antibody or antigen-binding fragment of any one of Items 1 to 21 H 、V L or V Hand V L Isolated nucleic acid molecules.

[0555] Item 26. The nucleic acid molecule of any one of Items 21 to 24, wherein

[0556] The V of the antibody or antigen-binding fragment H comprising the nucleic acid sequence shown in SEQ ID NO:44;

[0557] The V of the antibody or antigen-binding fragment H comprising the nucleic acid sequence shown in SEQ ID NO:45;

[0558] The V of the antibody or antigen-binding fragment L comprising the nucleic acid sequence shown in SEQ ID NO:47;

[0559] The V of the antibody or antigen-binding fragment H and V L Respectively include the nucleic acid sequences shown in SEQ ID NOs: 44 and 47;

[0560] The V of the antibody or antigen-binding fragment H and V L They include the nucleic acid sequences shown in SEQ ID NOs: 45 and 47, respectively.

[0561] Item 27. The nucleic acid molecule of any one of Items 24 to 26, operably linked to a promoter.

[0562] Item 28. An expression vector comprising the nucleic acid molecule of any one of Items 24 to 27.

[0563] Item 29. The expression vector of Item 28, wherein the expression vector is a viral vector.

[0564] Item 30. The expression vector of Item 29, wherein the viral vector is an adeno-associated viral vector.

[0565] Item 31. A host cell comprising the nucleic acid molecule of Item 27, particularly wherein the host cell is a eukaryotic host cell such as a human host cell.

[0566] Item 32. A pharmaceutical composition for treating HIV-1 infection, comprising:

[0567] A therapeutically effective amount of the antibody, antigen-binding fragment, nucleic acid molecule or expression vector of any one of items 1 to 21; and

[0568] A pharmaceutically acceptable carrier.

[0569] Item 33. A method for producing an antibody or antigen-binding fragment that specifically binds to gp41, comprising:

[0570] incubating the host cell of item 31 under in vitro conditions sufficient to express the nucleic acid molecule or expression vector to produce the antibody or antigen-binding fragment; and

[0571] The antibody or antigen-binding fragment is purified.

[0572] Item 34. A method for detecting HIV-1 infection in a subject, comprising:

[0573] contacting a biological sample from the subject with the antibody or antigen-binding fragment of any one of items 1 to 23 under conditions sufficient to form an immune complex; and

[0574] The sample is detected for the presence of the immune complex, wherein the presence of the immune complex on the sample indicates that the subject has HIV-1 infection.

[0575] Item 35. A method for inhibiting or treating HIV-1 infection in a subject, comprising administering to the subject a therapeutically effective amount of the antibody, antigen-binding fragment, nucleic acid molecule or expression vector of any one of Items 1 to 30, thereby preventing or treating the HIV-1 infection.

[0576] Item 36. The method of Item 35, wherein the subject is at risk for HIV-1 infection.

[0577] Item 37. The method of Item 35, wherein the subject has HIV-1 infection.

[0578] Item 38. The method of Item 37, wherein the subject has AIDS.

[0579] Item 39. The method of any one of Items 35 to 38, further comprising administering to the subject an additional antibody, antigen-binding fragment, or a nucleic acid encoding the additional antibody or antigen-binding fragment, wherein the additional antibody or antigen-binding fragment specifically binds to HIV-1 Env and neutralizes HIV-1 infection.

[0580] Item 40. The method of Item 39, wherein the additional antibody is a VRC01 class antibody.

[0581] Item 41. The method of Item 40, wherein the VRC01 class antibody is VRC01 or VRC07-523.

[0582] Item 42. Use of the antibody, antigen-binding fragment, nucleic acid molecule, expression vector or pharmaceutical composition of any one of Items 1 to 30 or Item 32 for inhibiting or treating HIV-1 infection in a subject.

[0583] The present invention also relates to the following scheme:

[0584] Item 1. An isolated antibody comprising:

[0585] Heavy chain variable region (V H ), the heavy chain variable region includes V shown in SEQ ID NO: 75 H heavy chain complementarity determining region (HCDR) 1, HCDR2 and HCDR3; and light chain variable region (V L ), the light chain variable region includes V shown in SEQ ID NO:6 L light chain complementarity determining regions (LCDR) 1, LCDR2 and LCDR3 (10E8v4 S100cF CDRs); and

[0586] The antibody specifically binds to gp41 and neutralizes HIV-1.

[0587] Item 2. The isolated antibody of Item 1, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 7, 8, 77, 10, 11 and 12, respectively (10E8v4S100cF IMGT CDRs).

[0588] Item 3. The isolated antibody of Item 1 or Item 2, wherein

[0589] The V H including arginine, glutamic acid, serine, lysine, aspartic acid, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82c, 84, 87, 89, 105, and 110, respectively; and

[0590] The V L Alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid and aspartic acid residues are included at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83 and 85, respectively.

[0591] Item 4. The isolated antibody of any one of Items 1 to 3, comprising an arginine residue at kabat position 5.

[0592] Item 5. The isolated antibody of any one of Items 1 to 4, wherein the V H and V L They include amino acid sequences that are at least 90% identical to SEQ ID NO: 75 and 6 (10E8v4 S100cF), respectively.

[0593] Item 6. The isolated antibody of any one of Items 1 to 4, wherein the V H and V L They include amino acid sequences that are at least 90% identical to SEQ ID NO: 76 and 6 (10E8v4 V5R S100cF), respectively.

[0594] Item 7. The isolated antibody of Item 1, wherein the V H and V L They respectively include the amino acid sequences shown in SEQ ID NOs: 75 and 6 (10E8v4 S100cF).

[0595] Item 8. The isolated antibody of Item 1, wherein the V H and V L They respectively include the amino acid sequences shown in SEQ ID NOs: 76 and 6 (10E8v4 V5R S100cF).

[0596] Item 9. An isolated antibody comprising:

[0597] Heavy chain variable region (V H ), the heavy chain variable region includes V shown in SEQ ID NO:5 H heavy chain complementarity determining region (HCDR) 1, HCDR2 and HCDR3; and light chain variable region (V L ), the light chain variable region includes V shown in SEQ ID NO:6 L The light chain complementarity determining regions (LCDR) 1, LCDR2 and LCDR3 (10E8v4 CDRs), wherein

[0598] The V H including arginine, glutamic acid, serine, lysine, aspartic acid, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82C, 84, 87, 89, 105, and 110, respectively; and

[0599] The V L comprising alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid, and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively; and

[0600] The antibody specifically binds to gp41 and neutralizes HIV-1.

[0601] Item 10. The isolated antibody of Item 9, wherein the VH and V L They include amino acid sequences that are at least 90% identical to SEQ ID NO: 5 and 6 (10E8v4), respectively.

[0602] Item 11. The isolated antibody of Item 9, wherein the V H and V L They include the amino acid sequences shown in SEQ ID NOs: 5 and 6, respectively (10E8v4).

[0603] Item 12. The isolated antibody of any one of Items 1 to 8, wherein

[0604] (a) V H Relative to SEQ ID NO:76 further comprising one of E1R, E1W, G15R, S25W, D28W, D30W, D30R, N31W, N31R, P52bR, D72R or E81R substitutions (kabat numbering);

[0605] (b) V H Relative to SEQ ID NO:76, also including D72R, E1R D28W and P52bR substitutions (kabat numbering);

[0606] (c) V H Relative to SEQ ID NO:76 also includes a D28Y substitution (kabat numbering);

[0607] (d) V L Relative to SEQ ID NO:6 further comprising one of the substitutions D7W, A14R, Q17R, T18R, T20R, D26R, K51R, N53R, S65R, G68R, G77R or G100R (kabat numbering);

[0608] (e) V L Relative to SEQ ID NO: 6, also including D7W, D26R and G68R substitutions (kabat numbering);

[0609] (f) V H Relative to SEQ ID NO: 76, further comprising D72R, E1R D28W and P52bR substitutions (Kabat numbering), the V L Relative to SEQ ID NO: 6, also including D7W, D26R and G68R substitutions (kabat numbering);

[0610] (g) V H Relative to SEQ ID NO: 76, further comprising a P52bR substitution (kabat numbering), the V LRelative to SEQ ID NO: 6, also includes D7W and G68R substitutions (kabat numbering);

[0611] (h) V H and V L Also included with respect to SEQ ID NOs: 76 and 6, respectively, are one of the following paired substitutions: E1R and D7W; E1R and D26R; E1R and G68R; D28W and D7W; D28W and D26R; D28W and G68R; D28W and Q17R; D28W and T18R; D28W and T20R; D28W and K51R; D28W and G77R; P52bR and D7W; P52bR and D26R; P52bR and G68R; P52bR and Q17R; P52bR and T18R; P52bR and T20R; P52bR and K51R; P52bR and G77R; D72R and D7W; D72R and D26R; or D72R and G68R; and

[0612] The antibody specifically binds to gp41 and neutralizes HIV-1.

[0613] Item 13. The isolated antibody of any of the preceding items, wherein the antibody is dissolved in phosphate buffered saline, pH 7.4 at 20°C to a concentration of at least 5 mg / ml.

[0614] Item 14. The isolated antibody of any of the preceding items, wherein the antibody has an IC of 50 μg / ml or less. 50 Neutralizes at least 95% of the virus strains listed in FIG. 16 .

[0615] Item 15. The isolated antibody of any of the preceding items, comprising a human framework region.

[0616] Item 16. The isolated antibody of any of the preceding items, comprising a human constant region.

[0617] Item 17. The isolated antibody of any of the preceding items, wherein the antibody is IgG, IgM or IgA.

[0618] Item 18. The isolated antibody of any of the preceding items, comprising a recombinant constant region comprising a modification that increases binding to a neonatal Fc receptor.

[0619] Item 19. The isolated antibody of Item 18, wherein the antibody is IgG1, and the modifications that increase binding to the neonatal Fc receptor include M428L and N434S amino acid substitutions.

[0620] Item 20. An antigen-binding fragment of the antibody of any one of Items 1 to 19.

[0621] Item 21. The antigen-binding fragment of Item 20, wherein the antigen-binding fragment is Fv, Fab, F(ab') 2 , scFV or scFV 2 Fragment.

[0622] Item 22. A bispecific antibody comprising the antibody or antigen-binding fragment of any of the preceding items.

[0623] Item 23. The antibody or antigen-binding fragment of any of the preceding items, linked to an effector molecule or a detectable label.

[0624] Item 24. The antibody or antigen-binding fragment of Item 23, wherein the detectable label is a fluorescent, enzymatic or radioactive label.

[0625] Item 25. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment of any one of Items 1 to 24.

[0626] Item 26. V encoding the antibody or antigen-binding fragment of any one of Items 1 to 24 H 、V L or V H and V L Isolated nucleic acid molecules.

[0627] Item 27. The nucleic acid molecule of any one of Items 25 to 26, wherein:

[0628] The V H comprising the nucleic acid sequence shown in SEQ ID NO:45 (H6-511-4mut), SEQ ID NO:78 (H6-511-4mut S100cF) or SEQ ID NO:79 (H6-511-4mut V5R S100cF); or

[0629] The V L comprising the nucleic acid sequence shown in SEQ ID NO:47 (rL3-6mut); or

[0630] The V H and V L They include the nucleic acid sequences shown in SEQ ID NOs: 45 and 47 (10E8v4), 78 and 47 (10E8v4 S100cF), or 79 and 47 (10E8v4 V5R, S100cF).

[0631] Item 28. The nucleic acid molecule of any one of Items 25 to 27, operably linked to a promoter.

[0632] Item 29. An expression vector comprising the nucleic acid molecule of any one of Items 25 to 28.

[0633] Item 30. A host cell comprising the nucleic acid molecule of any one of Items 25 to 29, particularly wherein the host cell is a eukaryotic host cell such as a human host cell.

[0634] Item 31. A pharmaceutical composition for treating HIV-1 infection, comprising:

[0635] A therapeutically effective amount of the antibody, antigen-binding fragment, nucleic acid molecule or expression vector of any one of items 1 to 29; and

[0636] A pharmaceutically acceptable carrier.

[0637] Item 32. A method for producing an antibody or antigen-binding fragment that specifically binds to gp41, comprising:

[0638] incubating the host cell of item 30 under in vitro conditions sufficient to express the nucleic acid molecule or expression vector to produce the antibody or antigen-binding fragment; and

[0639] The antibody or antigen-binding fragment is purified.

[0640] Item 33. A method for detecting HIV-1 infection in a subject, comprising:

[0641] contacting a biological sample from the subject with the antibody or antigen-binding fragment of any one of items 1 to 24 under conditions sufficient to form an immune complex; and

[0642] The sample is detected for the presence of the immune complex, wherein the presence of the immune complex on the sample indicates that the subject has HIV-1 infection.

[0643] Item 34. A method for inhibiting or treating HIV-1 infection in a subject, comprising administering to the subject a therapeutically effective amount of the antibody, antigen-binding fragment, nucleic acid molecule or expression vector of any one of Items 1 to 29, thereby preventing or treating the HIV-1 infection.

[0644] Item 35. The method of Item 34, wherein the subject is at risk for infection with HIV-1.

[0645] Item 36. The method of Item 34, wherein the subject has HIV-1 infection.

[0646] Item 37. The method of Item 36, wherein the subject has AIDS.

[0647] Item 38. The method of any one of Items 34 to 37, further comprising administering to the subject an additional antibody, antigen-binding fragment, or a nucleic acid encoding the additional antibody or antigen-binding fragment, wherein the additional antibody or antigen-binding fragment specifically binds to HIV-1 Env and neutralizes HIV-1 infection.

[0648] Item 39. The method of Item 38, wherein the additional antibody is a VRC01 class antibody.

[0649] Item 40. The method of Item 39, wherein the VRC01 class antibody is VRC01 or VRC07-523.

[0650] Item 41. Use of the antibody, antigen-binding fragment, nucleic acid molecule, expression vector or pharmaceutical composition of any one of Items 1 to 29 or Item 31 for inhibiting or treating HIV-1 infection in a subject.

[0651] III. Embodiment

[0652] The following examples are provided to illustrate specific features of certain embodiments, but the scope of the claims should not be limited to these exemplified features.

[0653] Example 1: Optimized 10E8 Antibody

[0654] This example illustrates the identification of modified 10E8 antibodies with improved combinations of neutralization, solubility, and autoreactivity properties compared to the parental 10E8 antibody.

[0655] The broadly neutralizing antibody 10E8 was isolated from HIV-1 infected donor N152 and recognizes a helix in the membrane-proximal region (MPER) just before the transmembrane region of the HIV-1 gp41 glycoprotein. 10E8 neutralizes 98% of a wide range of HIV-1 isolates with a 50% inhibitory concentration (IC 50 ) is about 0.32 μ / ml, which is significantly more efficient and broad-spectrum than other antibodies that bind to the MPER of gp41, such as 2F5 and 4E10 antibodies. For a description of the 10E8 antibody, see Huang et al. (Nature, 491: 406-412, 2012).

[0656] Previous efforts to improve the neutralizing properties of the 10E8 antibody identified the 10E8 V H The serine residue at kabat position 74 of HC6 was selected as a potential mutation site (see, U.S. Patent Publication 2014 / 0348785A1, Figure 44). For example, the 10E8 antibody with S74W substitution improved 10E8 neutralization by 3 to 5 fold (see U.S. Publication 2014 / 0348785A1, Figure 45). However, when the S74W substitution was incorporated into the HC6 V H(discussed below) and compared with 10E8 V L When paired, the resulting antibody was autoreactive by the cardiolipin antibody assay (see U.S. Publication 2014 / 0348785A1, FIG. 56 ), suggesting that antibodies with this substitution may not be optimal for therapeutic use.

[0657] Many clonal variants of the 10E8 heavy and light chains were identified by next generation sequencing of donor N152 samples and evaluated for function by pairing with complementary wild-type 10E8 chains (see pages 104 to 107 and Figures 50 to 52 of U.S. Publication No. 2014 / 0348785A1). Among the many identified clonal variants were HC6 V H (called gVRC-H2 dN152 and provided as SEQ ID NO: 154 in U.S. Publication No. 2014 / 0348785A1), H6 V H (called gVRC-H6 dN152 and provided as SEQ ID NO: 158 in U.S. Publication No. 2014 / 0348785A1), H8 V H (called gVRC-H8 dN152 and provided as SEQ ID NO: 160 in U.S. Publication No. 2014 / 0348785A1), L10 V L (called gVRC-L10 dN152 and provided as SEQ ID NO: 173 in U.S. Publication No. 2014 / 0348785A1), and L19 V L (called gVRC-L19 dN152 and provided as SEQ ID NO: 192 in U.S. Publication No. 2014 / 0348785A1).

[0658] 10E8 antibody V H Additional modification of HC6-S74Y V H (provided as SEQ ID NO: 192 in U.S. Publication No. 2014 / 0348785A1), and HC6-S74Y-511 V H (referred to as HC6_S77Y_hp_L02 and provided as SEQ ID NO: 201 in U.S. Publication No. 2014 / 0348785A1). L Modification of rL3 V L (referred to as "L3," or "10E8gL03" and provided as SEQ ID NO: 152 in U.S. Publication No. 2014 / 0348785A1).

[0659] Despite the improvements to the 10E8 antibody described above, there remains a need for additional 10E8 variants that are highly soluble, lack autoreactivity, and retain (or improve) the neutralizing capacity of wild-type 10E8.

[0660] The above antibody V H and V L The amino acid sequence of the domain is provided in Figures 1A to 1E middle. Figure 1A and 1B The CDRs of these heavy and light chain variable regions mapped by IMGT are shown. Figure 1C and Figure 1D The Kabat-mapped CDRs of these heavy and light chain variable regions are shown. Examples of amino acid sequences of the heavy and light chain variable regions discussed in this example are listed in Table 2.

[0661] Table 2. 10E8 variant sequences

[0662]

[0663] These modified 10E8 antibodies were essentially produced as previously described (see, e.g., Wu et al., Science, 333(6049):1593–1602, 2011 and Zhu et al., Front Microbiol 3(2012):315, 2012). Briefly, antibodies were expressed by co-transfecting Expi293F cells with equal amounts of paired heavy and light chain plasmids and purified using a recombinant protein A column (GE Healthcare). Unless otherwise indicated by the context, all antibody assays described in the Examples utilized variant 10E8 antibodies (with one or more amino acid substitutions in the variable region) in IgG format and were produced as described above.

[0664] One method of evaluating antibody solubility is to determine the turbidity of a solution containing the antibody by measuring the optical density OD of the solution at 350 nm. H and rL3 V L Antibodies, including HC6-S74Y-511 V H and rL3 V L The turbidity of the solution of the antibody or VRC01 antibody (which is highly soluble in aqueous solution) was measured. Briefly, these antibodies were expressed in animal cells and the resulting supernatant was passed through a protein A column. The column was washed and the bound antibody was eluted from the column using an elution buffer (10 mM glycine, pH 2.7) and dripped into a collection tube containing a 1 M Tris-HCl, pH 8.5 neutralization buffer. Figure 2As shown in , the eluate containing the 10E8 antibody or the HC6-S74Y / rL3 antibody exhibited substantially higher OD350 readings compared to the VRC01 antibody. This finding suggests that these two antibodies tend to aggregate and are less soluble than the VRC01 antibody. However, as determined by OD350, the HC6-S74Y-511 / rL3 antibody was significantly less turbid (i.e., more soluble) than the 10E8 and HC6-S74Y / rL3 antibodies ( Figure 2 ) and are equally effective in neutralizing HIV-1 ( Figure 3 ).

[0665] Additional heavy chain and light chain combinations were tested using a turbidity assay as a solubility screen ( FIG. 4 ). As shown in FIG. 4 , as determined by turbidity at OD350, the heavy chain and light chain combinations including H8 V H and L10 V L or L19V L or H6V H and L10 V L The antibodies in combination exhibited improved solubility.

[0666] To further improve the solubility of H6 and H8-based antibodies, V H The L72, I75 and F77 residues of the 10E8 V H The hydrophobic patch of (identified from the structure of the 10E8 / MPER complex, described in Huang et al., Nature, 491:406-412, 2012). Figure 4A As shown in FIG. 5A , compared to unmodified 10E8 antibody or modified 10E8 antibody including H6 / L10, H8 / L10 or H8 / L19, as determined by turbidity at OD350, the 10E8 antibody comprising H8-511 V H and L10 V L or H6-511 V H and L10 V L The antibody showed greatly improved solubility. H and L10 V L or H6-511 V H and L10 V L Each of the combinations was approximately 10-fold less effective in neutralizing HIV-1 strains ( Figure 4B ). This hydrophilic substitution is expected to reduce the overall hydrophobicity and increase the V H The solubility of the domains, but the drastic reduction in neutralization potency and breadth was not anticipated.

[0667] Designed H6-511 VH Additional modifications of were made in an attempt to improve HIV-1 neutralization without a corresponding decrease in antibody solubility. Figure 5 As shown in Figure 1, residues at kabat positions 28, 31, 52, and 98 are close to the epitope binding region of 10E8. H Kabat positions 28, 31, 52, and 98 are occupied by asparagine, aspartic acid, serine, and histidine residues, respectively. H (When with rL3 V L In the 1970s, 1980s, 1990s, 2000s, 2010s, 2011s, 2012s, 2013s, 2014s, 2015s, 2016s, 2017s, 2018s, 2019s, 2020s, 2021s, 2022s, 2023s, 2024s, 2025s, 2026s, 2027s, 2028s, 2029s, 2030s, 2040s, 2050s, 2060s, 2070s, 2080s, 2090s, 2110s, 2120s, 2091s, 2092s, 2093s, H The corresponding residue substitutions are from H6-511 V H Various combinations of Kabat positions 28, 31, 52, and 98 were used to generate a series of mutations with rL3 V L Paired V H domains and tested for solubility and neutralization. Figure 6 As shown in , all mutants exhibited improved solubility as determined by turbidity at OD350 compared to the unmodified 10E8 antibody. In addition, one of the modified antibodies, H6-511-4mut (the 7th, which includes H98Y, N28D, D31N, S52T substitutions compared to H6-511) broadly neutralized HIV-1 strains ( Figure 7 ). H Compared to the H6-511-4mut V H Includes Q3R, A61E, P62S, E64K, L72D, S74T, I75K, F77T, L(82C)V, M84T, S87T, Y98H, R105Q, and T110I replacements.

[0668] like Figure 8 Shown in, including L10 V L The modified 10E8 antibodies were identified as highly soluble (comparing unmodified 10E8 to 10E8H / L10). L The modified 10E8 antibody lacks the broad neutralization properties of the parent 10E8 antibody ( Figure 4B ). In contrast, including 10E8 V H Paired rL3 V L The modified 10E8 antibody was a broad-spectrum and potent neutralizer of HIV-1 infection, but was inferior to the L10 V L The antibody is soluble.

[0669] Therefore, a screening assay was performed to determine whether a gene with rL3 V L HIV-1 neutralization properties and L10 V L The solubility of the modified V L The L10 V L and rL3 V L There are differences in the sequence at 12 positions (see Figure 1). Antibodies with substitutions at each of these positions were screened for solubility and neutralization properties. Fig. 9 , containing rL3 V with S1A, Y2S, T8P, G9A, G16K and R17Q substitutions L Paired 10E8 V H The modified 10E8 antibody is significantly less turbid than the corresponding 10E8 WT or other rL3 variants. rL3V with S1A, Y2S, T8P, G9A, G16K and R17Q substitutions L The variant is called "rL3-6mut" and is identical to the natural 10E8 V L The comparison includes S1A, Y2S, E7D, T8P, G9A, G16K, R17Q, I45V, V58I, S76T, D83E and E85D.

[0670] Includes rL3-6mut V L Paired H6-511-4mut V H The antibody was significantly more soluble (as measured by turbidity assay) than the 10E8 wt antibody or the H6-511-4mut / rL3 antibody ( Fig.10 ). In addition, H6-511-4mut V H Each of the H6-511-4mut / rL3 antibodies had an IC smaller than 10E8 wt. 50 and IC 80 Value for neutralizing HIV-1.

[0671] Fig.12 A summary of the development process of HC6-S74Y-511 / rL3 (variant 1), H6-511-4mut / rL3-6mut (variant 4), and HC6-S74Y-511 / rL3-6mut (variant 5) antibodies is provided. The nomenclature, sequence, and substitutions of these variant antibodies compared to wild-type 10E8 are summarized in Table 3.

[0672] Table 3. 10E8 variants

[0673]

[0674] Additional assays were performed to further analyze the solubility, autoreactivity, and neutralization of the HC6-S74Y-511 / rL3, H6-511-4mut / rL3-6mut, and HC6-S74Y-511 / rL3-6mut antibodies (Figures 13 to 16).

[0675] Kinetic concentration. Kinetic concentration assays were performed to evaluate the solubility of HC6-S74Y-511 / rL3, H6-511-4mut / rL3-6mut, and HC6-S74Y-511 / rL3-6mut antibodies. Three milliliters (concentration 0.35 OD) of each variant in phosphate buffered saline, pH 7.4 were loaded into a centrifugal concentrator with a molecular weight cutoff of 30,000 Daltons and centrifuged at 4000 g for 20 minutes ( Fig.13A After concentration, the volume of each modified antibody was smaller than that of wild-type 10E8, among which the volume of H6-511-4mut / rL3-6mut and HC6-S74Y-511 / rL3-6mut antibodies was significantly smaller than that of 10E8 antibody ( Fig. 13B It was also observed that the protein concentration of the H6-511-4mut / rL3-6mut and HC6-S74Y-511 / rL3-6mut antibody solutions increased significantly ( Fig. 13C ).

[0676] Dynamic Light Scattering. HC6-S74Y-511 / rL3, H6-511-4mut / rL3-6mut, and HC6-S74Y-511 / rL3-6mut antibodies were evaluated by dynamic light scattering. Dynamic light scattering is a useful technique for determining the uniformity of the size of polymers dissolved in a solution. Monodisperse solutions are solutions containing polymers (proteins) of the same size. Polydisperse solutions contain polymers of different sizes. For protein solutions containing aggregates, the dynamic light scattering assay will identify them as polydisperse solutions. Fig.14 As shown in , antibody solutions containing wild-type 10E8 were polydisperse, while antibody solutions containing HC6-S74Y-511 / rL3, H6-511-4mut / rL3-6mut or HC6-S74Y-511 / rL3-6mut antibodies were monodisperse. These findings show that these variant antibodies do not aggregate in solution.

[0677] Autoreactivity. A common property of the previously characterized MPER mAbs 2F5 and 4E10 is that they cross-react with self-antigens, which are subsequently known to react with self-antigens present on HIV-1 naive HEp-2 cells (Haynes et al., Science 308, 1906-1908, 2005; Huang et al., Nature, 491: 406-412, 2012). Therefore, the autoreactivity of HC6-S74Y-511 / rL3, H6-511-4mut / rL3-6mut and HC6-S74Y-511 / rL3-6mut antibodies was tested by HEp-2 cell staining. Reactivity to HIV-1 negative human epithelial (HEp-2) cells was determined by indirect immunofluorescence on slides using Evans Blue and FITC-conjugated goat anti-human IgG (Zeus Scientific) as counterstains, essentially as described previously (Haynes et al., Science, 308(5730):1906–1908, 2005). Fig.15 As shown in , HC6-S74Y-511 / rL3, H6-511-4mut / rL3-6mut, and HC6-S74Y-511 / rL3-6mut antibodies showed almost no staining of HEp-2 cells compared to the known autoreactive antibody VRC07-G54W. Lower staining intensity was observed with H6-511-4mut / rL3-6mut and HC6-S74Y-511 / rL3-6mut antibodies than with the HC6-S74Y-511 / rL3 antibody.

[0678] Neutralization. To further evaluate the neutralization breadth and potency of HC6-S74Y-511 / rL3, H6-511-4mut / rL3-6mut and HC6-S74Y-511 / rL3-6mut antibodies, a previously described pseudovirus neutralization assay (see Wu et al., Science, 329(5993):856-861, 2010, which is incorporated herein by reference in its entirety) was tested on a large panel of 200 HIV-1 pseudoviruses. Briefly, the monoclonal antibodies were serially diluted 5-fold with Dulbecco's modified Eagle's medium 10% FCS (Gibco), and 10 μl was incubated with 40 μl of pseudovirus in a 96-well plate at 37°C for 30 min, then TZM-bl cells were added, and the plate was incubated for 48 h. The assay was then developed using a luciferase assay system (Promega) and relative light units (RLU) were read on a luminometer. HIV-1 Env pseudovirus was generated by co-transfecting 293T cells with a 2:1 ratio of the pSG3δEnv backbone and a second plasmid expressing HIV-1 Env. 72 hours after transfection, the supernatant containing the pseudovirus was collected and frozen at -80°C until future use. Neutralization curves were fit by nonlinear regression using the 5-parameter hill slop equation as described. IC 50 and IC 80 Values ​​are reported as the antibody concentration required to inhibit infection by 50% or 80%, respectively.

[0679] 10E8, HC6-S74Y-511 / rL3, H6-511-4mut / rL3-6mut, and HC6-S74Y-511 / rL3-6mut antibodies neutralized 98% of the viruses in this large panel, with a median IC 50 About 0.4μg / ml ( Figures 16A to 16C ), median IC 80 About 2.0 μg / ml ( Figures 16D to 16F ).

[0680] Serum half-life. The serum half-life of the 10E8v4 antibody was evaluated in macaques. Antibodies comprising the heavy and light chain variable regions of the 10E8 or 10E8v4 antibodies were generated using IgG1 constant regions or IgG1 constant regions with an "LS" mutation and injected IV into macaques to evaluate serum half-life. Fig.17 As shown, the 10E8v4 antibody (with or without LS substitution in the constant region) had a significantly increased half-life compared to the parental 10E8 antibody.

[0681] In summary, the potency, breadth, and autoreactivity of the HC6-S74Y-511 / rL3, H6-511-4mut / rL3-6mut, and HC6-S74Y-511 / rL3-6mut antibodies were similar to those of the unmodified 10E8 antibody, but the modified 10E8 antibodies were much more soluble in aqueous solution, especially the H6-511-4mut / rL3-6mut and HC6-S74Y-511 / rL3-6mut antibodies.

[0682] Example 2: Optimized 10E8 Antibody

[0683] This example describes a series of modified 10E8 antibodies developed using a surface-matrix approach, where single surface mutations were evaluated for neutralization potency and manufacturability. To improve 10E8, a human antibody capable of neutralizing 98% of HIV-1 isolates, 280 surface mutants were generated that added hydrophobic changes (Phe / Trp), hydrophilic protrusions (Arg), large steric protrusions (N-linked glycans), and side chain removals (polyglycines). For potency, these mutants were evaluated by neutralization.

[0684] Hydrophobic substitutions at heavy chain residue 100c were observed to increase neutralization. The 10E8 variant (10E8v4-100cW) including the S100cW substitution produced the broadest and most potent HIV-1 neutralizing antibodies described to date. In addition, polyglycine substitutions including modifications of light chain residue 32 were observed to enhance homogeneity in size exclusion chromatography, an effect also achieved by the introduction of a disulfide bond in this region.

[0685] 1. Surface Matrix Method

[0686] Macromolecular function often depends heavily on surface interactions. Although alanine scanning or Arg scanning has been used to understand function, these scans have not been used to directly improve function. Specific protein surface changes that may enhance function are selected.

[0687] 280 10E8 surface mutants were generated that incorporated hydrophobic changes (Phe / Trp - enhances binding to hydrophobic patches), hydrophilic protrusions (Arg - aids solubility or interaction with membranes), large steric protrusions (N-linked polysaccharides - aids solubility or aids steric inhibition of entry), and side chain removals (polyglycine - aids solubility or removes steric constraints on recognition). These 10E8 mutants were screened for HIV-1 neutralization as described above using a 9-isolate HIV-1 panel, and turbidity was assessed as described above for all mutants except the Phe / Trp changes.

[0688] Table 4. Surface scanning of 10E8 antibody

[0689]

[0690]

[0691] In the above table, "turbidity improvement>x-fold" indicates that the actual turbidity of the variant in PBS, pH 7.4 is reduced x-fold compared to the turbidity of the 10E8 antibody. For the glycan scan, the residue shown (Ri) was replaced by Asn and the residue two positions away from the residue shown at the c-terminus (Ri+2) was replaced by Ser or Thr to add a glycan at position Ri.

[0692] A summary of the targeted mutations and corresponding changes in neutralization profiles is provided in the table below.

[0693] Table 5. Summary of certain 10E8 amino acid substitutions that enhance HIV-1 neutralization and / or reduce turbidity.

[0694]

[0695] 2. Substitution at Kabat position 32 of the light chain

[0696] When 10E8, 10E8 variant 4, and 10E8 variant 5 were purified by gel chromatography, a rare "double peak" was observed (see Fig.18A Although this doublet did not appear to affect neutralization breadth or potency or antibody solubility, additional modifications of the 10E8 antibody sequence were performed to determine if this doublet could be removed. Based on the structure of 10E8 bound to the MPER peptide, a number of amino acid substitutions were determined, including V L Kabat position 32. Unexpectedly, replacing V with almost any other amino acid L The tyrosine residue at position 32 of Kabat removes this "double peak" ( Fig.18B and 18C ). The resulting 10E8 variants were tested for HIV-1 neutralization using a panel of 9 HIV-1 isolates (specifically selected to represent diversity). As noted in the table below, 10E8 variant 4 antibodies that also include Y32W have improved HIV-1 neutralization properties compared to 10E8 variant 4, and variants with the Y32F substitution are nearly as effective as 10E8 variant 4.

[0697] Table 6. Neutralization of 10E8 variants

[0698]

[0699]

[0700] Further in V LSeveral 10E8 variant 4 antibodies including substitutions at Kabat position 32 were assayed for turbidity ( Fig.18D ). 10E8 variant 4 having Y32A, Y32F, Y32I, Y32L, Y32M, Y32P, Y32V, or Y32W substitutions each had a turbidity profile similar to that of 10E8 variant 4.

[0701] 2. 10E8v4-100cW

[0702] The surface matrix mutagenesis assay discussed above identified that a hydrophobic change at the heavy chain kabat position 100c improved HIV-1 neutralization potency (S100cF substitution). Antibody surface analysis suggested potential membrane interactions and that a Trp substitution at kabat position 100c may also be of interest. Therefore, the S100cW substitution was introduced into 10E8v4 and analyzed for HIV-1 neutralization ( Fig.19 ). 10E8v4-100cW was tested on a panel of 208 HIV-1 isolates and was shown to be the most potent and broadest antibody to date.

[0703] ANA Hep-2 staining analysis and anti-cardiolipin ELISA were performed to test the autoreactivity of several 10E8 variants. The Hep-2 staining assay was performed as described above. The anti-cardiolipin ELISA was performed as described by Asokan et al., J. Virol., doi: 10.1128 / JVI.02097-15, 2015). The results of these assays are summarized in the table below.

[0704] Table 7. Autoreactivity of 10E8 variants

[0705] Antibody ANA HEp2 staining assay Anti-cardiolipin ELISA 10E8 wt No responsiveness No responsiveness 10E8v4 No responsiveness No responsiveness 10E8v4 w / LS heavy chain No responsiveness No responsiveness 10E8v4 s100cW No responsiveness No responsiveness 10E8v4 Y32W No responsiveness No responsiveness 10E8v4 Y32F No responsiveness No responsiveness 10E8v4 S100cW / Y32F No responsiveness No responsiveness 10E8v4 S100cW / Y32W Reactivity (1+) No responsiveness 10E8v4 S100cF No responsiveness No responsiveness 10E8v4 S100cF / Y32F No responsiveness No responsiveness 10E8v4 S100cF / Y32W No responsiveness No responsiveness 10E8v4 S100cY No responsiveness No responsiveness 10E8v4 S100cY / Y32F No responsiveness No responsiveness 10E8v4 S100cY / Y32W No responsiveness No responsiveness

[0706] 3. 10E8v4-100cW-DS

[0707] An atypical 10E8v4 100cW purification profile was observed by size exclusion chromatography. Therefore, purification profiles of several additional 10E8 variants were determined by size exclusion chromatography. These variants included:

[0708] 1. 10E8v4 S100cF

[0709] 2. In V L 10E8v4S100cF with A, D, E, F, H, I, K, L, M, N, P, Q, R, T, V or W substitution at kabat position 32

[0710] 3. 10E8v4 S100cW

[0711] 4. In V L 10E8v4S100cW with A, D, E, F, H, I, K, L, M, N, P, Q, R, T, V or W substitution at kabat position 32

[0712] 5. In V H 10E8v4 with D, E, I, K, M, N, P, Q, T or V substitution at kabat position 100c

[0713] 6. 10E8v4-DS(V H Y100eC and V L S30C replacement)

[0714] 7. 10E8v4 S100cW-DS

[0715] 8. 10E8v4-HCDR3 (HCDR3 deleted)

[0716] Of these modified 10E8 antibodies, 10E8v4 S100cP and 10E8v4 HCDR3 showed typical size exclusion chromatography-based antibody profiles, and 10E8v4 S100cF / Y32N, 10E8v4 S100cW / Y32N, 10E8v4-DS, and 10E8v4 S100cW-DS showed profiles close to typical size exclusion chromatography-based antibody profiles. The remaining antibodies eluted from the size exclusion column as multiple peaks. A summary of the size exclusion chromatography profiles for the 10E8v4, 10E8v4 / Y32F, 10E8v4 S100cA, 10E8v4 with HCDR3 deletion, 10E8v4-DS, and 10E8v4 S100cW-DS antibod...

Claims

1. An isolated antibody comprising: Heavy chain variable region (V H ), the heavy chain variable region includes V shown in SEQ ID NO: 5 H heavy chain complementarity determining region (HCDR) 1, HCDR2 and HCDR3; and light chain variable region (V L ), the light chain variable region includes V shown in SEQ ID NO: 6 L light chain complementarity determining region (LCDR) 1, LCDR2 and LCDR3; wherein The V H including arginine, glutamic acid, serine, lysine, aspartic acid, threonine, lysine, threonine, valine, threonine, threonine, tyrosine, glutamine, and isoleucine residues at kabat positions 3, 61, 62, 64, 72, 74, 75, 77, 82c, 84, 87, 89, 105, and 110, respectively; and The V L including alanine, serine, aspartic acid, proline, alanine, lysine, glutamine, valine, isoleucine, threonine, glutamic acid, and aspartic acid residues at kabat positions 1, 2, 7, 8, 9, 16, 17, 45, 58, 76, 83, and 85, respectively; wherein the antibody specifically binds to gp41 and neutralizes HIV-1; and The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are selected from: (1) V shown in SEQ ID NO: 5 determined according to Kabat numbering H HCDR1, HCDR2 and HCDR3, and V shown in SEQ ID NO: 6 determined according to Kabat numbering L LCDR1, LCDR2 and LCDR3; (2) V shown in SEQ ID NO: 5 determined according to Chothia numbering H HCDR1, HCDR2 and HCDR3, and V shown in SEQ ID NO: 6 determined according to Chothia numbering L LCDR1, LCDR2 and LCDR3; and (3) V shown in SEQ ID NO: 5 determined according to IMGT numbering H HCDR1, HCDR2 and HCDR3, and V represented by SEQ ID NO: 6 determined according to IMGT numbering L LCDR1, LCDR2 and LCDR3.

2. The isolated antibody of claim 1, wherein the V H and V L Respectively comprising amino acid sequences having at least 90% identity to SEQ ID NO: 5 and 6, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are as defined in claim 1.

3. The isolated antibody of claim 1 or 2, wherein the V H and V L They include the amino acid sequences shown in SEQ ID NOs: 5 and 6, respectively.

4. The isolated antibody of claim 1 or 2, further comprising a V5R mutation in VH according to Kabat numbering.

5. The isolated antibody of claim 1 or 2, comprising a human framework region and / or a human constant region.

6. The isolated antibody of claim 1 or 2, wherein the antibody is IgG, IgM or IgA.

7. The isolated antibody of claim 1 or 2, comprising a recombinant constant region comprising a modification that increases binding to a neonatal Fc receptor.

8. The isolated antibody of claim 7, wherein the antibody is IgG1 and the modifications that increase binding to the neonatal Fc receptor comprise M428L and N434S amino acid substitutions.

9. An antigen-binding fragment of the antibody of any one of claims 1 to 8, wherein the antigen-binding fragment comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3.

10. The antigen-binding fragment of claim 9, wherein the antigen-binding fragment is a Fv, Fab, F(ab')2, scFV or scFV2 ​​fragment.

11. A conjugate comprising the antibody of any one of claims 1 to 8 or the antigen-binding fragment of claim 9 or 10 linked to a detectable label.

12. The conjugate of claim 11, wherein the detectable label is a fluorescent, enzymatic or radioactive label.

13. An isolated nucleic acid molecule encoding the antibody of any one of claims 1-8 or the antigen-binding fragment of claim 9 or 10.

14. A V sequence encoding the antibody of any one of claims 1 to 8 or the antigen-binding fragment of claim 9 or 10 H and V L Isolated nucleic acid molecules.

15. The isolated nucleic acid molecule of claim 14, operably linked to a promoter.

16. An expression vector comprising the nucleic acid molecule of claim 14 or 15.

17. A host cell comprising the nucleic acid molecule of claim 14 or 15 or the expression vector of claim 16.

18. The host cell of claim 17, wherein the host cell is a eukaryotic host cell.

19. The host cell of claim 18, wherein the host cell is a human host cell.

20. A pharmaceutical composition for use in a method of inhibiting or treating HIV-1 infection, comprising: A therapeutically effective amount of the antibody of any one of claims 1 to 8, the antigen-binding fragment of claim 9 or 10, the conjugate of claim 11 or 12, the nucleic acid molecule of any one of claims 13 to 15, or the expression vector of claim 16; and A pharmaceutically acceptable carrier.

21. A method for producing an antibody or antigen-binding fragment that specifically binds to gp41, comprising: Incubating the host cell of any one of claims 17 to 19 under in vitro conditions sufficient to express the nucleic acid molecule or expression vector to produce the antibody or antigen-binding fragment; as well as The antibody or antigen-binding fragment is purified.

22. Use of the antibody of any one of claims 1 to 8 or the antigen-binding fragment of claim 9 or 10 in the preparation of a medicament for use in a method for detecting HIV-1 infection in a subject, the method comprising: contacting a biological sample from the subject with the antibody of any one of claims 1 to 8 or the antigen-binding fragment of claim 9 or 10 under conditions sufficient to form an immune complex; and The sample is detected for the presence of the immune complex, wherein the presence of the immune complex on the sample indicates that the subject has HIV-1 infection.

23. Use of the antibody of any one of claims 1 to 8, the antigen binding fragment of claim 9 or 10, the conjugate of claim 11 or 12, the nucleic acid molecule of any one of claims 13-15 or the expression vector of claim 16 in the preparation of a medicament for inhibiting or treating HIV-1 infection in a subject.

24. The use of claim 23, wherein the subject is at risk of infection with HIV-1.

25. The use of claim 23, wherein the subject has HIV-1 infection.

26. The use of claim 25, wherein the subject suffers from AIDS.

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