Human immunodeficiency virus neutralizing antibodies

Abstract

The present invention relates to human immunodeficiency virus neutralizing antibodies. The present invention provides novel anti-HIV antibodies with improved therapeutic properties, related pharmaceutical compositions and methods of use thereof.

Description

[0001] This application is a divisional application of the Chinese invention patent application with the application date of December 14, 2016, application number 201680079823.5, and invention name “Human Immunodeficiency Virus Neutralizing Antibodies”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 267,652, filed December 15, 2015, which is incorporated herein by reference in its entirety.

[0004] Statement regarding sequence listing

[0005] The sequence table associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence table is GILE_112_01WO_ST25.txt. The text file generated on December 13, 2016 is approximately 640KB and is submitted electronically via EFS-Web. Technical Field

[0006] The present invention relates to antibodies and antigen-binding fragments thereof for the treatment and prevention of human immunodeficiency virus (HIV) infection. In particular, the present invention provides novel anti-HIV antibodies and antigen-binding fragments thereof, broadly including neutralizing anti-HIV antibodies and antigen-binding fragments thereof, pharmaceutical compositions comprising these antibodies and fragments thereof, and methods of using these antibodies and fragments thereof to reduce HIV replication and treat and prevent HIV infection. Background Art

[0007] Human immunodeficiency virus (HIV) infection and related diseases are major public health problems worldwide. Recently approved treatments for HIV infection target viral reverse transcriptase, protease and integrase, but HIV resistance to these existing drugs, long-term toxicity and patients' lack of adherence to daily dosing regimens have proven to be problems associated with these treatments. Therefore, it is important to discover and develop new HIV drugs.

[0008] WO2012 / 030904 describes human anti-HIV antibodies derived from memory B cells of HIV-infected donors that are able to inhibit infection by HIV-1 species from multiple clades. However, the therapeutic use of these antibodies is limited due to problems associated with immunogenicity, pharmacokinetics, antigenic specificity, effector function, and manufacturing. Therefore, there is a need in the art for new anti-HIV antibodies with favorable properties for therapeutic use. Summary of the invention

[0009] In particular, the present invention provides compositions and methods for treating or preventing HIV.

[0010] In one embodiment, the present invention includes an isolated monoclonal antibody or antigen-binding fragment thereof comprising one or more heavy chain complementary determining regions (CDRs) and one or more light chain CDRs of the PGT121 LO6 antibody (using Kabat, IMGT, Chothia or Honegger numbering). In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof comprising heavy chain complementary determining regions 1-3 (CDRs 1-3) listed in SEQ ID NOs: 362, 364 and 367 and light chain CDRs 1-3 listed in SEQ ID NOs: 395, 396 and 397, wherein the antibody or antigen-binding fragment thereof comprises zero to eight (or zero to four) amino acid substitutions within the CDRs, and wherein the antibody or antigen-binding fragment thereof comprises one or more of the following: an IgG1m17 allotype heavy chain; a λ2 light chain; a heavy chain constant region comprising one or more of the following amino acid substitutions: Ala at position 236, Asp at position 239, Leu at position 330, Glu at position 332, Leu at position 428 and Ser at position 434 (using EU numbering); and a heavy chain variable region comprising one or more of the following: Ser-Ser-Val or Thr-Gly-Val at positions 82a-82c, Gln at position 39, Asn at position 60, His at position 68, any of Lys, His or Thr at position 105, Leu at position 2, Ala at position 32 and Ala at position 95 (using Kabat numbering). In a specific embodiment, the antibody or antigen-binding fragment thereof comprises two or more heavy chain complementary determining regions (CDRs) and two or more light chain CDRs of the PGT121 LO6 antibody (using Kabat, IMGT, Chothia or Honegger numbering). In a specific embodiment, the antibody or antigen-binding fragment thereof comprises all three heavy chain complementary determining regions (CDRs) and all three light chain CDRs of the PGT121 LO6 antibody (using Kabat, IMGT, Chothia or Honegger numbering). In a specific embodiment, the monoclonal antibody or its antigen-binding fragment comprises an IgG1m17 allotype heavy chain. In a specific embodiment, the heavy chain constant region comprises Lys at position 214, Glu at position 356, Met at position 358, and Ala at position 431 (using EU encoding). In a specific embodiment, the monoclonal antibody comprises a heavy chain constant region (Fc) of any of the following antibodies: PGT121.42, PGT121.43, PGT121.60, PGT121.61, PGT121.54, PGT121.55, PGT121.64, and PGT121.65.In a specific embodiment of any antibody or antigen-binding fragment thereof described herein, the heavy chain constant region (Fc) is shown in any one of SEQ ID NO: 252, 255, 266, 267, 268, 269, 272 and 273. In a specific embodiment, the monoclonal antibody or antigen-binding fragment thereof comprises a λ2 light chain. In certain embodiments, the light chain comprises one or more of the following amino acid replacements (using Kabat numbering): Arg at position 67b, Pro at position 67c, and Lys at position 103. In one embodiment, the light chain comprises one or more of the following amino acid replacements (using. Figure 1In a specific embodiment, the monoclonal antibody comprises a light chain of any of the following antibodies: PGT121.42, PGT121.43, PGT121.60, PGT121.61, PGT121.54, PGT121.55, PGT121.64 and PGT121.65. In a specific embodiment of any antibody or antigen-binding fragment thereof described herein, the light chain is shown in any one of SEQ ID NOs: 338, 341, 352, 353, 354, 355, 358 and 359. In a specific embodiment, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising one or more of the following amino acid substitutions (using EU numbering): Ala at position 236, Asp at position 239, Leu at position 330, Glu at position 332, Leu at position 428, and Ser at position 434. In a specific embodiment, the constant region comprises Ala at position 236, Asp at position 239, Leu at position 330, and Glu at position 332. In a specific embodiment, the heavy chain constant region comprises Leu at position 428 and Ser at position 434. In certain embodiments, the heavy chain constant region comprises Ala at position 236, Asp at position 239, Leu at position 330, Glu at position 332, Leu at position 428, and Ser at position 434 (using EU numbering). In a specific embodiment, the monoclonal antibody or antigenic fragment thereof comprises the heavy chain constant region of any of the following antibodies: PGT121.42, PGT121.43, PGT121.60, PGT121.61, PGT121.54, PGT121.55, PGT121.64 and PGT121.65. In certain embodiments, the monoclonal antibody or antigenic fragment thereof comprises a heavy chain variable region (Fab) comprising one or more of the following amino acid substitutions: Ser-Ser-Val or Thr-Gly-Val at positions 82a-82c, Gln at position 39, Asn at position 60, His at position 68, any one of Lys, His or Thr at position 105, Leu at position 2, Ala at position 32 and Ala at position 95 (using Kabat numbering). In a specific embodiment, the heavy chain variable region comprises Ser-Ser-Val or Thr-Gly-Val at positions 82a-82c (using Kabat numbering). In a specific embodiment, the heavy chain variable region comprises Asn at position 60, His at position 68, and Lys, His or Thr at position 105 (using Kabat numbering).In certain embodiments, the heavy chain variable region (Fab) comprises: Ser-Ser-Val or Thr-Gly-Val at positions 82a-82c, Gln at position 39, Asn at position 60, His at position 68, and Lys, His or Thr at position 105 (using Kabat numbering). In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region provided in Table 1. In a specific embodiment, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region provided in Table 1. In a specific embodiment, the monoclonal antibody or antigen-binding fragment thereof comprises a light chain or variable region thereof provided in Table 1.

[0011] In a related embodiment, the invention includes an isolated polynucleotide encoding a monoclonal antibody or antigen-binding fragment thereof of the invention, or a heavy chain or light chain of the monoclonal antibody.

[0012] In yet another related embodiment, the present invention comprises a vector comprising a polynucleotide of the present invention.

[0013] In another embodiment, the invention includes a cell comprising a polynucleotide or vector of the invention. In a specific embodiment, the cell is a mammalian, bacterial or yeast cell.

[0014] In yet another embodiment, the present invention includes a pharmaceutical composition comprising a monoclonal antibody or fragment thereof of the present invention, a polynucleotide of the present invention, or a vector of the present invention. In a specific embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient, or diluent.

[0015] In another related embodiment, the present invention includes a method for treating or preventing human immunodeficiency virus (HIV) in a subject in need thereof, comprising providing an effective amount of a pharmaceutical composition of the present invention to the subject. In a specific embodiment, a second therapeutic agent is also provided to the subject. In certain embodiments, the second therapeutic agent is an antiviral agent.

[0016] In yet another embodiment, the present invention includes a method of producing a monoclonal antibody or antigen-binding fragment thereof of the present invention, comprising recombinantly expressing the monoclonal antibody or antigen-binding fragment thereof in a cell of the present invention.

[0017] The present invention also relates to the following items.

[0018] 1. An isolated monoclonal antibody or antigen-binding fragment thereof, comprising heavy chain complementary determining regions 1-3 (CDRs 1-3) set forth in SEQ ID NOs: 362, 364, and 367 and light chain CDRs 1-3 set forth in SEQ ID NOs: 395, 396, and 397, wherein the antibody or antigen-binding fragment thereof comprises zero to four amino acid substitutions within the CDRs, and wherein the antibody or antigen-binding fragment thereof comprises one or more of the following:

[0019] a. IgG1m17 allotype heavy chain;

[0020] b.λ2 light chain;

[0021] c. a heavy chain constant region comprising one or more of the following amino acid substitutions: Ala at position 236, Asp at position 239, Leu at position 330, Glu at position 332, Leu at position 428, and Ser at position 434 (using EU numbering); and

[0022] d. a heavy chain variable region comprising one or more of the following: Ser-Ser-Val or Thr-Gly-Val at positions 82a-82c, Gln at position 39, Asn at position 60, His at position 68, any of Lys, His or Thr at position 105, Leu at position 2, Ala at position 32 and Ala at position 95 (using Kabat numbering).

[0023] 2. The monoclonal antibody or antigen-binding fragment thereof of item 1, which comprises an IgG1m17 allotype heavy chain.

[0024] 3. The monoclonal antibody or antigen-binding fragment thereof of item 2, wherein the heavy chain constant region comprises Lys at position 214, Glu at position 356, Met at position 358 and Ala at position 431 (using EU numbering).

[0025] 4. The monoclonal antibody or antigen-binding fragment thereof according to item 3, which has a heavy chain constant region (Fc) listed in any one of SEQ ID NOs: 252, 255, 266, 267, 268, 269, 272 and 273.

[0026] 5. The monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 4, wherein the light chain is λ2.

[0027] 6. The monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 5, wherein the light chain comprises one or more of the following amino acid substitutions (using Kabat numbering): Arg at position 67b, Pro at position 67c, and Lys at position 103.

[0028] 7. The monoclonal antibody or antigen-binding fragment thereof of any one of items 1 to 6, which has a light chain listed in any one of SEQ ID NOs: 338, 341, 352, 353, 354, 355, 358, 359.

[0029] 8. The monoclonal antibody or antigen-binding fragment thereof of any one of items 1 to 7, wherein the heavy chain constant region comprises one or more of the following amino acid substitutions (using EU numbering): Ala at position 236, Asp at position 239, Leu at position 330, Glu at position 332, Leu at position 428, and Ser at position 434.

[0030] 9. The monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 8, wherein the heavy chain constant region comprises Ala at position 236, Asp at position 239, Leu at position 330, and Glu at position 332.

[0031] 10. The monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 9, wherein the heavy chain constant region comprises Leu at position 428 and Ser at position 434.

[0032] 11. The monoclonal antibody or antigen-binding fragment thereof of any one of items 1 to 10, wherein the heavy chain constant region comprises Ala at position 236, Asp at position 239, Leu at position 330, Glu at position 332, Leu at position 428 and Ser at position 434 (using EU numbering).

[0033] 12. The monoclonal antibody or antigen-binding fragment thereof of any one of items 1 to 11, which has a heavy chain constant region listed in any one of SEQ ID NOs: 252, 255, 266, 267, 268, 269, 272 and 273.

[0034] 13. The monoclonal antibody or antigen-binding fragment thereof of any one of items 1-12, wherein the heavy chain variable region (Fab) comprises one or more of the following amino acid replacements: Ser-Ser-Val or Thr-Gly-Val at positions 82a-82c, Gln at position 39, Asn at position 60, His at position 68, any one of Lys, His or Thr at position 105, Leu at position 2, Ala at position 32 and Ala at position 95 (using Kabat numbering).

[0035] 14. The monoclonal antibody or antigen-binding fragment thereof of claim 13, wherein the heavy chain variable region comprises Ser-Ser-Val or Thr-Gly-Val at positions 82a-82c (using Kabat numbering).

[0036] 15. The monoclonal antibody or antigen-binding fragment thereof according to item 13 or item 14, wherein the heavy chain variable region comprises Asn at position 60, His at position 68, and Lys, His or Thr at position 105 (using Kabat numbering).

[0037] 16. The monoclonal antibody or antigen-binding fragment thereof of any one of items 1-15, wherein the heavy chain variable region (Fab) comprises: Ser-Ser-Val or Thr-Gly-Val at positions 82a-82c, Gln at position 39, Asn at position 60, His at position 68 and Lys, Thr or His at position 105 (using Kabat numbering).

[0038] 17. The monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 15, which has a heavy chain variable region provided in Table 1.

[0039] 18. The monoclonal antibody or antigen-binding fragment thereof of item 17, which has a heavy chain constant region provided in Table 1.

[0040] 19. The monoclonal antibody or antigen-binding fragment thereof according to item 17 or item 18, which has a light chain provided in Table 1.

[0041] 20. An isolated polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 19, or the heavy chain or light chain of the monoclonal antibody.

[0042] 21. A vector comprising the polynucleotide of item 20.

[0043] 22. A cell comprising the polynucleotide of item 20 or the vector of item 21.

[0044] 23. The cell of item 22, wherein the cell is a mammalian, bacterial or yeast cell.

[0045] 24. A pharmaceutical composition comprising the monoclonal antibody or a fragment thereof according to any one of items 1 to 19, the polynucleotide according to item 20, or the vector according to item 21.

[0046] 25. The pharmaceutical composition of item 24, comprising the monoclonal antibody or fragment thereof of any one of items 1 to 19 and a pharmaceutically acceptable carrier, excipient or diluent.

[0047] 26. A method for treating or preventing human immunodeficiency virus (HIV) in a subject in need thereof, comprising providing an effective amount of the pharmaceutical composition of item 24 or item 25 to the subject.

[0048] 27. The method of claim 26, wherein a second therapeutic agent is provided to the subject.

[0049] 28. The method of claim 27, wherein the second therapeutic agent is an antiviral agent.

[0050] 29. A method for producing the monoclonal antibody or antigen-binding fragment thereof of any one of items 1 to 19, comprising recombinantly expressing the monoclonal antibody or antigen-binding fragment thereof in the cell of item 22 or item 23. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The amino acid sequences of the heavy chain variable domain and light chain variable domain of the PGT-121LO6 antibody are shown, with the corresponding Kabat number annotation shown below each amino acid. Amino acid deletions are indicated by "-". Amino acid insertions are indicated by lowercase letters after the amino acid number.

[0052] Figure 2 is a graph showing the Tm of the Fab domain determined by DSF for PGT121 WT and selected variants. All variants tested had improved thermal stability compared to PGT121 WT, as indicated by higher Tm.

[0053] Figure 3 is a graph showing paired analysis of unique variants that differ only at the indicated amino acid, revealing mutations that stabilize or destabilize the Fab domain. The change in Tm of various PGT121 mutants compared to the Tm of PGT121 WT is shown.

[0054] Figure 4 is a graph showing the Fab melting temperatures of the Fab domains of selected PGT121 variants determined by DSC.

[0055] Figure 5 is a graph showing the fold change in gp120 ELISA EC50 for selected PGT121 variants compared to PGT121 LO6 WT. The dashed line at an EC50 ratio of 1 indicates no change in affinity. PGT121 LO6 WT is boxed. For a given gp120 strain (gp120BaL or gp120TRO as indicated), variants with an EC50 ratio > 1 have improved antigen binding affinity compared to PGT121 WT, while variants with an EC50 ratio < 1 have decreased antigen binding affinity compared to PGT121 WT for a given gp120 strain.

[0056] Figure 6is a graph showing the fold change in gp140 ELISA EC50 for PGT121 variants compared to PGT121 LO6 WT. The dashed line at an EC50 ratio of 1 indicates no change in affinity. PGT121 LO6 WT is boxed. For a given gp140 strain (gp140 BaL or gp140 SHIV SF162P3 as indicated), variants with an EC50 ratio > 1 have improved antigen binding affinity compared to PGT121 LO6 WT, while variants with an EC50 ratio < 1 have decreased antigen binding affinity compared to PGT121 LO6 WT for a given gp120 strain.

[0057] Figure 7 is a graph showing the EC50 (nM) values ​​of PGT121 variants assayed against recombinant gp120 from 7 unique HIV strains (BaL, pRHPA4259, qh0692, 6535, pCAAN5342, pWITO4160 and AC10.0). In contrast to PGT121 WT, there were no statistically significant differences in gp120 binding between the specific variants PGT121.42, PGT121.60, PGT121.61 and PGT121.65.

[0058] Figures 8A-8H Figures showing the results of ELISA using various recombinant human FcγR extracellular domains and PGT121 WT and Fc and Fab variants are provided (8A, FcγRI; 8B, FcγRIII-176F; 8C, FcγRIII-176V; 8D, FcγRII-167H; 8E, FcγRIIA-167R; 8F, FcγRIIB; 8G, FcγRIIIB-NA1; 8H, FcγRIIIB-NA2). Biotinylated human FcγR extracellular domains were captured on 384-well plates coated with neutravidin at 0.5ug / mL. 12-point ELISA titrations were performed using the indicated antibodies, and the data were fitted with a 4-parameter dose response curve and EC50 values ​​were calculated. The gray line is the geometric mean of the EC50 values ​​determined in multiple unique experiments (each data point represents a unique experiment performed in duplicate).

[0059] Fig. 9 PGT121 WT and Fab variants are shown to bind to recombinant HIV Env in transfected human cells. Recombinant HIV Env BaL was transfected in HEK293T cells. Transfected cells were incubated with different concentrations of the indicated antibodies. After flow cytometry analysis, the collected MFIs were fitted to nonlinear regression dose-response curves.

[0060] Fig.10The fold change of recombinant HIV Env binding EC50 of PGT121 variants compared to PGT121 WT is shown. The dashed line at an EC50 ratio of 1 indicates no change in affinity. For a given recombinant HIV Env strain (BaL or US657 as shown), variants with an EC50 ratio>1 have improved antigen binding affinity compared to PGT121 WT, while variants with an EC50 ratio<1 have reduced antigen binding affinity compared to PGT121 WT for a given recombinant HIV Env strain.

[0061] Fig.11 pH hold screen results for PGT121 and variants in WT Fc background are shown. The changes in % monomer (wherein monomer is defined as a single complete IgG molecule) shown by SEC after elution from protein A resin after 1 hour low pH hold at pH 3.5 are plotted for each antibody in buffers with different NaCl contents shown. Values ​​below 0% represent a reduction in antibody monomers during low pH hold screening and a corresponding increase in aggregate content. Dotted and dashed lines represent values ​​for PGT121 WT (framed) in buffers containing 0 mM and 10 mM NaCl, respectively. Variants with improved or reduced performance relative to PGT121 LO6 are shown.

[0062] Fig.12 pH retention screening of PGT121 variants in DEALLS Fc background is shown. DEALLS Fc constructs contain the following amino acid substitutions compared to PGT121 LO6: G236A, S239D, A330L, I332E, M428L and N434S. Changes in the % monomer (wherein monomer is defined as a single complete IgG molecule) shown by SEC after 1 hour low pH retention at pH 3.5 in the formulation buffer are plotted for each antibody in the buffer with different NaCl contents shown. Values ​​below 0% represent a reduction in antibody monomers during the low pH retention screening process and a corresponding increase in aggregate content. Dotted and dashed lines represent the mean values ​​of PGT121 WT (framed) in buffers containing 0 mM and 40 mM NaCl, respectively. PGT121 WT (framed) with WT Fc is shown together with PGT121.56 (which contains PGT121 WT Fab combined with DEALLS Fc) for reference. Other variants tested contained variable domain mutations and showed improved low pH stability in this screen.

[0063] Fig.13Effector cell activation by PGT121 WT and Fc variants is shown, including those with the following amino acid substitutions compared to PGT121 WT (EU numbering): S239D and I332E (DE); G236A, S239D and I332E (DEA); S239D, A330L and I332E (DEL); G236A, S239D, A330L and I332E (DEAL); and S239D, H268F, S324T and I332E (FTDE). Recombinant HIV Env 92HT593 was transfected in HEK293T cells. Transfected cells were incubated with different concentrations of the antibodies shown and the engineered Jurkat T cells expressing human FcgRIIIA coupled to the NFAT luciferase reporter. Activation of effector cells by antibodies was measured as luciferase activity.

[0064] Fig.14 is a graph showing the fold change in antibody-dependent effector cell activation of PGT121 Fc variants compared to PGT121 WT, including Fig.13 and another comprising the following amino acid substitutions (EU numbering) compared to PGT121 WT: G236A, S267E, H268F, S324T and I332E (EFTEA). Fig.14 The antibody-dependent effector cell activation is represented by the AUC of the non-regressive dose-response curves shown in . The dotted line at a ratio of 1 indicates no change in antibody activity. Variants with an AUC ratio > 1 for a given recombinant HIV Env strain (BaL or 92HT593 as shown) have increased activity compared to PGT121 WT, while variants with an AUC ratio < 1 for a given recombinant HIV Env strain have decreased activity compared to PGT121 WT.

[0065] Fig.151 is a graph showing the fold change in antibody-dependent effector cell activation of PGT121 Fab variants with DEAL Fc mutations compared to PGT121 WT Fab variants with DEAL Fc mutations. Antibody-dependent effector cell activation is represented by the AUC of the non-regressive dose-response curve of a given antibody. The dotted line at a ratio of 1 indicates that there is no change in antibody activity. Variants with an AUC ratio>1 for a given recombinant HIV Env strain (such as US657 or 92HT593 as shown) have increased activity compared to PGT121 WT Fab, while variants with an AUC ratio<1 for a given recombinant HIV Env strain have reduced activity compared to PGT121 WT Fab. It is noteworthy that when quantified by the area under the curve (AUC), the activity of certain antibodies of the present invention is 10 times stronger than that of the PGT-121 antibody.

[0066] Fig.16 is a graph showing the fold change in AUC in an effector cell activation assay of HEK293 cells transfected with Env HT593 using the PGT121.60 and PGT121.61 mutants compared to the PGT121.42 mutant.

[0067] Fig.17 Target CD4 that showed enhanced HIV infection by PGT121.42 + Figure 3. ADCC of T cells. Primary CD4 T cells infected with the virus isolate US657 by effector-enhanced PGT121.42 and WT PGT121. + Representative dose response curve for ADCC of T cells. Assays were performed in the presence of 10 mg / mL serum IgG.

[0068] Fig.18A and 18B The enhanced monocyte and PBMC-mediated HIV infection targeting CD4 by PGT121.42 in the presence of 1 mg / mL serum IgG was shown. + Figure 3. T cell killing of PGT121.42 and PGT121 donor CD4 T cells infected in vitro with virus isolate US657 using isolated autologous PBMCs (A) or autologous monocytes (B). + Representative dose-response curves for T cells.

[0069] Fig.19A and 19B19A) or R5 tropism (R5 isolate; 19B) of a group of isolates with dual and / or mixed tropism (DM and X4 isolates, 19A) or R5 tropism (R5 isolate; 19B) of PGT121 and variants are shown. For each antibody variant, the activity against any given isolate is expressed relative to PGT121 (variant IC50 / PGT121 IC50). Each point represents a single isolate. The bar represents the median IC50 of the isolate with interquartile range.

[0070] Fig. 20 is a graph showing the neutralizing activity of variants against a large panel of clinical isolates using the Monogram PhenoSense neutralization assay. Each circle represents a single isolate. For each antibody, the activity against a given isolate is shown relative to PGT121 (IC95 PGT121 / IC95 variant).

[0071] Fig.21 is a graph showing the neutralization of PGT121 variants against a panel of 5 HIV-1 isolates using an analysis based on a CEM-NKr-CCR5-Luc reporter cell line. The horizontal line represents the geometric mean IC50 for each of the 5 isolates. It was observed that specific variants had improved neutralization potency (up to 2-fold) relative to PGT121 against several isolates (but not necessarily all isolates tested).

[0072] Fig. 22 is a graph showing the whole molecule immunogenicity evaluation of PGT121 WT and selected variants tested on primary cells from a panel of 50 donors. VRCO1 is an anti-HIV antibody, while A33 is a positive control that showed anti-drug antibody response in a Phase I clinical trial (Welt et al., 2003. Clin Cancer Res. 9:1338-1346).

[0073] Fig.23A and 23B is a graph showing FcRn ELISA pH 6.0 EC50 values ​​for PGT121 WT (23B) and selected variants (23A and 23B).

[0074] Fig.24 is a graph showing the serum concentration-time profiles of PGT121.1 (triangles) and PGT121.42 (circles) following 1 mg / kg IV bolus administration to naive cynomolgus monkeys (n=2). Each symbol is the measured concentration for each individual animal, while the bar represents the mean of two subjects.

[0075] Fig.25Graph showing serum concentration-time profiles of PGT121.3 (triangles), PGT121.42 (circles), PGT121 LS (open circles), and PGT121.60 (open squares) after 10 mg / kg IV administration to naive cynomolgus monkeys (n=3). Each symbol is the measured concentration from each individual animal, and the bars represent the mean of three subjects.

[0076] Fig.26 is a graph showing effector cell activation of PGT121 (open circles) and mutant PGT121.60 (filled circles) in an effector cell activation assay using HEK293 cells transfected with Evn HT593.

[0077] Fig. 27 Figure 2 shows the HIV infection target CD4 by a selected panel of PGT121 variants. + Figure 3 shows ADCC activity of T cells against primary CD4 T cells infected with virus isolate US657 and PGT121 variants enhanced by effectors. + T cell killing, representative ADCC dose-response curve.

[0078] Fig.28 is a graph showing the enhanced ADCC activity of PGT121 variant PGT121.60 compared to PGT121 in the presence of 5 mg / mL competing human serum IgG.Killing of CEM.NKr.CCR5.Luc CD4 reporter cell line infected with HIV-1 isolate US657 as shown using primary human NK cells from two healthy donors.

[0079] Fig.29A and 29B Improved efficacy of PGT121 variants against R5, X4 and D / M viruses was demonstrated. Fig.29A : Fold improvement in efficacy of individual PGT21 Fab variants vs. PGT121 WT against R5 virus and X4&D / M virus. Fig.29B : Higher efficacy fold or fold change (IC) of PGT121 variants compared to PGT121 WT against R4 virus or R5 virus 50 ). R5 virus = R5-tropic virus, preferentially uses the CCR5 receptor; X4 = R4-tropic virus, preferentially uses the CXCR4 receptor; D / M virus = dual / mixed, showing both R5 and X4 tropism. DETAILED DESCRIPTION

[0080] The present invention is based in part on the identification of novel neutralizing anti-HIV antibodies with advantageous properties for therapeutic use. The present invention provides these antibodies and antigen-binding fragments thereof, as well as related pharmaceutical compositions and methods of use thereof, e.g., for the treatment and prevention of HIV and related diseases and disorders.

[0081] Definitions and Abbreviations

[0082] The terms "a" and "an" mean one or more unless expressly stated otherwise.

[0083] "About" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In any embodiment discussed in the context of a numerical value used in conjunction with the term "about", it is specifically contemplated that the term about may be omitted.

[0084] Unless the context requires otherwise, throughout this specification and claims, the word "comprise" and variations such as, "comprises" and "comprising" are to be interpreted in an open, inclusive sense, i.e., as "including, but not limited to." Where the terms "comprises" or "comprising" are used herein, it is understood that the invention further encompasses embodiments in which these terms are replaced with "consisting of" or "consisting essentially of" or "composed of" or "consisting essentially of."

[0085] “Consisting of means including and limited to what follows the phrase “consisting of.” Thus, the phrase “consisting of” means that the listed elements are required or mandatory, and no other elements may be present.

[0086] "Consisting essentially of means including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of means that the listed elements are required or mandatory, but other elements are optional and may or may not be present depending on whether they affect the activity or action of the listed elements.

[0087] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0088] An "increased" or "enhanced" amount is typically a "statistically significant" amount and can include an increase of 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and greater than 1, e.g., 2.1, 2.2, 2.3, 2.4, etc.) in the amounts or levels described herein. It can also include at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 500%, or at least 1000% increase in the amounts or levels described herein.

[0089] A "reduced" or "decreased" or "less" amount is typically a "statistically significant" amount, and can include a 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) decrease in the amounts or levels described herein (including all integers and decimal points in between and greater than 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.). It can also include at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 500%, or at least 1000% reduction in the amounts or levels described herein.

[0090] A "composition" may include an active agent, e.g., a contrast agent, and a carrier (inert or active), e.g., a pharmaceutically acceptable carrier, diluent, or excipient. The composition may be a pharmaceutical composition. In certain embodiments, the composition is sterile, substantially free of endotoxins, or nontoxic to the recipient at the dosage or concentration used.

[0091] "Pharmaceutically acceptable carriers, diluents or excipients" include, but are not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier that has been approved by the U.S. Food and Drug Administration as acceptable for use in humans or livestock.

[0092] The terms "mammal" and "subject" include human and non-human mammals, such as, for example, humans, mice, rats, rabbits, monkeys, cows, pigs, sheep, horses, dogs, and cats.

[0093] The term "buffer" as used herein refers to a pharmaceutically acceptable excipient that stabilizes the pH of a pharmaceutical formulation. Suitable buffers are well known in the art. Suitable pharmaceutically acceptable buffers include, but are not limited to, acetate buffers, histidine buffers, citrate buffers, succinate buffers, tris-buffers, and phosphate buffers. In certain embodiments, the concentration of the buffer is about 0.01 mM to about 1000 mM, about 0.1 mM to about 1000 mM, about 0.1 mM to about 500 mM, about 0.1 to about 200 mM, about 0.1 to about 100 mM, about 1 mM to about 1000 mM, about 1 mM to about 500 mM, about 1 mM to about 200 mM, about 1 mM to about 100 mM, about 1 mM to about 50 mM, about 2 mM to about 60 mM, about 4 mM to about 60 mM, or about 4 mM to about 40 mM, about 5 mM to about 20 mM, or about 5 mM to about 25 mM.

[0094] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not occur.

[0095] "Pharmaceutical composition" refers to a preparation of a compound and a medium generally recognized in the art for delivering a biologically active compound to a mammal (eg, a human). Such a medium may therefore include any pharmaceutically acceptable carrier, diluent or excipient.

[0096] "Effective amount" or "therapeutically effective amount" refers to an amount of an antibody or antigen-binding fragment thereof of the present invention sufficient to achieve a therapeutic or beneficial result in a subject when administered to a cell, tissue or subject, alone or in combination with another therapeutic agent. The amount constituting an "effective amount" will vary depending on the antibody or antigen-binding fragment thereof and its specific use, as well as potentially depending on the condition and its severity, the mode of administration and the age of the subject to be treated, but can be routinely determined by one of ordinary skill in the art based on their own knowledge and this disclosure. A therapeutically effective dose further refers to an amount of an antibody or antigen-binding fragment thereof sufficient to treat, prevent or ameliorate an infection or disease condition or the progression of an infection or disease, and an amount sufficient to achieve an increase in the rate of treatment, cure, prevention or improvement of such a condition. When applied to a single antibody or antigen-binding fragment thereof administered alone, a therapeutically effective dose refers to that active ingredient alone. When applied to a combination, a therapeutically effective dose refers to the combined amount of the active ingredients that results in a therapeutic effect, whether administered in combination, sequentially or simultaneously.

[0097] As used herein, "treat," "treating," or "treatment" encompasses the treatment of a target disease, injury, or condition (e.g., HIV-1 infection) in a subject (e.g., a mammal, such as a human) suffering from the target disease or condition, and includes: (i) inhibiting the progression of the disease, injury, or condition, i.e., stopping its development; (ii) alleviating or relieving the disease, injury, or condition, i.e., causing the decline of the disease or condition; or (iii) alleviating the symptoms caused by the disease, injury, or condition. As used herein, the terms "disease," "disorder," and "disorder" are used interchangeably. As used herein, "inhibit," "treat," "treat," and "improve" are used interchangeably and refer to, for example, stagnation of symptoms, prolongation of survival, partial or complete improvement of symptoms, and partial or complete elimination of a condition, disease, or disorder.

[0098] As used herein, "preventing" or "preventing" includes (i) preventing or inhibiting the occurrence of a disease, injury or condition in a subject, particularly when such subject is susceptible to the condition but has not yet been diagnosed as having the condition; or (ii) reducing the likelihood that the disease, injury or condition will occur in a subject.

[0099] As used herein, the term "antibody" refers to a binding agent comprising a separation or reorganization of the necessary variable region sequences that specifically bind to an antigenic epitope. Therefore, an antibody is an antibody or fragment thereof in any form that exhibits a desired biological activity (e.g., in conjunction with a specific target antigen). Therefore, the term is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, nanobodies, double antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, including but not limited to scFv, Fab and Fab2, as long as they exhibit desired biological activity.

[0100] The term "human antibody" refers to an antibody containing sequences of human origin except for possible non-human CDR regions, and does not mean that the complete structure of an IgG molecule is present, only that the antibody has minimal immunogenicity in humans.

[0101] "Antibody fragments" comprise a portion of an intact antibody, e.g., the antigen binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (e.g., Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a designation that reflects the ability to crystallize readily. Pepsin treatment produces a F(ab')2 fragment that has two antigen-binding sites and is still able to cross-link antigen.

[0102] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. In this configuration, the three CDRs of each variable domain typically interact to define V H -V LThe antigen binding site on the surface of the dimer. Generally, the six CDRs confer antigen binding specificity to the antibody as a whole, although there are examples of maintaining antigen binding specificity when deleting or modifying one or more of the six CDRs, for example, by changing the amino acid sequence of one or more CDRs, for example, by amino acid insertion, deletion or substitution. In addition, even a single variable domain (or half of an Fv that contains only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, although with an affinity lower than that of the complete binding site. Residues other than those residues present in the CDRs may also be important or play a role in antigen binding and / or specificity, as shown for PGT121 and closely related somatic variants, which use residues in the light chain framework 3 to interact with the gp120 antigen (Julien et al., Science 342: 1477-83 (2013); Julien et al., PLOS Pathog. 9: e1003342 (2013)). These residues arise in part from an unusual three amino acid insertion that extends an otherwise short surface loop in PGT121 and related somatic variants (e.g., PGT122, PGT123, PGT124, PGT133, PGT1134) that contacts the N322-linked glycan and protein residues on HIV Env, effectively forming an additional (e.g., fourth) complementarity determining region (CDR) loop in the PGT121 light chain between LC CDRs 2 and 3.

[0103] The term "hypervariable region" refers to the amino acid residues of an antibody that are generally responsible for antigen binding. The hypervariable region generally includes amino acid residues from the "complementarity determining region" or "CDR" (e.g., V when numbered according to the Kabat numbering system). L Around residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in V H around residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the protease inhibitory factor (PFI) domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); and / or those from "hypervariable loops" (e.g., V according to the Chothia numbering system, V L Residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in V H26-32 (H1), 52-56 (H2), and 95-101 (H3) in the present invention; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); and / or those residues from the "hypervariable loop" VCDR (e.g., V L Residues 27-38 (L1), 56-65 (L2), and 105-120 (L3), and V H 27-38 (H1), 56-65 (H2) and 105-120 (H3) in; Lefranc, MP et al., Nucl. Acids Res. 27:209-212 (1999), Ruiz, M. et al., Nucl. Acids Res. 28:219-221 (2000)). Optionally, the antibody has symmetric insertions at one or more of the following points: V according to AHo; Honneger, A. and Plunkthun, AJ Mol. Biol. 309:657-670 (2001) numbering, V L 28, 36 (L1), 63, 74-75 (L2) and 123 (L3), and V H 28, 36 (H1), 63, 74-75 (H2) and 123 (H3).

[0104] The "Fab" fragment is the region on the antibody that binds to the antigen. It consists of a constant region and a variable region of each heavy and light chain. These domains form a paratope-antigen binding site at the amino terminus of the monomer. The two variable regions bind to the epitope on its specific antigen. The Fab fragment differs from the Fab' fragment in that several residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the hinge region of the antibody. Fab'-SH is the nomenclature herein for Fab' in which the cysteine ​​residues of the constant domains carry free thiol groups. F(ab')2 antibody fragments were originally produced as Fab' fragment pairs, which have hinge cysteines between the Fab' fragments. Other chemical couplings of antibody fragments are also known.

[0105] The "light chain" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on the amino acid sequence of their variable or constant domains. Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chains. There are five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

[0106] "Single-chain Fv" or "scFv" or "sFv" antibody fragments comprise the V H and V L domains, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a V H and V L The polypeptide linker between the domains enables the sFv to form the desired structure for antigen binding.

[0107] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments consist of two nucleotides connected to the same polypeptide chain (V H -V L ) in the light chain variable domain (V L )'s heavy chain variable region domain (V H ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and form two antigen-binding sites. Diabodies are more fully described in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0108] An "isolated" antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that has been identified and separated and / or recovered from a component of its natural environment. Contaminating components of its natural environment are substances that interfere with the diagnostic or therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody will be purified (1) to greater than 95% by weight of the antibody, as determined by the Lowry method, e.g., greater than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequencer, or (3) to homogeneity as determined by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver stain. An isolated antibody includes an antibody in situ within a recombinant cell, since at least one component of the antibody's natural environment will not be present. However, typically, an isolated antibody will be prepared by at least one purification step.

[0109] An antibody or antigen-binding fragment thereof that "specifically binds" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide is an antibody or antigen-binding fragment thereof that binds to that particular polypeptide or an epitope on a particular polypeptide and does not substantially bind to any other polypeptide or polypeptide epitope. In some embodiments, the antibodies of the present disclosure have a dissociation constant K of equal to or less than 100 nM, optionally less than 10 nM, optionally less than 1 nM, optionally less than 0.5 nM, optionally less than 0.1 nM, optionally less than 0.01 nM, or optionally less than 0.005 nM measured at a temperature of about 4°C, 25°C, 37°C, or 42°C. d , specifically binds to an antigen, e.g., HIV-1 gp120 polypeptide, in the form of a monoclonal antibody, scFv, Fab, or other antibody. The affinity of an antibody can be readily determined using conventional techniques, e.g., those described by Scatchard et al. (Ann. NY Acad. Sci. USA 51:660 (1949), ELISA analysis, biomembrane interference (BLI) analysis, and surface plasmon resonance (SPR) analysis. Immunoassay methods are commonly used, including, e.g., immunofluorescence-based analysis, such as immunohistochemistry (IHC) and / or fluorescence activated cell sorting (FACS), to determine and evaluate the binding properties of an antibody to its antigen, cell, or tissue.

[0110] As used herein, an "internalizing" antibody is an antibody that is taken up by a cell {i.e., enters a cell) when it binds to an antigen {e.g., a cell surface polypeptide or receptor) on a mammalian cell. Internalizing antibodies will of course include antibody fragments, human or chimeric antibodies, and antibody conjugates. For specific therapeutic applications, in vivo internalization is contemplated. The number of internalized antibody molecules is sufficient or suitable to kill cells or inhibit their growth, particularly infected cells. Depending on the efficacy of the antibody or antibody conjugate, in some cases, the uptake of a single antibody molecule into a cell is sufficient to kill the target cell to which the antibody binds. For example, certain toxins are highly efficient in killing, such that internalization of one toxin molecule conjugated to an antibody is sufficient to kill infected cells.

[0111] The term "antagonist" antibody is used in the broadest sense and includes antibodies that partially or completely block, inhibit or neutralize the biological activity of an epitope, polypeptide or cell to which it specifically binds. Methods for identifying antagonist antibodies may include contacting a polypeptide or cell to which a candidate antagonist antibody specifically binds with the candidate antagonist antibody and measuring a detectable change in one or more biological activities normally associated with the polypeptide or cell.

[0112] "Antibodies that inhibit the growth of infected cells" or "growth inhibitory" antibodies are antibodies that bind to infected cells that express or are capable of expressing HIV1 epitopes that are bound by antibodies and cause measurable growth inhibition of the cells. Preferred growth inhibitory antibodies inhibit the growth of infected cells by more than 20%, preferably about 20% to about 50%, and even more preferably, more than 50% (e.g., about 50% to about 100%) compared to a suitable control, which is typically an infected cell that is not treated with the tested antibody. Growth inhibition can be measured in cell culture at an antibody concentration of about 0.1 to about 30 μg / ml or about 0.5nM to about 200 nM, wherein growth inhibition is measured 1-10 days after the infected cells are exposed to the antibody. Growth inhibition of infected cells in vivo can be measured in various ways known in the art. If an antibody of about 1 μg / kg to about 100 mg / kg body weight is administered for the first time, within about 5 days to 3 months, preferably within about 5 to 30 days, resulting in a reduction in the percentage of infected cells or the total number of infected cells, the antibody is growth inhibitory in vivo.

[0113] An antibody that "induces apoptosis" is an antibody that induces programmed cell death, as determined by binding of annexin V, fragmentation of DNA, cell shrinkage, expansion of the endoplasmic reticulum, cell fragmentation, and / or the formation of membrane vesicles (called apoptotic bodies). Preferably, the cells are infected cells. Various methods can be used to assess cellular events associated with apoptosis. For example, phosphatidylserine (PS) translocation can be measured by annexin binding; DNA fragmentation can be assessed by DNA laddering; and nuclear / chromatin condensation and DNA fragmentation can be assessed by any increase in hypodiploid cells. Preferably, an antibody that induces apoptosis is an antibody that causes about 2 to 50 times, preferably about 5 to 50 times, and most preferably about 10 to 50 times annexin binding induction relative to untreated cells in an annexin binding assay.

[0114] Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or an amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis (e.g., antibody-dependent cell-mediated phagocytosis (ADCP)); downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0115] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted or exogenously administered Igs that bind to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. The antibodies "arm" the cytotoxic cells and are required for such killing. The primary cells for mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 4 on page 464 of Ravctch and Kinct, Annu. Rev. Immunol 9:457-92 (1991). In order to evaluate the ADCC activity of the target molecule, an in vitro ADCC assay can be performed, such as described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337. Useful effector cells for such an analysis include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the antibody or its antigen-binding fragment can be evaluated in vivo, for example, in an animal model, such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 95: 652-656 (1998).

[0116] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In certain embodiments, the FcR is a native sequence human FcR. In addition, a preferred FcR is an FcR (gamma receptor) that binds to an IgG antibody and includes receptors of the FcγRI, FcγRII and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains, and FcγRIIC, which includes an FcγRIIB extracellular domain fused to an activation cytoplasmic region. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see review by M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and deHaas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those identified in the future, are included in the term "FcR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), and which plays a role in the clearance of IgG from lysosomal degradation by FcRn-dependent recycling after endocytosis. FcRn binding after pinocytosis in endothelial cells has been shown to be important for maintaining an extended pharmacokinetic half-life of antibodies. Evaluation of pH-dependent human FcRn binding of antibodies in vitro can be performed to provide predictions of the potential for favorable clinical pharmacokinetics (Datta-Mannan and Wroblewski, Drug Metab. Dispos. 42:1867-1872 (2014)).

[0117] "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include PBMCs, NK cells, monocytes, cytotoxic T cells, and neutrophils; PBMCs and NK cells are preferred. Effector cells can be isolated from natural sources, for example, from blood.

[0118] "Complement dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) that binds to its cognate antigen. To assess complement activation, a CDC assay can be performed, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).

[0119] "Mammal" for the purpose of treating infection refers to any mammal, including humans, domestic and farm animals, research animals (such as mice, rats and primates), and zoo animals, sports animals or pet animals (such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc.). In a specific embodiment, the mammal is a human.

[0120] "Neutralizing antibodies" are antibodies that can neutralize the ability of the pathogen to initiate and / or maintain infection in a host and / or in a target cell in vitro. The present invention provides neutralizing monoclonal human antibodies and antigen-binding fragments thereof, wherein the antibodies recognize antigens from HIV, for example, gp120 polypeptides. In certain embodiments, "neutralizing antibodies" can inhibit entry of HIV-1 virus (e.g., SF162 and / or JR-CSF) with a neutralization index of >1.5 or >2.0 (Kostrikis LG et al. / Virol. 1996; 70(1): 445-458). "Broad-spectrum neutralizing antibodies" refer to antibodies that neutralize more than one HIV-1 virus species (from different clades and different strains within a clade) in a neutralization assay. Broad-spectrum neutralizing antibodies can neutralize at least 2, 3, 4, 5, 6, 7, 8, 9 or more different HIV-1 strains, the strains belonging to the same or different clades. In certain embodiments, the broadly neutralizing antibodies can neutralize multiple HIV-1 species belonging to at least 2, 3, 4, 5 or 6 different clades. In certain embodiments, the inhibitory concentration of the monoclonal antibody in a neutralization assay can be less than about 0.0001 μg / ml, less than about 0.001 μg / ml, less than about 0.01 μg / ml, less than about 0.1 μg / ml, less than about 0.5 μg / ml, less than about 0.1 μg / ml, less than about 0.5 μg / ml, less than about 1.0 μg / ml, less than about 5 μg / ml, less than about 10 μg / ml, less than about 25 μg / ml, less than about 50 μg / ml, or less than about 100 μg / ml to neutralize about 50% of the input virus.

[0121] HIV viruses are divided into specific groups, M, N, O and P, of which M is the "main" group and causes most of HIV / AIDS worldwide. Based on its genetic sequence, group M is further subdivided into subtypes (also called clades) with different prevalence in different geographic locations.

[0122] Group M "subtype" or "clade" is a subtype of HIV-1 group M defined by genetic sequence data. Examples of group M subtypes include subtype AK. Some subtypes are known to be more toxic or resistant to different drugs. There are also "circulating recombinant forms" or CRFs derived from the recombination between different subtype viruses, which are numbered separately. For example, CRF12_BF is a recombination between subtypes B and F. Subtype A is common in West Africa. Subtype B is the main form in Europe, the United States, Japan, Thailand and Australia. Subtype C is the main form in South Africa, East Africa, India, Nepal and parts of China. Subtype D is usually only seen in East Africa and Central Africa. Subtype E has never been identified as a non-recombinant, and is only recombined with subtype A as CRF01_AE. Subtype F has been found in Central Africa, South Africa and Eastern Europe. Subtype G (and CRF02_AG) has been found in Africa and Central Europe. Subtype H is limited to Central Africa. Subtype I was originally used to describe the strain now known as CRF04_cpx, where cpx indicates a "complex" recombination of several subtypes. Subtype J is found primarily in North, Central, and West Africa, while Caribbean subtype K is limited to the Democratic Republic of the Congo and Cameroon. These subtypes are sometimes further divided into sub-subtypes, such as A1 and A2, or F1 and F2. In 2015, strain CRF19, a recombinant of subtypes A, D, and G, with a subtype D protease, was found to be strongly associated with the rapid progression of AIDS in Cuba.

[0123] "HIV tropism" refers to the specificity of the HIV virus for a particular host cell, which is determined in part by the interaction of viral surface structures with receptors present on the host cell surface. The HIV tropism of a patient's virus can be measured by Trofile analysis.

[0124] HIV can infect a variety of cells, such as CD4+ helper T cells and macrophages expressing CD4 molecules on their surfaces. HIV-1 enters macrophages and T helper cells not only through the interaction of viral particle envelope glycoproteins (e.g., gp120) with CD4 molecules on target cells, but also through the interaction with its chemokine co-receptor. HIV-1 macrophage (M-tropic) strains or non-syncytial induction strains (NSI) use β-chemokine receptor CCR5 to enter, and therefore can replicate in macrophages and CD4+T cells. These strains are called R5 viruses. This CCR5 co-receptor is used by almost all primary HIV-1 isolates, regardless of viral genetic subtypes. T-tropic isolates, or syncytial induction (SI) strains replicate in primary CD4+T cells and in macrophages and use α-chemokine receptor CSCR4 to enter. These strains are called X4 viruses. Viruses that utilize only the CCR5 receptor are called R5, those that utilize only CXCR4 are called X4, and those that utilize both are called X4R5 or dual / mixed tropism. However, the use of co-receptors alone cannot explain viral tropism, because not all R5 viruses are able to utilize CCR5 on macrophages for productive infection.

[0125] The present invention also relates to "non-neutralizing antibodies," which in certain embodiments are antibodies that bind to one or more viral strains but do not neutralize the virus. However, in terms of Fc-mediated killing, non-neutralizing antibodies can still eliminate cells expressing viral antigens that are bound by the antibody but not neutralized by the antibody. Thus, in certain embodiments, the antibodies of the present invention can bind to viral antigens and eliminate virally infected cells without neutralizing the virus.

[0126] The term "nucleic acid molecule" refers to a polymeric form of nucleotides, and includes sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. In a specific embodiment, nucleotides refer to ribonucleotides, deoxyribonucleotides, or modified forms of any nucleotide type, and combinations thereof. The term also includes, but is not limited to, single-stranded and double-stranded forms of DNA. In addition, polynucleotides, such as cDNA or mRNA, can include any one or two naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide bonds. Nucleic acid molecules can be chemically or biochemically modified, or can contain non-natural or derived nucleotide bases, as readily understood by those skilled in the art. Such modifications include, for example, labeling, methylation, replacement of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), side-joining moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylating agents, and modified bonds (e.g., α-anomeric nucleic acids, etc.). The above terms are also intended to include any topological conformations, including single-stranded, double-stranded, partially double-stranded, triple-stranded, hairpin, circular, and padlock conformations. Reference to a nucleic acid sequence includes its complementary sequence, unless otherwise noted. Therefore, reference to a nucleic acid molecule having a specific sequence should be understood to include its complementary chain, having its complementary sequence. The term also includes codon-optimized nucleic acids.

[0127] The term "operably linked" refers to two or more nucleic acid sequence elements that are generally physically linked and functionally associated with each other. For example, a promoter is operably linked to a coding sequence if the promoter is capable of initiating or regulating the transcription or expression of the coding sequence, in which case the coding sequence is understood to be "under the control" of the promoter.

[0128] As used herein, "substitution" means that one or more amino acids or nucleotides are replaced by different amino acids or nucleotides, respectively.

[0129] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0130] "Isolated nucleic acid encoding an antibody or fragment thereof" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including these nucleic acid molecules in a single vector or separate vectors, and these nucleic acid molecules present in one or more locations in a host cell.

[0131] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that bind to the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0132] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom regardless of the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity screened or selected in the initially transformed cell are included herein.

[0133] Polynucleotide "variants", as the term is used herein, are polynucleotides that generally differ from the polynucleotides specifically disclosed herein by one or more substitutions, deletions, additions and / or insertions. Such variants may be naturally occurring or may be produced synthetically, for example, by modifying one or more polynucleotide sequences of the invention and evaluating one or more biological activities of the encoded polypeptide as described herein and / or using any of a variety of techniques known in the art.

[0134] Polypeptide "variants", as the term is used herein, are polypeptides that generally differ from the polypeptides specifically disclosed herein by one or more substitutions, deletions, additions and / or insertions. Such variants may be naturally occurring or may be produced synthetically, for example, by modifying one or more of the above polypeptide sequences of the invention and evaluating one or more biological activities of the polypeptide as described herein and / or using any of a variety of techniques known in the art.

[0135] The term "variant" can also refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations. In one embodiment, the molecule is an antibody. For example, somatic variants can include all related naturally occurring antibodies that are part of or derived from the same B cell lineage. Engineered variants can include all single mutations or combined mutations performed on an antibody.

[0136] The terms "PGT121", "PGT-121", "PGT121 WT", "PGT-121WT", "PGT121-WT", "PGT121.WT", "PGT121.1", "PGT121 LO6", "PGT121 L06", "PGT121 L06 WT", "PGT121 LO6WT", etc. are used interchangeably herein and refer to an antibody comprising a heavy chain and a light chain, wherein the heavy chain has the amino acid sequence listed in SEQ ID NO: 190 and the light chain has the amino acid sequence listed in SEQ ID NO: 276.

[0137] Modifications can be carried out in the structure of polynucleotides and polypeptides of the present invention, and still obtain functional molecules encoding variants or derivative polypeptides with desired properties. When it is desired to change the amino acid sequence of a polypeptide to form an equivalent or even improved variant or part of a polypeptide of the present invention, those skilled in the art will usually change one or more codons of the encoding DNA sequence.

[0138] For example, certain amino acids can replace other amino acids in the protein structure without appreciable loss of their ability to bind to other polypeptides (e.g., antigens) or cells. Since it is the binding ability and properties of a protein that define the biological functional activity of the protein, certain amino acid sequence substitutions can be made in the protein sequence and certainly in its underlying DNA coding sequence and still obtain a protein with similar properties. It is therefore contemplated that various changes can be made in the polypeptide sequences of the disclosed antibodies and antigen-binding fragments thereof, or in the corresponding DNA sequences encoding the polypeptides, without appreciable loss of their biological use or activity.

[0139] In many cases, a polypeptide variant will contain one or more conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted for another amino acid with similar properties, such that one skilled in the art of peptide chemistry would expect that the secondary structure and hydropathic nature of the polypeptide would not be substantially altered.

[0140] As described below, when comparing polynucleotides and polypeptide sequences, two sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence. Comparison between two sequences is usually performed by comparing the sequences over a comparison window to identify and compare sequence similarities in local regions. As used herein, a "comparison window" refers to a segment of at least about 20 consecutive positions, typically 30 to about 75, 40 to about 50 consecutive positions, wherein after the two sequences are optimally aligned, a sequence can be compared to a reference sequence of the same number of consecutive positions.

[0141] Optimal alignment of sequences for comparison can be achieved using the Megalign program in the Lasergene bioinformatics software suite (DNASTAR, Inc., Madison, WI) using default parameters. This program implements several alignment schemes described in the following references: Dayhoff, MO (1978) A model of evolutionary change in proteins-Matrices for detecting distant relationships. See Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol.5, Suppl.3, pp.345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp.626-645 Methods in Enzymology vol.183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM (1989) CABIOS 5:151-153; Myers, EW and Muller W. (1988) CABIOS 4:11-17; Robinson, ED (1971) Comb. Theor 77:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, PHA and Sokal, RR (1973) Numerical Taxonomy-the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, WJ and Lipman, DJ (1983) Proc. Natl. Acad., Sci.USA 80:726-730.

[0142] Alternatively, optimal alignment of sequences for comparison may be performed by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman (1988) J. Mol. Biol. 48:443, computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TEASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by inspection.

[0143] An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST2.0 can be used, for example, using the parameters described herein, to determine the percent sequence identity of the polynucleotides and polypeptides of the present invention. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information.

[0144] In an illustrative example, for nucleotide sequences, the cumulative score can be calculated using the parameters M (reward score for a pair of matching residues; always>0) and N (penalty score for mismatched residues; always<0). Extension of word hits in each direction stops when the following occurs: the cumulative alignment score decreases by the amount X from its maximum achieved value; the cumulative score reaches zero or less due to the accumulation of one or more negative scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11 and an expectation (E) of 10 and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) for alignments, with 50 (B), 10 expectations (E), M=5, N=-4, and comparison of both chains as defaults.

[0145] For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the following occurs: the cumulative alignment score decreases by the amount X from its maximum achieved value; the cumulative score reaches zero or lower due to the accumulation of one or more negative scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.

[0146] In one method, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein for optimal alignment of the two sequences, the portion of the polynucleotide or polypeptide sequence in the comparison window may include 20% or less, typically 5 to 15%, or 10 to 12% additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions). The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in the two sequences to generate the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the result by 100 to generate the percentage of sequence identity.

[0147] "Homology" refers to the percentage of residues in a polynucleotide or polypeptide sequence variant that are identical with the non-variant sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology.

[0148] "Developability" refers to the inherent chemical and biophysical properties of an antibody that make it suitable for commercial manufacturing and therapeutic use. These properties can include thermal stability (e.g., melting temperature), low pH stability (e.g., during viral inactivation procedures required during GMP production), solubility, viscosity, product homogeneity, and chemical stability (e.g., oxidation, deamidation, isomerization, cleavage, glycosylation, glycation, hydroxylation).

[0149] "Binding affinity" can refer to the binding dissociation constant (Kd) or the apparent affinity (eg, EC50) value.

[0150] "Percent aggregation" can refer to the percent loss of soluble protein monomer as determined by SEC. Thus, a percent change in monomer content with a negative value will indicate a loss of monomer and an increase in aggregation, while a percent change in monomer content with a positive value indicates an increase in monomer and a corresponding decrease in aggregation.

[0151] Antibodies and antigen-binding fragments thereof, nucleic acids, vectors and host cells

[0152] The present invention includes new antibodies and antigen binding fragments thereof. In certain embodiments, these antibodies and antigen binding fragments thereof bind and neutralize HIV-1, for example, cell-free HIV-1 virus. In certain embodiments, these antibodies and antigen binding fragments thereof bind HIV-1 antigens expressed on the cell surface and eliminate or kill cells. In various different embodiments, the antibody activates effector cells, for example, T cells expressing FcγRIIA. In certain embodiments, the Fc domain of the antibody binds to FcγR expressed on innate immune cells. In specific embodiments, they induce antibody-dependent cellular killing of HIV-1 infected cells mediated by natural killer (NK) cells, for example, via antibody-dependent cellular cytotoxicity (ADCC). In specific embodiments, they induce antibody-dependent cellular killing mediated by monocytes and peripheral blood mononuclear cells (PBMC), for example, via antibody-dependent cellular phagocytosis (ADCP) and / or granzyme- and perforin-mediated cytotoxicity or ADCC.

[0153] In certain embodiments, the antibodies and antigen-binding fragments thereof of the present invention relate to the antibody previously described as PGT-121LO6 in PCT Application Publication No. WO2012 / 030904. In certain embodiments, the antibodies and antigen-binding fragments thereof comprise the six CDRs present in the PGT-121LO6 antibody, as defined by one or more of the Kabat, IMGT or Chothia antibody numbering schemes. In certain embodiments, the antibodies and antigen-binding fragments thereof comprise at least one, at least two, at least three, at least four or at least five CDRs present in the PGT-121LO6 antibody. The PGT-121LO6 CDRs are provided in Table 1. In certain embodiments, the present invention includes a light chain (or an antigen-binding fragment thereof) or a heavy chain (or an antigen-binding fragment thereof) of an antibody of the present invention. In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention are not PGT-121LO6 antibodies or antigen-binding fragments thereof.

[0154] In certain embodiments, the antibodies and antigen-binding fragments thereof of the present invention relate to any variant or derivative of PGT-121LO6 described herein, for example, having a sequence shown in Table 1. In certain embodiments, the antibodies and antigen-binding fragments thereof comprise the six CDRs present in any of the antibodies shown in Table 1, as defined by one or more of the Kabat, IMGT or Chothia antibody numbering schemes. In certain embodiments, the antibodies and antigen-binding fragments thereof comprise at least one, at least two, at least three, at least four or at least five CDRs present in any of the antibodies provided in Table 1.

[0155] In certain embodiments, the antibodies and antigen-binding fragments thereof comprise CDRs, heavy chains and / or light chains comprising one or more amino acid modifications (e.g., insertions, deletions or substitutions) compared to PGT-121LO6. In various embodiments, one or more amino acid modifications impart one or more improved properties (e.g., therapeutic properties) to the antibodies or antigen-binding fragments thereof compared to PGT-121LO6. Without limitation, certain embodiments of one or more amino acid substitutions provide antibodies or antigen-binding fragments thereof with superior pharmacokinetic properties, improved serum stability (e.g., improved serum half-life), improved CDRs, and / or light chains compared to PGT-121LO6. max , improved binding affinity, improved effector function, improved HIV-1 neutralization, reduced immunogenicity and / or improved manufacturing efficiency or convenience of the antibody or antigenic fragment thereof. In various different embodiments, one or more amino acid modifications confer improved developability (e.g., improved Tm, improved stability during low pH virus inactivation procedures and / or removal of promiscuous N-linked glycans). In various different embodiments, by removing (e.g., by site-directed mutagenesis) experimentally identified T-cell epitopes, one or more amino acid modifications confer reduced immunogenicity (e.g., reduced response rate of ex vivo T-cell activation donors). Without limitation, specific embodiments of one or more amino acid modifications provide for superior pharmacokinetic properties, improved serum stability (e.g., improved serum half-life), improved C max , improved binding affinity, improved effector function, improved HIV-1 neutralization, reduced immunogenicity and / or improved manufacturing efficiency or convenience of the antibody or its remote binding fragment. In various embodiments, the mutation introduced to enhance any of the above-listed properties (e.g., reduced immunogenicity, enhanced therapeutic properties and / or antigen binding or enhanced developability) can be located in the CDR region, framework region, or framework region predicted to interact directly with the antigen (e.g., a framework region functionally equivalent to the CDR).

[0156] The crystal structure and experimental analysis of an antibody highly related to the PGT-121LO6 antibody (i.e., PGT-122) revealed that it uses amino acid residues outside the CDR to bind to the antigen (together with the CDR). For example, this antibody appears to have additional regions in the framework region that contact the antigen (see, e.g., Experimental Validation for PGT121 and related antibodies: Sok et al., 2013. PLOS Pathogens 9, e1003754). The high-resolution structure of PGT122 bound to the Env viral antigen has been determined (see, e.g., Julien, JP et al., 2013, Science 342, 14777-14783 and Pancera, M. et al., 2014, Nature 514, 455-461). The structure of PGT121 is described in Julien JP et al., 2013, PLOS Pathogens 9, e1003342 and Mouquet H et al., 2012, PNAS 109, E3268-E3277. The structure of PGT122 is described in Julien JP et al., 2013. PLOS Pathogens 9, e1003342. PDB ID 4JY5; and the structure of PGT123 is described in Julien JP et al., 2013, PLOS Pathogens 9, e1003342. The PGT122 and PGT123 antibodies are closely related to the PGT121 antibody, so the PGT122 / Env structure together with the knowledge of the PGT121, PGT122 and PGT123 structures can be used to very accurately model the structure of PGT121 bound to Env and predict with high confidence the residues of PGT121 involved in binding to Env. The predicted PGT121 contact residues based on similarity to the PGT122 and PGT122 / Env structures are provided below, with framework residues shown in bold:

[0157] HC (Kabat #): 33, 56, 58, 99, 100, 100A, 100B, 100C, 100D, 100E, 100G, 100I, 100J, 100K, 100L; and

[0158] LC(Kabat#): 28, 29, 30, 50, 51, 52, 66, 67, 67A, 67C, 91, 92, 93, 94, 95, 95A, 95B.

[0159] In addition, PGT-121LO6 antibody has been shown to bind many different antigen variants, for example, different virus strains, which can contact antibodies at unknown amino acid positions other than those listed above. Different virus strains have different Env (i.e., antigen) sequences and different glycosylation patterns, and even single Env sequences can have heterogeneous glycosylation patterns, thereby requiring broad-spectrum binding or neutralizing antibodies to recognize Env proteins of different HIV-1 variants or even different glycosylation patterns on the same Env protein. For example, the epitope of PGT121 is composed of Env V3 loops, especially N-linked polysaccharides at position N332. The V3 loop is the main determinant of cell tropism and viral clade. In 117 CCR5-tropic viruses of multiple clades, the presence of potential N-linked glycosylation (PNG) motifs in the viral DNA sequence encoding N332 polysaccharides is statistically significantly correlated with the susceptibility of neutralization of PGT121 in viruses of clades B, G, A, AC and AE. Of the 50 clade B Env sequences isolated from patients participating in a Gilead-sponsored clinical trial, 94% of CCR5-tropic Envs bearing the N332 PNG motif were susceptible to neutralization by PGT121, compared to only 26% of viruses that were not CCR5-tropic and were positive for the N332 PNG motif (P<0.0001). Thus, genetic determination of Env clade, tropism, and the presence of the N332 PNG motif are highly predictive of neutralization susceptibility by PGT121 and can be used as a marker to predict viral susceptibility to neutralization by PGT121 and its derivatives.

[0160] Moreover, given the diversity of amino acid sequences of different antigenic variants, it may be difficult to predict which amino acid residues in the variable region of an antibody are required for binding to various antigenic variants, and it may also be difficult or impossible to accurately predict a priori which amino acid residues of an antibody can be changed to give the antibody improved properties, such as reduced glycosylation, reduced immunogenicity, reduced isomerization, enhanced effector function, enhanced neutralization activity, or enhanced recombinant production. The present invention relates to the identification of specific amino acid modifications and combinations thereof that effectively enhance the therapeutic properties of the antibody and its antigen-binding fragments without substantially affecting its ability to bind to multiple antigenic variants and (in certain embodiments) its associated broad-spectrum neutralization properties. In certain embodiments, the antibodies of the present invention are neutralizing antibodies, e.g., broad-spectrum neutralizing antibodies, while in other embodiments, the antibodies of the present invention are non-neutralizing antibodies capable of eliminating cells expressing viral antigens that are bound by the antibody but not neutralized by the antibody.

[0161] In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention have better serum pharmacokinetics (e.g., improved serum half-life) than the PGT-121 LO6 antibody, e.g., after administration to a mammal. In other embodiments, the antibodies or antigen-binding fragments of the invention have comparable serum pharmacokinetics (e.g., improved serum half-life) than the PGT-121 LO6 antibody. In certain embodiments of the invention, serum pharmacokinetics (e.g., area under the concentration-time curve (AUC), clearance (CL), volume (V), half-life (t 1 / 2 ), maximum concentration (C max ) or minimum concentration (C min )). Analytical methods for measuring serum antibody levels and methods for calculating the resulting pharmacokinetics are known in the art. The amount of antibody present in serum can be determined by standard techniques, such as LC-MS / MS, ELISA, SPR, BLI, ECL (MSD), α-LISA or HTRF, for example, an ELISA using recombinant gp120 protein as described in Example 3 of PCT Application Publication No. WO2012 / 040904. In certain embodiments, the antibody serum concentration can be determined by capturing with recombinant BAL gp120 or SHIV gp140, and detecting with an anti-human IgG1 conjugate using electrochemiluminescence (ECL) detection using a mesoscale discovery (MSD) platform. Pharmacokinetics can be determined from the resulting antibody serum concentration-time profile using standard non-compartmental or multicompartmental pharmacokinetic analysis. In certain embodiments, the antibodies or antigen-binding fragments of the present invention have unchanged or improved pharmacokinetics compared to the PGT121 LO6 antibody after administration to a mammal (e.g., a human, rat or monkey). Improved pharmacokinetics through increased exposure (AUC), decreased clearance and increased half-life, increased C max or improved C min In certain embodiments, the pharmacokinetics are improved by at least 0%, 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000%.

[0162] In certain embodiments, the antibodies or antigen-binding fragments of the invention have improved serum stability (e.g., improved serum half-life) compared to the PGT-121LO6 antibody, for example, after administration to a mammal. Assays for measuring serum stability or antibody half-life are known in the art. In one embodiment, serum stability or serum half-life can be determined by measuring serum levels of the antibody at various time points after administration to a subject (e.g., a human or a test animal). The amount of antibody present in serum can be determined by standard techniques, such as LC-MS / MS, ELISA, SPR, BLI, ECL (MSD), α-LISA or HTRF, for example, an ELISA using recombinant gp120 protein as described in WO 2012 / 040904. In some embodiments, serum stability or serum half-life can be improved by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500% or at least 1000%. In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention has a serum half-life of at least 30 min, at least 1 hour, at least 4 hours, at least 8 hours, at least 12 hours, at least 16 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least one week, at least two weeks, at least three weeks, at least four weeks or at least two months. In some embodiments, the antibodies or antigen-binding fragments thereof of the invention may have improved serum stability at about 4°C, at about 5°C, at about 10°C, at about 15°C, at about 20°C, at about 25°C, at about 30°C, at about 37°C or higher compared to the PGT-121LO6 antibody.

[0163] In certain embodiments, the antibodies or antigen-binding fragments of the invention have an equal or improved or higher antigen-binding affinity, e.g., affinity for at least one HIV strain, than the PGT-121LO6 antibody. In a specific embodiment, the antigen is an intact membrane-bound HIV Env trimer expressed on the cell surface or viral envelope, a soluble gp140 fragment of Env, a soluble gp120 fragment of Env, or any smaller subdomain or engineered portion of Env containing all the necessary epitope-determining residues and structures required for PGT121 binding, from any HIV-1 strain described herein. Binding affinity can be readily determined using assays known and available in the art, such as ELISA, SPR, BLI, or flow cytometry.

[0164] In certain embodiments, the apparent binding affinity is determined by standard techniques, such as ELISA, for example, using recombinant gp120 or gp140 proteins as described in Example 3 of PCT Application Publication No. WO2012 / 030904, or as described herein. In certain embodiments, the binding affinity for at least one HIV strain is improved, for example, by at least 0%, 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 1000%, at least 1500%, at least 2000%, at least 2500%, at least 3000%, at least 5000%, or at least 10,000%. In certain embodiments, one or more strains include BaL, TRO, SHIV, SF162 P3, pRHPA4259, qh0692, 6535, pCAAN5342, pWITO4160, AC10.0, US92HT593, or U92US657. In some embodiments, one or more strains include BaL, TRO, SHIV SF162 P3, pRHPA4259, qh0692, 6535, pCAAN5342, pWITO4160, AC10.0, US92HT593, or U92US657. In specific embodiments, the antibodies and fragments thereof of the invention bind to HIV gp120 BaL with an EC50 of <20.0 nM, <10 nM, or <1 nM, as determined in a direct ELISA assay as described herein. In certain embodiments, the antibodies and fragments thereof of the invention bind to HIV gp120 BaL, gp140 BaL, gp140 SHIV SF162P3 or gp120 TRO with a lower ELISA EC50 value, e.g., 2-fold, 1.6-fold, 1.4-fold, 1.2-fold, 1-fold, 0.8-fold, 0.6-fold lower than PGT121 LO6. In certain embodiments, the antibodies and fragments thereof of the invention bind to HIV gp120 BaL, gp120 pRHPA4295, gp120 qh0692, gp120 6535, gp120 pCAAN5342, gp120 pWITO4160 or gp120 AC10.0 with an ELISA EC50 value that is 3-fold, 2.5-fold, 2-fold, 1.5-fold, 1-fold or 0.5-fold lower than PGT121 LO6.

[0165] In other embodiments, the binding affinity is determined by FACS using a human cell line expressing recombinant HIV Env. One or more strains include BaL, US657, HT593 or SHIV SF162 P3. In a specific embodiment, the binding affinity to at least one strain is improved by, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 1000%, at least 1500%, at least 2000%, at least 2500%, at least 3000%, at least 5000% or at least 10,000%. In certain embodiments, the antibodies and fragments thereof of the present invention bind to the HIV Env of at least one strain with an IC50 of <1.0 nM or <0.8 nM, as measured by ELISA. In certain embodiments, the antibodies and fragments thereof of the invention bind to at least one strain of HIV Env with an IC50 of about 0.1 uM to about 10 nM or about 0.1 uM to about 20 nM, as determined by ELISA. In certain embodiments, the antibodies and fragments thereof of the invention bind to at least one strain of HIV gp120 (BaL) with an EC50 of <20 nM, <10 nM, <5 nM, <2 nM, <1 nM, <0.5 nM, <0.2 nM, or <0.1 nM. In specific embodiments, the at least one strain comprises two or more, three or more, four or more, or five or more strains, e.g., any of those described herein.

[0166] In a specific embodiment, the antibody or antigen-binding fragment of the present invention has an improved binding affinity to FcγR compared to the PGT-121LO6 antibody. Binding affinity can be easily determined using analytical methods known and available in the art, such as ELISA, SPR or BLI. In a specific embodiment, binding affinity is determined by standard techniques, such as ELISA, for example, as described herein. In a specific embodiment, the binding affinity is improved by, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 1000%, at least 20 times, at least 30 times, at least 50 times, at least 100 times or at least 1000 times. In certain embodiments, the antibodies or fragments thereof of the invention bind to any of human FcγRI, human FcγRIIA-167H, human FcγRIIA-167R, human FcγRIIB, human FcγRIIIA-176V, human FcγRIII-176F, human FcγRIIIB-NA1, human FcγRIIIB-NA2, human FcRn at pH 7.0, or human FcRn at pH 6.0 with any of the following EC50s as determined by ELISA: <0.1 nM, <0.2 nM, <0.3 nM, <0.5 nM, <1.0 nM, <1.5 nM, <2.0 nM, <2.5 nM, <3.0 nM, <3.5 nM, <5 nM, <10 In certain embodiments, the antibodies or fragments thereof of the present invention bind to human FcgRI with an EC50 of 0.1 to 1 μM. In certain embodiments, the antibodies or fragments thereof of the present invention bind to human FcgRIIIA-176F with an EC50 of 0.1 to 10 nM. In certain embodiments, the antibodies or fragments thereof of the present invention bind to human FcgRIIIA-176V with an EC50 of 1 to 0.1 nM. In certain embodiments, the antibodies or fragments thereof of the present invention bind to human FcgRIIA-167H with an EC50 of 100 to 1 nM. In a specific embodiment, the antibodies or fragments thereof of the invention bind to human FcgRIIA-167R with an EC50 of 50 to 1 nM. In a specific embodiment, the antibodies or fragments thereof of the invention bind to human FcgRIIB with an EC50 of 1 μM to 10 nM. In a specific embodiment, the antibodies or fragments thereof of the invention bind to human FcgRIIIB-NA1 with an EC50 of 1 nM to 50 nM.In a specific embodiment, the antibodies or fragments thereof of the present invention bind to human FcgRIIIB-NA2 with an EC50 of 1 nM to 50 nM. In certain embodiments, the antibodies or fragments thereof of the present invention bind to human FcgRI with an EC50 of 0.5 to 1.5 times the EC50 determined for PGT121 LO6. In a specific embodiment, the antibodies or fragments thereof of the present invention bind to human FcγRI with an EC50 that is unchanged compared to PGT121 LO6. In certain embodiments, the antibodies or fragments thereof of the present invention bind to human FcgRIIIA-176F with an EC50 that is 0.5, 2, 4, 10, 15, 20, 50, or 100 times lower than the value determined for PGT121 LO6. In a specific embodiment, the antibodies or fragments thereof of the present invention bind to human FcgRIIIA-176F with an ELISA EC50 that is 10 to 20 times lower than PGT121 LO6.

[0167] In specific embodiments, the antibodies or antigen-binding fragments of the invention have improved binding affinity to FcRn at pH 6.0 when compared to the PGT-121LO6 antibody. In certain embodiments, the antibodies or antigen-binding fragments of the invention have improved binding affinity to FcRn at pH 6.0 when compared to the PGT-121LO6 antibody, and have reduced or similar binding at pH 7.4 or pH 7.0. Binding affinity can be readily determined using assays known and available in the art, such as ELISA, SPR, or BLI. In specific embodiments, binding affinity is determined by standard techniques, such as ELISA, e.g., as described herein. In certain embodiments, the binding affinity to FcRn is improved, for example, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 1000%, at least 20-fold, at least 30-fold, at least 50-fold, at least 100-fold or at least 1000-fold. In certain embodiments, the antibodies or fragments thereof of the invention bind to human FcRn pH 6.0 at <10 nM, <2.0 nM or <1.0 nM. In certain embodiments, the antibodies or fragments thereof of the invention bind to human FcRn at pH 7.0 with any of the following EC50s as determined by ELISA: <0.1 nM, <0.2 nM, <0.3 nM, <0.5 nM, <1.0 nM, <1.5 nM, <2.0 nM, <2.5 nM, <3.0 nM, <3.5 nM, <5 nM, <10 nM, <20 nM, <25 nM, <30 nM, <40 nM, <50 nM, <100 nM, <200 nM, <250 nM, <500 nM, <1 μM, <2 μM, <5 μM, <10 μM, <50 μM, <100 μM, or greater than or equal to 100 μM.

[0168] The manufacturing method for biotherapeutics requires a virus inactivation (VI) procedure, which is designed as a safety measure to remove any potential viral contaminants that may be present in the cell culture process for producing antibodies. This VI procedure is usually completed by keeping the purified antibody solution at a low pH (usually at or near a pH of 3.5) for an extended time (usually 1-3 hours). Some antibodies are known to form soluble or insoluble aggregates (10-20% or higher aggregate content) in this process, making them unsuitable for manufacturing. Therefore, the stability of antibodies at low pH (e.g., pH 3.5) is a key manufacturing attribute, and the improvement of low pH stability can enable the manufacture and production of certain antibodies for therapeutic use.

[0169] In certain embodiments, the antibodies or antigen-binding fragments of the invention have improved stability at low pH, as measured by % aggregation, i.e., when stored at low pH (e.g., pH 3.5), reduced aggregation is shown in various antibody storage and elution buffers when compared to PGT121 LO6. In a specific embodiment, when kept at pH 3.5 for 1 hour, it shows less than 80%, less than 50%, less than 10% aggregation. In a specific embodiment, when kept at pH 3.5 for 1 hour, it shows less than 10%, less than 5%, less than 2% or less than 1% or 0% measurable aggregation. In a specific embodiment, when kept at pH 3.5 for 1 hour, it shows an increase in monomer content (i.e., a decrease in aggregate content) of 1%, 2%, 5% or 10%.

[0170] In certain embodiments, the antibodies or antigen-binding fragments of the invention have improved or greater effector function compared to the PGT-121LO6 antibody. In specific embodiments, the effector function is improved, for example, by at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 1000%, at least 1.5 times, at least 2 times, at least 3 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 50 times, at least 100 times, at least 1000 times, or at least 10,000 times. In specific embodiments, the effector function is ADCC, while in other embodiments, the effector function is ADCP. In specific embodiments, the effector function of the antibodies or antigen-binding fragments of the invention ranges from no response (below the limit of detection) observed at 100 μg / ml (-667 nM) to potency in the 0.1 nM-1 μM range or the 0.1 nM-1 nM range.

[0171] Effector function can be measured using assays known or available in the art, including, for example, those described in the accompanying examples. In certain embodiments, ADCC is measured in an in vitro ADCC reporter assay using engineered cells, such as donor cells infected with various HIV strains; and ADCP is measured in an in vitro ADCP assay using donor cells infected with various HIV strains. In certain embodiments, ADCC is measured in an in vitro ADCC assay using primary NK effector cells from healthy donors and engineered CD4+ luciferase reporter T cell lines infected with various HIV strains; and ADCP is measured in an in vitro ADCP assay using donor cells infected with various HIV strains. In certain embodiments, antibody-dependent effector cell activation is measured using cells (e.g., T cells) expressing human FcγRIIIA in combination with NFAT-linked reporter gene Env expressing cells. In certain embodiments, antibody-dependent effector cell activation is determined using engineered cells (e.g., T cells) expressing human FcγRIIIA coupled to NFAT-connected reporter genes co-cultured with HIV Env expressing cells. In a specific embodiment, using primary CD4+T cells infected by HIV-1 and autologous effector NK cells from healthy donors (which express FcγRIIIA and mediate antibody-mediated killing of infected cells via granzyme- and perforin-mediated cytotoxicity (ADCC)), ADCC is determined in a cell-based assay, for example, as described in the attached examples. In certain embodiments, using CD4+T cells infected by HIV-1 as target cells and primary autologous PBMC or isolated monocytes as effector cells, antibody-dependent cell killing of monocytes- and PBMC-mediated is determined, for example, as described in the attached examples.

[0172] In certain embodiments, the antibodies or antigen-binding fragments of the present invention show improved or higher neutralization of HIV-1 compared to the PGT-121LO6 antibody. Neutralization can be determined using techniques known in the art. In a specific embodiment, HIV-1 is a specific strain, for example, any strain described herein, for example, HIV subtype B isolate. In a specific embodiment, neutralization is determined for multiple different strains, for example, as an average of several or many different strains. For example, an infectivity test based on CEM-NKr-CCR5-LucR (modified CD4 T-cell line) or JC53-BL (also known as TZM-bl, modified HeLa cell line) luciferase reporter cells (Li et al., 2005 Jvir, 79 (16), 10108-25) can be used to measure neutralization activity for a virus or a group of viruses. Depending on the virus used (pseudotyped or replication-competent virus), the test can be run as a single or multiple rounds of viral replication test. Alternatively, an infectivity test based on the U87.CCR5.CXCR4 cell line (modified CD4 T cell line) can be used, using a recombinant luciferase reporter virus pseudotyped with a patient's HIV envelope protein to determine neutralizing activity (Richman et al., 2003 PNAS, 100 (7), 4144-9). Examples of viruses that can be used include, but are not limited to, laboratory-used HIV-1BaL strains and subtype B clinical isolates 93HT593, 92US657, 92US712, and 92US727 (NIH AIDS Reagent Program). In certain embodiments, neutralization is measured as described in Example 4 of PCT Application Publication No. WO2012 / 030904, or as described in the accompanying Examples. In certain embodiments, neutralization of any particular strain or the average neutralization of multiple different strains is improved, for example, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 1000%, at least 20-fold, at least 30-fold, at least 50-fold, at least 100-fold, or at least 1000-fold or at least 10,000-fold. In certain embodiments, neutralization is improved for one or a few strains, but the average neutralization is not significantly improved for multiple strains tested. In specific embodiments, the antibody or fragment thereof has an IC50 for neutralizing BaL of <0.02 or ≤0.015; for HT593, <0.2 or <0.16; for US657, <0.1; for US712, <0.25 or ≤0.20; and / or for US727, <0.1 or <0.02 or ≤0.015.In one embodiment, the antibody or fragment thereof may have an IC<0.02 μg / ml or ≤0.015 μg / ml for neutralizing BaL. 50 ; for HT593, <0.2 μg / ml or <0.16 μg / ml; for US657, <0.1 μg / ml; for US712, <0.25 μg / ml or ≤0.20 μg / ml; and / or for US727, <0.1 μg / ml or <0.02 μg / ml or ≤0.015 μg / ml IC 50 In certain embodiments, the antibody or antigen-binding fragment thereof may have a neutralization IC of 0.0005 μg / mL to 10 μg / mL for a subtype B isolate. 50 In some embodiments, the antigens and antigen-binding fragments thereof may have a neutralization IC of 0.001 μg / mL to 3.3 μg / mL for subtype B DM and X4 isolates. 50 scope.

[0173] In certain embodiments, the antibodies or antigen-binding fragments of the invention have reduced or lower immunogenicity compared to the PGT-121LO6 antibody. In certain embodiments, immunogenicity is determined using assays known and available in the art or described in the accompanying Examples. In a specific embodiment, the immunogenicity is determined using the EpiScreen 50 donor in vitro peptide scanning T-cell activation assay as described (Baker et al., 2007, Drug Discovery Development 10(2):219-227). TM T-cell epitope mapping is used to determine the location of T-cell epitopes. In a specific embodiment, the relative immunogenicity of the functionalized antibody variants is determined using a 50 donor in vitro whole molecule T-cell activation and cytokine release assay as described (Jaber et al., 2007, J Pharm Biomed Anal. 43 (4): 125-1261) (which can be a prediction of clinical immunogenicity). In a specific embodiment, the donor response rate is reduced by, for example, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or at least 80% compared to the PGT-121LO6 antibody. In a specific embodiment, the EpiScreen donor response is <10%, <15%, <20%, <25%, <30% or <35%.

[0174] In a specific embodiment, the binding of the antibodies and fragments thereof of the invention to the EnV protein is predicted to involve regions of Env in or around the following residues (HIV Env HXB2 numbering): V3 loop (324-328, 330) and the associated N332 glycan and a portion of the V1-loop (135-137) and the associated N137 glycan, residues 415-417. Antibody fill-in predictions for Env binding involve residues in the following regions that make direct contacts to the antigen in the PGT-122-Env crystal structure (Kabat numbering): CDRH1 (33), CDRH2 (50, 56, 58), CDRH3 (99, 100, 100A-E, 100G, 100I, 100L), CDRL1 (28-30), CDRL2 (50-52), LFR3 (66, 67, 67A-C), and CDRL3 (93, 94, 95A-95B).

[0175] In some aspects, the antibodies provided herein can be conjugated or connected to treatment and / or imaging / detectable moieties. Methods for conjugating or connecting antibodies are well known in the art. The combination between the antibody and the label includes any manner known in the art, including, but not limited to, covalent and non-covalent interactions. In a non-limiting embodiment, the antibody can be combined with a chemotherapeutic agent that is toxic when delivered to a cancer cell, such as a toxin, a radionuclide, an iron-related compound, a dye, an imaging agent, a fluorescent label, or is delivered to a cancer cell. Alternatively, the antibody can be combined with a detectable label, such as a radionuclide, an iron-related compound, a dye, an imaging agent, or a fluorescent agent for target antigen immunodetection. Non-limiting examples of radioactive labels include, for example, 32P, 33P, 43K, 52Fe, 57Co, 64Cu, 67Ga, 67Cu, 68Ga, 71Ge, 75Br, 76Br, 77Br, 77As, 77Br, 81Rb / 81MKr, 87MSr, 90Y, 97Ru, 99Tc, 100Pd, 101Rh, 103Pb, 105Rh, 109Pd, 111Ag, 111In, 113In, 119Sb, 121Sn, 123I, 125I, 127Cs, 128Ba, 129Cs, 131I, 131Cs, 143Pr, 153Sm, 161Tb, 166Ho, 169Eu, 177Lu, 186Re, 188Re, 189Re, 191Os, 193Pt, 194Ir, 197Hg, 199Au, 203Pb, 211At, 212Pb, 212Bi and 213Bi. Non-limiting examples of toxins include, e.g., diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthus proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotonin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and tricothecenes, Clostridium perfringens perfringens) phospholipase C (PLC), bovine pancreatic ribonuclease (BPR), antiviral protein (PAP), abrin, cobra venom factor (CVF), gelonin (GEL), saponin (SAP), viscumin.The non-limiting examples of iron-related compounds include, for example, magnetic iron oxide particles, iron or ferrous particles, Fe2O3 and Fe3O4. Iron-related compounds and methods for labeling polypeptides, proteins and peptides can be found in, for example, U.S. Patents 4,101,435 and 4,452,773, and U.S. published applications 20020064502 and 20020136693, all of which are incorporated herein by reference in their entirety. In some embodiments, the antibody can be covalently or non-covalently combined with cytotoxins or other cell proliferation inhibitory compounds to confine the delivery of the agent to tumor cells. For example, the agent can be selected from medicaments, enzyme inhibitors, proliferation inhibitors, lysates, DNA or RNA synthesis inhibitors, membrane permeability modifiers, DNA metabolites, dichloroethyl sulfide derivatives, protein production inhibitors, ribosome inhibitors, apoptosis inducers and neurotoxins. In some embodiments, the antibody can be combined with agents useful in tumor imaging. Such reagents include: metals; metal chelators; lanthanides; lanthanide chelators; radioactive metals; radioactive metal chelators; positron emission cores; microbubbles (for ultrasound); liposomes; molecules microencapsulated in liposomes or nanospheres; single crystal iron oxide nanocompounds; magnetic resonance imaging contrast agents; light absorption, reflection and / or scattering agents; colloidal particles; fluorophores, such as near-infrared fluorophores. In many embodiments, such secondary functionality / parts will be relatively large, for example, at least 25amu in size, and in many cases, can be at least 50, 100 or 250amu in size. In some embodiments, secondary functionality is a chelating moiety for chelating metals, for example, a chelator for radioactive metals or paramagnetic ions. In other embodiments, it is a chelator for radionuclides that can be used for radiotherapy or imaging procedures. Radionuclides useful within the present invention include gamma-emitters, positron-emitters, Auger electron-emitters, X-ray emitters and fluorescence-emitters, with beta- or alpha-emitters being preferred for therapeutic use.Examples of radionuclides useful as toxins in radiation therapy include: 32P, 33P, 43K, 52Fe, 57Co, 64Cu, 67Ga, 67Cu, 68Ga, 71Ge, 75Br, 76Br, 77Br, 77As, 77Br, 81Rb / 81MKr, 87MSr, 90Y, 97Ru, 99Tc, 100Pd, 101Rh, 103Pb, 105Rh, 109Pd, 111Ag, 111In, 113In, 119Sb, 121Sn, 123I, 125I, 127Cs, 128Ba, 129Cs, 131I, 131Cs, 143Pr, 153Sm, 161Tb, 166Ho, 169Eu, 177Lu, 186Re, 188Re, 189Re, 191Os, 193Pt, 194Ir, 197Hg, 199Au, 203Pb, 211At, 212Pb, 212Bi and 213Bi. Preferred therapeutic radionuclides include 188Re, 186Re, 203Pb, 212Pb, 212Bi, 109Pd, 64Cu, 67Cu, 90Y, 125I, 131I, 77Br, 211At, 97Ru, 105Rh, 198Au and 199Ag, 166Ho or 177Lu. The conditions for the coordination of chelating agents to metals are described, for example, in Gasnow et al., U.S. Pat. Nos. 4,831,175, 4,454,106 and 4,472,509, each of which is incorporated herein by reference. In the present invention, "radionuclide" and "radiolabel" are interchangeable. 99Tc is a particularly attractive radionuclide for diagnostic applications because it is easily available in all nuclear medicine departments, is inexpensive, produces minimal patient radiation doses, and has ideal nuclear imaging properties. It has a half-life of six hours, which means that rapid targeting of technetium-labeled antibodies is desired. Therefore, in certain preferred embodiments, the modified antibody includes a chelator for technium. In yet other embodiments, the secondary functionality can be a radiosensitizer, for example, a portion that increases the sensitivity of cells to radiation. Examples of radiosensitizers include nitroimidazoles, metronidazole, and misonidazole (see: DeVita, VT, see Harrison's Principles of Internal Medicine, p.68, McGraw-Hill Book Co., NY, 1983, which is incorporated herein by reference). Modified antibodies including a radiosensitizer as an active portion are administered and localized to target cells. When an individual is exposed to radiation, the radiosensitizer becomes "activated" and causes cell death.

[0176] Antibody sequences

[0177] The antibodies of the present invention include one or more polypeptide sequences of the γ heavy chain and λ light chain of the various antibodies described herein, and variants and antigen-binding fragments thereof. In certain embodiments, the antibodies of the present invention (and variants thereof and antigen-binding fragments thereof) include the heavy chain variable region and / or light chain variable region of any of the various antibodies described herein. In a specific embodiment, the antibodies of the present invention, their variable regions and antigen-binding fragments thereof include one or more amino acid sequence modifications compared to the wild-type PGT-121LO6 antibody. Table 1 provides the amino acid sequences of the following PGT121 LO6 and illustrative antibodies of the present invention: heavy chain, heavy chain CDR 1-3 (Kabat, IMGT, Chothia and Honegger), light chain, light chain CDR 1-3 (Kabat, IMGT, Chothia and Honegger). The PGT121 variant antibodies shown in Table 1 are identified by PGT numbering, such as PGT121.15, and / or by descriptions of modifications compared to PGT LO6. PGT121 LO6 may also be referred to as PGT121 WT (wild type). Table 1 also provides the amino acid sequences of illustrative PGT122 antibodies of the present invention: heavy chain, heavy chain CDR 1-3 (Kabat, IMGT, Chothia and Honegger), light chain, light chain CDR 1-3 (Kabat, IMGT, Chothia and Honegger). Table 1 also provides the amino acid sequences of illustrative PGT123 antibodies of the present invention: heavy chain, heavy chain CDR 1-3 (Kabat, IMGT, Chothia and Honegger), light chain, light chain CDR 1-3 (Kabat, IMGT, Chothia and Honegger). In some embodiments, any antibody or antigen-binding fragment thereof of the present disclosure is modified to include one or more tags. In certain embodiments, the one or more tags include an avidin tag.

[0178] In certain embodiments, any heavy or light chain described herein further comprises a signal peptide at the N-terminus. In various embodiments, the signal peptide used is "natural" (derived from an antibody), endogenous (i.e., used by a naturally secreted protein such as albumin) or engineered (e.g., designed in silico for secretion of the encoded protein). In certain embodiments, the signal peptide has any of the following sequences: MDPKGSLSWRILLFLSLAFELSYG (SEQ ID NO: 1) or MSVPTQVLGLLLLWLTDARC (SEQ ID NO: 2). In certain embodiments, the heavy chain comprises the signal sequence: MDPKGSLSWRILLFLSLAFELSYG (SEQ ID NO: 1), while the light chain comprises the signal sequence: MSVPTQVLGLLLLWLTDARC (SEQ ID NO: 2).

[0179] In a specific embodiment, any antibody or antigen-binding fragment thereof of the present invention is glycosylated. In a specific embodiment, glycosylation is the native glycosylation present in the PGT-121LO6 antibody. In other embodiments, glycosylation is a modified glycosylation, for example, it can be introduced post-translationally or introduced by genetic engineering. In a specific embodiment, the antibody or antigen-binding fragment thereof is afucosylated, for example, at a glycosylation site present in the antibody or antigen-binding fragment thereof. Most approved monoclonal antibodies are IgG1 isotypes, in which two N-linked bifurcated complex-type oligosaccharides are bound to the Fc region. The Fc region exerts the effector function of ADCC through its interaction with the leukocyte receptors of the FcγR family. Afucosylated monoclonal antibody is an engineered monoclonal antibody so that the oligosaccharides in the Fc region of the antibody do not have any fucose units. When antibodies are afucosylated, they can have improved FcγIIIa binding and improved antibody-dependent cellular cytotoxicity (ADCC). In a specific embodiment, the antibody and antigen-binding fragment thereof is defucosylated and comprises the DEAL modification (ie, S239D, I332E, G236A and A330L according to EU numbering) in its Fc region.

[0180] The PGT-121LO6 antibody includes a gamma heavy chain having the amino acid sequence shown in Table 1. The variable region of the gamma heavy chain of the PGT-121LO6 antibody is underlined in Table 1. The heavy chain constant region (Fc) constitutes the non-underlined sequence shown in Table 1 for PGT-121LO6. The gamma heavy chain CDRs of PGT-121LO6 and illustrative antibodies and antigen-binding fragments thereof of the present invention are also provided in Table 1. The variable regions of the heavy and light chains of all illustrative antibodies and antigen-binding fragments thereof provided in Table 1 are underlined. The constant regions of the heavy and light chains of all illustrative antibodies and antigen-binding fragments thereof provided in Table 1 constitute the non-underlined sequence. In a specific embodiment, the antibodies and antigen-binding fragments of the present invention include one or more PGT-121LO6 gamma heavy chain CDRs, as defined by any of the Kabat, IMGT, Chothia or Honegger antibody numbering schemes. In a specific embodiment, the antibodies and antigen-binding fragments of the present invention include two or more or all three of these gamma heavy chain CDRs. In a specific embodiment, the antibodies and antigen-binding fragments of the present invention include all three of these γ heavy chain CDRs, wherein the CDRs collectively include one or more, e.g., one, two, three, four, five, six, seven or eight amino acid modifications, e.g., amino acid substitutions, deletions or insertions (as compared with the corresponding sequence of PGT121 LO6). In more embodiments, the antibodies and antigen-binding fragments of the present invention include one or more PGT-122γ heavy chain CDRs, as defined by any of the Kabat, IMGT, Chothia or Honegger antibody numbering schemes. In other further embodiments, the antibodies and antigen-binding fragments of the present invention include one or more PGT-123γ heavy chain CDRs, as defined by any of the Kabat, IMGT, Chothia or Honegger antibody numbering schemes. In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention include a heavy chain, a heavy chain variable region or a heavy chain constant region having a sequence of any of the antibodies shown in Table 1 or having at least 90%, at least 95%, at least 98% or at least 99% sequence identity or homology thereto.

[0181] The coding sequence of the polynucleotide sequence encoding the γ heavy chain of PGT-121LO6 is shown below: ATGAAACACCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGATG GGTCCTGTCACAGATGCAGTTACAGGAGTCGGGCCCCGGACTGGTGAAGCCTTCGGAAACCCTGTCCCTCACGTGCAGTGTGTCTGGTGCCTC CATAAGTGACAGTTACTGGAGCTGGATCCGGCGGTCCCCAGGGAAGGGACTTGAGTGGATTGGGTATGTCCACAAAAGCGGCGACACAAATT ACAGCCCCTCCCTCAAGAGTCGAGTCAACTTGTCGTTAGACACGTCC AAAAATCAGGTGTCCCTGAGCCTTGTGGCCGCGACCGCTGCGGACTCGGGCAAATATTATTGCGCGAGAACACTGCACGGGAGGAGAATTTA TGGAATCGTTGCCTTCAATGAGTGGTTCACCTACTTCTACATGGACGTCTGGGGCAATGGGACTCAGGTCACCGTCTCCTCAGCCTCCACCAAG GGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGC ACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACA TCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAG AGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTC ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTC CAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCC TGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAG CAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAA GAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA(SEQ ID NO:3)。

[0182] The coding region of the polynucleotide encoding the gamma heavy chain variable region of PGT-121LO6 is shown below: CAGATGCAGTTACAGGAGTCGGGCCCCGGACTGGTGAAGCCTTCGG AAACCCTGTCCCTCACGTGCAGTGTGTCTGGTGCCTCCATAAGTGACAGTTACTGGAGCTGGATCCGGCGGTCCCCAGGGAAGGGACTTGAGT GGATTGGGTATGTCCACAAAAGCGGCGACACAAATTACAGCCCCTCCCTCAAGAGTCGAGTCAACTTGTCGTTAGACACGTCCAAAAATCAG GTGTCCCTGAGCCTTGTGGCCGCGACCGCTGCGGACTCGGGCAAAT ATTATTGCGCGAGAACACTGCACGGGAGGAGAATTTATGGAATCGTTGCCTTCAATGAGTGGTTCACCTACTTCTACATGGACGTCTGGGGCAATGGGACTCAGGTCACCGTCTCCTCA (SEQ ID NO: 4).

[0183] The PGT-121LO6 antibody includes a lambda light chain having an amino acid sequence as shown in Table 1. The amino acid sequence of the lambda light chain variable region of the PGT-121LO6 antibody is shaded in Table 1. The variable regions of the other antibodies in Table 1 can be determined by comparison. The lambda light chain Kabat CDRs of PGT-121LO6 and illustrative antibodies and antigen-binding fragments thereof of the present invention are shown in Table 1. In a specific embodiment, the antibodies and antigen-binding fragments of the present invention include one or more PGT-121 LO6 lambda light chain CDRs, as defined by any of the Kabat, IMGT, Chothia or Honegger antibody numbering schemes. In a specific embodiment, the antibodies and antigen-binding fragments of the present invention include two or more, or all three of these light chain CDRs. In a specific embodiment, the antibodies and antigen-binding fragments of the present invention include all three of these light chain CDRs, wherein the CDRs collectively include one or more, e.g., one, two, three, four, five, six, seven or eight amino acid modifications, e.g., amino acid substitutions, deletions or insertions (as compared to the corresponding sequence of PGT121 LO6). In more embodiments, the antibodies and antigen-binding fragments of the invention include one or more PGT-122 light chain CDRs, as defined by any of the Kabat, IMGT, Chothia, or Honegger antibody numbering schemes. In still further embodiments, the antibodies and antigen-binding fragments of the invention include one or more PGT-123 light chain CDRs, as defined by any of the Kabat, IMGT, Chothia, or Honegger antibody numbering schemes. In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention include a light chain or light chain variable region having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity or homology with the sequence of any of the antibodies shown in Table 1.

[0184] The coding region of the polynucleotide encoding the λ light chain encoding PGT-121LO6 is shown below: ATGGCCTGGACCTTTCTCCTCCTCGGCCTCCTCTCTCACTGCACAGC CTCTGTGACCTCCGATATATCTGTGGCCCCAGGAGAGACGGCCAGGATTTCCTGTGGGGAAAAGAGCCTTGGAAGTAGAGCTGTACAATGGT ATCAACACAGGGCCGGCCAGGCCCCCTCTTTAATCATATATAATAAT CAGGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCCCTGACTCCCCTTTTGGGACCACGGCCACCCTGACCATCACCAGTGTCGAAG CCGGGGATGAGGCCGACTATTACTGTCATATATGGGATAGTAGAGT TCCCACCAAATGGGTCTTCGGCGGAGGGACCACGCTGACCGTGTTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTC TGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCA GCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAG CAGTGGAAGTCCCACAAAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCATAG(SEQ ID NO:19).

[0185] The coding region of the polynucleotide encoding the lambda light chain variable region of PGT-121LO6 is shown below: TCCGATATATCTGTGGCCCCAGGAGAGACGGCCAGGATTTCCTGTGG GGAAAAGAGCCTTGGAAGTAGAGCTGTACAATGGTATCAACACAGGGCCGGCCAGGCCCCCTCTTTAATCATATATAATAATCAGGACCGGCC CTCAGGGATCCCTGAGCGATTCTCTGGCTCCCCTGACTCCCCTTTTG GGACCACGGCCACCCTGACCATCACCAGTGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCATATATGGGATAGTAGAGTTCCCACCAAATGGGTCTTCGGCGGAGGGACCACGCTGACCGTGTTA (SEQ ID NO: 20).

[0186] In certain embodiments, the antibodies or fragments thereof of the present invention include one or more, two or more, three or more, four or more, five or more, or all six CDRs present in the PGT-121LO6 antibody, as defined by any of the Kabat, IMGT, Chothia, or Honegger numbering schemes. In specific embodiments, the antibodies and antigen-binding fragments of the present invention include all six of these CDRs, wherein the CDRs collectively include one or more, e.g., one, two, three, four, five, six, seven, or eight amino acid modifications, e.g., amino acid substitutions, deletions, or insertions. In certain embodiments, the antibodies or fragments thereof of the present invention include one or more, two or more, three or more, four or more, five or more, or all six CDRs present in any of the antibodies shown in Table 1. In other embodiments, one or more CDRs of PGT-121LO6 are replaced, for example, by one or more corresponding CDRs of another anti-HIV antibody. In a specific embodiment, one or more CDRs of PGT-121LO6 are replaced by one or more corresponding CDRs from PGT-122, PGT-123, PGT-124, PGT-133 or PGT-134 antibodies described in PCT Publication WO2012 / 030904. Corresponding CDRs are CDRs at the same position in other antibodies, such as heavy chain CDRs 1, 2 and 3, and light chain CDRs 1, 2 and 3, so replacing with corresponding CDRs means replacing heavy chain CDR1 with a different heavy chain CDR1, replacing light chain CDR1 with a different CDR1, etc. The CDR sequences of these antibodies are provided below:

[0187] Table 2. CDRs of PGT antibodies

[0188]

[0189]

[0190]

[0191] The antibodies and antigen-binding fragments thereof of the present invention include one or more amino acid modifications compared to PGT-121LO6. These may include any of a variety of different types of modifications, including, for example, allotype modifications, Fc modifications, Fab modifications, and modifications of selected residues, such as glycosylation sites. Non-limiting examples of modifications present in certain embodiments of the antibodies and antigen-binding fragments thereof of the present invention are described herein and are shown in the sequences listed in Table 1.

[0192] i. Allotypic modification

[0193] IgG antibodies exist in various allotypes and isoallotypes. In a specific embodiment, the antibodies and antigen-binding fragments thereof of the invention comprise an IgG1 heavy chain having an allotype of G1m1; nG1m2; G1m3; G1m17,1; G1m17,1,2; G1m3,1 or G1m17. Each of these allotypes or isoallotypes is characterized by the following amino acid residues (EU numbering) at the indicated positions within the IgG1 heavy chain constant region (Fc):

[0194] G1m1:D356,L358;

[0195] nG1m1:E356,M358;

[0196] G1m3: R214, E356, M358, A431;

[0197] G1m17,1:K214,D356,L358,A431;

[0198] G1m17,1,2:K214, D356, L358, G431;

[0199] G1m3,1:R214,D356,L358,A431; and

[0200] G1m17:K214, E356, M358, A431.

[0201] The PGT-121LO6 antibody comprises an IgG1m3 allotype. In specific embodiments, the antibodies and antigen-binding fragments thereof of the invention comprise an IgG1m3 allotype, while in other embodiments, the allotype is modified compared to the PGT-121LO6 antibody. In specific embodiments, the allotype is nG1m1, G1m3; G1m17,1; G1m17,1,2; G1m3,1; or G1m17. In certain embodiments, the allotype is G1m17. In specific embodiments, the antibody or antigen-binding fragment thereof comprises an IgG1 heavy chain constant region comprising one of the following amino acid sequences, wherein representative allotype-determining residues are shown in bold:

[0202] IgG1m3:

[0203] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 41);

[0204] IgG1m17,1:

[0205] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 42);

[0206] IgG1m17,1,2:

[0207] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSLSPGK(SEQ ID NO: 43);

[0208] IgG1m3.1:

[0209] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD OR

[0210] IgG1m17:

[0211] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 45).

[0212] In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise an IgG1 heavy chain comprising the following amino acid sequence of the allotype IgG1m17:

[0213] QMQLQESGPGLVKPSETLSLTCSVSGASISDSYWSWIRRSPGKGLEWIG YVHKSGDTNYSPSLKSRVNLSLDTSKNQVSLSLVAATAADSGKYYCARTLHGRRIYGIVAFNEWFTYFYMDVWGNGTQVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 46).

[0214] In a specific embodiment, the antibodies and antigen-binding fragments thereof of the invention comprise a kappa light chain having an allotype selected from Km1; Km2; or Km3. Each of these allotypes is characterized by the following amino acid residues (EU numbering) at the indicated positions within the IgG1 light chain:

[0215] Km1:V153,L191;

[0216] Km1,2:A153;L191; and

[0217] Km3:A153,V191.

[0218] In a specific embodiment, the antibody or antigen-binding fragment thereof comprises an IgG1 kappa light chain comprising one of the following amino acid sequences, wherein representative allotype-determining residues are shown in bold:

[0219] Km1:

[0220] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNVLQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:47);

[0221] Km1,2:

[0222] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:48); or

[0223] Km3:

[0224] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 49).

[0225] Each individual human comprises seven to eleven different lambda light chain genes encoding a light chain selected from the group consisting of lambda 1, lambda 2, lambda 3, lambda 4, lambda 5, lambda 6, and lambda 7. In a specific embodiment, the antibodies and antigen-binding fragments thereof of the present invention comprise a lambda light chain selected from the group consisting of lambda 1, lambda 2, lambda 3, lambda 4, lambda 5, lambda 6, and lambda 7. In a specific embodiment, the antibodies and antigen-binding fragments thereof of the present invention comprise a lambda light chain selected from the group consisting of lambda 1, lambda 2, lambda 3, and lambda 7. In a specific embodiment, the antibody or antigen-binding fragment thereof comprises an IgG1 lambda light chain comprising one of the following amino acid sequences, wherein representative lambda determining residues are shown in bold:

[0226] IGLC1:

[0227] GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSP VKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO:50);

[0228] IGLC2:

[0229] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPV KAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO:51);

[0230] IGLC3:

[0231] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPA KAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVE KTVAPTECS(SEQ ID NO:52);

[0232] IGLC7:

[0233] GQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSP VKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAECS (SEQ ID NO: 53).

[0234] In a specific embodiment, the light chain comprises the amino acid sequence:

[0235] SDISVAPGETARISCGEKSLGSRAVQWYQHRAGQAPSLIIYNNQDRPSGI PERFSGSPDSPFGTTATLTITSVEAGDEADYYCHIWDSRVPTKWVFGGGTTLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWK ADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 54).

[0236] In a specific embodiment, an antibody or antibody binding fragment thereof of the invention comprises any one of the light chain sequences listed in Table 1 or a sequence having at least 90%, at least 95%, at least 98% or at least 99% identity thereto.

[0237] In a specific embodiment, the antibody or antigen-binding fragment thereof comprises an IgG1 heavy chain having an IgGm17 allotype and a λ2 light chain.

[0238] In certain embodiments, the light chain comprises one or more of the following amino acid substitutions (compared to PGT121 LO6, using Figure 1 67b, Pro at position 67c, and Lys at position 103. In certain embodiments, the light chain comprises the following amino acid substitutions (compared to PGT121 LO6, using Figure 1 Kabat numbering scheme in ): Arg at position 67b and Pro at position 67c.

[0239] ii. Fc modification

[0240] In certain embodiments, the antibodies and antigen-binding fragments thereof include one or more amino acid sequence modifications in the heavy chain constant region (Fc) compared to the PGT-121LO6 antibody. In specific embodiments, these modifications increase the stability of the modified antibody or antigen-binding fragment thereof or increase binding affinity compared to the PGT-121LO6 antibody. In specific embodiments, some of these modifications or a combination thereof surprisingly increase antibody effector function or neutralizing activity. The sequences of certain illustrative heavy chain sequences including one or more Fc modifications and antibodies including such modified heavy chain sequences are shown in Table 1.

[0241] In certain embodiments, one or more modifications are selected from the following Fc amino acid replacements (compared to PGT121 LO6; EU numbering) or a combination thereof: L234F; L235E; G236A; S239D; F243L; D265E; S267E; H268F; R292P; N297Q; S298A; S324T; I332E; S239D; A330L; L234F; L235E; P331S; F243L; Y300L; V305I; P396L; S298A; E333A; K334A; E345R; L235V; F243L; R292P; Y300L; P396L; M428L; E430G; N434S; G236A, S267E, H268F, S324T and I332E; G236A, S239D and I332E; S239D, A330L, I332E; L234F, L235E and P331S; F243L, R292P, Y300L, V305I and P396L; G236A, H268F, S324T and I332E; S239D, H268F, S324T and I332E; S298A, E333A and K334A; L235V, F243L, R292P, Y300L and P396L; S239D, I332E; S239D, S298A and I332E; G236A, S239D, I332E, M428L and N434S; G236A, S239D, A330L, I332E, M428L and N434S; S239D, I332E, G236A and A330L; M428L and N4343S; M428L, N434S; G236A, S239D, A330L and I332E; and G236A and I332E.

[0242] In a specific embodiment, antibodies and Fabs thereof include two or more, three or more, four or more, five or more or six or more modified Fc amino acid residues. In certain embodiments, antibodies and Fabs thereof include S239D, I332E, G236A and A330L mutations, which are collectively referred to as DEAL. In certain embodiments, antibodies and Fabs thereof include M248L and N434S mutations, which are collectively referred to as LS. In certain embodiments, antibodies and Fabs thereof include S239D, I332E, G236A, A330L, M428L and N434S mutations (DEALLS). In a specific embodiment, antibodies and Fabs thereof of the present invention include one of the heavy chain constant region sequences present in any of the antibodies shown in Table 1 or variants having at least 90%, at least 95%, at least 98% or at least 99% sequence identity or homology therewith.

[0243] In certain embodiments, the antibodies and antigen-binding fragments are defucosylated. In some embodiments, the antibodies and antigen-binding fragments include one or more tags. In certain embodiments, the one or more tags include an avidin tag.

[0244] iii. Fab modification

[0245] In certain embodiments, the antibodies and antigen-binding fragments thereof include one or more amino acid sequence modifications in the heavy chain variable region or light chain variable region (Fab) compared to the PGT-121LO6 antibody. In specific embodiments, these modifications improve the stability of the modified antibody or its antigen-binding fragment or improve the binding affinity compared to the PGT-121LO6 antibody. In certain embodiments, these modifications reduce the immunogenicity, isomerization, glycosylation or oxidation of the antibody or its antigen-binding fragment. In specific embodiments, the modification results in the removal of potential isomerization, glycosylation or oxidation sites from the antibody or its antigen-binding fragment. In certain embodiments, the amino acid substitution results in the translation of the improved antibody or its antigen-binding fragment. In certain embodiments, these modifications improve the % recovery of monomeric IgG from the low pH virus inactivation procedure. In certain embodiments, these modifications improve the HIV neutralization efficacy of the antibody or its antigen-binding fragment when measured against a single HIV strain or a subset of related HIV strains compared to the PGT-121LO6 antibody.

[0246] In a specific embodiment, the modification includes amino acid replacement within one or more potential glycosylation sites in the PGT-121LO6 antibody. The N-linked glycosylation site may include the sequence NX (S / T), wherein X is any amino acid residue (except proline), and wherein N is glycosylated. In a specific embodiment, the glycosylation site is located at residues 58, 68 and 105 (Kabat numbering) of the PGT-121LO6 heavy chain variable region sequence. In a specific embodiment, compared to PGT-121LO6, the antibody or antigen-binding fragment thereof of the present invention includes one or more amino acid replacements within residues 58-60, 68-70 and 105-107 (Kabat numbering) of the heavy chain Fab, which eliminates the NX (S / T) glycosylation consensus sequence. In certain embodiments, one or more amino acid replacements occur at positions 60, 68 or 105 (Kabat numbering). In a specific embodiment, the amino acid replacement removes or destroys the glycosylation site at the modified position. In certain embodiments, antibodies or antigen-binding fragments thereof of the invention include two or more or three or more amino acid substitutions at these glycosylation sites. In one embodiment, they include amino acid substitutions that remove or destroy all three of these glycosylation sites.

[0247] In a specific embodiment, the modification includes amino acid replacements within one or more potential immunogenic regions in the PGT-121LO6 antibody. In a specific embodiment, the potential immunogenic region is located at residues 82a-82c of the heavy chain variable region of PGT-121LO6. In a specific embodiment, the antibody or antigen-binding fragment thereof of the present invention includes one or more amino acid replacements within residues 82a-82c (Kabat numbering) of the heavy chain Fab compared to PGT-1121LO6. In certain embodiments, one or more amino acid replacements occur at positions 82a-82c (Kabat numbering) of the heavy chain Fab compared to PGT-121LO6. In a specific embodiment, amino acid replacement reduces the immunogenicity of the antibody or its antigen-binding fragment. In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention includes two or more or three or more amino acid replacements in this immunogenic region. In one embodiment, they include amino acid replacements of three residues in this immunogenic region, for example, replacement of VAA present in PGT-121LO6, for example, replacement with SSV, TSV or TGV. In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise one or more amino acid substitutions that remove glycosylation sites, and wherein the immunogenicity is less than or equal to that of PGT-121LO6. In certain embodiments, the antibodies comprise one or more amino acid modifications, e.g., substitutions, within a determined or predicted immunogenic region corresponding to any of the PGT-121LO6 regions shown in any one of Tables 14-16.

[0248] In specific embodiments, the antibody comprises modifications of any combination of heavy chain residues 2, 32, 60, 68, 82a-82c, 95 or 105 and / or light chain residues 67b, 67c or 103 that result in reduced donor responses in the whole molecule T-cell activation assay described herein.

[0249] In certain embodiments, the antibody or antigen-binding fragment thereof comprises one or more amino acid modifications, e.g., substitutions, to improve translation. For example, the substitution may be a substitution of an amino acid encoded by a rare codon, or for which only a limited amount of the corresponding tRNA is present in the cell in which the protein is expressed. In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises one or more such modifications, or amino acid substitutions, e.g., compared to PGT-121LO6. In a specific embodiment, the antibody or antigen-binding fragment thereof comprises one or more amino acid substitutions of M2, A28, R39, N68, S81, V82a, A82b, A82c, or N105 (Kabat numbering) in the heavy chain variable region of the PGT-121LO6 antibody. In certain embodiments, R39 of PGT-121 LO6 is, for example, substituted with Q.

[0250] In certain embodiments, the antibody or its antigen-binding fragment includes one or more amino acid replacements to enhance the stability of the Fab domain, as measured by melting temperature. In a specific embodiment, the antibody or its antigen-binding fragment includes one or more of the following amino acid replacements in the heavy chain: R39Q, S60N and VAA (82a-82c) SSV (Kabat numbering). As shown in the attached examples, the antibodies of the present invention show enhanced thermal stability compared to PGT-121LO6, as measured by the increase in the Tm of the Fab domain determined by DSF. In a specific embodiment, the antibody or its antigen-binding fragment includes amino acid replacements of R39Q, S60N and VAA (82a-82c) TGV (Kabat numbering) in the heavy chain. In a specific embodiment, the antibody or its antigen-binding fragment includes amino acid replacements of VAA (82a-82c) TGV (Kabat numbering) in the heavy chain. In a specific embodiment, the antibody or its antigen-binding fragment includes amino acid replacements of VAA (82a-82c) TGV (Kabat numbering) in the heavy chain.

[0251] In certain embodiments, the antibody or antigen-binding fragment thereof comprises one or more amino acid substitutions to enhance the stability of the Fab domain at low pH, thereby preventing aggregation during the low "pH hold" procedure used to inactivate potential viruses derived from mammalian cell culture during the purification of antibodies (e.g., PGT-121 LO6). In a specific embodiment, the antibody or antigen-binding fragment thereof comprises an amino acid substitution of one or more of the following heavy chain mutations: Q1E, M2V, M2L, A27G, S32A, R39Q, S60N, N68T, N68H, S81K, VAA(82a-82c)SSV, VAA(82a-82c)TSV, VAA(82a-82c)TGV, K89T, T95A, N105K, N105H, N105Q, N105T; light chain mutations K95cN or S45V; or a combination of heavy chain mutations R39Q plus light chain mutations H38Q. In a specific embodiment, the antibody or antigen-binding fragment thereof comprises the addition of SYVLTQP or SSVTSYV to the N-terminus of the light chain, as well as combinations of these additions with any of the above substitutions. As described in the accompanying examples, the antibodies of the invention exhibit enhanced stability at low pH compared to PGT-121 LO6, which enables GMP production of these molecules. This can be found in WHO Technical Report, Series No. 924, 2004; Jacob and Frech. 2004. Scale up of antibody purification: from laboratory scale to production, p. 101-132., see G. Subramanian (ed.), Antibodies, vol. 1. Production and Purification. Kluwer Academic / Plenum Publishers, New York, NY.

[0252] In certain embodiments, the antibodies or antigen-binding fragments thereof include one or more amino acid substitutions to enhance HIV neutralization potency and breadth. As shown in the accompanying examples, certain antibodies of the invention exhibit statistically significant increases in breadth and neutralization potency (~2-3 fold) compared to PGT-121LO6 or other antibodies.

[0253] In certain embodiments, one or more modifications are selected from the following heavy chain Fab mutations (Kabat numbering) or a combination thereof (compared to PGT121 LO6): Q1E; M2V; M2L; A27G; S32A; R39Q; S60N; N68T; N68H; S81K; V82aT; V82aS; A82bG; A82bS; A82cV; K89T; T95A; W100jA; W100jL; N105K; N105Q; N105T; N105H; S60A; N68A; N105A; N105H; N105T; S60N, N68T In some embodiments, the heavy chain Fab comprises the following mutations (Kabat numbering): S60N, N68H, VAA82a-cTGV and N105T. In some embodiments, the heavy chain Fab comprises the following mutations (Kabat numbering): S60N, N68H, VAA82a-cSSV and N105K. In some embodiments, the heavy chain Fab comprises the following mutations (Kabat numbering): S60N, N68H, VAA82a-cSSV and N105K. In some embodiments, the heavy chain Fab comprises the following mutations (Kabat numbering): S60N, N68H, VAA82a-cSSV and N105H. In certain embodiments, the heavy chain Fab comprises the following mutations (Kabat numbering): M2L, S32A, S60N, N68H, VAA82a-cTGV, T95A and N105T. In certain embodiments, the heavy chain Fab comprises the following mutations (Kabat numbering): R39Q, S60N, N68H, VAA82a-cSSV and N105K.

[0254] In a specific embodiment, the antibodies and antigen-binding fragments thereof of the invention comprise one or more, two or more, three or more, or four or more amino acid substitutions in the heavy chain Fab, wherein the amino acid substitutions are selected from the following substitution combinations (EU numbering): V82aS, A82bS, A82cV and N105K; N68H, V82aS, A82bS and A82cV; N68H, V82aS, A82bS, A82cV and N105K; S60N, V82aS, A82bS and A82cV; S60N, N105K; S60N and N68H; S60N, N68H and N105K; R39Q; R39Q, N68H and N105K; R39Q and N68H; R39Q and N105K; R39Q and S60N; R39Q, S60N and N105K; R39Q, S60N and N68H; R39Q, S60N, N68H and N105K; V82aS, A82bS and A82cV; S60N, N68H, VAA82a-cTGV and N105T; S60N, N68H, VAA82a-cSSV and N105K; S60N, N68H, VAA82a-cSSV and N105H; M2L, S32A, S60N, N68H, VAA82a-cTGV, T95A, and N105T; and R39Q, S60N, N68H, VAA82a-cSSV, and N105K.In some embodiments, the antibodies and antigen-binding fragments thereof of the invention comprise one or more, two or more, three or more, four or more, five or more or six or more amino acid substitutions in the heavy chain Fab, wherein the amino acid substitutions are selected from the following substitution combinations (EU numbering): V82aS, A82bS, A82cV and N105K; N68H, V82aS, A82bS and A82cV; N68H, V82aS, A82bS, A82cV and N105K; S60N, V82aS, A82bS and A82cV; S60N, N105K; S60N and N68H; S60N, N68H and N105K; R39Q; R39Q, N68H and N105K; R39Q and N68H; R39Q and N105K; R39Q and S60N; R39Q, S60N and N105K; R39Q, S60N and N68H; R39Q, S60N, N68H and N105K; V82aS, A82bS and A82cV; S60N, N68H, VAA82a-cTGV and N105T; S60N, N68H, VAA82a-cSSV and N105K; S60N, N68H, VAA82a-cSSV and N105H; M2L, S32A, S60N, N68H, VAA82a-cTGV, T95A and N105T; and R39Q, S60N, N68H, VAA82a-cSSV and N105K. In a specific embodiment, any of these antibodies or their antigen-binding fragments further include one or more, two or more, three or more, four or more or five or more of the following Fc mutations (EU numbering): G236A, S239D, A330L, I332E, M428L and N434S. In a specific embodiment, they include four, five, six or seven of these amino acid replacements in the heavy chain Fab. In a specific embodiment, they further include all of the following amino acid replacements (EU numbering): G236A, S239D, A330L, I332E, M428L and N434S. In a specific embodiment, they further include G1m17 allotypes. In some embodiments, the antibodies and antigen-binding fragments thereof of the present invention include six heavy chain variable domain mutations. In other embodiments, the antibodies and antigen-binding fragments thereof of the present invention include two light chain variable domain mutations. In certain embodiments, the antibodies and antigen-binding fragments thereof of the present invention include six heavy chain variable domain mutations and two light chain variable domain mutations. Embodiments of the invention include any combinatorial variants derived from any of the mutations described herein.

[0255] In a specific embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises one or more amino acid modifications in the light chain compared to PGT121 LO6. In a specific embodiment, the one or more modifications are selected from the following light chain mutations: P67bR, F67cR and T103K (Kabat numbering). In one embodiment, one or more modifications are selected from the following light chain amino acid replacements: P67bR, F67cR and T103K (Kabat numbering). In a specific embodiment, the antibody or antigen-binding fragment thereof comprises the following light chain mutations: P67bR, F67cR and T103K. In certain embodiments, one or more modifications are selected from the following light chain Fab mutations (Kabat numbering) or a combination thereof: SYVLTQP at the N-terminus; SSVTSYV at the N-terminus; S45T; or K95cN. In certain embodiments, one or more modifications are selected from the following light chain Fab mutations (Kabat numbering) or a combination thereof: SYVLTQP at the N-terminus; SSVTSYV at the N-terminus; S45T; or K95cN.

[0256] In certain embodiments, the antibody or antigen-binding fragment thereof comprises one or more modifications selected from the following combinations of heavy chain Fab mutations and light chain Fab mutations (Kabat numbering): HC: R39Q and LC: H38Q; HC: S60N, N68T and N105K and LC: SYVLTPQ at the N-terminus; HC: S60N, N68H, VAA82a-cTGV, N105T and LC: P67bR and F67cP; HC: S60N, N68H, VAA82a-cTGV, N105T and LC: P67bR, F67cP and T103K; HC: M2L, S32A, S60N, N68H, VAA82a-cTGV, N105T and LC: P67bR and F67cP; or HC: M2L, S32A, S60N, N68H, VAA82a-cTGV, N105T and LC: P67bR, F67cP, and T103K. In one embodiment, the antibody or antigen-binding fragment thereof comprises one or more modifications selected from the following heavy chain Fab mutations and light chain Fab mutations (Kabat numbering) combination: HC: R39Q and LC: H38Q; HC: S60N, N68T and N105K and LC: SYVLTPQ at the N-terminus; HC: S60N, N68H, VAA82a-cTGV, N105T and LC: P67bR and F67cP; HC: S60N, N68H, VAA82a-cTGV, N105T and LC: P67bR, F67cP and T103K; HC: M2L, S32A, S60N, N68H, VAA82a-cTGV, N105T and LC: P67bR and F67cP; HC: M2L, S32A, S60N, N68H, VAA82a-cTGV, N105T and LC: P67bR and F67cP; N68H, VAA82a-cTGV, N105T and LC: P67bR, F67cP and T103K; HC: S60N, N68H, VAA82a-cSSV, N105K and LC: P67bR and F67cP. In specific embodiments, the antibody or antigen-binding fragment thereof has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with the corresponding region of the PGT121 LO6 antibody. In more embodiments, it includes all six CDRs in the PGT121 LO6 antibody according to Kabat numbering.

[0257] In certain embodiments, the antibodies and antigen-binding fragments include two or more, three or more, four or more, five or more, or six or more of any Fab mutation described herein or shown in any of the PGT121 LO6 mutant sequences provided in Table 1. In certain embodiments, any of these antibodies and antigen-binding fragments thereof further include one or more of the following heavy chain constant region mutations: S239D, I332E, G236A, and A330L (EU numbering). In certain embodiments, any of these antibodies and antigen-binding fragments thereof include M428L and N434S mutations. In certain embodiments, any of the antibodies and antigen-binding fragments thereof include S239D, I332E, G236A, A330L, M428L, and N434S mutations.

[0258] In a specific embodiment, the antibody or fragment thereof of the present invention comprises any of the heavy chain sequences, constant regions or variable regions thereof, or light chain sequences, constant regions or variable regions thereof shown in Table 1, or a variant having at least 90%, at least 95%, at least 98% or at least 99% sequence identity therewith. In a specific embodiment, the antibody and antigen-binding fragment thereof of the present invention comprises one of the heavy chain Fab or variable domain sequences shown by underlining in Table 1. In a specific embodiment, the antibody and antigen-binding fragment thereof of the present invention comprises any heavy chain shown in Table 1. In certain embodiments, the antibody or fragment thereof of the present invention comprises both the heavy chain sequence and the light chain sequence present in any antibody shown in Table 1, i.e., the combination present in any clone designation. In a specific embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain (or fragment thereof) having a sequence shown in Table 1, and a light chain (or fragment thereof) having a sequence shown in Table 1, wherein the heavy chain and the light chain are not present in the same antibody in Table 1. Any heavy chain (or fragment thereof) shown in Table 1 can be combined with any light chain (or fragment thereof) shown in Table 1.

[0259] In a specific embodiment, the antibody or fragment thereof of the invention comprises one or more modifications in the light chain variable region compared to PGT-121LO6. In certain embodiments, the modification is selected from the following amino acid substitutions: P67bR, F67cP or T103K.

[0260] In a specific embodiment, the antibody or fragment thereof of the invention comprises one of the light chain variable regions underlined in Table 1 or shown below:

[0261] SDISVAPGETARISCGEKSLGSRAVQWYQHRAGQAPSLIIYNNQDRPSGI PERFSGSPDSPFGTTATLTITSVEAGDEADYYCHIWDSRVPTKWVFGGGTTLTVLG(SEQ ID NO:55);

[0262] SDISVAPGETARISCGEKSLGSRAVQWYQHRAGQAPSLIIYNNQDRPSGI PERFSGSPDSPFGTTATLTITSVEAGDEADYYCHIWDSRVPTKWVFGGGTKLTVLG(SEQ ID NO:56);

[0263] SDISVAPGETARISCGEKSLGSRAVQWYQHRAGQAPSLIIYNNQDRPSGI PERFSGSPDSRPGTTATLTITSVEAGDEADYYCHIWDSRVPTKWVFGGGTTLTVLG(SEQ ID NO:57);

[0264] SDISVAPGETARISCGEKSLGSRAVQWYQHRAGQAPSLIIYNNQDRPSGI PERFSGSPDSRPFGTTATLTITSVEAGDEADYYCHIWDSRVPTKWVFGGGTKLTVLG (SEQ ID NO: 58).

[0265] In certain embodiments, the present invention includes antigen-binding fragments of antibodies. In certain embodiments, there are advantages to using antibody fragments rather than complete antibodies. For example, the smaller size of the fragment allows for rapid clearance and can lead to improved access to certain tissues such as solid tumors. Examples of antibody fragments include: Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibodies; nanobodies, and multispecific antibodies formed from antibody fragments.

[0266] In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv). See WO 93 / 16185; U.S. Patent Nos. 5,571,894; and 5,587,458. Fv and sFv are the only substances with intact combining sites from which the constant regions have been removed. Therefore, they are suitable for reduced nonspecific binding during in vivo use. sFv fusion proteins can be constructed to produce fusions of effector proteins at the amino or carboxyl termini of sFv. See Antibody Engineering, ed., Borrebaeck, supra. Antibody fragments can also be linear antibodies, for example, as described in U.S. Patent No. 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

[0267] In a specific embodiment, the antibody or antigen-binding fragment thereof comprises the heavy chain complementarity determining region (CDR) and light chain CDR (defined by any numbering scheme) of PGT121 LO6, wherein the antibody or antigen-binding fragment thereof comprises one or more, two or more, three or more, or all four of the following features:

[0268] (A) the heavy chain is an allotype other than IgG1m3 (e.g., G1m17, G1m1, nG1m1, G1m2, nG1m2, G1m17,1, G1m17,1,2, or G1m3,1), wherein in a specific embodiment, the allotype is an IgG1m17 allotype;

[0269] (B) light chain is λ2;

[0270] (C) the heavy chain constant region (Fc) comprises one or more amino acid substitutions at position 236, position 239, position 330, position 332, position 428, and position 434 compared to the Fc of PGT-121LO6, wherein in a specific embodiment, the one or more amino acid substitutions are selected from: Ala at position 236, Asp at position 239, Leu at position 330, Glu at position 332, Leu at position 428, and Ser at position 434; and

[0271] (D) the heavy chain variable region (Fab) includes one or more amino acid replacements in a glycosylation site present in the Fab of PGT-121LO6 or an immunogenic region in the Fab of PGT-121LO6, wherein in a specific embodiment, the glycosylation site present in the Fab of PGT-121LO6 is located at positions 58-60, 68-70 and 105-107, and the immunogenic region in the Fab of PGT-121LO6 is located at positions 82a-82c, and wherein in certain embodiments, the one or more amino acid replacements are selected from: Ser-Ser-Val at positions 82a-82c, Thr-Gly-Val at positions 82a-82c, Gln at position 39, Asn at position 60, His at position 68, Lys at position 105, Thr at position 105 and His at position 105.

[0272] In a specific embodiment, the antibody or antigen-binding fragment thereof includes any of the following combinations of the features identified above: A and B; A and C; A and D; B and C; B and D; C and D; A, B and C; A, B and D; A, C and D; B, C and D; or A, B, C and D.

[0273] In certain embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the heavy chain is of the IgG1m17 allotype.

[0274] In a specific embodiment of any of the antibodies or antigen-binding fragments thereof described herein, the heavy chain constant region (Fc) includes Ala at position 236, Asp at position 339, Leu at position 330, Glu at 332, Leu at 428, and Ser at position 434.

[0275] In certain embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the heavy chain constant region (Fc) comprises Lys at position 214, Glu at position 356, Met at position 358, and Ala at position 431.

[0276] In certain embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the heavy chain constant region (Fc) comprises the heavy chain sequence of PGT121.33.

[0277] In one embodiment of any antibody or antigen-binding fragment thereof described herein, the heavy chain constant region (Fc) comprises the heavy chain sequence of PGT121.42, PGT121.43, PGT121.60, PGT121.61, PGT121.54, PGT121.55, PGT121.64 and PGT121.65. In other embodiments of any antibody or antigen-binding fragment thereof described herein, the heavy chain constant region (Fc) is shown in any one of SEQ ID NO: 252, 255, 266, 267, 268, 269, 272 and 273.

[0278] In specific embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the light chain is λ2. In specific embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the light chain comprises Ala at position 6, Ser at position 8, Ile at position 30, Ser at position 46, Val at position 49, Ala at position 5, Thr at position 57, Arg at position 83, Gln at position 88, and Thr at position 103. In specific embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the light chain comprises any of the light chain sequences shown in Table 1, or a variable region thereof. In some embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the light chain region comprises the light chain sequences of PGT121.42, PGT121.43, PGT121.60, PGT121.61, PGT121.54, PGT121.55, PGT121.64, and PGT121.65. In certain embodiments of any of the antibodies or antigen-binding fragments thereof described herein, the light chain is set forth in any one of SEQ ID NOs: 338, 341, 352, 354, 355, 358, and 359.

[0279] In a specific embodiment of any antibody or antigen-binding fragment thereof described herein, the heavy chain variable region (Fab) includes amino acid replacements at two or more or all three glycosylation sites present in IPGT-121LO6. In a specific embodiment, the heavy chain variable region (Fab) includes one or more of the following: Ser-Ser-Val at positions 82a-82c, Thr-Gly-Val at positions 82a-82c, Gln at position 39, Asn at position 60, His at position 68, Lys at position 105, Thr at position 105, and His at position 105. In a specific embodiment of any antibody or antigen-binding fragment thereof described herein, the heavy chain variable region (Fab) includes Ser-Ser-Val at positions 82a-82c or Thr-Gly-Val at positions 82a-82c. In a specific embodiment, the heavy chain variable region (Fab) comprises Asn at position 60, His at position 68, Lys at position 105, Thr at position 105, and His at position 105. In a specific embodiment, the heavy chain variable region (Fab) comprises: Ser-Ser-Val at positions 82a-82c, Thr-Gly-Val at positions 82a-82c, Gln at position 39, Asn at position 60, His at position 68, Lys at position 105, Thr at position 105, and His at position 105. In a specific embodiment of any of the antibodies or antigen-binding fragments thereof described herein, the heavy chain variable region comprises any of the heavy chain variable regions shown in Table 1.

[0280] In a specific embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain (or a fragment thereof) and / or a light chain (or a fragment thereof) having a sequence shown in Table 1. In a specific embodiment, it has a combination of any heavy chain and any light chain shown in Table 1. In a specific embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain (or a fragment thereof) listed in SEQ ID NOs: 404, 423, 425, 427, 429 and 449. In a specific embodiment, the antibody or antigen-binding fragment thereof comprises a light chain (or a fragment thereof) listed in SEQ ID NOs: 415, 424, 426, 428, 430 and 450. In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof comprising heavy chain complementary determining regions 1-3 (CDR 1-3) listed in SEQ ID NOs: 363, 405 and 406 and light chain CDR 1-3 listed in SEQ ID NOs: 416, 417 and 418. In some embodiments, the present disclosure provides antibodies or antigen-binding fragments thereof comprising heavy chain complementary determining regions 1-3 (CDR1-3) listed in SEQ ID NOs: 362, 364 and 367 and light chain CDR1-3 listed in SEQ ID NOs: 395, 396 and 397. In some embodiments, the present disclosure provides antibodies or antigen-binding fragments thereof comprising heavy chain complementary determining regions 1-3 (CDR1-3) listed in SEQ ID NOs: 362, 366 and 367 and light chain CDR1-3 listed in SEQ ID NOs: 395, 396 and 397. In some embodiments, the present disclosure provides antibodies or antigen-binding fragments thereof comprising heavy chain complementary determining regions 1-3 (CDR1-3) listed in SEQ ID NOs: 431, 432 and 433 and light chain CDR1-3 listed in SEQ ID NOs: 442, 443 and 444.

[0281] The present invention also includes nucleic acid molecules encoding antibodies of the present invention or their antigen-binding fragments. In a specific embodiment, the nucleic acid molecule encodes an antibody light chain (or its fragment) or an antibody light chain (or its fragment) or both. In certain embodiments, the nucleic acid encodes a bivalent antibody or its fragment. In a specific embodiment, the nucleic acid is DNA, cDNA or mRNA. In a specific embodiment, the nucleic acid molecule is codon optimized to enhance expression in a host cell.

[0282] The present invention further includes polypeptide variants of the antibodies and antigen-binding fragments thereof disclosed herein, including, for example, complete antibodies, scFv, heavy chains, light chains, V H Area and V LIn certain embodiments, the invention includes polypeptide variants having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homology to the polypeptides described herein, including any of those shown in Table 1.

[0283] The present invention further includes nucleic acid molecules encoding polypeptide variants of the antibodies and antigen-binding fragments thereof disclosed herein, as well as nucleic acid molecules encoding antibodies or antigen-binding fragments thereof (such as, for example, complete antibodies, scFvs, heavy chains, light chains, V H Area and V L In certain embodiments, the present invention includes polynucleotide variants having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% homology to the polynucleotides described herein.

[0284] In some embodiments of nucleic acid molecules, they (or its part or subregion) can be codon optimized.Codon optimization method is known in the art and can be used for the codon frequency in for example matching target and host organism, ensure correct folding, bias GC content to improve mRNA stability or reduce secondary structure, minimize the tandem repeat codon or base extension (base run) that may damage gene construction or expression, customize transcription and antisense control region, insert or remove protein transport (trafficking) sequence, modify ribosome binding site and mRNA degradation site, regulate translation rate to allow the various domains of protein to correctly fold or reduce or eliminate the problematic secondary structure in polynucleotide.Codon optimization tools, algorithms and services are known in the art, and its limiting examples include the service from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and / or proprietary method.In one embodiment, optimization algorithm is used to optimize nucleotide sequence.The example that the codon for each amino acid selected is provided in table 3.

[0285] Table 3. Codon usage

[0286]

[0287]

[0288] The present invention also includes vectors comprising nucleic acid molecules of the present invention. The vector can be of any type, for example, a recombinant vector, such as an expression vector. The vector includes, but is not limited to, plasmids, cosmids, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs), and vectors derived from bacteriophages or plant or animal (including human) viruses. The vector may include a replication origin recognized by the proposed host cell, and in the case of an expression vector, a promoter and other regulatory sequences recognized by the host cell. In a specific embodiment, the vector includes a polynucleotide encoding an antibody or antigen-binding fragment thereof of the present invention operably connected to a promoter and optional other regulatory elements. Certain vectors are capable of autonomous replication in the host into which they are introduced (for example, a vector with a bacterial replication origin can be replicated in bacteria). Other vectors can be integrated into the host's genome when introduced into the host, and thus replicated with the host genome. The vector includes, but is not limited to, those suitable for the recombinant production of the antibody and antigen-binding fragment thereof of the present invention, and those suitable for introduction into a subject or its cell in need, so as to provide the antibody or antigen-binding fragment thereof of the present invention to the subject.

[0289] The selection of vector depends on the recombination procedure followed and the host used. The introduction of vector into the host cell can be achieved particularly by calcium phosphate transfection, viral infection, DEAE-dextran mediated transfection, lipofectamin transfection or electroporation. The vector can replicate autonomously or can replicate with the chromosome into which they have been integrated. In some embodiments, the vector comprises one or more selection markers. The selection of markers can depend on the host cell selected. These include, but are not limited to kanamycin, neomycin, puromycin, hygromycin, zeocin, thymidine kinase gene (HSV-TK) from herpes simplex virus and dihydrofolate reductase gene (dhfr) from mice. The disclosure also encompasses vectors comprising one or more nucleic acid molecules encoding antibodies and antigen-binding fragments thereof described herein (which are operably connected to one or more nucleic acid molecules encoding proteins or peptides that can be used to separate antibodies and fragments thereof). These proteins or peptides include, but are not limited to glutathione-S-transferase, maltose binding protein, metal-binding polyhistidine, green fluorescent protein, luciferase and beta-galactosidase.

[0290] In a specific embodiment, the vector is pcDNA TM 3.1+ (ThermoFisher, MA), which has the following sequence:

[0291] GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACA ATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACA ACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGT TGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTC ATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGAC GTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCA GTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATT ACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAAT GGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGG TGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACC CAAGCTGGCTAGCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCG ACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATA AAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAG ACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGA GGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCG TGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTA AATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCA TCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTT CTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCT ATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTA ATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCA ACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACT CCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGC CTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAA GATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCC GTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGAC CGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGA CGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGT ATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGG CTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGAC GAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGA TGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACAT AGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCA CGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGG AATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTAT AATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATG TATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCG TAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGG GGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAA TCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGC GAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGA ATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCAT AGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTC CAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGA CTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACT ACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAAC AAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACG CGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGT CATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCT GACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTG TCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATA CCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGC ATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAA AACGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCT TACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACT CAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTT TAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTG AGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTATT GAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCG AAAAGTGCCACCTGACGTC (SEQ ID NO: 59).

[0292] In a specific embodiment, the heavy and light chain coding sequences are cloned into a vector backbone cut with NheI and EcoRI. In certain cases when the expressed antibody is histidine-tagged, the following coding sequence is attached to the 3' end of the last codon of the heavy chain constant region: - CAC CAT CAC CAT CAC CAT CAC CAT (SEQ ID NO: 60) - stop codon.

[0293] The present invention also provides host cells comprising nucleic acids or vectors of the present invention. Any of a variety of host cells can be used. In one embodiment, the host cell is a prokaryotic cell, for example, Escherichia coli. In another embodiment, the host cell is a eukaryotic cell, for example, a mammalian cell, such as a Chinese hamster ovary (CHO) cell, a COS cell, a BHK cell, a NSO cell or a Bowes melanoma cell. Examples of human host cells are particularly HeLa, 911, AT1080, A549, 293 and HEK293T cells.

[0294] The present invention includes methods for producing antibodies and antigen-binding fragments thereof. In certain embodiments, they are produced recombinantly or by chemical synthesis. For example, antibodies and fragments thereof can be produced synthetically or by enzymatic or chemical cleavage of complete immunoglobulins, or they can be genetically engineered by recombinant DNA techniques. Such production methods are well known in the art and described in, for example, Antibodies: A Laboratory Manual, edited by E. Harlow and D. Lane (1988), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, which is incorporated herein by reference. Antibodies and antigen-binding fragments can be produced synthetically or by enzymatic or chemical cleavage of complete immunoglobulins, or they can be genetically engineered by recombinant DNA techniques. Illustrative production methods are well known in the art and described in, for example, Antibodies: A Laboratory Manual, edited by E. Harlow and D. Lane (1988), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Various techniques have been developed for producing antibody fragments. Traditionally, these fragments are produced by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al., Science, 229: 81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and scFv antibody fragments can all be expressed in E. coli and secreted from E. coli, thus allowing large amounts of these fragments to be easily produced. Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell cultures. Fab and F(ab')2 fragments with increased in vivo half-life that include salvage receptor binding epitope residues are described in U.S. Pat. No. 5,869,046. Other techniques for the production of antibodies and antibody fragments will be apparent to those skilled in the art.

[0295] In one embodiment, the antibody or its antigen binding fragment is produced by isolating the antibody or its antigen binding fragment from a host cell comprising an expression vector encoding the antibody or its antigen binding fragment. In certain embodiments, the method further comprises culturing the host cell under conditions suitable for expression of the antibody or its antigen binding fragment and / or further comprises introducing an expression vector encoding the antibody or its antigen binding fragment into the host cell.

[0296] Pharmaceutical composition

[0297] The present invention also includes a pharmaceutical composition comprising an antibody or its antigen-binding fragment as described herein or a polynucleotide encoding an antibody or its antigen-binding fragment as described herein and a pharmaceutically acceptable diluent, carrier or excipient. In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an antibody, its antigen-binding fragment or polynucleotide.

[0298] Various pharmaceutically acceptable diluents, carriers and excipients and technologies for preparing and using pharmaceutical compositions are known to those skilled in the art according to the disclosure of the present invention. Illustrative pharmaceutical compositions and pharmaceutically acceptable diluents, carriers and excipients are also described in Remington: The Science and Practice of Pharmacy, 20th edition, (Lippincott, Williams & Wilkins 2003). In a particular embodiment, each carrier, diluent or excipient is "acceptable" in the sense that it is compatible with other ingredients of the pharmaceutical composition and harmless to the subject. Typically, pharmaceutically acceptable carriers are aqueous pH buffers. Some examples of materials that can be used as pharmaceutically acceptable carriers, diluents, or excipients include: water; buffers, e.g., phosphate-buffered saline; sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; and other nontoxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0299] In some embodiments, the pharmaceutical composition is sterile.In some embodiments, the pharmaceutical composition has a pH in the range of 4.5 to 8.5, 4.5 to 6.5, 6.5 to 8.5, or about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0 or about 8.5 pH. In one embodiment, the pharmaceutical composition has a molar osmotic pressure concentration in the range of 240-260 or 250-330mOsmol / L. In some embodiments, the pharmaceutical composition is isotonic or close to isotonic. In some embodiments, the pharmaceutical composition is formulated for intravenous administration and has an antibody or fragment thereof of 10-100 mg / ml, 10-50mg / ml, 20 to 40mg / ml or about 30mg / ml. In certain embodiments, the pharmaceutical composition is formulated for subcutaneous injection and has a concentration of 50-500 mg / ml, 50-250 mg / ml, or 100 to 150 mg / ml of the antibody or fragment thereof, and a viscosity of less than 50 cP, less than 30 cP, less than 20 cP, or about 10 cP. In specific embodiments, the pharmaceutical composition is liquid or solid. In specific embodiments, the pharmaceutical composition is formulated for parenteral administration, e.g., intravenous, subcutaneous, or oral administration.

[0300] The formulation and delivery method of the pharmaceutical composition are generally varied depending on the site and disease to be treated. Exemplary formulations include, but are not limited to, those suitable for parenteral administration (e.g., intravenous, intraarterial, intramuscular or subcutaneous administration), including formulations encapsulated in micelles, liposomes or drug release capsules (active agents incorporated into a biocompatible coating designed for slow release); orally administrable formulations; formulations for topical use, such as creams, ointments and gels; and other formulations, such as inhalants, aerosols and sprays.

[0301] How to use

[0302] The present invention provides methods for treating and preventing HIV infection or related diseases or conditions in subjects in need thereof, comprising providing an effective amount of an antibody or antigen-binding fragment thereof as described herein, or a polynucleotide encoding the antibody or antigen-binding fragment thereof, to a subject in need thereof. The polynucleotide may be present in a vector, for example, a viral vector. In a specific embodiment, the related disease or condition is caused by HIV infection. In a specific embodiment, it is acquired immunodeficiency syndrome (AIDS). In a specific embodiment, the subject is a virologically contained HIV-infected mammal, while in other embodiments, the subject is an untreated HIV infects mammals. In certain embodiments, untreated subjects have 10 3 Up to 10 5In some embodiments, the subject is a mammal, for example, a human. In some embodiments, the subject has been diagnosed with HIV (for example, HIV-1 or HIV-2) infection or associated disease or condition, for example, AIDS, or is considered to be in the risk of producing HIV (for example, HIV-1 or HIV-2) infection or associated disease or condition (for example, AIDS). Subjects in HIV-related diseases or disease risks include patients who have been exposed to infected personnel or have been exposed to HIV in some other ways. The use of preventive agents can be carried out before presenting HIV-related diseases or disease characteristic symptoms, so that disease or disease is prevented, or alternatively, its progress is delayed.

[0303] The present invention further provides a method for preventing or inhibiting the increase of HIV viral titer, viral replication, viral proliferation, or HIV viral DNA, HIV proviral DNA, or HIV viral protein in a subject. In one embodiment, the method comprises providing an antibody or its antigen binding fragment described herein, or a polynucleotide encoding the antibody or its antigen binding fragment, to a subject in need thereof, which effectively prevents the increase of HIV titer, viral replication, or HIV protein amount of one or more HIV strains or isolates in the subject. In certain embodiments, the method further comprises measuring the amount of HIV virus or proviral DNA or protein at one or more time points, for example, before and after providing the subject with the antibody or antibody binding fragment of the present invention. Methods and biomarkers for determining the amount of HIV viral or proviral DNA or protein in a subject are known and available in the art and are described, for example, in Siliciano, JD et al., Curr Opin. HIV AIDS (2010) 5(6):491-7 and Rouzioux, C. et al., Curr Opin HIV AIDS (2013) 8(3):170-5, each of which is incorporated herein by reference in its entirety.

[0304] In certain aspects, the antibodies disclosed herein can be used, for example, in methods of inhibiting certain viruses (such as the HIV isolates described herein), prophylactically inhibiting or preventing infection by certain viruses (such as the HIV isolates described herein), detecting certain viruses (such as the HIV isolates described herein) in a sample, inhibiting certain viruses (such as the HIV isolates described herein), and diagnosing certain viruses (such as the HIV isolates described herein).

[0305] For in vivo treatment of mammalian subjects (e.g., humans), a pharmaceutical composition comprising an antibody or its antigen binding fragment of the present invention can be administered or provided to a subject. When used for in vivo treatment, the antibody of the present invention and its antigen binding fragment are usually administered or provided to a patient in a therapeutically effective amount (i.e., eliminating or reducing the amount of the patient's viral load and / or viral reservoir). According to known methods, such as, but not limited to, intravenous administration, for example, as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical or inhalation routes, the antibody or its antigen binding fragment is administered or provided to a mammalian subject, for example, a human. The antibody or its antigen binding fragment can be administered parenterally (when possible, at the target cell site) or intravenously. In certain embodiments, it is preferred that the antibody or its antigen binding fragment be administered intravenously or subcutaneously. In a specific embodiment, the pharmaceutical composition of the present invention is administered systemically, parenterally or topically to a patient or subject.

[0306] In a specific embodiment, for parenteral administration, the antibody or antigen-binding fragment thereof is combined with a pharmaceutically acceptable parenteral vehicle and formulated into a unit dose injection form (solution, suspension, emulsion). Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles, such as fixed oils and ethyl oleate, may also be used. Liposomes may be used as carriers. The vehicle may contain small amounts of additives, such as substances that enhance isotonicity and chemical stability, for example, buffers and preservatives. In certain embodiments, the antibody or antigen-binding fragment thereof is formulated in such a vehicle at a concentration of about 1 mg / ml to about 200 mg / ml, about 1 mg / ml to about 100 mg / ml, about 1 mg / ml to about 50 mg / ml, about 1 mg / ml to about 25 mg / ml, about 1 mg / ml to about 10 mg / ml, e.g., about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 25 mg / ml, about 50 mg / ml, about 100 mg / ml, about 150 mg / ml or about 200 mg / ml.

[0307] Dosage and dosage regimen depend on the multiple factors that the doctor easily determines, such as the nature of infection, the characteristics of the experimenter and the history of the experimenter.In a specific embodiment, the amount of the antibody or its Fab applied to or provided to the experimenter is in the range of about 0.1mg / kg to about 50mg / kg of the experimenter's body weight.According to the type and severity of infection, in some embodiments, the antibody or its Fab of about 0.1 mg / kg to about 50mg / kg body weight (for example, about 0.1-15mg / kg / agent) can be provided to the patient as the initial candidate dose, no matter for example by one or more separate administrations, or by continuous infusion.Can be by conventional methods and analysis and based on the standard known to the doctor or other persons skilled in the art to easily monitor the progress of treatment.

[0308] In certain embodiments, the amount of the drug may be sufficient to obtain a C of ≤ 0.1 μg / mL, ≤ 0.5 μg / ml, ≤ 1 μg / ml, ≤ 10 μg / ml, ≤ 20 μg / ml, ≤ 30 μg / ml, ≤ 40 μg / ml, ≤ 50 μg / ml, ≤ 75 μg / ml, ≤ 100 μg / ml, ≤ 200 μg / ml, ≤ 300 μg / ml, or ≤ 500 μg / mL. trough In some embodiments, the antibody or fragment thereof of the present invention can be provided to a subject in an amount sufficient to obtain 1 μg / mL to 100 μg / mL of C trough In certain embodiments, the antibody or fragment thereof of the invention is provided to a subject in an amount sufficient to obtain a C of >1 μg / ml, >5 μg / ml, >10 μg / ml, >50 μg / ml, >100 μg / ml, >200 μg / ml, >300 μg / ml, >400 μg / ml, >500 μg / ml, or ≥1000 μg / mL. max In some embodiments, the antibody or fragment thereof of the present invention may be provided to a subject in an amount sufficient to obtain a C max The antibodies or fragments thereof of the present invention are provided to the subject in an amount sufficient to provide the subject with the antibody or fragments thereof.

[0309] In certain embodiments, the antibody of the present invention or its antigen binding fragment is combined with one or more other therapeutic agents for treating HIV infection or related diseases or conditions to provide to the subject. In certain embodiments, a method for treating or preventing HIV infection in a mammal (e.g., a person) suffering from infection or at risk of infection is provided, comprising combining a therapeutically effective amount of an antibody disclosed herein or its fragment or its pharmaceutically acceptable salt with a therapeutically effective amount of one or more (e.g., one, two, three, one or two or one to three) other therapeutic agents to be administered to a person. In one embodiment, a method for treating HIV infection in a person suffering from infection or at risk of infection is provided, comprising combining a therapeutically effective amount of an antibody disclosed herein or its fragment or its pharmaceutically acceptable salt with a therapeutically effective amount of one or more (e.g., one, two, three, one or two or one to three) other therapeutic agents to be administered to a person.

[0310] In certain embodiments, the present disclosure provides a method for treating HIV infection, comprising administering to a patient in need thereof a therapeutically effective amount of an antibody or fragment thereof disclosed herein, or a pharmaceutically acceptable salt thereof, in combination with a therapeutically effective amount of one or more other therapeutic agents useful for treating HIV infection.

[0311] In certain embodiments, two or more antibodies disclosed herein or fragments thereof or pharmaceutically acceptable salts thereof may be provided to a subject. In a specific embodiment, the two or more antibodies or fragments thereof may have different specificities, for example, each may preferentially bind to, inhibit or neutralize different HIV strains or combinations of strains from each other. In certain embodiments, the antibody may include any of the following: one of PGT-121LO6 or its variants (including somatic variants PGT122, PGT123, PGT124, 10-1074, PGT133 or PGT134) (or any variant described herein); PGT145 or one of its variants; PG16 or one of its variants; PG9 or one of its variants; PGT151 or one of its variants; or any other combination of bNAbs that provide complementary binding, neutralization or infected cell killing activity. In a specific embodiment, the HIV strain is a branch B strain.

[0312] The antibodies or fragments thereof disclosed herein can be combined with one or more other therapeutic agents at any antibody or fragment dose (e.g., 50 mg to 1000 mg of compound). In addition, the antibodies or fragments thereof disclosed herein can be combined with one or more other therapeutic agents at any antibody or fragment dose (e.g., about 0.1 mg / kg to about 50 mg / kg subject body weight or 50 mg to 4000 mg of compound).

[0313] In one embodiment, a pharmaceutical composition is provided comprising an antibody or fragment thereof disclosed herein, or a pharmaceutically acceptable salt thereof, in combination with one or more (e.g., one, two, three, one or two, or one to three) other therapeutic agents and a pharmaceutically acceptable carrier, diluent, or excipient.

[0314] In one embodiment, a kit is provided comprising an antibody or fragment thereof disclosed herein, or a pharmaceutically acceptable salt thereof, in combination with one or more (eg, one, two, three, one or two, or one to three) other therapeutic agents.

[0315] In the above embodiments, the other therapeutic agent can be an anti-HIV agent. For example, in some embodiments, the other therapeutic agent is selected from HIV protease inhibitors, HIV non-nucleoside or non-nucleotide reverse transcriptase inhibitors, HIV nucleoside or nucleotide reverse transcriptase inhibitors, HIV integrase inhibitors, HIV non-catalytic site (or allosteric) integrase inhibitors, HIV entry inhibitors (e.g., CCR5 inhibitors, gp41 inhibitors (i.e., fusion inhibitors) and CD4 adhesion inhibitors), CXCR4 inhibitors, gp120 inhibitors, G6PD and NADH-oxidase inhibitors, HIV vaccines, HIV maturation inhibitors, latent reversal agents (e.g., histone deacetylase inhibitors, proteasome inhibitors, protein kinase C (PKC) activators and BRD4I inhibitors), compounds targeting HIV capsid ("capsid" inhibitors; e.g., capsid polymerization inhibitors or capsid disruption compounds, HIV nucleocapsid p7 (NCp7) inhibitors, HIV p24 capsid protein inhibitors), pharmacokinetic enhancers, immune-based therapies (e.g., Pd-1 modulators, Pd-L1 modulators, toll-like receptor modulators, IL-15 agonists), HIV antibodies, bispecific antibodies, and “antibody-like” therapeutic proteins (e.g., Fab derivatives), including those targeting HIV gp120 or gp41, combination drugs for HIV, HIV p17 matrix protein inhibitors, IL-13 antagonists, peptidyl-prolyl cis-trans isomerase A modulators, protein disulfide isomerase inhibitors, complement C5a receptor antagonists, DNA methyltransferase inhibitors, HIV vif gene modulators, Vif dimerization antagonists, HIV-1 viral infection factor inhibitors, TAT protein inhibitors, HIV-1 Nef modulators, Hck tyrosine kinase modulators, mixed lineage kinase-3 (MLK-3) inhibitors, HIV-1 splicing inhibitors, Rev protein inhibitors, integrin antagonists, nuclear protein inhibitors, splicing factor modulators, COMM domain-containing protein 1 modulators, HIV ribonuclease H inhibitors, Retrocyclin modulators, CDK-9 inhibitors, dendritic ICAM-3 grabbing non-integrin 1 inhibitors, HIV GAG protein inhibitors, HIV POL protein inhibitors, complement factor H regulators, ubiquitin ligase inhibitors, deoxycytidine kinase inhibitors, cyclin-dependent kinase inhibitors, proprotein convertase PC9 stimulators, ATP-dependent RNA helicase DDX3X inhibitors, reverse transcriptase initiation complex inhibitors, HIV gene therapy, PI3K inhibitors, such as WO 2013 / 006738 (Gilead Sciences0, US 2013 / 0165489 (University of Pennsylvania), WO 2013 / 091096A1 (Boehringer Ingelheim), WO 2009 / 062285 (Boehringer Ingelheim), US20140221380 (Japan Tobacco), US20140221378 (Japan Tobacco), WO 2010 / 130034 (Boehringer Ingelheim), WO 2013 / 159064 (Gilead Sciences), WO Compounds of those disclosed in WO 2012 / 145728 (Gilead Sciences), WO 2012 / 003497 (Gilead Sciences), WO 2014 / 100323 (Gilead Sciences), WO 2012 / 145728 (Gilead Sciences), WO 2013 / 159064 (Gilead Sciences), WO 2012 / 003498 (Gilead Sciences), and WO 2013 / 006792 (Pharma Resources), and other drugs for treating HIV,and combinations thereof. ,

[0316] In certain embodiments, the additional therapeutic agent is selected from HIV protease inhibitors, HIV non-nucleoside or non-nucleotide reverse transcriptase inhibitors, HIV nucleoside or nucleotide reverse transcriptase inhibitors, HIV integrase inhibitors, HIV non-catalytic site (or allosteric) integrase inhibitors, pharmacokinetic enhancers, and combinations thereof.

[0317] In certain embodiments, the other therapeutic agent is a latency reversal agent (LRA), for example, a TLR7 agonist. In other embodiments, the other therapeutic agent is a latency reversal agent (LRA), for example, a TLR8 agonist. Examples of TLR agonists include, but are not limited to, Vesatolimod. Other examples include, but are not limited to, compounds described in U.S. Patent No. 8,367,670 and compounds described in U.S. Patent Application Publication No. 2016-0289229. In one embodiment, the antibodies of the present invention can be combined with a TLR7 agonist such as Vesatolimod. In another embodiment, the antibodies of the present invention can be combined with a TLR8 agonist. In one embodiment, the other therapeutic agent is a TLR modulator. TLR modulators can include modulators of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13. Examples of TLR3 modulators include rintatolimod, multi-ICLC, Apoxxim, IPH-33, MCT-465, MCT-475 and ND-1.1. Examples of TLR7 modulators include GS-9620, GSK-2245035, imiquimod, resiquimod, DSR-6434, DSP-3025, IMO-4200, MCT-465, MEDI-9197, 3M-051, SB-9922, 3M-052, Limtop, TMX-30X, TMX-202, RG-7863, RG-7795 and compounds disclosed in US20100143301 (Gilead Sciences), US20110098248 (Gilead Sciences) and US20090047249 (Gilead Sciences). Examples of TLR8 modulators include motolimod, resiquimod, 3M-051, 3M-052, MCT-465, IMO-4200, VTX-763, VTX-1463, and US20140045849 (Janssen), US20140073642 (Janssen), WO2014 / 056953 (Janssen), WO2014 / 076221 (Janssen), WO2014 / 128189 (Janssen), US20140350031 (Janssen), WO2014 / 023813 (Janssen), US20080234251 (Array Biopharma), US20080306050 (Array Biopharma), US20100029585 (VentirxPharma), Compounds disclosed in US20110092485 (Ventirx Pharma), US20110118235 (Ventirx Pharma), US20120082658 (Ventirx Pharma), US20120219615 (Ventirx Pharma), US20140066432 (Ventirx Pharma), US20140088085 (Ventirx Pharma), US20140275167 (Novira Therapeutics) and US20130251673 (Novira Therapeutics).Examples of TLR9 modulators include BB-001, BB-006, CYT-003, IMO-2055, IMO-2125, IMO-3100, IMO-8400, IR-103, IMO-9200, agatolimod, DIMS-9054, DV-1079, DV-1179, AZD-1419, leftolimod (MGN-1703), litenimod, and CYT-003-QbG10.

[0318] Without wishing to be bound by any particular theory, it is believed that LRAs can increase cell surface Env expression, thereby enhancing latent cell killing via effector function enhanced by the antibodies of the invention. In certain embodiments, the other therapeutic agents include one or more antiretroviral therapies (ARTs). In a specific embodiment, the ART is a combination ART (cART), such as a highly active ART (HAART). In a specific embodiment, the ART includes one or more of a nucleoside reverse transcriptase inhibitor (NRTI), a non-nucleoside reverse transcriptase inhibitor (NNRTI), a protease inhibitor (PI), an entry inhibitor, or an HIV integrase inhibitor. Examples of NRTIs include, but are not limited to: Zidovudine (Retrovir, AZT); Didanosine (Videx, Videx EC, ddI); Stavudine (Zerit, d4T); Lamivudine (Epivir, 3TC); Tenofovir, a nucleotide analog (Viread, TDF); Combivir (a combination of zidovudine and lamivudine); Trizivir (a combination of zidovudine, lamivudine, and abacavir); Emtricitabine (Emtriva, FTC); Truvada (a combination of emtricitabine and tenofovir); and Epzicom (a combination of abacavir and lamivudine). Examples of NNRTIs include, but are not limited to: Nevirapine (Viramune, NVP); Delavirdine (Rescriptor, DLV); Efavirenz (Sustiva or Stocrin, EFV, also part of Atripla); Etravirine (Intelence, ETR); and Rilpivirine (Edurant, RPV, also part of Complera or Epivlera).Examples of PIs include, but are not limited to, saquinavir (Invirase, SQV); indinavir (Crixivan, IDV); ritonavir (Norvir, RTV); nelfinavir (Viracept, NFV); amprenavir (Agenerase, APV); lopinavir / ritonavir (Kaletra or Aluvia, LPV / RTV); atazanavir (Reyataz, ATZ); fosamprenavir (Lexiva, Telzir, FPV); tipranavir (Aptivus, TPV); and darunavir (Prezista, DRV). Examples of entry inhibitors include, but are not limited to, Enfuvirtide (Fuzeon, ENF, T-20) and Maraviroc (Selzentry or Celsentri, MVC). Examples of HIV integrase inhibitors include, but are not limited to, Raltegravir (Isentress, RAL); Elvitegravir (EVG, part of the combination Stribild) and Dolutegravir (Tivicay, DTG).

[0319] In certain embodiments, the antibodies or fragments thereof disclosed herein are formulated into tablets, which may optionally contain one or more other compounds useful for treating HIV. In certain embodiments, the tablets may contain another active ingredient for treating HIV, such as HIV protease inhibitors, HIV non-nucleoside or non-nucleotide reverse transcriptase inhibitors, HIV nucleoside or nucleotide reverse transcriptase inhibitors, HIV integrase inhibitors, HIV non-catalytic site (or allosteric) integrase inhibitors, pharmacokinetic enhancers, and combinations thereof.

[0320] In certain embodiments, such tablets are suitable for once daily administration. In certain embodiments, the other therapeutic agent is selected from one or more of the following:

[0321] (1) Selected from (Efavirenz + Tenofovir Disoproxil Fumarate + Emtricitabine), ( Rilpivirine + tenofovir disoproxil fumarate + emtricitabine), (Elvitegravir + cobicistat + tenofovir disoproxil fumarate + emtricitabine), dolutegravir + abacavir sulfate + lamivudine, (Dolutegravir + Abacavir + Lamivudine), Lamivudine + Nevirapine + Zidovudine, Dolutegravir + Rilpivirine, Dolutegravir + Rilpivirine Hydrochloride, Atazanavir Sulfate + Cobicistat, Atazanavir + Cobicistat, Darunavir + Cobicistat, Efavirenz + Lamivudine + Tenofovir Disoproxil Fumarate, Tenofovir Alafenamide Hemifumarate + Emtricitabine + Cobicistat + Elvitegravir, Tenofovir Alafenamide Hemifumarate + Emtricitabine, Tenofovir Alafenamide + emtricitabine, tenofovir alafenamide hemifumarate + emtricitabine + rilpivirine, tenofovir alafenamide + emtricitabine + rilpivirine, Vacc-4x + romidepsin, darunavir + tenofovir alafenamide hemifumarate + emtricitabine + cobicistat, APH-0812, raltegravir + lamivudine, ( Lopinavir + ritonavir), atazanavir sulfate + ritonavir, (zidovudine + lamivudine, ZT + 3TC), ( Abacavir sulfate + lamivudine, ABC + 3TC), (Abacavir sulfate + Zidovudine + Lamivudine, ABC + AZT + 3TC), (tenofovir disoproxil fumarate + emtricitabine, TDF + FTC), doravirine + lamivudine + tenofovir disoproxil fumarate, doravirine + lamivudine + tenofovir disoproxil, tenofovir + lamivudine, and lamivudine + tenofovir disoproxil fumarate combination drugs;

[0322] (2) an HIV protease inhibitor selected from amprenavir, atazanavir, fosamprenavir, fosamprenavir calcium, indinavir, indinavir sulfate, lopinavir, ritonavir, nelfinavir, nelfinavir mesylate, saquinavir, saquinavir mesylate, tipranavir, brecanavir, darunavir, DG-17, TMB-657 (PPL-100) and TMC-310911;

[0323] (3) HIV non-nucleoside or non-nucleotide reverse transcriptase inhibitors selected from the group consisting of delavirdine, delavirdine mesylate, nevirapine, etravirine, dapivirine, doravirine, rilpivirine, efavirenz, KM-023, VM-1500, lentinan, and AIC-292;

[0324] (4) Selected and HIV nucleoside or nucleotide reverse transcriptase inhibitors EC (didanosin, ddl), zidovudine, emtricitabine, didanosin, stavudine, zalcitabine, lamivudine, censavudine, abacavir, abacavir sulfate, elvucitabine, alovudine, phosphazid, fozivudine tidoxil, apricitabine, KP-1461, fosalvudine tidoxil, tenofovir, tenofovir disoproxil, tenofovir disoproxil fumarate, tenofovir disoproxil hemifumarate, tenofovir alafenamide, tenofovir alafenamide hemifumarate, tenofovir alafenamide fumarate, adefovir, adefovir dipivoxil, and festinavir;

[0325] (5) an HIV integrase inhibitor selected from the group consisting of curcumin, curcumin derivatives, chicoric acid, chicoric acid derivatives, 3,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid derivatives, aurintricarboxylic acid, aurintricarboxylic acid derivatives, caffeic acid phenethyl ester, caffeic acid phenethyl ester derivatives, tyrphostin, tyrphostin derivatives, quercetin, quercetin derivatives, raltegravir, elvitegravir, dolutegravir and cabotegravir;

[0326] (6) HIV non-catalytic site or allosteric integrase inhibitor (NCINI) selected from CX-05168, CX-05045 and CX-14442;

[0327] (7) HIV gp41 inhibitors selected from enfuvirtide, sifuvirtide and albuvirtide;

[0328] (8) HIV entry inhibitors selected from cenicriviroc;

[0329] (9) HIV gp120 inhibitors selected from Radha-108 (Receptol) and BMS-663068;

[0330] (10) CCR5 inhibitors selected from aplaviroc, vicriviroc, maraviroc, cenicriviroc, PRO-140, Adaptavir (RAP-101), nifeviroc (TD-0232), TD-0680 and vMIP (Haimipu);

[0331] (11) CD4 adhesion inhibitors, for example, Fostemsavir (BMS-663068);

[0332] (12) an inhibitor of a post-binding event required for entry selected from ibalizumab;

[0333] (12) a CXCR4 inhibitor selected from plerixafor, ALT-1188, vMIP and Haimipu;

[0334] (13) a pharmacokinetic enhancer selected from cobicistat and ritonavir;

[0335] (14) selected from dermaVir, interleukin-7, plaquenil (hydroxychloroquine), proleukin (aldesleukin, IL-2), interferon alpha, interferon alpha-2b, interferon alpha-n3, pegylated interferon alpha, interferon gamma, hydroxyurea, mycophenolate mofetil (MPA) and its ester derivative mycophenolate mofetil (MMF), WF-10, ribavirin, IL-2, IL-12, polymer polyethyleneimine (PEI), Gepon, VGV-1, MOR-22, BMS-936559, toll-like receptor modulators (TLR1, TLR2, TLR3, TLR4, immune-based therapeutics of TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13), rintatolimod, and IR-103;

[0336] (15) selected from peptide vaccines, recombinant subunit protein vaccines, live vector vaccines, DNA vaccines, virus-like particle vaccines (pseudovirion vaccines), CD4-derived peptide vaccines, vaccine combinations, rgp120 (AIDSVAX), ALVAC HIV (vCP1521) / AIDSVAX B / E (gp120) (RV144), monomeric gp120 HIV-1 subtype C vaccine (Novartis), Remune, ITV-1, Contre Vir, Ad5-ENVA-48, DCVax-001 (CDX-2401), PEP-6409, Vacc-4x, Vacc-C5, VAC-3S, multiclade DNA recombinant adenovirus-5 (rAd5), Pennvax-G, VRC-HIV MAB060-00-AB, AVX-101, AVX-201, HIV-LAMP-vax, Ad35, Ad35-GRIN, NAcGM3 / VSSP ISA-51, multi-ICLC adjuvanted vaccine, TatImmune, GTU-multiHIV (FIT-06), AGS-004, gp140[δ]V2.TV1+MF-59, rVSVIN HIV-1 gag vaccine, SeV-Gag vaccine, AT-20, DNK-4, Ad35-GRIN / ENV, TBC-M4, HIVAX, HIVAX-2, NYVAC-HIV-PT1, NYVAC-HIV-PT4, DNA-HIV-PT123, rAAV1-PG9DP, GOVX-B11, GOVX-B21, ThV-01, TUTI-16, VGX-3300, TVI-HIV-1, Ad-4 (Ad4-env clade C+Ad4-mGag), HIV vaccines EN41-UGR7C, EN41-FPA2, PreVaxTat, TL-01, SAV-001, AE-H, MYM-V101, CombiHIVvac, ADVAX, MYM-V201, MVA-CMDR, MVATG-17401, ETV-01, CDX-1401, rcAd26.MOS1.HIV-Env, and DNA-Ad5 gag / pol / nef / nev (HVTN505);

[0337] (16) HIV antibodies, bispecific antibodies, and “antibody-like” therapeutic proteins (e.g. Fab derivatives), including BMS-936559, TMB-360, and those targeting HIV gp120 or gp41 selected from bavituximab, UB-421, C2F5, C2G12, C4E10, C2F5+C2G12+C4E10, 3-BNC-117, PGT145, PGT121, MDX010 (ipilimumab), VRC01, A32, 7B2, 10E8, VRC-07-523, and VRC07;

[0338] (17) selected from histone deacetylase inhibitors, such as romidepsin, vorinostat, panobinostat; proteasome inhibitors, such as Velcade; protein kinase C (PKC) activators, such as Indolactam, Prostratin, Ingenol B and DAG-lactone, Ionomycin, GSK-343, PMA, SAHA, BRD4 inhibitors, IL-15, JQ1, disulfram and latency reversal agents of amphotericin B;

[0339] (18) HIV nucleocapsid p7 (NCp7) inhibitors selected from azodicarbonamide;

[0340] (19) HIV maturation inhibitors selected from BMS-955176 and GSK-2838232;

[0341] (20) selected from idelalisib, AZD-8186, buparlisib, CLR-457, pictilisib, neratinib, rigosertib, Ligosertin sodium, EN-3342, TGR-1202, alpelisib, duvelisib, UCB-5857, taselisib, XL-765, gedatolisib, VS-5584, copanlisib, CAI orotic acid, perifosine, RG-7666, GSK-2636771, DS-7423, panulisib, GSK-2269557, GSK-2126458, CUDC-907, PQR-309, INCB-040093, pilaralisib, BAY-1082439, puquitinib mesylate, PI3K inhibitors of SAR-245409, AMG-319, RP-6530, ZSTK-474, MLN-1117, SF-1126, RV-1729, sonolisib, LY-3023414, SAR-260301, and CLR-1401;

[0342] (21) Compounds disclosed in WO 2004 / 096286 (Gilead Sciences), WO 2006 / 110157 (Gilead Sciences), WO 2006 / 015261 (Gilead Sciences), WO 2013 / 006738 (Gilead Sciences), US 2013 / 0165489 (University of Pennsylvania), US20140221380 (Japan Tobacco), US20140221378 (Japan Tobacco), WO 2013 / 006792 (Pharma Resources), WO 2009 / 062285 (Boehringer Ingelheim), WO 2010 / 130034 (Boehringer Ingelheim), WO 2013 / 091096A1 (Boehringer Ingelheim), WO 2013 / 159064 (Gilead Sciences), WO 2012 / 145728 (Gilead Sciences), WO2012 / 003497 (Gilead Sciences), WO2014 / 100323 (Gilead Sciences), WO2012 / 145728 (Gilead Sciences), WO2013 / 159064 (Gilead Sciences), and WO 2012 / 003498 (Gilead Sciences); and

[0343] (22) selected from BanLec, MK-8507, AG-1105, TR-452, MK-8591, REP 9, CYT-107, alisporivir, NOV-205, IND-02, metenkefalin, PGN-007, Acemannan, Gamimune, Prolastin, 1,5-dicaffeoylquinic acid, BIT-225, RPI-MN, VSSP, Hlviral, IMO-3100, SB-728-T, RPI-MN, VIR-576, HGTV-43, MK-1376, rHIV7-shl-TAR-CCR5RZ, MazF gene therapy, BlockAide, ABX-464, SCY-635, naltrexone, Other drugs used to treat HIV include AAV-eCD4-Ig gene therapy, TEV-90110, TEV-90112, deferiprone, and PA-1050040 (PA-040).

[0344] In certain embodiments, an antibody disclosed herein or its fragment or its pharmaceutically acceptable salt is combined with one, two, three, four or more other therapeutic agents. In certain embodiments, a compound disclosed herein or its pharmaceutically acceptable salt is combined with two other therapeutic agents. In other embodiments, a compound disclosed herein or its pharmaceutically acceptable salt is combined with three other therapeutic agents. In more embodiments, a compound disclosed herein or its pharmaceutically acceptable salt is combined with four other therapeutic agents. The one, two, three, four or more other therapeutic agents can be different therapeutic agents selected from the same therapeutic agent category, and / or they can be selected from different therapeutic agent categories. In a specific embodiment, a compound disclosed herein or its pharmaceutically acceptable salt is combined with HIV nucleoside or nucleotide reverse transcriptase inhibitors and HIV non-nucleoside reverse transcriptase inhibitors. In another specific embodiment, a compound disclosed herein or its pharmaceutically acceptable salt is combined with HIV nucleoside or nucleotide reverse transcriptase inhibitors and HIV protease inhibitors. In another embodiment, the compound disclosed herein or its pharmaceutically acceptable salt is combined with HIV nucleoside or nucleotide reverse transcriptase inhibitor, HIV non-nucleoside reverse transcriptase inhibitor and HIV protease inhibitor. In other embodiments, the compound disclosed herein or its pharmaceutically acceptable salt is combined with HIV nucleoside or nucleotide reverse transcriptase inhibitor, HIV non-nucleoside reverse transcriptase inhibitor and pharmacokinetic enhancer. In certain embodiments, the compound disclosed herein or its pharmaceutically acceptable salt is combined with at least one HIV nucleoside reverse transcriptase inhibitor, integrase inhibitor and pharmacokinetic enhancer. In another embodiment, the compound disclosed herein or its pharmaceutically acceptable salt is combined with two HIV nucleoside or nucleotide reverse transcriptase inhibitors.

[0345] In certain embodiments, the compounds disclosed herein, or pharmaceutically acceptable salts thereof, are combined with one, two, three, four or more other therapeutic agents selected from (dulutegravir + abacavir + lamivudine), dolutegravir + abacavir sulfate + lamivudine, raltegravir, raltegravir + lamivudine, (tenofovir disoproxil fumarate + emtricitabine, TDF + FTC), maraviroc, enfuvirtide, ( Abacavir sulfate + lamivudine, ABC + 3TC), (Abacavir sulfate + Zidovudine + Lamidine, ABC + AZT + 3TC), Adefovir, Adefovir dipivoxil, (Elvitegravir + Cobicistat + Tenofovir Disoproxil Fumarate + Emtricitabine), Rilpivirine, Rilpivirine Hydrochloride, ( Rilpivirine + tenofovir disoproxil fumarate + emtricitabine), cobicistat, atazanavir sulfate + cobicistat, atazanavir + cobicistat, darunavir + cobicistat, (Efavirenz + Tenofovir Disoproxil Fumarate + Emtricitabine), Atazanavir, Atazanavir Sulfate, Dolutegravir, Elvitegravir, ( Lopinavir + ritonavir), ritonavir, emtricitabine, atazanavir sulfate + ritonavir, darunavir, lamivudine, Prolastin, fosamprenavir, fosamprenavir calcium, efavirenz, (zidovudine + lamivudine, AZT + 3TC), etravirine, nelfinavir, nelfinavir mesylate, interferon, didanosine, stavudine, indinavir, indinavir sulfate, tenofovir + lamivudine, zidovudine, nevirapine, saquinavir, saquinavir mesylate, aldesleukin, zalcitabine, tipranavir, amprenavir, delavirdine, delavirdine mesylate, Radha-108 (Receptol), Hlviral, lamivudine + tenofovir disoproxil fumarate, efavirenz + lamivudine + tenofovir disoproxil fumarate, phosphazid, lamivudine + nevirapine + zidovudine, abacavir, abacavir sulfate, tenofovir, tenofovir disoproxil, tenofovir disoproxil fumarate, darunavir + cobicistat, atazanavir sulfate + cobicistat, atazanavir + cobicistat, tenofovir alafenamide and tenofovir alafenamide hemifumarate. In some embodiments, the compounds disclosed herein or pharmaceutically acceptable salts thereof are combined with one, two, three, four or more other therapeutic agents selected from (efavirenz, tenofovir disoproxil fumarate, and emtricitabine); ( rilpivirine, tenofovir disoproxil fumarate, and emtricitabine); (elvitegravir, cobicistat, tenofovir disoproxil fumarate, and emtricitabine); (tenofovir disoproxil fumarate and emtricitabine; TDF+FTC); (tenofovir alafenamide and emtricitabine); (tenofovir alafenamide, emtricitabine, and rilpivirine); (tenofovir alafenamide, emtricitabine, cobicistat, and elvitegravir); adefovir; adefovir dipivoxil; cobicistat; emtricitabine; tenofovir; tenofovir disoproxil; tenofovir disoproxil fumarate; tenofovir alafenamide; tenofovir alafenamide hemifumarate; (dolutegravir, abacavir, and lamivudine); dolutegravir, abacavir sulfate, and lamivudine; raltegravir; raltegravir and lamivudine; maraviroc; enfuvirtide; ( lopinavir and ritonavir); (zidovudine and lamivudine; AZT+3TC); ( abacavir sulfate and lamivudine; ABC+3TC); (abacavir sulfate, zidovudine and lamivudine; ABC+AZT+3TC); rilpivirine; rilpivirine hydrochloride; atazanavir sulfate and cobicistat; atazanavir and cobicistat; darunavir and cobicistat; atazanavir; atazanavir sulfate; dolutegravir; elvitegravir; ritonavir; atazanavir sulfate and ritonavir; darunavir; lamivudine; prolastin; fosamprenavir; fosamprenavir calcium; efavirenz; etravirine; nelfinavir; nelfinavir mesylate; interferon; didanosine; stavudine; indinavir; indinavir sulfate; tenofovir and lamivudine; zidovudine; nevirapine; Saquinavir; saquinavir mesylate; aldesleukin; zalcitabine; tipranavir; amprenavir; delavirdine; delavirdine mesylate; Radha-108 (receptol); lamivudine and tenofovir disoproxil fumarate; efavirenz, lamivudine and tenofovir disoproxil fumarate; phosphazid; lamivudine, nevirapine and zidovudine; abacavir; and abacavir sulfate. Those skilled in the art will recognize that the other therapeutic agents listed above may be included in more than one of the above listed categories. It is not intended that a particular category be used to limit the functionality of those compounds listed in those categories.

[0346] In a specific embodiment, a compound disclosed herein, or a pharmaceutically acceptable salt thereof, is combined with abacavir sulfate, tenofovir, tenofovir disoproxil, tenofovir disoproxil fumarate, tenofovir disoproxil hemifumarate, tenofovir alafenamide, or tenofovir alafenamide hemifumarate.

[0347] In a specific embodiment, a compound disclosed herein, or a pharmaceutically acceptable salt thereof, is combined with tenofovir, tenofovir disoproxil, tenofovir disoproxil fumarate, tenofovir alafenamide, or tenofovir alafenamide hemifumarate.

[0348] In a specific embodiment, a compound disclosed herein, or a pharmaceutically acceptable salt thereof, is combined with a first additional therapeutic agent selected from abacavir sulfate, tenofovir, tenofovir disoproxil, tenofovir disoproxil fumarate, tenofovir alafenamide, and tenofovir alafenamide hemifumarate and a second additional therapeutic agent selected from emtricitabine and lamivudine.

[0349] In a specific embodiment, a compound disclosed herein, or a pharmaceutically acceptable salt thereof, is combined with a first additional therapeutic agent selected from tenofovir, tenofovir disoproxil, tenofovir disoproxil fumarate, tenofovir alafenamide, and tenofovir alafenamide hemifumarate and a second additional therapeutic agent, wherein the second additional therapeutic agent is emtricitabine.

[0350] In certain embodiments, the compounds disclosed herein or their pharmaceutically acceptable salts are combined with 5-30 mg of tenofovir alafenamide fumarate, tenofovir alafenamide hemifumarate or tenofovir alafenamide and 200 mg of emtricitabine. In certain embodiments, the compounds disclosed herein or their pharmaceutically acceptable salts are combined with 5-10; 5-15; 5-20; 5-25; 25-30; 20-30; 15-30; or 10-30 mg of tenofovir alafenamide fumarate, tenofovir alafenamide hemifumarate or tenofovir alafenamide and 200 mg of emtricitabine. In certain embodiments, the compounds disclosed herein or their pharmaceutically acceptable salts are combined with 10 mg of tenofovir alafenamide fumarate, tenofovir alafenamide hemifumarate or tenofovir alafenamide and 200 mg of emtricitabine. In certain embodiments, a compound disclosed herein, or a pharmaceutically acceptable salt thereof, is combined with 25 mg of tenofovir alafenamide fumarate, tenofovir alafenamide hemifumarate, or tenofovir alafenamide and 200 mg of emtricitabine. As disclosed herein, the compounds (e.g., compounds of Formula (I)) can be combined with the agents provided herein at any compound dose (e.g., 50 mg to 500 mg of the compound), as if each dose combination is specifically and separately listed. As disclosed herein, the compounds (e.g., compounds of Formula (I)) can be combined with the agents provided herein at any compound dose (e.g., 1 mg to 500 mg of the compound), as if each dose combination is specifically and separately listed.

[0351] In certain embodiments, a compound disclosed herein or a pharmaceutically acceptable salt thereof is combined with 200-400 mg of tenofovir disoproxil fumarate, tenofovir disoproxil hemifumarate or tenofovir disoproxil and 200 mg of emtricitabine. In certain embodiments, a compound disclosed herein or a pharmaceutically acceptable salt thereof is combined with 200-250; 200-300; 250-350; 250-400; 350-400; 300-400; or 250-400 mg of tenofovir disoproxil fumarate, tenofovir disoproxil hemifumarate or tenofovir disoproxil and 200 mg of emtricitabine. In certain embodiments, a compound disclosed herein or a pharmaceutically acceptable salt thereof is combined with 300 mg of tenofovir disoproxil fumarate, tenofovir disoproxil hemifumarate or tenofovir disoproxil and 200 mg of emtricitabine. Antibodies or fragments thereof as disclosed herein can be combined with a pharmaceutical agent provided herein in any antibody or fragment dosage (e.g., 50 mg to 500 mg antibody or fragment thereof), as each dosage combination is specifically and individually listed. Compounds as disclosed herein (e.g., compounds of formula (I)) can be combined with a pharmaceutical agent provided herein in any compound dosage (e.g., 1 mg to 500 mg compound), as each dosage combination is specifically and individually listed.

[0352] In certain embodiments, when an antibody or fragment thereof disclosed herein is combined with one or more other therapeutic agents as described above, the components of the composition are administered as a simultaneous or sequential regimen. When administered sequentially, the composition may be administered in two or more administrations.

[0353] In certain embodiments, the antibodies or fragments thereof disclosed herein are combined with one or more other therapeutic agents in a unit dosage form for simultaneous administration to a patient, for example, as a solid dosage form for oral administration.

[0354] In certain embodiments, the antibodies or fragments thereof disclosed herein are administered together with one or more other therapeutic agents. Co-administration of antibodies or fragments thereof disclosed herein with one or more other therapeutic agents generally refers to the simultaneous or sequential administration of antibodies or fragments thereof disclosed herein and one or more other therapeutic agents, such that a therapeutically effective amount of antibodies or fragments thereof disclosed herein and one or more other therapeutic agents are present in the patient's body at the same time.

[0355] Co-administration includes administration of a unit dose of an antibody or fragment thereof disclosed herein before or after administration of a unit dose of one or more other therapeutic agents, for example, administration of an antibody or fragment thereof disclosed herein within a few seconds, minutes, or hours of administration of one or more other therapeutic agents. For example, in some embodiments, a unit dose of an antibody or fragment thereof disclosed herein is first administered, followed by administration of a unit dose of one or more other therapeutic agents within a few seconds or minutes. Alternatively, in other embodiments, a unit dose of one or more other therapeutic agents is first administered, followed by administration of a unit dose of an antibody or fragment thereof disclosed herein within a few seconds or minutes. In some embodiments, a unit dose of an antibody or fragment thereof disclosed herein is first administered, followed by administration of a unit dose of one or more other therapeutic agents after a period of several hours (e.g., 1-12 hours). In other embodiments, a unit dose of one or more other therapeutic agents is first administered, followed by administration of a unit dose of an antibody or fragment thereof disclosed herein after a period of several hours (e.g., 1-12 hours).

[0356] The combined administration can be co-administration (using separate pharmaceutical compositions or a single pharmaceutical composition), or sequential administration in either order, with an optional period of time during which both (or all) therapeutic agents simultaneously exert their biological activities. Such combined treatment can result in a synergistic therapeutic effect. In certain embodiments, it is desirable to co-administer an antibody of the invention with another antibody or antigen-binding fragment thereof directed against another antigen associated with an infectious agent.

[0357] In addition to administering the antibody or antigen-binding fragment protein of the protein to the subject, the present invention also provides a method for providing the antibody by gene therapy. This administration of nucleic acids encoding antibodies is included in the statement "administering an effective amount of an antibody or its antigen-binding fragment". See, for example, PCT patent application publication WO96 / 07321 involving the use of gene therapy to produce intracellular antibodies. In a specific embodiment, the nucleic acid includes one or more polynucleotides encoding an antibody or its antigen-binding fragment. In certain embodiments, the polynucleotide encodes scFv. In a specific embodiment, the polynucleotide includes DNA, cDNA or RNA. In certain embodiments, the polynucleotide is present in a vector, for example, a viral vector.

[0358] Example

[0359] The application can be better understood by reference to the following non-limiting examples, which are provided as illustrative embodiments of application. The following examples are presented to more fully illustrate embodiments of the present invention, but should never be construed as limiting the wide range of the application. Although some embodiments of the application have been shown and described herein, it will be clear that such embodiments are only provided as examples. Those skilled in the art will now make various changes, variations and substitutions without departing from the present invention. For example, some analyses can be carried out under different conditions and can generally produce the result in the range of reported values. It should be understood that the various alternatives of the embodiments described herein can be used to implement the described methods.

[0360] Example 1: Method for synthesizing antibodies

[0361] For all antibodies described herein, the coding sequences of the variable regions of the variable heavy (VH) and variable light (VL) chains, including respective signal peptides and Kozak sequences, were codon-optimized for Homo sapiens expression and were prepared by GeneArt TM The signal peptide of mouse heavy chain (mewsrvfifl lsvtagvhsq vqlqqsgaelvrpgtsvkvs ckasgyaftn yliewvkqrp gqglewigvi npgsggtnyn ekfkgkatlt adkssstaymqlssltseds avyfcarsyy gydwfaywgq gtlvtvsa (SEQ ID NO: 61); GenBank accession number no. AF04502.1) and the signal peptide of mouse light chain (MRCLAEFLGLLVLWIPGAIGDIVMTQAAPSVPVTPGESVSISCRSSTS LLHSSGKHRLYWFLQRPGQSPQLLIYYMSNLASGVPDRFSGSGSGTDFT LRISRVEAEDFGVYYCMQSLEYP (SEQ ID NO:62); GenBank accession no.ADK97503.1) were used as the corresponding signal peptides for the heavy chain (HC) and light chain (LC), respectively. Cloning: The DNA fragments encoding VH and VL together with their respective constant regions were cloned into the mammalian expression vector pcDNA3.1.

[0362] For antibodies in Expi293F TMFor transient expression in cells, the manufacturer's protocol was followed. Briefly, 2.5 × 10 cells / mL in 1 liter of Expi293 medium were transfected with 0.6 μg of light chain and 0.4 μg of heavy chain plasmid using 2.7 ml of Expifectamine transfection reagent. 9 After transfection, the cultures were incubated in a humidified incubator at 37°C and 8% CO2 for 5-6 days. When the viability reached approximately 60%, the cell-free supernatant was collected for purification.

[0363] The expressed IgG was purified by protein A affinity followed by dialysis. R The resin was balanced in PBS pH 7.4, and the resin volume used was consistent with the expression expected for the 20 mg / ml capacity. The conditioned medium collected by 0.2 um filtration was passed through the resin at 0.7 × Col vol / min. When loading was completed, a washing step of 10 × Col vol PBS pH 7.4 was carried out. This was followed by a pH 3.6 elution step, followed by immediate neutralization to pH 5.5. This collection was dialyzed into PBS pH 7.4 or 20 mM sodium acetate, 9% sucrose and 0.02% Tween 20 pH 5.5, and 0.2 um filtered and stored at 4 ° C.

[0364] Other similar methods can also be used to generate the antibodies described herein, including those described in PCT Application Publication No. WO2012 / 030904.

[0365] Example 2: In vitro characterization of anti-HIV-1 neutralizing antibodies

[0366] Antibodies are characterized using a variety of tests to determine their melting temperature, resistance to aggregation during low pH hold procedures, target binding and HIV neutralization activity and breadth, direct binding to FcRn and FcgR, effector cell activation and killing activity, and immunogenicity potential.

[0367] Melting temperature

[0368] The thermal stability of PGT-121LO6 and antibodies of the invention was determined using differential scanning fluorimetry (DSF) or differential scanning calorimetry (DSC) (Niesen FH et al., 2007, Nature Protocol 2:2212-2221, Garber E and Demarest SJ. 2007. Biochem and Biophys Res Commun 355:751-757). In both analyses, changes in the unfolding transition of the Fab domain were monitored to determine the effect of each different point mutation introduced into PGT121 WT.

[0369] DSF measures the thermal unfolding transition temperature (Tm) of proteins based on the change of the fluorescence intensity of the environmentally sensitive dye SYPRO Orange. Using ViiA7 real-time PCR instrument (Life Technologies, Grand Island, NY) in a formulation buffer containing PBS, pH 7.4 or 20 sodium acetate, 9% sucrose, pH 5.5, DSF is performed in a 96-well MicroAmp fast reaction plate. The antibody at a concentration of 1 mg / mL is mixed with a 1:1000 diluted Sypro Orange fluorescent probe (all concentrations are final concentrations after mixing) in a volume of 25 μL per well. By increasing the temperature from 20 ° C to 95 ° C at a rate of 1 ° C / min, thermal denaturation is performed within 5-10 min of dye addition. Fluorescence intensity data (excitation at 490nm and emission using ROX filters at 600-630nm) are collected at 0.07 ° C intervals, and the first-order derivative method is used to analyze and calculate Tm using Protein Thermal Shift Software (Invitrogen). In this method, Tm is the temperature corresponding to the maximum of the first derivative of the DSF melting curve. For antibodies with multiple melting transitions, Tm1 and Tm2 refer to the melting temperatures for the first and second discrete melting transitions, respectively. The results of the DSF analysis are shown in Figure 2 and 3 middle.

[0370] DSC experiments were performed using a MicroCal VP-Capillary DSC (GE Healthcare, Piscataway, NJ). The samples were buffer exchanged into 20 mM sodium acetate, 9% sucrose pH 5.5 formulation buffer and diluted to a final concentration of 1 mg / mL. For each measurement, the formulation buffer was used as a reference solution. Thermal denaturation of the samples was achieved by increasing the temperature of the protein solution from 25°C to 95°C at a scan rate of 1°C / min. The data were analyzed after subtracting the reference buffer and subtracting the linear baseline. Three transition melting temperatures for each sample were obtained after fitting each thermogram to a Non-2 state model with three transitions using Origin 7 software (OriginLab v7.0552). Figure 4 The Tm of the Fab domain determined by DSC for selected PGT121 variants is shown.

[0371] Table 4 summarizes illustrative mutations that improve the thermal stability of the PGT121 antibodies of the invention.

[0372] Table 4. Mutations that improve the thermal stability of PGT121

[0373] Mutation (Kabat number) Average increase in Tm (℃) V82aS, A82bS, A82cV 1.1 QUR 1.0 S60N 2.2

[0374] Post-translational modifications of PGT121 and variants

[0375] PGT121 LO6 has three consensus glycosylation sites in the heavy chain variable region. Although the presence of glycosylation at the third site (N105-linked, Kabat numbering) has been previously confirmed (Mouquet et al., 2012. PNAS 109: E3268-E3277), the glycosylation status of the other sites is unknown. Glycosylation, including glycosylation on the Fc or Fab domains, can be heterogeneous and have an impact on PK, drug safety and immunogenicity (Jones et al., 2007. Glycobiology 17: 529-540, Alessandri et al., 2012. mAbs 4: 509-520, Goetze et al., 2011. Glycobiology 21: 949-959, Chung et al., 2008. NEJM 358: 1109-1117). The Fab glycan content in PGT121 and selected variants was analyzed in a mass spectrometry peptide mapping analysis similar to those described in (Zhang Z. 2009. Anal. Chem. 81: 8354-8364, Shah et al., 2014. J. Am. Soc. Mass Spectrom. 25: 999-1011). The results of this analysis revealed detectable and heterogeneous glycosylation at both N68 (5.5% glycosylated) and N105 (92.4% glycosylated), as shown in Tables 5 and 6. Peptide mapping analysis of N68 glycosylation of PGT121 WT showed that N68 was approximately 3.7% glycosylated, with multiple glycol-forms present as shown (nomenclature consistent with IMGT rules, -N = loss of GlcNAc; bN = forked GlcNAc). Man5-containing glycans are implicated in poor pharmacokinetic properties (Goetze et al., 2011. Glycobiology 21:949-959).

[0376] Table 5. Glycosylation of N68 of PGT121 LO6

[0377]

[0378] Table 6. Glycosylation of N105 of PGT121 LO6

[0379]

[0380] Binds gp120 antigen and Fc receptor

[0381] The in vitro binding characteristics of the antibodies to both antigen (HIV gp120) and Fc binding receptors (FcγR, FcRn) were determined using enzyme-linked immunosorbent assay (ELISA). For gp120 / gp140 ELISA analysis, 25 μL of recombinant gp120 protein at 1 μg / mL in PBS pH 7.4 was coated onto 384-well NUNC Maxisorp plates with gentle rocking overnight at 4°C, the plates were blocked with 5% BSA in PBS pH 7.4 for 1 hour, the plates were washed 4 times with PBS pH 7.4 + 0.05% Tween 20 (PBST), a maximum antibody concentration of 300 nM was used, 25 μL of a three-fold dilution series of antibodies in PBS pH 7.4 + 1% BSA were incubated in the wells with shaking at 600 RPM for 1 hour at room temperature, the plates were washed 4 times with PBST, 25 μL of HRP-conjugated Thermo goat anti-human IgG (H+L) polyclonal diluted 1:10,000 in PBS were incubated in the wells for 1 hour at room temperature with shaking at 600 RPM, the plates were washed 4 times with PBST, 25 μL TMB substrate was added to the wells, the plates were incubated at room temperature for 120 seconds, 25 μL of 1 M HCl was added to the wells, the absorbance at 450 nm was read on a Spectramax plate reader, and the resulting points were fit using nonlinear regression to determine the ELISA EC50 values.

[0382] Multiple Env sequences were used to evaluate binding of gp120 and / or gp140 forms, including BaL, TRO, SHIV, SF162 P3, pRHPA4259, qh0692, 6535, pCAAN5342, pWITO4160, and AC10.0. The apparent EC50 values ​​from these analyses are shown below for PGT121 LO6 WT and selected PGT121 variants described herein, and their relative binding affinities are shown (see Tables 7 and 8). Figure 5 and 6 It was demonstrated that some single-site PGT121 variants made for other reasons (e.g., to improve low pH stability, improve immunogenicity, remove glycosylation sites, etc.) resulted in decreased target binding affinity, while others improved target binding affinity. Figure 7 gp120 of selected PGT121 variants are shown. These results demonstrate that only certain mutations do not negatively affect gp120 binding affinity.

[0383] Table 7. ELISA EC50 values ​​of PGT121 WT and selected variants against gp120 BaL and gp120 TRO

[0384] Variant Name EC50 TRO(nM) EC50 BaL(nM) PGT121.30 1.142 5.694 PGT121.26 1.342 5.981 PGT121.32 1.713 6.066 PGT121.28 1.306 6.706 PGT121.31 1.090 7.057 PGT121.29 1.388 6.914 PGT121.19 1.497 7.111 PGT121.25 1.366 7.434 PGT121.11 1.137 7.805 PGT121.20 1.498 7.560 PGT121.27 1.090 7.970 PGT121.12 1.106 8.221 PGT121.17 1.178 8.243 PGT121.21 1.877 7.881 PGT121WT 1.327 8.512 PGT121.2 1.260 9.143 PGT121.8 1.654 8.780 PGT121.7 1.396 9.093 PGT121.23 2.089 8.583 PGT121.4 1.859 9.090 PGT121.1 1.282 9.799 PGT121.18 1.489 9.832 PGT121.15 1.127 10.240 PGT121.10 1.297 10.150 PGT121.16 1.263 10.780 PGT121.9 1.399 13.080 PGT121.3 5.582 11.860 PGT121.22 4.120 13.700 PGT121.5 1.927 16.370 PGT121.14 2.429 21.710 PGT121.13 5.802 30.660

[0385] Table 8. ELISA EC50 values ​​of PGT121 WT and selected variants against gp140 BaL and gp140 SHIV SF162P3

[0386] Variant Name EC50(nM)gp140BaL EC50(nM)gp140 SHIV SF162P3 PGT121.46 0.52 0.43 PGT121.39 0.50 0.40 PGT121.52 0.50 0.39 PGT121.36 0.48 0.41 PGT121.48 0.55 0.50 PGT121.44 0.60 0.42 PGT121.33 0.46 0.34 PGT121.45 0.52 0.42 PGT121.57 0.54 0.46 PGT121.40 0.50 0.41 PGT121.35 0.58 0.49 PGT121.49 0.55 0.41 PGT121WT 0.60 0.45 PGT121.51 0.53 0.41 PGT121.56 0.59 0.48 PGT121.37 0.50 0.42 PGT121.38 0.55 0.37 PGT121.50 0.57 0.47 PGT121.42 0.52 0.45 PGT121.47 0.64 0.49 PGT121.54 0.63 0.55 PGT121.43 0.60 0.50 PGT121.34 0.54 0.42 PGT121.41 0.56 0.51 PGT121.55 0.58 0.49 PGT121.53 0.64 0.53 PGT121.13 1.32 0.94

[0387] For FcγR ELISA analysis, the antigen used was a C-terminal Avitag TM The extracellular domain of human FcγR has a single biotin conjugated to the bacterial BirA protein. To perform this assay, 384-well Neutravidin TM ELISA plates (Pierce TM ) were incubated in the wells with 25 μL of 0.5 μg / mL FcγR diluted in PBS pH 7.4 + 1% bovine serum albumin (BSA) for 1 hour with shaking at 600 RPM, the plates were washed 5 times with PBST, 25 μL of a 3.5-fold dilution series of antibodies in PBS pH 7.4 + 1% BSA were incubated in the wells with shaking at 600 RPM for 1 hour at room temperature, the maximum antibody concentration was 5000 nM, the plates were washed 5 times with PBS pH 7.4 + 0.05% Tween 20 (PBST), 25 μL of horseradish peroxide (HRP)-conjugated goat F(ab')2 anti-human F(ab')2 polyclonal (Jackson ImmunoResearch) diluted 1:5,000 in PBS pH 7.4 + 1% BSA was incubated in the wells with shaking at 600 RPM for 30 minutes at room temperature, the plates were washed 5 times with PBST, 25 μL TMB substrate was added to the wells, the plates were incubated at room temperature for 120 seconds, 25 μL of 1 M HCl was added to the wells, the absorbance at 450 nM was read on a Spectramax plate reader, and the resulting points were fitted using nonlinear regression using methods known in the art to determine the ELISA EC50 values. The EC50 values ​​determined for PGT121 WT and variants in these analyses are shown in Fig. 8A Similar experiments were performed using other recombinant human FcγRs to determine the EC50 of PGT121 WT and variants including various point mutations, and the results are shown in Figures 8B-8H middle.

[0388] Binding to recombinant HIV Env in transfected human cell lines

[0389] Flow cytometry was used to evaluate the binding of antibodies to recombinant HIV Env expressed on the surface of transfected HEK293 cell lines. Three Env sequences were used for binding studies, including BaL, US92HT593 and U92US657. For binding analysis, recombinant HIV Env constructs were transfected in HEK293T cells. Cells were collected 48 hours after transfection and incubated for 1 hour with serially diluted antibodies at 4 degrees. The cells were then washed and incubated with a second goat anti-human IgG Alexa488-coupled antibody. Binding was then detected and quantified by flow cytometry. The measured MFI values ​​were fitted to nonlinear regression dose-response curves to determine EC50 values. The apparent EC50 values ​​from these analyses for PGT121 LO6 WT and representative PGT121 variants described herein are shown in Fig. 9 In Fig.10 Relative binding affinities are shown.

[0390] Low pH-induced aggregation

[0391] In order to screen the protein aggregation induced by low pH, as the protein aggregation induced by industrial virus inactivation procedure in antibody production process, two different procedures are used by antibody at pH 3.5 for 1 hour.In the first procedure, first protein is dialyzed to 25mM NaOAc (containing or not containing 10mM NaCl) pH 5, concentrated to 16mg / ml, and then with the 0.5M acetic acid containing or not containing 10mM NaCl, sample is titrated to pH 3.5, and the final protein concentration of targeting 10mg / mL.Subsequently sample is incubated at room temperature for 1 hour, neutralized to pH 5 with 2M Tris, kept for 2 hours at room temperature again, and analyzed by size exclusion chromatography (SEC) subsequently.The result of the first procedure carried out to selected PGT121 mutant in the presence or absence of NaCl is shown in Fig.11 In the second procedure, duplicate antibody solution samples of 9 mg / mL in 20 mM sodium acetate pH 5.5, 9% sucrose were titrated to 0 or 40 mM NaCl by adding 10% v / v 20 mM NaOAc, 9% sucrose, 0.44 M NaCl. Next, 0.5 M acetic acid was added to a target pH of 3.5 in the presence or absence of 40 mM NaCl, and the samples were incubated for 1 hour at room temperature, neutralized with 20% v / v 2M Tris base, kept at room temperature for another 2 hours, and then analyzed by SEC. The percentage of monomer peaks on SEC was analyzed before and after the low pH hold procedure, and the change in monomer content was analyzed to identify antibody variants with increased aggregation / reduced monomer content. The results of the second procedure are shown in Fig.12 middle.

[0392] Antibody-dependent effector cell activation

[0393] The ability of the antibody to activate Jurkat cells expressing human FcγRIIIA coupled to a NFAT luciferase reporter was determined using HEK293 cells expressing Env. Three Env sequences were used for reporter testing, including 92HT593, 92US657, and BaL. Transfected cells were collected 48 hours after transfection and incubated with the antibody and the effector cells for 6 hours at 37 degrees. The activation of effector cells was represented and measured as a luciferase signal. The collected data were fitted to a nonlinear regression dose-response curve, and the activity was quantified by the area under the curve (AUC). The results are shown in Figure 13-16 When quantified by the area under the curve (AUC), the activity of certain antibodies of the invention was 10-fold greater than the activity of the PGT-121 antibody.

[0394] NK-mediated antibody-dependent cellular cytotoxicity (ADCC)

[0395] Antibody-dependent killing of natural killer (NK) cells was evaluated using a primary cell-based assay system with infected primary CD4+T cells and purified autologous effector NK cells obtained from healthy donors. NK cells express activating FcγRIIIA and mediate antibody-dependent killing of infected cells through granzyme- and perforin-mediated cytotoxicity (ADCC). To mimic the latent CD4+T cell pool (where cell surface antigen expression is predicted to be very low), target cells were generated by infecting resting primary CD4+T cells by spinfection. In addition, to reproduce physiological conditions, ADCC was evaluated in the presence of 10 mg / ml nonspecific human serum IgG that competes with effector Ab for FcγR binding. Using flow cytometry, CD4 expressing p24 was expressed in 1% IgG and 1% IgG was expressed in 1% IgG. 低 Antibody-dependent killing of NK cells was measured by % reduction of T cells.

[0396] PGT-121 mediates killing of infected CD4+T cells in the absence of competing nonspecific serum IgG, but the overall extent of killing is substantially reduced by the presence of competing nonspecific serum IgG. In contrast, in the presence of competing nonspecific IgG, the antibodies of the present invention mediate killing of US657-infected target cells. As quantified by the area under the curve (AUC) calculated for ADCC dose response, the ADCC activity of certain antibodies is 10 times higher than that of PGT-121. Similar levels of enhancement were observed when using Bal-infected target cells. In the presence of competing nonspecific human serum IgG, the antibodies of the present invention are more active than PGT121 due to their enhanced binding to FcγRIII on effector cells. These results are shown in Fig.17 and Tables 9-11. In Tables 9-11, the analysis was performed in the presence of 10 mg / mL nonspecific human serum IgG. 1 μg / mL effector mAb = 6.7 nM. Other results are shown in Fig.26 , 27 and 28 and Tables 20-21.

[0397] Table 9. CD4 expression of PGT121.42 and PGT121 against HIV-1 strain US657 infection + ADCC activity of primary NK cells mediated by T cells

[0398]

[0399]

[0400] Table 10. CD4 expression of PGT121.42 and PGT121 against HIV-1 strain BaL infection + ADCC activity of primary NK cells mediated by T cells

[0401]

[0402] Table 11. PGT121.42 and PGT121 against HIV-1 strain HT593 infected CD4 + ADCC activity of primary NK cells mediated by T cells

[0403]

[0404] Monocyte- and PBMC-mediated antibody-dependent cell killing

[0405] Using HIV-1 infected primary resting CD4 + The ability of antibodies to mediate antibody-dependent cellular killing was studied in vitro using T cells as target cells and primary autologous PBMC or isolated monocytes as effector cells. PBMC, especially CD14 + Monocytes and CD56 + NK cells, express activating FcγR I, IIA, and IIIA, and can mediate antibody-dependent killing of infected cells by phagocytosis (antibody-dependent cellular phagocytosis; ADCP) as well as granzyme- and perforin-mediated cytotoxicity (ADCC). The target CD4 + T cells, primary CD14 + Mononuclear effector cells and PBMCs were obtained from healthy donors.

[0406] In two donors infected in vitro with two independent virus isolates, the antibodies of the present invention showed significantly improved monocyte-mediated killing of HIV-1 infected CD4 T cells compared with PGT-121, and were significantly more effective than PGT-121. In the four donors examined using virus isolate US657, they also showed significantly improved efficacy and maximum killing of HIV-1 infected cells compared with PGT-121 in the PBMC effect test. For two donors, they showed killing in the case of PTG-121 inactivity. In the other two donors, they showed an Emax higher than PGT-121. The observed efficacy improvement ranges from about 2- to 25-fold for donors in which PGT-121 is active to >2000 for donors in which PGT-121 is inactive. The data are shown in Figure 18 and Tables 12, 24 and 25.

[0407] Table 12: Parameters for monocyte- and PBMC-mediated PGT121.42-dependent cell killing

[0408]

[0409] Virus neutralizing activity

[0410] PGT121 is a highly potent neutralizing antibody with broad coverage of HIV subtype B isolates (IC50 0.03 μg / ml, 80% breadth). Env-based binding studies described herein demonstrate comparable activity of PGT-121 and antibodies of the invention. The neutralization potency (measured as IC50 or IC95) and breadth (% of isolates neutralized in the panel tested) of PGT121 and its variants were examined using two different published assay formats: i) an assay based on the CEM-NKr-CCR5-Luc reporter cell line (Li et al., 2005. J Vir 79(16):10108-10125), which is compatible with screening for antibodies against pseudotyped as well as replication-competent HIV isolates; and ii) the Monogram HIV PhenoSense neutralization assay (Monogram Biosciences), which uses a luciferase reporter virus pseudotyped with the HIV Env variant of interest (Richman et al., 2003. PNAS 100(7):4144-4149). In the reporter cell line-based CEM-NKr-CCR5-LucR neutralization assay, which is a multi-cycle viral replication assay (Spenlehauer et al., 2001. Virology, doi:10.1006 / viro.2000.0780), antibodies were screened against a panel of five replication-competent clinical isolates, including the laboratory-adapted HIV-1BaL strain and subtype B isolates 93HT593, 92US657, 92US712, and 92US727 amplified from patient plasma samples (NIH AIDS Reagent Program).

[0411] For the five viruses tested, it was observed that the neutralization potency of the antibodies of the invention was comparable to that of PGT-121, indicating that the modifications present in these antibodies had minimal effects on the recognition and binding of antigenic determinants compared to PGT-121 (Table 13 below with CEM-NKr-CCR5-Luc cells). Other variants (e.g., PGT121.60, PGT121.61) exhibited a 2-3 fold increase in neutralization potency against this limited group of viruses compared to PGT121.

[0412] Table 13. Neutralization activity of PGT121 and selected variants against HIV-1 strains BaL, HT593, US657, US712 and US727 as observed using a CEM.NKr.CCR5.Luc based assay. Data represent the average of 2 to 3 replicates

[0413]

[0414] In Monogram neutralization analysis, the plasma viral RNA isolated from HIV+ART original viremia patients amplifies Env (gp160) coding region and is cloned into expression vectors so that the virus quasispecies distribution present in the patient's plasma sample is maintained. Subsequently, expression vectors are used to produce HIV-1 pseudovirus colonies expressing the Env protein of patient origin. Two groups of clade B clinical isolates were produced for Monogram neutralization analysis: Group 1 (Monogram clinical isolate group) includes 63 isolates from Monogram laboratory collection, and includes 33 or more CCR5- tropism viruses, 15 or more CXCR4- tropisms (X4) and 15 or more dual-mixed (DM) tropism viruses; and Group 2 (Gilead clinical isolate group) includes 142 subtype B viruses isolated from the pre-ARC baseline plasma samples of the original HIV patients of the ART enrolled in the clinical trial, and includes 113 CCR5 tropism (R5) viruses, 28 dual or mixed tropism (DM) viruses and a CXCR4 tropism (X4) virus. HIV-1Env is known to show significant diversity in patient isolates (between clades and within clades), and a group of viruses from the Monogram laboratory collection are also used to analyze the neutralizing activity of PGT121 and variants for viruses representing non-B clades. The Monogram HIV PhenoSense neutralization analysis is used to analyze a large patient isolate collection, so that the width and effectiveness of PGT121 and the variants produced can be more carefully typed. The results are shown in Figures 19-21, 29 and Tables 22-25. The results show that variants of PGT121 (such as PGT121.60) show enhanced neutralizing activity for selected viruses.

[0415] These experiments have demonstrated the unexpected improvement of PGT121 enhanced by Fc in X4-tropism HIV neutralization.Except CD4, HIV can utilize two co-receptors-CXCR4 or CCR5 for entering T cells.Co-receptor binding is mediated by Env, and Env is the target of the broad spectrum neutralizing antibody described herein.Different HIV strains with different sequences therefore preferentially utilize CXCR4 (referred to as X4-tropism), CCR5 (referred to as R5-tropism) or both (referred to as X4 / R5 or dual tropism).The virus pool that demonstrates both R5 and X4 tropism (referred to as dual-mixing or DM) can contain the mixture of R5, X4 and or dual tropism strains.PGT121 usually shows the sensitivity (low efficacy and width) of the difference for X4 strain isolate, thereby preferentially neutralizing R5 tropism virus. Addition of the Fc mutation DEAL+LS to PGT121 (PGT121.56) specifically enhanced its neutralization activity against DM- and X4-tropic viruses (median IC 50 Although some PGT121 Fab variants (e.g., PGT121.13 and PGT121.22) exhibited reduced neutralization potency against R5 DM and X4 viruses, several engineered PGT121 Fab variants carrying the DEAL+LS Fc mutations (including PGT121.56 with WT Fab) were more effective in neutralizing DM and X4 viruses compared to R5 viruses (P<0.0001) ( Figures 29A-29B ). This was highly unexpected since HIV neutralization was thought to be mediated only by the Fab domain and not the Fc domain. In the R5 isolate, a 2- to 3-fold increase in neutralization was observed in approximately 46% of the isolates tested. The DEAL+LS mutation is present in certain antibodies and fragments thereof of the invention. Other modifications introduced into PGT121.56 further improved the neutralizing activity of selected variants (PGT121.42, PGT121.60, etc.), but not necessarily all.

[0416] As IC 95 The coverage width was calculated by the percentage of viruses neutralized at ≤15 μg / ml. 95 Potency was determined by the median IC95 value of virus ≤15 μg / ml ( Fig. 20And Table 24). When tested against a group of two HIV-1 isolates (including a total of 89 clade B isolates), the antibodies of the present invention did not show a loss of neutralizing activity compared to PGT121 (data not shown). The potency of some antibodies was almost the same as PGT121, with a slightly improved neutralization breadth. The neutralization spectrum is also used as an alternative evaluation of the ability of antibodies to recognize and bind to diverse Env antigens from a wide range of HIV-1 clinical isolates relative to PGT-121. Data from a variety of different antibody typings indicate that antibodies with reduced neutralization potency also show reduced ADCC activity (data not shown), indicating a positive correlation between the neutralization activity and ADCC activity of the antibody, and supporting the use of neutralization breadth as an alternative for ADCC breadth evaluation.

[0417] Immunogenicity

[0418] Three methods were used to evaluate immunogenicity and guide engineering to remove immunogenic motifs in PGT121. Computer simulation prediction tools were used to identify sites of potential immunogenicity risk in the PGT121 antibody, and engineering work was also guided to improve manufacturability (e.g., removing glycosylation sites, improving low pH stability) while preventing the introduction of new T cell epitopes. Based on this analysis, modifications to the framework regions were made in the antibodies of the present invention to reduce immunogenicity, which has a low risk of affecting functional activity. In addition, to further identify potential immunogenic motifs within the variable domains of an antibody of the present invention, an in vitro human T cell activation assay, EpiScreen TM (Antitope, Ltd., Cambridge, UK). CD4 T cells induced by overlapping 15-amino acid peptides derived from antibodies and KLH (keyhole limpet hemocyanin, positive control) in 50 healthy donors representing various HLV haplotypes + T cell response uses H-thymidine incorporation analysis to measure T cell proliferation. This analysis enables the location of specific T cell epitopes in primary antibody sequences to guide antibody engineering. The classification of the relative immunogenicity of T cell epitopes for test antibodies is also provided. The predicted or actual immunogenic epitopes identified in these analyses are shown in Tables 14-16 (the computer simulation prediction of T cell epitopes is shown in gray, and the amino acid variants designed to reduce T cell epitopes are shown in white). The data using donor cells are shown to reduce immunogenicity in these analyses by some mutations, while others do not.

[0419] Table 14. In silico prediction of T cell epitopes in PGT-121LO6 HC and amino acid variants designed to reduce the T cell epitope content in PGT-121LO6 HC

[0420]

[0421]

[0422]

[0423] Table 15. In silico prediction of T cell epitopes in PGT-121LO6 LC and amino acid variants designed to reduce the T cell epitope content in PGT-121LO6 LC

[0424]

[0425]

[0426] Table 16. In vitro T cell epitope mapping screening results of PGT-121LO6 HC and LC and variants designed to reduce immunogenicity

[0427]

[0428]

[0429] To evaluate the clinical immunogenicity risk of selected antibody variants, EpiScreen TM Time course T cell analysis (Antitope, Ltd., Cambridge, UK) is used to measure T cell activation induced by complete antibodies. Full molecule analysis was performed as described in (Baker and Jones 2007. Curr. Opin. Drug Discov. Devel. 10: 219-227). Therefore, this analysis not only takes into account T cell epitope content, but also takes into account the processing of natural IgG. Different from computer simulation and peptide scanning analysis, full molecule ex vivo T cell activation analysis can provide an evaluation of the relative clinical risk of a given antibody, and in some cases, can be used to predict clinical immunogenicity rates, as described in (Baker and Jones 2007 Curr. Opin. Drug Discov. Devel. 10: 219-227).

[0430] Many clinical stage antibodies were run in this analysis, and antibodies that showed little or no clinical immunogenicity had scores close to or below 10% in this analysis (dotted line), while antibodies that showed high clinical immunogenicity such as Alemtuzumab and Infliximab showed scores in the 25-40% range (Baker and Jones 2007. Curr. Opin. Drug Discov. Devel. 10:219-227). PGT121.42, PGT121.60, PGT121.61, and PGT121.65 showed reduced donor response rates compared to PGT121 WT, supporting the reduced clinical immunogenicity risk of these variants.

[0431] FcRn binding

[0432] Neonatal Fc receptor (FcRn) is a IgG molecule that has been shown to play a major role in regulating the pharmacokinetics of humans and preclinical species. After endocytosis, at acidic pH (<6.5), FcRn binds the Fc portion of IgG with high affinity. IgG bound to FcRn circulates back into the extracellular space, where IgG binding affinity decreases at physiological pH, and IgG is released back into the circulation. Free IgG that is not cleared by the FcRn pathway is degraded into endogenous amino acids in the lysosome. The relative binding affinity characteristics of IgG and FcRn at pH 6.0 / 7.0 become a very determined correlation for the half-life classification of IgG in vivo and the design features for pharmacokinetic optimization.

[0433] The binding of antibodies to FcRn of various species at different pH was determined. 96-well Maxisorp plates were coated with 100ul of 5μg / ml FcRn. The plates were incubated overnight at 4°C and then blocked with 4% skim milk for 2hr at room temperature after washing 3 times with 0.05% Tween 20 wash buffer. The plates were incubated for 1hr at room temperature with 3-fold serial dilutions of primary antibody. The plates were then washed 3 times and 100μL of Fab-anti-human Fab-HRP or goat anti-human IgG-HRP secondary antibodies diluted in 4% skim milk were added. The plates were then incubated for 50min at room temperature, washed three times, and 100μL of fresh TMB substrate was added. The plates were developed on the bench for 3 minutes using gentle shaking. The plates were quenched with 100μL 1M HCl, shaken briefly, and read at A450 on a spectramax m5 plate reader.

[0434] Relative to PGT-121, antibodies of the invention comprising LS mutations in the Fc region of IgG that interacts with FcRn show a significant improvement in FcRn binding at pH 6.0, while having a lower impact on binding at a neutral pH of 7.0, as indicated by the ratio of pH 7.0 / 6.0 for human FcRn. The improved binding is attributed to the presence of the LS mutations and is predicted to provide an extended half-life in humans relative to PGT-121. The data are shown in Table 17 and Fig.23A and 23B middle.

[0435] Table 17. Human FcRn binding data for PGT121 and variants

[0436]

[0437] This data shows a significant improvement of PGT121.60 and 61 relative to PGT121.56 or PGT121.42. PGT121.56 is a WT Fab with DEAL+LS Fc. This suggests that the Fab mutations in PGT121.60 and 61 improve FcRn binding. PGT121.64 and PGT121.65 did not show this improvement, suggesting that the Fab modifications in these two variants may actually reduce FcRn binding.

[0438] Distribution in the body

[0439] PGT121 and several antibodies from the present application were assayed to characterize their basic pharmacokinetic characteristics to ensure that the Fab / Fc modifications present in the antibodies of the present invention enhance and do not significantly interfere with the inherent pharmacokinetic properties of PGT121. In two male natural cynomolgus monkeys (n=2), the in vivo disposition of PGT121 and several other antibodies of the present invention was characterized after a single intravenous (IV) 1.0 kg / kg dose. Serum samples were collected from the monkeys and analyzed using a bioanalytical method (described herein) to determine the serum concentration-time profile, and the mean serum pharmacokinetic parameters were determined by non-compartmental pharmacokinetic analysis (NCA). The pharmacokinetic concentration-time profile of PGT121, compared to the illustrative antibody PGT121.42 of the present invention, is depicted in Fig.24 middle.

[0440] In a separate study, the intrinsic pharmacokinetic properties of PGT121, PGT121 LS, and new batches of PGT121.42 and PGT121.60 were characterized in three male naive cynomolgus monkeys (n=3) after a single IV 10.0 mg / kg. Serum samples were collected and analyzed using the bioanalytical method (described herein) to determine serum concentration-time profiles, and mean serum pharmacokinetic parameters were determined by non-compartmental pharmacokinetic analysis (NCA). The pharmacokinetic concentration-time profiles of PGT121, PGT121.LS, PGT121.42, and PGT121.60 for a 10 mg / kg IV dose are depicted in Fig.25 middle.

[0441] Mean serum pharmacokinetic parameters for PGT121, PGT121.42, PGT121.43, PGT121.60 and PGT121.61 were determined from non-compartmental pharmacokinetic analysis of concentration-time profiles and are described in Table 18. All antibodies of the invention tested in vivo had comparable or improved pharmacokinetics (as defined herein) relative to PGT121.

[0442] Table 18: Pharmacokinetic parameters of PGT121 and variants after IV administration (1 mg / kg) in naive cynomolgus monkeys (n=2)

[0443]

[0444] The mean serum pharmacokinetic parameters of PGT121, PGT121 LS, PGT121.42 and PGT121.60 were determined from non-compartmental pharmacokinetic analysis of concentration-time profiles and are described in Table 19. All antibodies of the invention tested in vivo had comparable or improved pharmacokinetics (as defined herein) relative to PGT121. The results of additional features are shown in Tables 20-27. Table 23 shows that PGT121.60 exhibits improved efficacy against the tested viruses compared to PGT121. Table 24 shows that some PGT121 variants such as PGT121.60, PGT121.64 and PGT121.65 exhibited improved efficacy in all tested virus isolates (see also Fig. 20 ). This suggests that the modifications made (probably modifications made to antigen contact residues outside the CDRs) improved neutralization potency. Table 25 illustrates that PGT121.60 exhibits substantially improved neutralization activity against viruses representing both B and non-B subtypes compared to PGT121.

[0445] Table 19: Pharmacokinetic parameters of PGT121, PGT121 LS, PGT121.42 and PGT121.60 following IV administration (10 mg / kg) in naive cynomolgus monkeys (n=3)

[0446]

[0447] Table 20: Selected PGT121 variant sets against primary CD4 T cells infected with 2 HIV-1 strains US657 or HT539 + ADCC activity of primary NK cells mediated by T cells

[0448]

[0449] Table 21: Primary NK cell-mediated ADCC activity of PGT121 and PGT121.60 against CEM.NKr.CCR5.Luc cells infected with three HIV-1 isolates

[0450]

[0451]

[0452] Table 22: Neutralization activity of PGT121 and PGT121.60 against subtype B virus (IC 50 )

[0453]

[0454] Table 23: Neutralization efficacy of PGT121 and PGT121.60 against subtype B virus

[0455]

[0456] Table 24: Neutralization efficacy and coverage of PGT121 and selected variants against 92 subtype B viruses

[0457]

[0458]

[0459] Table 25: Neutralization activity of PGT121 and PGT121.60 against multiple clades of viruses

[0460]

[0461]

[0462]

[0463] Table 26. Killing of HIV-infected primary CD4 T cells against six HIV-1 strains

[0464]

[0465] nc: non-computable

[0466] Table 27. Killing of HIV-infected primary CD4 T cells against HIV-1 strain 92US657

[0467]

[0468] All of the above-referenced U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification and / or listed in the Application Data Sheet are incorporated herein by reference in their entirety.

[0469] The present invention may be embodied in other specific forms without departing from the structures, methods and other essential features broadly described herein and required hereinafter. The embodiments described are considered to be illustrative in all respects and not restrictive. Therefore, the scope of the present invention is determined by the appended claims, rather than the previous description. All changes within the equivalent meaning and scope of the claims are included within their scope.

[0470] Table 1

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

[0508]

[0509]

[0510]

[0511]

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524]

[0525]

[0526]

[0527]

[0528]

[0529]

[0530]

[0531]

[0532]

[0533]

[0534]

[0535]

[0536]

[0537]

[0538]

[0539]

[0540]

[0541]

[0542]

[0543]

[0544]

[0545]

[0546]

[0547]

[0548]

[0549]

[0550]

[0551]

[0552]

[0553]

[0554]

[0555]

[0556]

[0557]

[0558]

[0559]

[0560]

[0561]

[0562]

[0563]

[0564]

[0565]

[0566]

[0567]

[0568]

[0569]

[0570]

[0571]

[0572]

[0573]

[0574]

[0575]

[0576]

[0577]

[0578]

[0579]

[0580]

[0581]

[0582]

[0583]

[0584]

[0585]

[0586]

[0587]

[0588]

[0589]

[0590]

[0591]

[0592]

[0593]

[0594]

[0595]

[0596]

[0597]

[0598]

[0599]

[0600]

[0601]

[0602]

[0603]

[0604]

[0605]

[0606]

[0607]

[0608]

[0609]

[0610]

[0611]

[0612]

[0613]

[0614]

[0615]

[0616]

[0617]

[0618]

[0619]

[0620]

[0621]

[0622]

[0623]

[0624]

[0625]

[0626]

[0627]

[0628]

[0629]

[0630]

[0631]

[0632]

[0633]

[0634]

[0635]

[0636]

[0637]

[0638]

[0639]

[0640]

[0641]

[0642]

[0643]

[0644]

[0645]

[0646]

[0647]

[0648]

[0649]

[0650]

[0651]

[0652]

[0653]

[0654]

[0655]

[0656]

[0657]

[0658]

[0659]

[0660]

[0661]

[0662]

[0663]

[0664]

[0665]

[0666]

[0667]

[0668]

[0669]

[0670]

[0671]

[0672]

[0673]

[0674]

[0675]

[0676]

[0677]

[0678]

[0679]

[0680]

[0681]

[0682]

[0683]

[0684]

[0685]

[0686]

[0687]

[0688]

[0689]

[0690]

[0691]

[0692]

[0693]

[0694]

[0695]

[0696]

[0697]

[0698]

[0699]

[0700]

[0701]

[0702]

[0703]

[0704]

[0705]

Claims

1. A pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof that binds to human immunodeficiency virus gp120, and a pharmaceutically acceptable carrier comprising an aqueous pH buffer and a sugar, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises: i. the heavy chain variable region set forth in SEQ ID NO: 273 and the light chain variable region set forth in SEQ ID NO: 359; ii. the heavy chain variable region set forth in SEQ ID NO: 252 and the light chain variable region set forth in SEQ ID NO: 338; iii. the heavy chain variable region set forth in SEQ ID NO: 255 and the light chain variable region set forth in SEQ ID NO: 341; iv. a heavy chain variable region set forth in SEQ ID NO: 266 and a light chain variable region set forth in SEQ ID NO: 352; v. the heavy chain variable region set forth in SEQ ID NO: 267 and the light chain variable region set forth in SEQ ID NO: 353; or vi. the heavy chain variable region set forth in SEQ ID NO: 272 and the light chain variable region set forth in SEQ ID NO:

358.

2. The pharmaceutical composition according to claim 1, further comprising a heavy chain constant region comprising Ala at position 236, Asp at position 239, Leu at position 330, Glu at position 332, Leu at position 428 and / or Ser at position 434, using EU numbering.

3. The pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable carrier comprises acetate buffer, histidine buffer, citrate buffer, succinate buffer, tris-buffer or phosphate buffer.

4. The pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable carrier comprises a sugar selected from the group consisting of lactose, glucose and sucrose.

5. The pharmaceutical composition of claim 1, wherein the pharmaceutically acceptable carrier comprises phosphate buffered saline or sodium acetate; and a sugar selected from the group consisting of lactose, glucose and sucrose.

6. The pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable carrier comprises sodium acetate and sucrose.

7. The pharmaceutical composition of claim 1, wherein the pharmaceutically acceptable carrier comprises phosphate buffered saline.

8. The pharmaceutical composition of claim 1, further comprising one or more other therapeutic agents.

9. The pharmaceutical composition according to claim 8, wherein the one or more other therapeutic agents are anti-HIV agents and / or TLR7 agonists.

10. The pharmaceutical composition of any one of claims 8 to 9, wherein the one or more additional therapeutic agents comprise one or more broadly neutralizing antibodies that provide complementary binding, neutralizing or infected cell killing activities.

11. Use of the pharmaceutical composition of any one of claims 1 to 10 in the preparation of a medicament for treating or preventing human immunodeficiency virus infection in a subject.

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