Broadly neutralizing anti-HIV-1 antibodies and methods of use thereof

Abstract

To provide broadly neutralizing and potent anti-HIV-1 antibodies, kits, and methods of use thereof.SOLUTION: The present invention provides an isolated anti-HIV-1 antibody or an antigen binding portion thereof, comprising (i) a heavy chain variable region that comprises CDRH1, CDRH2, and CDRH3, wherein the CDRH1, CDRH2 and CDRH3 comprise specific sequences and (ii) a light chain variable region that comprises CDRL1, CDRL2 and CDRL3, wherein the CDRL1, CDRL2 and CDRL3 comprise specific sequences.SELECTED DRAWING: None

Description

[Technical Field]

[0001] I. Federal Funding Statement This invention was made with government support under Grant No. AI100148 awarded by the National Institutes of Health (NIH). The U.S. Government has certain rights in this invention.

[0002] II. Technical Field of the Invention The field of this invention is broadly neutralizing anti-HIV-1 antibodies (bNabs) and methods of use thereof. [Background technology]

[0003] Human immunodeficiency virus ("HIV") is a lentivirus (family Retroviridae) that infects humans. Over time, if left untreated, HIV infection leads to acquired immunodeficiency syndrome (AIDS), a condition in which progressive dysfunction of the immune system can lead to life-threatening opportunistic infections (e.g., toxoplasmosis) and cancers (e.g., Kaposi's sarcoma), ultimately resulting in death. Without treatment, the average survival time after initial infection is a total of 9-11 years. HIV / AIDS is a major global health crisis, considered a pandemic according to the CDC. As of 2014, approximately 37 million people were infected with HIV, resulting in approximately 1.2 million deaths that year.

[0004] HIV has several characterized subtypes, including HIV-1 and HIV-2. HIV-1 is more virulent, more infectious, and prevalent worldwide, while HIV-2 is less virulent, less infectious, and currently only endemic in West Africa. HIV-1 is the more clinically important target because it accounts for the majority of HIV infections. HIV-1 has several subgroups, including group M, group N, group O, and group P. Group M represents the "major lineage" and accounts for approximately 90% of HIV infections. It is further divided into subtypes A through K, and occasionally into subsubtypes (e.g., A1, A2, etc.). The significant diversity of HIV-1, driven by a high mutation rate, makes it difficult to effectively treat HIV. To combat this problem, researchers have attempted to develop what are known as broadly neutralizing antibodies ("bNAbs"). Broadly neutralizing antibodies are so designated for their ability to neutralize multiple strains of HIV, including multiple HIV-1 virus strains, such as those disclosed in U.S. Patent Application Publication No. 2014 / 0328862, the entire contents of which are incorporated herein by reference. A small proportion of HIV-1-infected individuals develop bNAbs, which typically develop over a 1-3 year period during which circulating virus strains and antibodies coevolve. However, these bNAbs typically fail to neutralize coexisting autologous virus due to antibody-mediated selection against susceptible virus strains. Therefore, there is an urgent need for novel broadly neutralizing anti-HIV antibodies that exhibit high efficacy against HIV-1 infection. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] US Patent No. 2014 / 0328862 Summary of the Invention

[0006] The present invention relates to multiple potent, broadly neutralizing anti-HIV-1 antibodies and methods of use thereof. Accordingly, in some embodiments, the invention includes an anti-HIV-1 antibody. In some embodiments, the anti-HIV-1 antibody is a broadly neutralizing anti-HIV-1 antibody (bNab) or an antigen-binding portion thereof. In some embodiments, the anti-HIV-1 antibody includes NC37 and an antigen-binding portion thereof. In some embodiments, the anti-HIV-1 antibody includes BG1 and an antigen-binding portion thereof. In some embodiments, the anti-HIV-1 antibody includes BG18 and an antigen-binding portion thereof. In some embodiments, the invention includes variants of BG18 and antigen-binding portions thereof. In some embodiments, the variants of BG18 include one of 354BG8, 354BG18, 354BG42, 354BG33, 354BG129, 354BG188, 354BG411, and 354BG426, or antigen-binding portions thereof. In some embodiments, the anti-HIV-1 antibody or antigen-binding portion thereof comprises a monoclonal antibody (mAb). In some embodiments, the anti-HIV-1 antibody, or antigen-binding portion thereof, comprises a recombinant antibody. In some embodiments, the anti-HIV-1 antibody, or antigen-binding portion thereof, comprises a recombinantly produced antibody. In some embodiments, the anti-HIV-1 antibody, or antigen-binding portion thereof, comprises a human antibody. In some embodiments, the anti-HIV-1 antibody, or antigen-binding portion thereof, comprises a bispecific antibody.

[0007] In some embodiments, the anti-HIV-1 antibody, or antigen-binding portion thereof, comprises a variable heavy chain region. In some embodiments, the variable heavy chain region comprises one of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, and 89. In some embodiments, the variable heavy chain region has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to one of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, and 89. In some embodiments, one or more of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, and 89 have conservative substitutions.

[0008] In some embodiments, the anti-HIV-1 antibody, or antigen-binding portion thereof, comprises a variable light chain region. In some embodiments, the variable light chain region comprises one of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, and 93. In some embodiments, the variable light chain region has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to one of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, and 93. In some embodiments, one or more of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, and 93 have conservative substitutions.

[0009] In some embodiments, the anti-HIV-1 antibody, or antigen-binding portion thereof, comprises at least one complementarity-determining region (CDR) within the variable heavy chain region, wherein at least one CDR comprises one of SEQ ID NOs: 2-4, 10-12, 18-20, 26-28, 34-36, 42-44, 50-52, 58-60, 66-68, 74-76, 82-84, and 90-92. In some embodiments, at least one CDR has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to one of SEQ ID NOs: 2-4, 10-12, 18-20, 26-28, 34-36, 42-44, 50-52, 58-60, 66-68, 74-76, 82-84, and 90-92. In some embodiments, one or more of SEQ ID NOs: 2-4, 10-12, 18-20, 26-28, 34-36, 42-44, 50-52, 58-60, 66-68, 74-76, 82-84, and 90-92 have conservative substitutions.

[0010] In some embodiments, the anti-HIV-1 antibody, or antigen-binding portion thereof, comprises at least one complementarity-determining region (CDR) within the variable light chain region, wherein at least one CDR comprises one of SEQ ID NOs: 6-8, 14-16, 22-24, 30-32, 38-40, 46-48, 54-56, 62-64, 70-72, 78-80, 86-88, and 94-96. In some embodiments, at least one CDR has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to one of SEQ ID NOs: 6-8, 14-16, 22-24, 30-32, 38-40, 46-48, 54-56, 62-64, 70-72, 78-80, 86-88, and 94-96. In some embodiments, one or more of SEQ ID NOs: 6-8, 14-16, 22-24, 30-32, 38-40, 46-48, 54-56, 62-64, 70-72, 78-80, 86-88, and 94-96 have conservative substitutions.

[0011] In some embodiments, the present invention relates to a kit comprising a first antibody. In some embodiments, the first antibody is an anti-HIV-1 antibody or antigen-binding portion thereof. In some embodiments, the anti-HIV-1 antibody or antigen-binding portion thereof comprises a broadly neutralizing anti-HIV-1 antibody (bNab). In some embodiments, the broadly neutralizing anti-HIV-1 antibody is any anti-HIV-1 antibody according to the present invention. In some embodiments, the anti-HIV-1 antibody specifically binds to HIV-1 or an antigenic fragment thereof. In some embodiments, the anti-HIV-1 antibody neutralizes HIV-1 upon binding. In some embodiments, the kit contains a second antibody. In some embodiments, the second antibody comprises an anti-HIV-1 antibody or antigen-binding portion thereof that specifically binds to HIV-1 or an antigenic fragment thereof. In other embodiments, the second antibody specifically binds to the first antibody or antigen-binding portion thereof. In some embodiments, the first antibody or antigen-binding portion thereof is bound to a substrate. In some embodiments, the second antibody or antigen-binding portion thereof is bound to a substrate. In some embodiments, the first antibody or antigen-binding portion thereof is detectably labeled. In some embodiments, the second antibody, or antigen-binding portion thereof, is detectably labeled. In some embodiments, at least one of the first antibody, or antigen-binding portion thereof, and the second antibody, or antigen-binding portion thereof, is detectably labeled. In some embodiments, the detectable label comprises a reporter molecule. In some embodiments, the reporter molecule comprises a fluorescent molecule. In some embodiments, the reporter comprises a radioactive label. In other embodiments, the detectable label comprises an enzyme. In some embodiments, the kit includes a substrate for the enzyme. In some embodiments, addition of the substrate to the enzyme generates a detectable signal. In some embodiments, the detectable signal comprises a colored soluble product. In some embodiments, the radioactive label comprises I-125. In some embodiments, the enzyme comprises horseradish peroxidase. In some embodiments, the substrate for the enzyme comprises TMB. In some embodiments, the kit is capable of detecting HIV-1, or an antigenic fragment thereof, in a sample.In some embodiments, the kit is capable of quantifying the amount of HIV-1 or an antigenic fragment thereof present in a sample.

[0012] In some embodiments, the kit further comprises an HIV agent. In some embodiments, the HIV agent is selected from the group consisting of a non-nucleoside reverse transcriptase inhibitor (NNRTI), a nucleoside reverse transcriptase inhibitor (NRTI), a protease inhibitor (PI), a fusion inhibitor, a CCR5 antagonist / entry inhibitor, an integrase strand transfer inhibitor (INSTI), and combinations thereof. In some embodiments, the HIV agent is in unit dosage form. In some embodiments, the anti-HIV-1 antibody, or antigen-binding portion thereof, is in unit dosage form. In some embodiments, the unit dosage form is a unit-dose injectable form. In some embodiments, the HIV agent is stored in the same container as the anti-HIV-1 antibody, or antigen-binding portion thereof. In some embodiments, the HIV agent is stored in a separate container. In some embodiments, the kit further comprises one or more additional anti-HIV-1 antibodies, or antigen-binding portions thereof. In some embodiments, the one or more additional anti-HIV-1 antibodies, or antigen-binding portions thereof, comprise any anti-HIV-1 antibody of the present invention. In some embodiments, the one or more additional anti-HIV-1 antibodies, or antigen-binding portions thereof, comprise any anti-HIV-1 antibody of the present invention. The HIV-1 antibody or antigen-binding portion thereof is selected from the group consisting of NC37, NC133, NC102, AC40, AC41, AC72, and combinations thereof. In some embodiments, the kit includes instructions for use. In some embodiments, the kit includes one or more pharmaceutically acceptable carriers or preservatives.

[0013] In some embodiments, the present invention relates to methods for detecting HIV-1 or an antigenic fragment thereof present in a sample. In some embodiments, the method includes obtaining a sample containing HIV-1 or an antigenic fragment thereof. In some embodiments, the method includes contacting the sample with an anti-HIV-1 antibody or antigen-binding portion thereof. In some embodiments, the method includes detecting the presence of specific binding of the anti-HIV-1 antibody or antigen-binding portion thereof to HIV or an antigenic fragment thereof. In some embodiments, the method includes quantifying the amount of HIV-1 or an antigenic fragment thereof present in the sample. In some embodiments, the sample is a biological sample. In some embodiments, the anti-HIV-1 antibody or antigen-binding portion thereof is an anti-HIV-1 antibody or antigen-binding portion thereof according to any aspect of the present invention. In some embodiments, the anti-HIV-1 antibody or antigen-binding portion thereof comprises a monoclonal antibody. In some embodiments, the anti-HIV-1 antibody or antigen-binding portion thereof comprises a recombinant antibody. In some embodiments, the anti-HIV-1 antibody or antigen-binding portion thereof is recombinantly produced. In some embodiments, the anti-HIV-1 antibody or antigen-binding portion thereof comprises a human antibody. In some embodiments, detecting the presence of specific binding is achieved by immunoassay. In some embodiments, detecting the presence of specific binding is accomplished by a competitive immunoassay.

[0014] In another embodiment, the present invention relates to methods for diagnosing an individual as having an HIV-1 infection. In some embodiments, the method comprises identifying an individual having or suspected of having an HIV-1 infection. In some embodiments, the method comprises obtaining a sample containing HIV-1 or an antigenic fragment thereof from the individual. In some embodiments, the method comprises contacting the sample with an anti-HIV-1 antibody or antigen-binding portion thereof. In some embodiments, the anti-HIV-1 antibody or antigen-binding portion thereof is an anti-HIV-1 antibody or antigen-binding portion thereof according to any aspect of the present invention. In some embodiments, the method comprises detecting the presence of specific binding of the anti-HIV-1 antibody or antigen-binding portion thereof to HIV or an antigenic fragment thereof. In some embodiments, the method comprises diagnosing the individual as having an HIV-1 infection. In some embodiments, the individual is diagnosed with AIDS. In some embodiments, the sample is a biological sample. In some embodiments, the anti-HIV-1 antibody or antigen-binding portion thereof comprises a monoclonal antibody. In some embodiments, the anti-HIV-1 antibody or antigen-binding portion thereof comprises a recombinant antibody. In some embodiments, the anti-HIV-1 antibody or antigen-binding portion thereof is recombinantly produced. In some embodiments, the anti-HIV-1 antibody or antigen-binding portion thereof comprises a human antibody. In some embodiments, detecting the presence of specific binding is achieved by immunoassay. In some embodiments, detecting the presence of specific binding is achieved by competitive immunoassay.

[0015] In some embodiments, the present invention relates to methods of treating HIV-1 infection in an individual in need thereof. In some embodiments, the method of treating HIV-1 infection comprises administering at least one anti-HIV-1 antibody, or antigen-binding portion thereof, to an individual having or suspected of having HIV-1 infection. In some embodiments, the at least one anti-HIV-1 antibody, or antigen-binding portion thereof, comprises a broadly neutralizing anti-HIV-1 antibody (bNab), or antigen-binding portion thereof. In some embodiments, the at least one anti-HIV-1 antibody, or antigen-binding portion thereof, comprises any anti-HIV-1 antibody, or antigen-binding portion thereof, according to the present invention. In some embodiments, the method comprises, prior to the administering step, identifying the individual as having HIV-1 infection. In some embodiments, the anti-HIV-1 antibody, or antigen-binding portion thereof, comprises a monoclonal antibody. In some embodiments, the anti-HIV-1 antibody comprises a recombinant antibody. In some embodiments, the anti-HIV-1 antibody, or antigen-binding portion thereof, is recombinantly produced. In some embodiments, the anti-HIV-1 antibody, or antigen-binding portion thereof, comprises a human antibody. In some embodiments, the method further comprises administering at least one additional anti-HIV-1 antibody, or antigen-binding portion thereof. In some embodiments, the at least one additional anti-HIV-1 antibody, or antigen-binding portion thereof, comprises a broadly neutralizing anti-HIV-1 antibody (bNab), or antigen-binding portion thereof. In some embodiments, the at least one anti-HIV-1 antibody, or antigen-binding portion thereof, comprises any anti-HIV-1 antibody, or antigen-binding portion thereof, according to the present invention. In some embodiments, the additional anti-HIV-1 antibody, or antigen-binding portion thereof, comprises a monoclonal antibody. In some embodiments, the additional anti-HIV-1 antibody, or antigen-binding portion thereof, comprises a recombinant antibody. In some embodiments, the additional anti-HIV-1 antibody, or antigen-binding portion thereof, is recombinantly produced. In some embodiments, the method further comprises administering an HIV agent.In some embodiments, the HIV agent is selected from the group consisting of non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleoside reverse transcriptase inhibitors (NRTIs), protease inhibitors (PIs), fusion inhibitors, CCR5 antagonists / entry inhibitors, integrase strand transfer inhibitors (INSTIs), and combinations thereof. In some embodiments, the anti-HIV-1 antibody or antigen-binding portion thereof may be co-administered with one or more additional anti-HIV-1 antibodies or antigen-binding portions thereof and / or one or more HIV agents. In other embodiments, the anti-HIV antibody or antigen-binding portion thereof is administered before the additional one or more anti-HIV-1 antibodies or antigen-binding portions thereof and / or one or more HIV agents. In still other embodiments, the anti-HIV-1 antibody or antigen-binding portion thereof is administered after the additional one or more anti-HIV-1 antibodies or antigen-binding portions thereof and / or one or more HIV agents. In still yet other embodiments, the anti-HIV-1 antibody or antigen-binding portion thereof may be administered between the additional anti-HIV-1 antibodies or antigen-binding portions thereof and / or one or more HIV agents.

[0016] In another embodiment, the present invention relates to a passive vaccine. In some embodiments, the vaccine comprises at least one anti-HIV-1 antibody or antigen-binding portion thereof. In some embodiments, the anti-HIV-1 antibody or antigen-binding portion thereof comprises a broadly neutralizing anti-HIV-1 antibody (bNab) or antigen-binding portion thereof. In some embodiments, the anti-HIV-1 antibody or antigen-binding portion thereof comprises any anti-HIV-1 antibody or antigen-binding portion thereof according to the present invention. In some embodiments, the anti-HIV-1 antibody or antigen-binding portion thereof comprises a monoclonal antibody. In some embodiments, the anti-HIV-1 antibody or antigen-binding portion thereof comprises a recombinant antibody. In some embodiments, the anti-HIV-1 antibody or antigen-binding portion thereof is recombinantly produced. In some embodiments, the passive vaccine further comprises an adjuvant. In some embodiments, the passive vaccine further comprises a pharmaceutically acceptable excipient, preservative, and / or carrier. In another embodiment, the present invention relates to a method of preventing HIV-1 infection in an individual in need thereof. In some embodiments, the method comprises administering to the individual a passive vaccine composition according to any aspect of the present invention.

[0017] In some embodiments, the present invention relates to nucleic acids. In some embodiments, the nucleic acids comprise a nucleotide sequence encoding one or more heavy and / or light chains of an anti-HIV-1 antibody of the present invention. In some embodiments, the nucleic acids comprise a nucleotide sequence encoding one or more of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 99, 73, 77, 81, 85, 89, and 93. In some embodiments, the nucleic acids have conservative substitutions. In some embodiments, the nucleic acids comprise a nucleotide sequence encoding one or more CDRs of an anti-HIV-1 antibody of the present invention. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding one or more of SEQ ID NOs: 2-4, 6-8, 10-12, 14-16, 18-20, 22-24, 26-28, 30-32, 34-36, 38-40, 42-44, 46-48, 50-52, 54-56, 58-60, 62-64, 66-68, 70-72, 74-76, 78-80, 82-84, 86-88, 90-92, and 94-96. In some embodiments, the nucleic acid has conservative substitutions.

[0018] In some embodiments, the present invention relates to a vector or vector system containing one or more nucleic acid sequences encoding the heavy chain and / or light chain and / or CDRs of one or more of the anti-HIV-1 antibodies or antigen-binding portions thereof of the present invention. In some embodiments, the vector or vector system comprises a nucleic acid sequence encoding a variable heavy chain region and a nucleic acid sequence encoding a variable light chain region. In some embodiments, the nucleic acid sequence encoding the heavy chain is on the same vector as the nucleic acid sequence encoding the light chain. In some embodiments, the nucleic acid sequence encoding the variable heavy chain region is on a different vector from the nucleic acid sequence encoding the variable light chain region. In some embodiments, the vector or vector system is a plasmid or plasmids. In some embodiments, the vector or vector system is a phage vector or phage vectors. In some embodiments, the phage vector is a gamma phage. In some embodiments, the vector or vectors are cosmids or cosmids. In some embodiments, the vector or vector system is a recombinant chromosome or chromosomes. In some embodiments, the vector system is a combination of different vectors. In some embodiments, expression of the different nucleic acid sequences can occur simultaneously. In other embodiments, expression of the different nucleic acid sequences can be separately inducible. In another embodiment, the present invention relates to a vector or vector system containing one or more nucleic acid sequences encoding one or more complementarity determining regions (CDRs) of one or more heavy and / or light chains of one or more anti-HIV-1 antibodies of the present invention. [Brief explanation of the drawings]

[0019] [Figure 1A-C]Figure 1A depicts the HIV-1 antibody repertoire in donor EB354. Figure 1A: Viral load values ​​measured from 2002 to 2015. The time points at which neutralizing antibodies were isolated are indicated on the graph. The time points at which plasma was collected are marked with downward arrows. Figure 1B: Heat map showing IC50 values ​​in the TZM-bl assay for purified serum IgG from four time points (indicated by arrows in (1A)) tested against a panel of HIV-1 strains. All neutralization assays were performed in duplicate. Figure 1C: Top panel: IgG+ memory B cells pre-enriched using CD19 were stained with CD19 and three non-native HIV-1 baits: 2CC core, gp140YU2 fold-on trimer, and gp14092UG37.8+gp140CZA79012 (gp140A+C) fold-on trimer, along with one native bait, BG505.SOSIP-AviB. The population within the square inset represents cells that were single-cell sorted, and the numbers indicate the percentage of CD19+ / bait+ cells among all IgG+ cells. Bottom panel: Pie charts represent total Ig sequences amplified from single-sorted cells from the corresponding FACS plot shown at the top. The numbers in the center of the circles represent the total number of antibodies. Empty slices represent sequences that appeared only once and had no clonal associates. Slices represent antibody clones and are proportional to the number of sequences for each clone. Clones marked with an asterisk exhibited tier 2 neutralization and are represented by mutants NC37, BG18, and BG1. Figure 1D: Top panel: Heatmap showing the neutralization potency of monoclonal antibodies NC37, BG1, and BG18 based on IC50 values ​​in the TZM-bl assay. Bottom panel: Neutralization potency of NC37, BG1, and BG18 against donor polyclonal IgG obtained in 2014, as well as YU2WT, YU2N280Y, YU2NN332K, and YU2NN160K based on IC50 values ​​in the TZM-bl assay. All TZM-bl assays were performed in duplicate.Figure 1E: Heat map of neutralization potency based on IC50 in the TZM-bl assay for antibodies NC37, BG1, BG18, and the 1:1:1 combination ("comb.") tested against a panel of 120 Tier 2 HIV-1 pseudoviruses. The geometric mean and percent broadness of all viruses neutralized are shown in the bottom panel. Neutralization assays were performed in duplicate. Figure 1F: Range curve based on values ​​in (1E). [Figure 1D-F]Figure 1A depicts the HIV-1 antibody repertoire in donor EB354. Figure 1A: Viral load values ​​measured from 2002 to 2015. The time points at which neutralizing antibodies were isolated are indicated on the graph. The time points at which plasma was collected are marked with downward arrows. Figure 1B: Heat map showing IC50 values ​​in the TZM-bl assay for purified serum IgG from four time points (indicated by arrows in (1A)) tested against a panel of HIV-1 strains. All neutralization assays were performed in duplicate. Figure 1C: Top panel: IgG+ memory B cells pre-enriched using CD19 were stained with CD19 and three non-native HIV-1 baits: 2CC core, gp140YU2 fold-on trimer, and gp14092UG37.8+gp140CZA79012 (gp140A+C) fold-on trimer, along with one native bait, BG505.SOSIP-AviB. The population within the square inset represents cells that were single-cell sorted, and the numbers indicate the percentage of CD19+ / bait+ cells among all IgG+ cells. Bottom panel: Pie charts represent total Ig sequences amplified from single-sorted cells from the corresponding FACS plot shown at the top. The numbers in the center of the circles represent the total number of antibodies. Empty slices represent sequences that appeared only once and had no clonal associates. Slices represent antibody clones and are proportional to the number of sequences for each clone. Clones marked with an asterisk exhibited tier 2 neutralization and are represented by mutants NC37, BG18, and BG1. Figure 1D: Top panel: Heatmap showing the neutralization potency of monoclonal antibodies NC37, BG1, and BG18 based on IC50 values ​​in the TZM-bl assay. Bottom panel: Neutralization potency of NC37, BG1, and BG18 against donor polyclonal IgG obtained in 2014, as well as YU2WT, YU2N280Y, YU2NN332K, and YU2NN160K based on IC50 values ​​in the TZM-bl assay. All TZM-bl assays were performed in duplicate.Figure 1E: Heat map of neutralization potency based on IC50 in the TZM-bl assay for antibodies NC37, BG1, BG18, and the 1:1:1 combination ("comb.") tested against a panel of 120 Tier 2 HIV-1 pseudoviruses. The geometric mean and percent broadness of all viruses neutralized are shown in the bottom panel. Neutralization assays were performed in duplicate. Figure 1F: Range curve based on values ​​in (1E). [Figure 2A-D]Figure 2A shows the sequence and structural analysis of the BG18 bNab. Figure 2A: Alignment of the heavy chain amino acid sequences of 10-1074, PGT121, and BG18. Asterisks indicate gaps in the alignment. Arrows indicate framework positions where all three antibodies are mutated compared to their respective germline genes. CDRs H1, H2, and H3 are indicated. Figure 2B: Alignment of the light chain sequences as in (A) of 10-1074, PGT121, and BG18. Figure 2C: Left panel: Ribbon diagram of the VH (dark gray) and VL (light gray) domains of the BG18 Fab as solved at 1.3 Å resolution. Center panel: Surface diagram of the BG18 variable domain. Right panel: The CDRH3 conformation is stabilized by hydrogen bonds (shown as dashed lines) within the loops. Interacting residues are shown as sticks, and the remainder of CDRH3 and the neighboring CDRH1 are shown as ribbon diagrams. Figure 2D: Panel: Ribbon diagram showing the superimposition of the variable domains of BG18 (purple), PGT121 (green), and 10-1074 (blue). The CDRH3 of BG18 is highlighted in red, and the CDRH3 loops of PGT121 and 10-1074 are maroon. Right panel: The VLs of BG18 (light pink) and PGT121 (light green) are shown after superimposition of the VHs. The CDRL2 of BG18 is disordered in the crystal and is shown as a dashed line. 10-1074 is closely related to PGT121 and has been omitted for clarity. Figure 2E: Surface diagram of BG18 and PGT121. Figure 2F: Single-particle EM structure (gray transparent density) of BG505 SOSIP.664 bound to BG18 and 179NC75 Fab. Coordinates of models of BG505 SOSIP.664, BG18 Fab, and 179NC75 Fab (CH103 Fab: PDB 4JAM) were fitted to the EM density as described in Example 1 below. 2D class averages are shown. Figure 2G: Comparison of the angles of the BG505 bond adopted by BG18 Fab relative to Fabs of 10-1074, PGT122 (PDB 5FYJ), PGT135 (PDB 4JM2), and PGT128 (PDB 5ACO). To facilitate comparison, the density of 179NC75 Fab was subtracted from the BG18-179NC75-BG505 map. [Figure 2E-G]Figure 2A shows the sequence and structural analysis of the BG18 bNab. Figure 2A: Alignment of the heavy chain amino acid sequences of 10-1074, PGT121, and BG18. Asterisks indicate gaps in the alignment. Arrows indicate framework positions where all three antibodies are mutated compared to their respective germline genes. CDRs H1, H2, and H3 are indicated. Figure 2B: Alignment of the light chain sequences as in (A) of 10-1074, PGT121, and BG18. Figure 2C: Left panel: Ribbon diagram of the VH (dark gray) and VL (light gray) domains of the BG18 Fab as solved at 1.3 Å resolution. Center panel: Surface diagram of the BG18 variable domain. Right panel: The CDRH3 conformation is stabilized by hydrogen bonds (shown as dashed lines) within the loops. Interacting residues are shown as sticks, and the remainder of CDRH3 and the neighboring CDRH1 are shown as ribbon diagrams. Figure 2D: Panel: Ribbon diagram showing the superimposition of the variable domains of BG18 (purple), PGT121 (green), and 10-1074 (blue). The CDRH3 of BG18 is highlighted in red, and the CDRH3 loops of PGT121 and 10-1074 are maroon. Right panel: The VLs of BG18 (light pink) and PGT121 (light green) are shown after superimposition of the VHs. The CDRL2 of BG18 is disordered in the crystal and is shown as a dashed line. 10-1074 is closely related to PGT121 and has been omitted for clarity. Figure 2E: Surface diagram of BG18 and PGT121. Figure 2F: Single-particle EM structure (gray transparent density) of BG505 SOSIP.664 bound to BG18 and 179NC75 Fab. Coordinates of models of BG505 SOSIP.664, BG18 Fab, and 179NC75 Fab (CH103 Fab: PDB 4JAM) were fitted to the EM density as described in Example 1 below. 2D class averages are shown. Figure 2G: Comparison of the angles of the BG505 bond adopted by BG18 Fab relative to Fabs of 10-1074, PGT122 (PDB 5FYJ), PGT135 (PDB 4JM2), and PGT128 (PDB 5ACO). To facilitate comparison, the density of 179NC75 Fab was subtracted from the BG18-179NC75-BG505 map. [Figure 3A-E] Figure 3A: Maximum likelihood molecular phylogenetic tree of single-genome env gene sequences from donor EB354 collected in 2006, 2010, 2013, and 2014. Red asterisks indicate clades with bootstrap support of 90% or greater. Three major sequence groups are arbitrarily designated clusters A, B, and C. Three triangles indicate env sequences lacking N-linked glycosylation sites. Dashed boxes indicate VOC cultures. Numbers in parentheses represent the number of viral env VOC sequences. Figure 3B: Scatter plots showing pairwise nucleotide sequence diversity of plasma env sequences obtained in 2010, 2013, and 2014. Each point represents the pairwise genetic difference between two sequences at a given time point. P values ​​were determined using a two-sample U-statistic-based Z-test; ***p<0.0005. Figure 3C: Heatmap showing IC50 values ​​in the TZM-bl assay of BG18, BG1, NC37, and IgG purified from the same time point as the viral env gene against autologous pseudoviruses using the EB354 env sequence. Stars indicate pseudoviruses that were resistant to all three autologous bNAbs. Antibody 3BNC117 (described in U.S. Patent No. 2014 / 0328862) was used as a control. The circles on the left correspond to the time points at which the sequences were obtained, as shown in (3A). Neutralization assays were performed in duplicate and repeated at least twice. Gray boxes indicate assays not performed. Figure 3D: Pie charts show the susceptibility of autologous pseudoviruses using the EB354 env sequence to each of the three autologous bNAbs at the various time points analyzed. The numbers in the center of the circles represent the number of env pseudoviruses tested, and the various slices are proportional to the number of pseudoviruses. bNAbs are indicated on the left, and the time points at which samples were collected are indicated above the graph. The bars below the circles indicate the time points at which antibody transcripts were detected by PCR. Figure 3E: IC50 values ​​in the TZM-bl assay for BG18, BG1, NC37, and the control bNAb 3BNC117 against VOCs obtained from EB354 CD4 T cells obtained in 2014 and 2015. [Figure 4A-C] Figure 4A shows the treatment of HIV-1YU2-infected human mice. Viral load values ​​for four (BG18) and five (NC37) HIV-1YU2-infected human mice before and after monotherapy with BG18 (top panel) or NC37 (bottom panel). The gray shaded areas on the graph indicate the period during which BG18 (top) or NC37 (bottom) was administered. The bold lines indicate the geometric mean values ​​for each treatment experiment. Figure 4B shows the amino acid sequence alignment of gp120 sequences derived from cloned viruses 21 days after treatment with BG18 (top) or NC37 (bottom). Each horizontal bar represents the sequence of a single gp120 clone aligned to HIV-1YU2. Amino acid substitutions are indicated as checkmarks. The mouse IDs from which the sequences were obtained are indicated above the vertical bars. A magnification of the boxed area is shown to the right of each panel. Figure 4C: Pie chart showing recurrent mutations in gp120 for BG18 (top) and NC37 (bottom) compared to the wild-type HIV-1 YU2 sequence. The various slices are proportional to the number of sequences carrying the mutation. The number in the center of the circle represents the total number of cloned sequences. The white slice for NC37 indicates the absence of recurrent mutations. Figure 4D: Viral load values ​​in seven hu mice before and after treatment with the BG18 + NC37 + BG1 combination. Mice T1 and T2 are untreated control mice (white symbols). Mice T3 to T7 were administered the BG18 + NC37 + BG1 combination (black symbols). The shaded area indicates the period during which the bNAb combination was administered. The mean of the geometric means is shown in bold, dashed for the untreated group, and solid for the treated group. Figure 4E: Log10 difference in viral load compared to day 0 (before antibody administration). Asterisks indicate approximate values. [Figure 4D-E]Figure 4A shows the treatment of HIV-1YU2-infected human mice. Viral load values ​​for four (BG18) and five (NC37) HIV-1YU2-infected human mice before and after monotherapy with BG18 (top panel) or NC37 (bottom panel). The gray shaded areas on the graph indicate the period during which BG18 (top) or NC37 (bottom) was administered. The bold lines indicate the geometric mean values ​​for each treatment experiment. Figure 4B shows the amino acid sequence alignment of gp120 sequences derived from cloned viruses 21 days after treatment with BG18 (top) or NC37 (bottom). Each horizontal bar represents the sequence of a single gp120 clone aligned to HIV-1YU2. Amino acid substitutions are indicated as checkmarks. The mouse IDs from which the sequences were obtained are indicated above the vertical bars. A magnification of the boxed area is shown to the right of each panel. Figure 4C: Pie chart showing recurrent mutations in gp120 for BG18 (top) and NC37 (bottom) compared to the wild-type HIV-1 YU2 sequence. The various slices are proportional to the number of sequences carrying the mutation. The number in the center of the circle represents the total number of cloned sequences. The white slice for NC37 indicates the absence of recurrent mutations. Figure 4D: Viral load values ​​in seven hu mice before and after treatment with the BG18 + NC37 + BG1 combination. Mice T1 and T2 are untreated control mice (white symbols). Mice T3 to T7 were administered the BG18 + NC37 + BG1 combination (black symbols). The shaded area indicates the period during which the bNAb combination was administered. The mean of the geometric means is shown in bold, dashed for the untreated group, and solid for the treated group. Figure 4E: Log10 difference in viral load compared to day 0 (before antibody administration). Asterisks indicate approximate values. [Figure 5] Figure 5 shows the results of a competitive ELISA. Equal amounts of each neutralizing antibody were assayed for binding to BG505 SOSIP.664 in an ELISA in the presence of increasing amounts of various competing antibodies. The black line indicates binding of biotinylated antibody in the absence of competition. Figure 5A: BG18, Figure 5B: BG1. [Figure 6A-D]Figure 6 shows a sequence comparison between BG18, PGT121, and 10-1074. Figure 6A: Dendrogram of the heavy chain sequences of PGT121, 10-1074, and nine BG18 clonal variants, and their predicted germlines. Figure 6B: Alignment of heavy chain CDRs. Positions common to PGT121 / 10-1074 and BG18 variants are highlighted. Figure 6C: Same as (6A), except for the light chain. Figure 6D: Alignment of light chain CDRs. Positions common to PGT121 / 10-1074 and BG18 variants are highlighted. [Figure 7] The electrostatic potential of BG18 is shown. The electrostatic surface potential of the Fab was calculated using APBS, and the surface map was displayed and shaded in UCSF Chimera. The predicted binding interface is outlined by a black dotted line. The approximate occupancy areas on the surface of PGT121 and 10-1074 Fab of gp120 glycans bound to Asn137gp120, Asn156gp120, and Asn332gp120 are indicated by black triangles. [Figure 8A-C] Figure 8A shows the NC37 Fab-gp120 complex structure. Figure 8A: The 2.7 Å crystal structure of the NC37 Fab-gp120 complex superimposed on the 8ANC134 Fab-gp120 complex structure (PDB 4RX4) reveals similar gp120 binding orientations for the two antibodies. Figure 8B: A close-up of the Fab-gp120 interface shows the extended CDRH3 of NC37 compared to the 8ANC134 CDRH3. Figure 8C: A side view of the BG505 trimer structure in surface view (PDB 4TVP) with predicted epitopes for NC37, 8ANC134, and NIH45-46 shown on one protomer of the trimer. The longer CDRH3 of NC37 suggests a relatively large contact area on the adjacent protomer (light gray) of the trimer. [Figure 9A-B]Figure 9 shows BG18 antibody binding to a synthetic HIV-1 V3 glycopeptide. Biotin-tagged V3 glycopeptides were immobilized on a neotravidin chip, and BG8 IgG was used as the analyte. Figure 9A: Interaction between BG18 and JR-FL mini-V3 glycopeptide bearing a Man9GlcNAc2 glycan at N301. Weak binding was observed. Figure 9B: Interaction between BG18 and JR-FL mini-V3 glycopeptide bearing a Man9GlcNAc2 glycan at N332. Weak binding was observed. Figure 9C: Interaction between BG18 IgG and JR-FL mini-V3 glycopeptide bearing a biantennary complex N-glycan at N301. No binding was observed. Figure 9D: Interaction between BG18 IgG and JR-FL mini-V3 glycopeptide bearing a biantennary complex N-glycan at N332. No binding was observed. Figure 9E: Interaction between BG18 IgG and A244 mini-V3 glycopeptide bearing a Man9GlcNAc2 glycan at N334. Weak binding was observed. Figure 9F: Interaction between BG18 IgG and CAP45 mini-V3 glycopeptide bearing a Man9GlcNAc2 glycan at N334. Weak binding was observed. [Figure 9C-D]Figure 9 shows BG18 antibody binding to a synthetic HIV-1 V3 glycopeptide. Biotin-tagged V3 glycopeptides were immobilized on a neotravidin chip, and BG8 IgG was used as the analyte. Figure 9A: Interaction between BG18 and JR-FL mini-V3 glycopeptide bearing a Man9GlcNAc2 glycan at N301. Weak binding was observed. Figure 9B: Interaction between BG18 and JR-FL mini-V3 glycopeptide bearing a Man9GlcNAc2 glycan at N332. Weak binding was observed. Figure 9C: Interaction between BG18 IgG and JR-FL mini-V3 glycopeptide bearing a biantennary complex N-glycan at N301. No binding was observed. Figure 9D: Interaction between BG18 IgG and JR-FL mini-V3 glycopeptide bearing a biantennary complex N-glycan at N332. No binding was observed. Figure 9E: Interaction between BG18 IgG and A244 mini-V3 glycopeptide bearing a Man9GlcNAc2 glycan at N334. Weak binding was observed. Figure 9F: Interaction between BG18 IgG and CAP45 mini-V3 glycopeptide bearing a Man9GlcNAc2 glycan at N334. Weak binding was observed. [Figure 9E-F]Figure 9 shows BG18 antibody binding to a synthetic HIV-1 V3 glycopeptide. Biotin-tagged V3 glycopeptides were immobilized on a neotravidin chip, and BG8 IgG was used as the analyte. Figure 9A: Interaction between BG18 and JR-FL mini-V3 glycopeptide bearing a Man9GlcNAc2 glycan at N301. Weak binding was observed. Figure 9B: Interaction between BG18 and JR-FL mini-V3 glycopeptide bearing a Man9GlcNAc2 glycan at N332. Weak binding was observed. Figure 9C: Interaction between BG18 IgG and JR-FL mini-V3 glycopeptide bearing a biantennary complex N-glycan at N301. No binding was observed. Figure 9D: Interaction between BG18 IgG and JR-FL mini-V3 glycopeptide bearing a biantennary complex N-glycan at N332. No binding was observed. Figure 9E: Interaction between BG18 IgG and A244 mini-V3 glycopeptide bearing a Man9GlcNAc2 glycan at N334. Weak binding was observed. Figure 9F: Interaction between BG18 IgG and CAP45 mini-V3 glycopeptide bearing a Man9GlcNAc2 glycan at N334. Weak binding was observed. [Figure 10]Figure 10A shows single-genome env sequencing of plasma viruses obtained from donor EB354. Figure 10A shows the pie chart representing all env sequences amplified by single-genome PCR for each collection time point. The total number of sequences obtained from each time point is indicated in the center of the circle, and the various slices are proportional to the number of sequences. Figure 10B shows the maximum likelihood molecular phylogenetic tree of single-genome env gene sequences from donor EB354 collected in 2006, 2010, 2013, and 2014. Asterisks indicate clades with bootstrap support of 90% or greater. The table on the right shows the TCID50 and IC50 values ​​in the TZM-bl assay for BG18, BG1, NC37, and the control antibody 3BNC117 against autologous pseudoviruses pseudotyped with the EB354 env sequence. (C) Comparison of TCID50 values ​​for pseudoviruses with EB354 env sequences that were resistant to all three bNAbs, in contrast to pseudoviruses with EB354 env sequences that were sensitive to at least one bNAb. P value (t-test) = 0.0016. [Figure 11A-C] Figure 11A shows treatment of HIV-1YU2-infected hu mice with BG8. Figure 11A: Viral load values ​​in four hu mice before and after BG8 administration. The gray shaded areas on the graph indicate the period during which the antibody was administered. The bold line indicates the geometric mean value. Figure 11B: Amino acid sequence alignment of gp120 sequences from cloned viruses 21 days after treatment. Each gray horizontal bar represents the sequence of a single gp120 clone aligned to HIV-1YU2. Amino acid substitutions are indicated as black checkmarks. The mouse ID from which the sequence was obtained is indicated above the black vertical bar. A magnified view of the boxed area is shown on the right side of each panel. Figure 11C: Pie chart showing recurrent mutations in gp120 compared to the wild-type HIV-1YU2 sequence after BG8 treatment. The number in the center of the circle represents the total number of cloned sequences. Slices represent the most consistently mutated regions of gp120 and are proportional to the number of sequences carrying the mutation. [Figure 12A]Viral load in HIVYU2-infected, antibody-treated humanized mice. Viral load values ​​in HIVYU2-infected, humanized mice before and after monotherapy with BG18 (Figure 12A) and NC37 (Figure 12B). The shaded areas on the graphs indicate the period during which the antibodies were administered. Each graph shows two independent experiments, indicated by circles (first experiment) and squares (second experiment). Black and white shapes represent treated and control mice, respectively. The dashed and thick solid lines represent the average values ​​for control and treated mice, respectively. [Figure 12B] Viral load in HIVYU2-infected, antibody-treated humanized mice. Viral load values ​​in HIVYU2-infected, humanized mice before and after monotherapy with BG18 (Figure 12A) and NC37 (Figure 12B). The shaded areas on the graphs indicate the period during which the antibodies were administered. Each graph shows two independent experiments, indicated by circles (first experiment) and squares (second experiment). Black and white shapes represent treated and control mice, respectively. The dashed and thick solid lines represent the average values ​​for control and treated mice, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0020] A.Definition Anti-HIV-1 antibodies, as disclosed herein, can take one of many forms in the art. Antibodies are defined in part by the antigens they bind; thus, an "anti-HIV-1 antibody," as described herein, is any such antibody that specifically binds to at least one epitope found on the viral envelope of human immunodeficiency virus 1 ("HIV-1"), such as within gp120 and / or gp140. An antibody is understood in the art to be a glycoprotein, or an antigen-binding portion thereof, comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The heavy chain is composed of a heavy chain variable region (VH) and heavy chain constant regions (CH1, CH2, and CH3). The light chain is composed of a light chain variable region (VL) and light chain constant region (CL). Both the heavy and light chain variable regions comprise framework regions (FWR) and complementarity-determining regions (CDRs). The four FWR regions are relatively conserved, while the CDR regions (CDR1, CDR2, and CDR3) represent hypervariable regions and are arranged from the NH2-terminus to the COOH-terminus as follows: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens, while the constant regions may mediate the binding of the immunoglobulin to host tissues or factors, depending on the isotype. It is known in the art that it is possible to manipulate monoclonal and other antibodies and use recombinant DNA technology techniques to create other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques can be developed by introducing DNA encoding the immunoglobulin variable region or CDRs of an antibody into the constant region, or constant region plus framework regions, of a different immunoglobulin.

[0021] As used herein, the term "antibody" (Ab) is used in the broadest sense and specifically includes any immunoglobulin, whether natural or partially or wholly synthetically produced, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific and polyreactive antibodies), and antibody fragments. Thus, the term "antibody," as used in any context herein, is meant to include, but is not limited to, any specific binding member, immunoglobulin class and / or isotype (e.g., IgG1, IgG2a, IgG2b, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE), as well as biologically relevant fragments thereof, including, but not limited to, Fab, F(ab'), scFv (single chain or related entities), and (scFv)2.

[0022] As used herein, the term "antibody fragment" may include antibody fragments obtained using techniques readily known and available to those skilled in the art, as outlined herein. Thus, in addition to the definition of "antibody" above, the term "antibody" may further encompass any polypeptide or protein comprising a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. These may be derived from natural sources or may be partially or wholly synthetically produced. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, diabodies, and linear antibodies. As used herein, the terms "antigen-binding portion," "antigen-binding fragment," or "Fab" may refer to the region of an antibody that binds to an antigen. Those skilled in the art will understand that Fab is composed of one constant and one variable domain from each of the antibody's heavy and light chains.

[0023] As used herein, the term "monoclonal antibody" or "mAb" may refer to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor natural mutations. Monoclonal antibodies may include chimeric, humanized, recombinant, and human antibodies. Methods for producing monoclonal antibodies are well known in the art, and some specific examples discussed below include hybridoma technology, recombinant antibody production by, e.g., phage display or yeast display technology, production by transgenic mice, and single B-cell culture. Methods comprising amplifying heavy and / or light chains of antibody genes (e.g., plasmids or cosmids) by PCR (or similar techniques) in vitro or in bacterial and / or mammalian and / or yeast systems are considered within the scope of the present invention.

[0024] As used herein, the term "conservative sequence modification" or "conservative substitution" may refer to an amino acid modification to a target epitope of the present invention that does not significantly affect or alter the binding characteristics of an anti-HIV-1 antibody to the epitope. Such conservative modifications include amino acid substitutions, additions, and deletions. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a target epitope to which an anti-HIV-1 antibody of the invention specifically binds, e.g., an epitope on the viral envelope of HIV-1, e.g., an epitope on gp120 and / or gp140, can be substituted with other amino acid residues from the same side chain family, and antibodies of the invention can be tested against the target epitope, e.g., using functional assays described herein or otherwise known in the art.

[0025] The term "biological sample" refers to a sample obtained from an organism (e.g., a patient) or from components (e.g., cells) of an organism. A sample can be any biological tissue, cell, or fluid. A sample can be a "clinical sample," which is a sample derived from a subject, such as a human patient. Such samples include, but are not limited to, saliva, sputum, blood, blood cells (e.g., white blood cells), body fluids, lavage fluid, pancreatic juice, gastric juice, feces, CSF, lympho-amniotic fluid, plasma, semen, bone marrow, and tissue or fine needle biopsy samples, urine, stool, peritoneal fluid, and pleural fluid, or cells therefrom, and any combination thereof. Biological samples can also include sections of tissue, such as frozen sections taken for histological purposes. Biological samples can also be referred to as "patient samples." Biological samples can also include substantially purified or isolated proteins, membrane preparations, or cell cultures.

[0026] The term "bispecific antibody" refers to an artificial immunoglobulin construct composed of fragments of two different monoclonal antibodies that bind to two different antigens. There are several different types of bispecific antibodies, including, but not limited to, trifunctional antibodies and chemically conjugated Fabs. The anti-HIV-1 antibodies of the present invention may comprise bispecific antibodies, which may comprise fragments of one or more different anti-HIV-1 antibodies of the present invention, e.g., BG18 and BG1, or one or more anti-HIV-1 antibodies of the present invention and a known anti-HIV-1 antibody, e.g., BG18 and 3BNC117 (described in U.S. Patent No. 2014 / 0328862), or BG18 and VRC01, described in U.S. Patent No. 8,637,036. Bispecific anti-HIV bNabs and methods for making and using them are described in PCT / US16 / 64713, which is incorporated herein by reference.

[0027] As used herein, the term "effective amount" or "therapeutically effective amount" may refer to an amount of a compound or agent capable of producing a medically desirable result in a treated subject. Therapeutic methods can be performed in vivo or ex vivo, alone or in combination with other drugs or therapies. A therapeutically effective amount can be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or route of administration.

[0028] The terms "specific binding," "selective binding," "selectively binds," and "specifically binds" can refer to an antibody that binds to an epitope on a predetermined antigen but not to other antigens. Typically, an antibody binds to an epitope on a predetermined antigen, e.g., an epitope on the viral envelope of HIV-1, e.g., gp120, as the analyte and an antibody as the ligand, with a binding affinity of about 10, as determined by equilibrium dialysis or surface plasmon resonance (SPR) techniques in a BIACORE® 2000 surface plasmon resonance instrument, or by Scatchard analysis of the binding of the antibody to antigen-positive cells. -6 Less than m, e.g., about 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 The equilibrium dissociation constant (K D ) and (ii) binds to a given antigen with an affinity that is at least twice its affinity for binding to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein).

[0029] The term "homology" as used herein may refer to the presence of a shared structure between two compositions. In the context of proteins, the term "homology" may refer to the amount of overlap (e.g., expressed as a percentage) between two or more amino acid and / or peptide sequences. In the context of nucleic acids, the term may refer to the amount of overlap (e.g., expressed as a percentage) between two or more nucleic acid sequences. As used herein, the percent (%) homology between two sequences is equal to the percent identity between the two sequences. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap (i.e., % homology = number of identical positions / total number of positions × 100), which must be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be achieved using a mathematical algorithm. Such homology is well represented in the art through local alignment tools and / or algorithms, e.g., BLAST and / or BLAST 2.0, and may include pairwise alignments, multiple sequence alignment methods, structural alignment methods and / or phylogenetic analysis methods.

[0030] As used herein, the terms "co-administration," "co-administered," and "in combination with" can refer to the administration of at least two agents or therapies to a subject. In some embodiments, the co-administration of two or more agents / therapies is simultaneous. In other embodiments, a first agent / therapy is administered before a second agent / therapy. One of ordinary skill in the art will appreciate that the formulation and / or route of administration of the various formulations / therapies used may vary. For example, the anti-HIV-1 antibodies of the present invention may be co-administered with each other, with other antibodies, e.g., other broadly neutralizing antibodies, such as the broadly neutralizing anti-HIV antibodies disclosed in U.S. Patent No. 2014 / 0328862, e.g., 3BNC117 or VRC01 described in U.S. Patent No. 8,637,036, the entire contents of which are incorporated herein by reference, or, optionally, with other HIV agents. Co-administration may or may not occur simultaneously, e.g., one or more anti-HIV-1 antibodies of the invention may be administered before or after another anti-HIV-1 antibody of the invention (or other anti-HIV antibody or other HIV agent), or may be administered at the same or substantially the same time. Those skilled in the art will understand that co-administration refers to administration with other compounds and / or therapies as part of a patient's regimen. It will be understood that the time period is meant to describe the period and is not meant to be limiting.

[0031] The term "HIV agent" as used herein may refer to a therapy for the treatment of HIV that does not include anti-HIV-1 antibodies per se. For example, non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleoside reverse transcriptase inhibitors (NRTIs), protease inhibitors (PIs), fusion inhibitors, CCR5 antagonists / entry inhibitors, integrase strand transfer inhibitors (INSTIs), and combinations thereof are all non-antibody-based therapeutic treatments that can be co-administered with one or more anti-HIV-1 antibodies of the present invention and / or additional anti-HIV-1 antibodies, and therefore are considered "HIV agents." Those skilled in the art will understand that this list should not be considered exhaustive, and that as the state of the art advances and additional treatments become available, they will be considered to fall within the scope of this definition.

[0032] As used herein, the term "carrier" can include pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed to them at the dosages and concentrations used. Often, physiologically acceptable carriers are pH-buffered aqueous solutions. Examples of physiologically acceptable carriers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants, including but not limited to ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as but not limited to serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as but not limited to polyvinylpyrrolidone; amino acids, such as but not limited to glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including but not limited to glucose, mannose, or dextrins; chelating agents, such as but not limited to EDTA; sugar alcohols, such as but not limited to mannitol or sorbitol; salt-forming counterions, such as but not limited to sodium; and / or non-ionic surfactants, such as but not limited to TWEEN; polyethylene glycol (PEG), and PLURONICS.

[0033] The term "treating" a disease or "treatment" of a disease refers to carrying out a protocol that may include administering one or more drugs to a patient (human or otherwise) with the goal of alleviating the signs or symptoms of the disease. Alleviation can occur before signs or symptoms of the disease appear, as well as after their appearance. Thus, "treating" or "treatment" includes "preventing" a disease or "prevention" of a disease. The term "prevention" or "preventing" refers to protective and / or preventative measures, where the purpose is to prevent or suppress a targeted pathological condition or disorder. For example, in the case of infection with immunodeficiency virus 1 (HIV-1), "preventing" or "preventing" can occur in situations where a course of treatment is undertaken to prevent or stall infection by HIV-1, such as through passive vaccination. Such "preventing" or "prophylaxis" also occurs, for example, in the case of latent HIV-1 infection in an individual who is seropositive but does not exhibit any symptoms (i.e., has not progressed to symptoms of AIDS), where the objective is to prevent active infection and / or cure said HIV-1 infected patient. Furthermore, "treating" or "treatment" does not require complete alleviation of signs or symptoms, does not require a cure, and particularly includes protocols that have a small but favorable effect on the patient.

[0034] As used herein, the term "epitope" can refer to a region of an antigen to which an antibody or T cell binds, for example, but not limited to, a glycoprotein, e.g., gp120, or a region of the viral envelope of HIV-1, including a region on a glycoprotein. An "antigen" refers to a substance that elicits an immunological response or binds to the product of that response.

[0035] As used herein, the term "purified" or "isolated" antibody, peptide, polypeptide, or protein may refer to a peptide, polypeptide, or protein that has been separated from other proteins, lipids, and nucleic acids with which it is naturally associated. The polypeptide / protein may constitute at least 10% by dry weight of a purified preparation (i.e., any percentage between 10% and 100%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 99%). Purity can be measured by any appropriate standard method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. Isolated polypeptides / proteins (e.g., anti-HIV-1 antibodies) described herein can be produced by recombinant DNA technology.

[0036] B. Broadly neutralizing anti-HIV-1 antibodies The present invention relates to broadly neutralizing anti-HIV-1 antibodies (bNabs) and antigen-binding portions thereof. In particular, the present invention relates to several different monoclonal antibodies, BG18, NC37, and BG1, their antigen-binding portions, including but not limited to their complementarity-determining regions (CDRs), and derivatives and variants thereof, which exhibit broad and potent neutralization of HIV-1 in vivo (see Example 1 below). Thus, the anti-HIV-1 antibodies of the present invention have potential therapeutic utility, alone or in combination with each other, or with other bNabs, or in combination with other HIV therapies, as discussed herein.

[0037] BG18, NC37, and BG1 each have distinct, non-overlapping epitopes. NC37 recognizes CD4bs. NC37 and its clonal variants are V H 1-2 and V H However, structural analysis shows that NC37 binds Env to V. HThis suggests that NC37 recognizes CD4bs in a 1-46 manner. Without wishing to be bound by theory, it is predicted that the long CDRH3 of NC37 contacts adjacent promoters on the trimer, thereby recognizing a quaternary trimer epitope whose core overlaps with CD4bs.

[0038] BG1 binds to the V1V2 region of Env, another frequent target of bNAbs generated during natural infection. BG1 is the first antibody (bNab) of this class isolated from a clade B-infected donor. Its potency is roughly comparable to members of the VRC26 antibody family, such as those disclosed in NaDoria-Rose et al., Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies. Nature 509, 55-62 (2014), but less potent than PG9 / 16 and PGDM1400 bNAbs. Like other V1V2 bNAbs isolated to date, BG1 possesses a long, tyrosine-rich CDRH3.

[0039] BG18, the most potent of the three, NC37 and BG1, has Asn332 at the base of the V3 loop. gp120 directed against the central glycan patch, which is located at position Asn332 gp120 , Asn301 gp120 , Asn386 gp120 , Asn392 gp120 , Asn137 gp120 , Asn156 gp120PGT121-124, described in L.M. Walker et al., "Broad neutralization coverage of HIV by multiple highly potent antibodies," Nature 477, 466-470 (2011), and H. Mouquet et al., "Complex-type N-glycan recognition by potent broadly neutralizing HIV antibodies," Proceedings of the National Academy of Sciences of the United States of America. A number of monoclonal bNAbs have been isolated that bind both protein and carbohydrate moieties at this site, including 10-1074, described in [109, E3268-3277 (2012)]. All of these bNAbs were isolated from clade A donors and have a binding site at Asn332. gp120 They can be further classified into those that rely only on the glycans (10-1074 and PGT124) and those that make additional contacts with surrounding glycans in the V3 glycan patch (PGT121-123). BG18 and its clonal variants are characterized by their interaction with Asn332. gp120 Although similar to 10-1074 in its dependence on V3, BG18 is more potent than published anti-V3 bNAbs and was the first isolated from a clade B donor.

[0040] BG18 differs from previously characterized members of this class by the strikingly distinct orientation of the CDRH3 and light chain domains. Furthermore, BG18 differs from other members of this antibody group in that it has a relatively short CDRH3 and no insertions or deletions. Without wishing to be bound by theory, this suggests that BG18-like antibodies may be relatively easily elicited. In the absence of a high-resolution structure of the BG18-Env complex, it is difficult to predict how BG18's relatively short CDRH3 interacts with Env. Structural examination of the Env complex with PGT122 and 10-1074 Fab demonstrates that they adopt a similar approach angle, nearly perpendicular to the gp120 promoter within the Env trimer. Although the BG18-Env EM structure does not provide sufficient resolution to resolve the detailed interactions, it nonetheless demonstrates that BG18 approaches Env from a different angle, which is shifted by up to 40° toward the gp120 promoter compared to the approach angles of 10-1074 and PGT122. BG18 clone variants and derivatives, including 354BG8, 354BG18, 354BG42, 354BG33, 354BG129, 354BG188, 354BG411, and 354BG426, as well as their full-length sequences, heavy and light chain complementarity determining regions (CDRs), are shown in Table 1 below. A dendrogram of BG18 variants is shown in Figure 6 and discussed in Example 1 below. BG1 clone variants and derivatives, including BG1, BG22, and BG47, as well as their full-length sequences, heavy and light chain complementarity determining regions (CDRs), are shown in Table 2 below.

[0041] [Table 1-1]

[0042] [Table 1-2]

[0043] [Table 1-3]

[0044] [Table 2]

[0045] Those skilled in the art will understand that the complementarity-determining regions ("CDRs") of most antibodies, such as NC37, BG1, including all variants of BG1, and BG18, including all variants of BG18, primarily determine the biological activity of the antibody, with CDRH3 forming the most distinctive paratope on the antibody. Thus, the antibodies of the present invention may comprise NC37, its CDRs, BG1, its CDRs, and BG18 and its CDRs, including all variants of NC37, BG1, and BG18 and their respective CDRs. Those skilled in the art will also understand that the antibodies of the present invention may contain conservative substitutions involving the CDRs and still fall within the scope of the present invention. Accordingly, some embodiments of the present invention relate to antibodies and antigen-binding portions thereof that express the CDRs listed in Tables 1 and 2.

[0046] For example, some embodiments of the present invention relate to antibodies or antigen-binding portions thereof having a heavy chain that comprises or has a degree of homology, e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% and any intervening range of homology, to one or more of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81 and 89. One or more of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81 and 89 may have conservative substitutions. Some embodiments of the invention relate to antibodies, or antigen-binding portions thereof, having a light chain comprising one or more of SEQ ID NOs: 5, 13, 21, 20, 37, 45, 53, 61, 69, 77, 85, and 93, or having a degree of homology, e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% and any intervening range therein, to one or more of SEQ ID NOs: 5, 13, 21, 20, 37, 45, 53, 61, 69, 77, 85, and 93. One or more of SEQ ID NOs: 5, 13, 21, 20, 37, 45, 53, 61, 69, 77, 85, and 93 may have conservative substitutions.

[0047] IgG heavy chains are typically about 400 to about 600 amino acids in size, more typically about 450 to about 550 amino acids in size. The variable region of each heavy chain is typically about 100 to 140 amino acids in length, most typically about 110 to 130 amino acids, regardless of antibody class. Light chains are typically about 210 to 220 amino acids in length, more typically 211 to 217 amino acids in length. The light chain variable region is typically about 100 to 120 amino acids in length.

[0048] Some embodiments of the present invention relate to antibodies, or antigen-binding portions thereof, having one or more CDRs in the heavy chain that comprise or have a degree of homology, e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% and any intervening range of homology, to one or more of SEQ ID NOs: 2-4, 10-12, 18-20, 26-28, 34-36, 42-44, 50-52, 58-60, 66-68, 74-76, 82-84, and 90-92. One or more of SEQ ID NOs: 2 to 4, 10 to 12, 18 to 20, 26 to 28, 34 to 36, 42 to 44, 50 to 52, 58 to 60, 66 to 68, 74 to 76, 82 to 84, and 90 to 92 may have conservative substitutions.

[0049] Some embodiments of the invention relate to antibodies, or antigen-binding portions thereof, having one or more CDRs in the light chain that comprise or have a degree of homology, e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% and any intervening range of homology, to one of SEQ ID NOs: 6-8, 14-16, 22-24, 30-32, 38-40, 46-48, 54-56, 62-64, 70-72, 78-80, 86-88 and 94-96. One or more of SEQ ID NOs: 6-8, 14-16, 22-24, 30-32, 38-40, 46-48, 54-56, 62-64, 70-72, 78-80, 86-88, and 94-96 may have conservative substitutions.

[0050] Furthermore, antibodies of the present invention may be composed of antigen-binding portions, e.g., CDRs, from different individual bNabs, including, but not limited to, bNabs of the present invention. Purely by way of example, an engineered derivative may comprise a light chain from one of NC37, BG1, or BG18, including any variants, and a heavy chain from a different bNab, including one of NC37, BG1, or BG18, including any variants. Alternatively, an engineered derivative may comprise a light chain from one variant of BG18 and a heavy chain from another variant of BG18. Those skilled in the art will appreciate that recombinant antibody production methods allow for a substantial number of recombinant antibodies, each of which is expressly contemplated within the scope of this disclosure. Thus, bNabs of the present invention may be produced by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the present invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). After isolation, the DNA is placed into an expression vector, which is then transfected into host cells, such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, that do not normally produce immunoglobulin proteins, and the monoclonal antibody is synthesized in the recombinant host cells. The DNA may also be modified, for example, by covalently linking immunoglobulin coding sequences to all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can replace the constant domains of an antibody of the invention, or can replace the variable domains of one antigen-binding site of an antibody of the invention to create a chimeric bivalent antibody.

[0051] After production by recombinant or other means, anti-HIV-1 antibodies may be purified or isolated so that the antibodies are, for example, substantially free of any serum, supernatant, or other cell culture or related substances. Methods for antibody purification are known in the art and may include selective enrichment or specific isolation methods. For example, affinity purification, such as class-specific affinity, may be used. Fractionation methods may be used initially to aid in isolating a subset of sample proteins, including immunoglobulins. Typically, antigen-specific affinity is applied to isolate antibodies that bind to the antigen. The antibodies may then be combined with carriers, buffers, diluents, solvents, and / or preservatives, etc., useful for producing a pharmaceutically acceptable anti-HIV-1 antibody composition.

[0052] The antibody can be a monovalent antibody. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of an immunoglobulin light chain and a modified heavy chain. The heavy chain is generally truncated at a point within the Fc region to prevent heavy chain cross-linking. Alternatively, the relevant cysteine ​​residues are replaced with other amino acid residues or deleted to prevent cross-linking. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to generate fragments, particularly Fab fragments, can be achieved using routine techniques known in the art.

[0053] Human monoclonal antibodies can be produced using a variety of techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for preparing human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. After challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This technique is described, for example, in U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, as well as in the following scientific publications: Marks et al., Bio / Technology 10, 779-783 (1992); Lonberg et al., Nature 368, 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93(1995).

[0054] Some embodiments of the present invention relate to vectors and vector systems containing nucleotide sequences encoding the variable regions of the CDRs of the anti-HIV-1 antibodies of the present invention, such as, but not limited to, NC37, BG1, and BG18, including any variants and antigen-binding portions thereof. Vectors may be, but are not necessarily, plasmids. Other recombinant vectors are known in the art and may include, for example, phage vectors, such as lambda phage vectors, other viral vectors, such as non-replicating adenoviral vectors, cosmids, and / or artificial chromosomes. Vector systems may or may not be separately inducible, i.e., they may have different promoter and / or repressor elements. Common to most engineered vectors are origins of replication, multiple cloning sites, and selectable markers, so long as the vector (e.g., vector system, including multiple plasmids) containing the system is considered within the scope of the present invention. It is well within the skill of one of ordinary skill in the art to derive nucleic acid sequences from a given peptide sequence and clone them into the system of choice. Thus, in some embodiments, the present invention relates to vectors or vector systems containing one or more nucleic acid sequences encoding one or more of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 73, 77, 81, 85, 89 and 93. In some embodiments, the present invention relates to vectors or vector systems containing one or more nucleic acid sequences encoding one or more CDRs of one or more heavy and / or light chains of one or more of the anti-HIV-1 antibodies of the invention, e.g., one or more nucleotide sequences encoding one or more of SEQ ID NOs: 2-4, 6-8, 10-12, 14-16, 18-20, 22-24, 26-28, 30-32, 34-36, 38-40, 42-44, 46-48, 50-52, 54-56, 58-60, 62-64, 66-68, 70-72, 74-76, 78-80, 82-84, 86-88, 90-92, and 94-96, and combinations thereof.These nucleotides encoding the heavy / light chains and / or CDRs may have conservative substitutions, and for those encoding CDRs, may share a degree of homology (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) with the nucleotide sequence regardless of such conservative substitutions. The vectors or vector systems of the present invention may be introduced into one or more cells (i.e., cells are "transformed" by the vector), and such means are known in the art.

[0055] C. Pharmaceutical Formulation and Delivery One embodiment of the present invention relates to pharmaceutical compositions comprising at least one anti-HIV-1 antibody or antigen-binding portion thereof of the present invention, and methods for their use in treating patients in need thereof. Patients may have a latent or active infection with HIV-1. The anti-HIV-1 antibody or antigen-binding portion thereof utilized in these compositions and methods can be any anti-HIV-1 antibody or antigen-binding portion thereof of the present invention, although particularly useful are anti-HIV antibodies or antigen-binding portions thereof including BG18 or variants thereof, including recombinantly produced variants and / or engineered derivatives thereof.

[0056] Pharmaceutically acceptable anti-HIV-1 antibody or antigen-binding portion thereof compositions suitable for administration to patients contain an effective amount of one or more anti-HIV-1 antibodies or antigen-binding portions thereof in a formulation that maintains biological activity while also promoting maximum stability during storage within an acceptable temperature range. Depending on the desired formulation, the pharmaceutical composition may also include a pharmaceutically acceptable diluent, a pharmaceutically acceptable carrier, and / or a pharmaceutically acceptable excipient, or any such vehicle typically used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solution, dextrose solution, and Hank's solution. The amount of excipient useful in the pharmaceutical compositions or formulations of the present invention is an amount that helps to uniformly distribute the antibody throughout the composition so that it can be uniformly dispersed upon delivery to a subject in need thereof. The excipient may also help to dilute the antibody to a concentration that provides the desired beneficial palliative or curative effect while simultaneously minimizing adverse side effects that may result from excessively high concentrations. The excipient may also have a preservative effect. Thus, for antibodies with high physiological activity, more excipients will be used, whereas for any active ingredient that exhibits relatively low physiological activity, less excipients will be used.

[0057] Pharmaceutically acceptable compositions can be in liquid or solid form. Solid formulations are typically, but not necessarily, lyophilized and then converted into a solution prior to administration for either single or multiple doses. Formulations should not be exposed to extreme temperatures or pH to avoid thermal denaturation. Therefore, it is essential to formulate antibody compositions of the present invention within a biologically relevant pH range. Liquid formulations, particularly those stored for extended periods between formulation and administration, often require buffered solutions to maintain the appropriate pH range during storage. Typically, both liquid and solid formulations require storage at low temperatures (usually 2-8°C) to maintain stability over extended periods. Formulated antibody compositions, particularly liquid formulations, may contain effective concentrations (usually <1% w / v) of bacteriostatic agents to prevent or minimize proteolytic degradation during storage, including, but not limited to, benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and / or propylparaben. Bacteriostatic agents may be contraindicated for some patients. Therefore, lyophilized formulations can be reconstituted in solutions with or without such components. Additional components may be added to buffered liquid or solid antibody formulations, including, but not limited to, sugars (including, but not limited to, polyhydroxy hydrocarbons such as sorbitol, mannitol, glycerol, and dulcitol and / or disaccharides such as sucrose, lactose, maltose, or trehalose) as cryoprotectants, and, optionally, related salts (including, but not limited to, NaCl, KCl, or LiCl). Such antibody formulations, particularly liquid formulations intended for long-term storage, depend on a useful range of total osmolality to promote long-term stability at temperatures between 2 and 8°C or higher, while also making the formulation useful for parenteral injection. For example, although not necessarily required, a useful range of total osmolality (total number of molecules in solution) can be from about 200 mOs / L to about 800 mOs / L. It will be apparent that the amount of cryoprotectant, such as sucrose or sorbitol, will depend on the amount of salt in the formulation to maintain the total osmolality of the solution within the appropriate range.Thus, salt-free formulations may, but do not necessarily, contain from about 5% to about 25% sucrose.

[0058] Alternatively, salt-free sorbitol-based formulations may, but do not necessarily, contain sorbitol in the range of about 3% to about 12%. Salt-free formulations may require an increased range of each cryoprotectant to maintain effective osmolality levels. These formulations may also contain divalent cations (including, but not limited to, MgCl, CaCl, and MnCl) and non-ionic surfactants (such as, but not limited to, polysorbate 80 (Tween 80®), polysorbate 60 (Tween 60®), polysorbate 40 (Tween 40®), and polysorbate 20 (Tween 20®), polyoxyethylene alkyl ethers such as, but not limited to, Brij 58®, Brij 35®, and others, such as Triton X-100®, Triton X 114®, NP40®, Span 85, and the Pluronic series of non-ionic surfactants (e.g., Pluronic 121)). Any combination of such components, including the possible inclusion of a bacteriostatic agent, may be useful for loading the antibody-containing formulations of the invention. The anti-HIV-1 antibodies or antigen-binding portions thereof of the present invention may also be "chemical derivatives," which refer to antibodies containing additional chemical moieties (e.g., pegylation) that are not normally part of an immunoglobulin molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively, these moieties may attenuate undesirable side effects of the base molecule or reduce the toxicity of the base molecule.

[0059] For in vivo treatment, a pharmaceutical formulation comprising at least one anti-HIV antibody or antigen-binding portion thereof of the present invention is administered or provided to a patient. When used for in vivo therapy, the anti-HIV antibody or antigen-binding portion thereof of the present invention is administered to a patient in a therapeutically effective amount (i.e., an amount that eliminates or reduces the total viral load, as described in Example 1 below). The antibody is administered to a human patient according to known methods, for example, intravenous administration, e.g., as a bolus or by continuous infusion over a long period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. The antibody or antigen-binding portion thereof can be administered parenterally, if possible, to the target cell site, or intravenously. In some embodiments, the antibody is administered intravenously or subcutaneously. The therapeutic composition of the present invention can be administered to a patient or subject systemically, parenterally, or topically. The above parameters for assessing successful treatment and improvement of disease can be readily measured by routine procedures familiar to physicians.

[0060] For parenteral administration, the anti-HIV-1 antibody or its antigen-binding portion may be combined with a pharmaceutically acceptable parenteral vehicle to be formulated into a unit-dose injectable form (solution, suspension, emulsion). Examples of such vehicles include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles include, but are not limited to, fixed oils and ethyl oleate. Liposomes can be used as carriers. The vehicle may contain small amounts of additives, such as substances that enhance isotonicity and chemical stability, such as buffers and preservatives.

[0061] The anti-HIV-1 antibodies, or antigen-binding portions thereof, of the present invention can be administered to a host by any method, strategy, and / or combination available in the art in an amount sufficient to provide therapeutic treatment against HIV-1. These compositions may be provided to an individual by a variety of routes known in the art, particularly parenteral routes, including, but not limited to, intravenous (IV), intramuscular (IM), or subcutaneous (SC) administration, with IV administration being standard in the art for therapeutic antibody administration. These compositions may be administered as split or multiple doses (i.e., administration of the antibody at staggered times by maintaining sterility of the formulation throughout the treatment regime).

[0062] The dose and dosage regimen will depend on various factors readily determined by a physician, such as the nature of the infection, e.g., its therapeutic index, the patient, and the patient's medical history. Generally, a therapeutically effective amount of antibody is administered to a patient. In some embodiments, the amount of antibody administered is within the range of about 0.001 mg / kg to about 100 mg / kg (patient body weight), and any range therebetween. Depending on the type and severity of the infection, about 0.1 mg / kg to about 50 mg / kg body weight (e.g., about 0.1 to 15 mg / kg / dose) of antibody is an initial candidate dose for administration to a patient, e.g., in one or more divided doses or by continuous infusion. The anti-HIV-1 antibody can be delivered at a relatively low volume rate, e.g., but not necessarily, about 0.001 ml / day to 10 ml / day, to minimize tissue damage or trauma near the site where the formulation is released. Depending on the specific biological agent, the formulation may be released at a low dose, for example, about 0.01 μg / hour or 0.1 μg / hour, 0.25 μg / hour, 1 μg / hour, generally up to about 200 μg / hour, or may be delivered at a volume rate of, for example, about 0.001 mL / day to about 1 mL / day, for example, 0.01 micrograms per day to about 20 milligrams per day. The dosage depends on several factors, such as the potency, bioavailability, and toxicity of the active ingredient (e.g., an anti-HIV-1 antibody or antigen-binding portion thereof) used, and the patient's requirements. The progress of this therapy is easily monitored by conventional methods and assays and based on criteria known to physicians or other skilled artisans. The above parameters for assessing the success of treatment and improvement against disease are easily measurable by routine procedures familiar to physicians.

[0063] Specific embodiments of delivery vehicles for anti-HIV-1 antibodies of the present invention include PLGA microspheres, as discussed herein and further known in the art, and polymer-based non-degradable vehicles, including poly(ethylene-co-vinyl acetate; PEVAc). Furthermore, controlled release and localized delivery of antibody-based therapeutic products is reviewed in Grainger et al., 2004, Expert Opin. Biol. Ther. 4(7):1029-1044. Suitable microcapsules that can encapsulate antibodies also include hydroxymethylcellulose or gelatin microcapsules and polymethylmethacrylate microcapsules prepared by coacervation techniques or interfacial polymerization. See PCT Publication WO 99 / 24061, entitled "Method for Producing IGF-1 Sustained-Release Formulations," in which proteins are encapsulated in PLGA microspheres. This reference is incorporated herein by reference in its entirety. Additionally, microemulsion or colloidal drug delivery systems, such as liposomes and albumin microspheres, can also be used. Other preferred sustained-release compositions use bioadhesives to retain the antibody at the administration site. As noted above, sustained-release formulations can include biodegradable polymers into which the antibody is disposed, which can result in non-immediate release. Non-injectable devices may be referred to herein as "implants," "drug depot implants," "depot implants," "non-injectable depots," or similar terms. Typical depot implants include, but are not limited to, solid biodegradable and non-biodegradable polymeric devices (such as expanded polymers or coaxial rod-like devices), as well as numerous pump systems known in the art. Injectable devices are divided into bolus injections (drug release and dissipation after injection) and reservoir or depot injections, which provide a storage reservoir at the injection site, allowing for sustained release of the biologic over time. Depot implants may be surgically tethered to the delivery site to provide a suitable reservoir for extended release of the antibody over time.Such devices can deliver drug formulations in amounts therapeutically or prophylactically required for treatment over a preselected period of time. Depot implants can also protect the formulation from degradation by internal processes (such as proteases) during the treatment period. As known in the art, the term "sustained release" refers to the gradual (continuous or discontinuous) release of such agents from a block polymer matrix over an extended period of time. Regardless of the specific device, sustained release of the composition results in a localized, biologically effective concentration of the antibody. Sustained release of biologics can occur over periods of one day, several days, a week, or more, depending on the formulation, but most likely for one month or more, or up to about six months. Natural or synthetic polymers known in the art may be useful as depot implants due to their versatile degradation kinetics, safety, and biocompatibility. These copolymers can be engineered to improve the pharmacokinetics of the active ingredient, protect the drug from enzymatic attack, and degrade over time at the site of attachment or injection. Those skilled in the art will appreciate that there is ample teaching in the art for manipulating the properties of these copolymers, including their respective methods of manufacture, the catalysts used, and the final molecular weight of the sustained-release depot implant or injection. Natural polymers include, but are not limited to, proteins (e.g., collagen, albumin, or gelatin), polysaccharides (cellulose, starch, alginate, chitin, chitosan, cyclodextrin, dextran, hyaluronic acid), and lipids.Biodegradable synthetic polymers include, but are not limited to, various polyesters, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 22:547-556), polylactide ([PLA]; U.S. Pat. No. 3,773,919 and European Patent No. 058,481), polylactic acid polyglycolate (PLGA), e.g., as described above, polylactide-co-glycolide (see, e.g., U.S. Pat. No. 4,767,628 and U.S. Pat. No. 5,654,008), polyglycolide (PG), polyethylene glycol (PEG) conjugates of poly(α-hydroxy acid), polyorthoesters, polyaspirin, polyphosphagen, vinylpyrrolidone, polyvinyl alcohol (PVA), PVA-g-PLGA, PEGT-PBT copolymer (PolyActive), methacrylate, poly(N-isopropylacrylamide), PEO-PP O-PEO (pluronics), PEO-PPO-PAA copolymers, PLGA-PEO-PLGA, polyorthoesters (POEs), or any combination thereof (see, e.g., U.S. Pat. No. 6,991,654 and U.S. Patent Application Publication No. 20050187631, each of which is incorporated by reference in its entirety), hydrogels (see, e.g., Langer et al., 1981, J. Biomed. Mater. Res. 15:167-277; Langer, 1982, Chem. Tech. 12:98-105; non-degradable ethylene-vinyl acetate (e.g., ethylene-vinyl acetate discs and poly(ethylene-co-vinyl acetate)), degradable lactic acid-glycolic acid copolymers, e.g., Lupron Depot™, poly-D-(-)-3-hydroxybutyric acid (EP 133,988), hyaluronic acid gel (see, e.g., U.S. Pat. No. 4,636,524), alginate suspension, polyorthoester (POE), etc. Polylactide (PLA) and its copolymer with glycolide (PLGA) are well known in the art, with the commercialization of Lupron Depot™ approved in 1989 as the first parenteral sustained-release formulation utilizing a PLA polymer.Additional examples of products utilizing PLA and PLGA as excipients to achieve sustained release of active ingredients include Amidox (PLA; periodontal disease), Nutropin Depot (PLGA; with hGH), and Trelstar Depot (PLGA; prostate cancer). Other synthetic polymers included, but were not limited to, poly(c-caprolactone), poly(3-hydroxybutyric acid), poly(β-malic acid), and poly(dioxanone); polyanhydrides, polyurethanes (see WO 2005 / 013936), polyamides, cyclodextran, polyorthoesters, n-vinyl alcohol, polyethylene oxide / polyethylene terephthalate, polyphosphates, polyphosphonates, polyorthoesters, polycyanoacrylates, polyethylene glycols, polydihydropyrans, and polyacetals. Non-biodegradable devices include, but are not limited to, various cellulose derivatives (carboxymethylcellulose, cellulose acetate, cellulose acetate propionate, ethylcellulose, hydroxypropylmethylcellulose), silicone-based implants (polydimethylsiloxane), acrylic polymers (polymethacrylate, polymethylmethacrylate, polyhydroxy(ethylmethylacrylate), as well as polyethylene-co-(vinyl acetate), poloxamers, polyvinylpyrrolidone, poloxamine, polypropylene, polyamide, polyacetal, polyester, polyethylene-chlorotrifluoroethylene, polytetrafluoroethylene (PTFE or "Teflon™"), styrene butadiene rubber, polyethylene, polypropylene, polyphenylene oxide-polystyrene, poly-a-chloro-p-xylene, polymethylpentene, polysulfone, and other related biostable polymers. Suitable carriers for sustained-release depot formulations include, but are not limited to, microspheres, films, capsules, particles, gels, coatings, matrices, wafers, pills, or other pharmaceutical delivery compositions. Examples of such sustained-release formulations are described above.See also U.S. Patent Nos. 6,953,593, 6,946,146, 6,656,508, 6,541,033, and 6,451,346, the contents of each of which are incorporated herein by reference. The dosage form must be capable of delivering the drug formulation in an amount and concentration therapeutically required for treatment over a preselected period of time and must adequately protect the formulation from degradation by bodily processes during the treatment period. For example, the dosage form may be surrounded by an outer surface made of a material with properties to protect against degradation from metabolic processes and risks such as leakage, cracking, breakage, or distortion. This can prevent the dosage form contents from expelling in an uncontrolled manner under stresses that may be experienced during use, such as physical forces exerted on the drug release device or within a convective drug delivery device as a result of normal joint and other movements by the subject, or physical forces associated with pressure generated within the reservoir. The drug reservoir or other means for holding or containing the drug must also be of a material that avoids unintended reactions with the active agent formulation, and is preferably biocompatible (e.g., substantially non-reactive with the subject's body or bodily fluids when the dosage form is implanted). Generally, the anti-HIV-1 antibody will be administered to the individual over a period of at least 12 hours to at least 1 week, most likely via an implant designed to deliver the drug for at least 10, 20, 30, 100 days, or at least 4 months, or at least 6, 12, 24 months or more, as needed.

[0064] In some embodiments, the anti-HIV-1 antibodies, or antigen-binding portions thereof, of the present invention may be co-administered with one or more additional therapeutic agents for HIV-1, for example, one or more HIV agents (e.g., non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleoside reverse transcriptase inhibitors (NRTIs), protease inhibitors (PIs), fusion inhibitors, CCR5 antagonists / entry inhibitors, integrase strand transfer inhibitors (INSTIs), and combinations thereof), and / or additional anti-HIV-1 antibodies, or antigen-binding portions thereof, including, but not limited to, additional anti-HIV-1 antibodies, or antigen-binding portions thereof, of the present invention. As shown in Example 1 below, 1:1:1 administration of NC17, BG1, and BG18 was effective in reducing viral load in vivo. Without wishing to be bound by theory, the data indicate that monoclonal bNAbs and very low levels of neutralization-sensitive virus coexist in EB354, suggesting that the antibodies contribute to elite control in this individual. According to another embodiment, the present invention provides a passive vaccine or pharmaceutical composition containing at least one anti-HIV-1 antibody or antigen-binding portion thereof of the present invention and a pharmaceutically acceptable carrier. According to one embodiment, the vaccine or pharmaceutical composition is a composition containing at least one antibody described herein and a pharmaceutically acceptable excipient. The vaccine can include multiple antibodies having the properties described herein in any combination and can further include other anti-HIV-1 antibodies or antigen-binding portions thereof. Passive vaccines can include one or more pharmaceutically acceptable preservatives, carriers, and / or excipients known in the art. In other embodiments, the present invention provides an active vaccine or pharmaceutical composition containing HIV-1 or an antigenic fragment thereof. When HIV-1 is used, it can be attenuated. The HIV-1 can be heat-killed. When an antigenic fragment is utilized, it may be preferable, though not essential, to use glycoprotein gp120, although fragments of glycoprotein gp120 are acceptable.

[0065] The composition can be used in an immunogenic composition for immunizing animals. Such immunogenic compositions according to the present invention can be used in the preparation of vaccines. Preferably, prophylactic and / or therapeutic vaccines are produced. Therefore, within the scope of the present invention are immunogenic or vaccine compositions containing a pharmaceutically acceptable carrier and an effective amount of an antigen, as described above. The carrier used in the composition can be selected based on the method and route of administration and standard pharmaceutical practice. The composition can also contain an adjuvant. Examples of adjuvants include cholera toxin, E. coli heat-labile enterotoxin, liposomes, unmethylated DNA (CpG), or any other innate immune stimulating complex. Various adjuvants that can be used to further enhance the immunological response depend on the host species and include Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.

[0066] The vaccine preparation can be administered to a subject by itself or in the form of a pharmaceutical or therapeutic composition. The composition of the present invention and the adjuvant can be produced by conventional mixing, dissolving, granulating, dragee-making, elutriation, emulsifying, encapsulating, encapsulating or lyophilizing processes. The pharmaceutical composition can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries that facilitate the processing of the antigen of the present invention into a pharmaceutically usable preparation. The appropriate formulation depends on the selected administration route.

[0067] For injection, the vaccine preparation can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution, phosphate-buffered saline, or any other physiological saline buffer. The solution can contain formulatory agents such as suspending agents, stabilizers, and / or dispersing agents. Alternatively, the composition can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.

[0068] The amount of the composition to be administered will depend, as can be determined by one of skill in the art, on factors such as the particular antigen in the composition, whether an adjuvant is co-administered with the antigen, the type of co-administered adjuvant, the mode and frequency of administration, and the desired effect (e.g., protection or treatment). Determining an effective amount of a vaccine formulation for administration is well within the capabilities of one of skill in the art, especially in light of the detailed disclosure provided herein. The effective amount can be initially estimated from in vitro assays. For example, a dose can be formulated for an animal model using techniques well known in the art to achieve induction of an immune response. One of skill in the art would readily be able to optimize administration for any animal species based on the results described herein. Dosage and intervals may be adjusted individually. For example, when used as a vaccine, the vaccine formulation of the present invention may be administered in about one to three doses over a period of one to 36 weeks. Vaccination may continue indefinitely in some circumstances, particularly in the case of passive vaccination. Preferably, one or two doses are administered at intervals of about three weeks to about four months, followed by periodic booster vaccinations. Alternative protocols may be appropriate for individual animals. A suitable dose is an amount of vaccine preparation that, when administered as described above, is capable of raising an immune response in an immunized animal sufficient to protect the animal from infection for at least 4 to 12 months. Generally, the amount of antigen present in a dose ranges from about 1 pg to about 100 mg per kg of host, typically about 10 pg to about 1 mg, and preferably about 100 pg to about 1 pg per kg of host. Suitable dose ranges vary depending on the route of injection and the size of the subject, but are typically about 0.1 mL to about 5 mL. Additional booster doses can be administered as needed.

[0069] D. Kits and Diagnostic Methods One embodiment of the present invention relates to kits for detecting HIV-1 present in a sample. These kits may include an anti-HIV-1 antibody or antigen-binding portion thereof of the present invention and various reagents, such as reagents that aid in detecting binding between the anti-HIV-1 antibody and an epitope present on HIV-1, such as gp120 and / or gp140, or an antigenic fragment thereof.

[0070] The kit may be an in vitro assay such as an immunoassay, for example, an enzyme immunoassay (EIA), an enzyme-linked immunosorbent assay (ELISA), an ELISPOT (enzyme-linked immunospot), a radioimmunoassay (RIA), an immunofluorescence assay, and other assays known in the art, including, but not limited to, Western blot analysis and / or immunoprecipitation. In vitro assays may be competitive, indirect, as in a sandwich assay, or an antibody capture method. For example, in a direct ELISA, a buffered solution of an antigen, such as a sample containing HIV-1 or an antigenic fragment thereof, or a biological sample containing or suspected of containing HIV-1, is added to the wells of a microtiter plate, such as a 96-well plate. A solution of unreacted protein, such as bovine serum albumin or casein, is then added to the wells. Other common enzymes include alkaline phosphatase or β-D-galactosidase, although other enzymes are contemplated and are intended to be embodied by the present invention. An anti-HIV-1 antibody or antigen-binding portion thereof conjugated to a reporter molecule enzyme, such as, but not necessarily, horseradish peroxidase, is then added. A substrate for the enzyme is then added, thereby producing a detectable signal. For example, adding TMB to horseradish peroxidase produces a colored product, in which case the ELISA is a colorimetric assay. ELISAs can be operated in qualitative or quantitative formats. A qualitative result provides a simple positive or negative result (presence or absence) for the sample. The cutoff between positive and negative is determined by the analyst and may be statistical. Sandwich ELISAs generally follow the following protocol: A capture anti-HIV-1 antibody or antigen-binding portion thereof is bound (i.e., "immobilized") to a substrate, such as a microtiter plate. An antigen-containing sample (i.e., a sample containing HIV-1 or an antigenic fragment thereof) is then added to the substrate, at which point the antigen-containing sample is captured by the anti-HIV-1 antibody. The substrate is then washed to remove unbound antigen.A second anti-HIV-1 antibody or antigen-binding portion thereof is added, binding to a different epitope on HIV-1, such as a different epitope on gp120, or a different epitope on a different antigen, such as gp140. The second anti-HIV-1 antibody or antigen-binding portion thereof is conjugated to a reporter molecule, e.g., an enzyme, which can be any molecule that provides a detectable signal. The plate may be washed twice, and if the reporter molecule is an enzyme, a substrate, such as TMB, that provides a detectable signal may be added (also a colorimetric assay). A third common type of ELISA is the competitive ELISA. In these embodiments, an unlabeled anti-HIV-1 antibody or antigen-binding portion thereof is incubated in the presence of an antigen-containing sample, which is then added to the antigen-coated wells. The plate is washed to remove unbound antibody. A second antibody specific for the primary antibody, e.g., a secondary antibody specific for the anti-HIV-1 antibody, is added. The secondary antibody is conjugated to a reporter molecule, such as an enzyme (or any other molecule that can provide a detectable signal), as described herein. Some competitive ELISAs utilize a labeled antigen rather than a labeled antibody. The less antigen there is in the sample, the more labeled antigen is retained, resulting in a stronger detectable signal.

[0071] Another form of common in vitro assay is the radioimmunoassay (RIA), which typically involves binding a known amount of antigen to a radioactive tracer, such as I-125. 99 Others, such as Tc, are suitable for use, which is then mixed with a known amount of antibody specific to the antigen, such as an anti-HIV-1 antibody or its antigen-binding portion. A sample containing an unknown amount of antigen (e.g., a biological sample containing or suspected of containing HIV-1 or its antigenic fragment) is then added. This is direct competition for specific binding. As the concentration of unlabeled antigen increases, binding between the anti-HIV-1 antibody and the labeled standard decreases, which can be measured directly by measuring radioactivity. Other assays are known, and those skilled in the art will readily recognize their applicability.

[0072] In some embodiments, the present invention relates to methods for detecting HIV-1 or an antigenic fragment thereof in a sample. Such methods may utilize any of the assays described herein or other assays known in the art. Some of the assays described herein can quantify the amount of antigen present in a sample, and thus, in some embodiments, the present invention relates to methods for quantifying the amount of HIV-1 or an antigenic fragment thereof present in a sample, e.g., a biological sample.

[0073] Assays containing the anti-HIV-1 antibodies or antigen-binding portions thereof of the present invention may or may not be utilized for diagnostic purposes. Accordingly, in some embodiments, the present invention relates to diagnostic methods for using the anti-HIV-1 antibodies or antigen-binding portions thereof of the present invention. Due to the specificity of the anti-HIV-1 antibodies or antigen-binding portions thereof of the present invention, immunoassays containing the anti-HIV-1 antibodies or antigen-binding portions thereof of the present invention may be sufficient to diagnose individuals with active or latent HIV-1 infection. As long as the antibodies or antigen-binding portions thereof used are specific for HIV-1, the antibodies or antigen-binding portions thereof need not be limited to any particular epitope. For example, in a sandwich assay, a first anti-HIV-1 antibody or antigen-binding portion thereof may bind to a first epitope, such as on gp120, and a second anti-HIV-1 antibody or antigen-binding portion thereof (which binds to a reporter molecule) may bind to a second epitope, which may or may not be present on the same antigen. Thus, kits and methods used to detect or quantify the amount of HIV-1 or its antigenic fragments may contain BG18, NC37, and BG1, including their antigen-binding portions, and are considered within the scope of the present invention. Alternatively, any of BG18, NC37, and BG1, including their antigen-binding portions, as well as any other anti-HIV-1 antibodies, so long as they are suitable for incorporation into such kits / methods. It is understood that the anti-HIV-1 antibody or its antigen-binding portion used need not necessarily be capable of broadly neutralizing HIV-1 to be useful for detecting and / or quantifying HIV-1 present in a sample, or even for diagnostic purposes.

[0074] E. Equivalents Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, and any other stated or intervening value within that stated range, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding both of those included limits are also included in the invention.

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice and testing of the present invention, the preferred methods and materials are described herein. All publications mentioned herein are incorporated herein by reference in their entirety.

[0076] As used in this specification and the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise.

[0077] The term "about" refers to a range of values ​​that would not be interpreted by a person skilled in the art as being substantially different from the baseline value. For example, the term "about" can refer to values ​​within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the stated value, as well as intervening values ​​between such stated values.

[0078] The publications disclosed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0079] Each application and patent cited herein, and each literature or reference cited therein, patent or non-patent literature (including the prosecution of each issued patent; the "application cited literature"), and each PCT and foreign application or patent corresponding to and / or claiming priority from any of these applications and patents, and each literature cited or referenced within each application cited literature, are expressly incorporated herein by reference in their entirety. More generally, where a literature or reference is cited in the text, either in a bibliography before the claims or in the literature itself (the "references cited herein"), and each literature or reference cited within each of the references cited herein (including any manufacturer's specifications, instructions, etc.) are expressly incorporated herein by reference.

[0080] The following non-limiting examples serve to further illustrate the present invention.

[0081] VII. Working Examples 1. Coexistence of potent broadly neutralizing HIV-1 antibodies and antibody-sensitive virus in viremic controllers A. Method HIV-1 infected person EB354 We purified IgG from donor EB354, whose neutralization broadness and potency ranked in the top 1% of the HIV-1-infected long-term nonprogressors. Donor EB354 was diagnosed with HIV-1 clade B in 1986. Donor EB354 was treated with didanosine and stavudine between 1995 and 1998 but has not received antiretroviral therapy since then. Donor EB354 has been followed clinically frequently since 2002 (Table 3: HLA A*01:01, 24:02, B*27:05, 57:01, C*01:02, 06:02). In most individuals, strain-specific antibodies rapidly develop shortly after infection. This response is associated with the selection of resistant viral variants, which in some cases induce bNAbs. However, EB354 differs from the individuals studied in detail in that bNAb expression is associated with the rapid selection of autologous plasma viruses that are resistant to the coexisting bNAb. EB354 is important because susceptible and resistant virus strains coexist with bNAbs, and the resistant strains are unable to induce high levels of viremia because they are either only partially effective in some way or are suppressed by CD8+ T cells. Thus, bNAb-susceptible plasma viruses cannot escape immune pressure in this individual, resulting in a bNAb:virus equilibrium in which the virus persists but is unable to induce high levels of viremia. EB354 is unique in that it is both an elite controller and an elite neutralizer. HIV-1 elite controllers are infected individuals who maintain low viral loads for many years. These individuals are less likely to transmit the virus and remain AIDS-free for long periods of time. 85% of HIV-1 elite controllers have the HLA alleles B57*01 and / or B27*05. These alleles are associated with enhanced CD8+ T cell cytotoxic activity. Compared with viremia progressors (i.e., patients with high viral loads), elite controllers were less likely to develop bNAbs. In EB354, there was sufficient HIV-1 replication to support bNAb expression and affinity maturation, regardless of robust CD8+ T cell responses that may partially control the infection.As discussed herein, the bNAbs that account for the serological neutralizing activity of donor EB354 recognize several non-overlapping epitopes.

[0082] [Table 3]

[0083] B cell sorting and antibody isolation Bait from donor EB354 PBMCs was used as described in J.F. Scheid et al., A method for identification of HIV gp140-binding memory B cells in human blood. Journal of immunological methods 343, 65-67 (2009). + CD19 + IgG + Single-cell sorting of B cells was performed. Memory B cells were pre-enriched using anti-CD19 magnetic beads (MACS) and stained with the following four baits: gp120 2CC core protein as bait, gp140 YU2 , gp140 92UG37.8 (Clade A)+gp140 CZA79012 A 1:1 mixture of BG505 SOSIP.664 (clade C) and BG505 SOSIP.664 was used. Rescue primers were used to amplify both the heavy chain and Igλ genes, and normal primers were used for the IgK chain. Both PCR products were sequenced to identify Ig gene usage, CDR3, and V. H / V LThe number of somatic hypermutations (IgBLAST and IMGT) was analyzed. Purified and digested PCR products were cloned into human Igγ1, Igκ, or Igλ expression vectors as previously described in T. Tiller et al., "Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning." Journal of immunological methods 329, 112-124 (2008). Antibodies were generated by transient transfection of IgH, IgK, and IgL expression plasmids into exponentially growing HEK 293-6E cells as described in F. Klein et al., "Enhanced HIV-1 immunotherapy by commonly arising antibodies that target viral escape variants." The Journal of experimental medicine 211, 2361-2372 (2014).

[0084] Neutralization test HIV-1 neutralization was assessed using a luciferase-based TZM.bl cell assay. Briefly, enveloped pseudoviruses were incubated with five-fold serial dilutions of a single antibody and applied to TZM.bl cells carrying a luciferase reporter gene. After 48 hours, cells were lysed and luminescence was measured. IC 50 and IC 80 reflects the single antibody concentration that reduces relative luminescence units (RLU) by 50% and 80%, respectively. Range curves were constructed using an antibody database calculation tool.

[0085] Crystallization, X-ray data collection and structure determination Asn26 HCCrystals of BG18 Fab in which a potential N-linked glycosylation site at heavy chain position 26 was removed by mutating β to Gln showed superior size and morphology compared to crystals of wild-type BG18 Fab, suggesting the superiority of BG18. N26Q The structure was determined using Fab. BG18 N26Q Crystals of Fab (space group P21; a = 46.12 Å, b = 71.04 Å, c = 69.54 Å; β = 98.48°; one molecule per asymmetric unit) were obtained by mixing 0.2 μL of an 18 mg / mL protein solution with 0.2 mL of 0.1 M sodium acetate pH 4.5, 26.8% (v / v) polyethylene glycol (PEG) 400, and 13.4% (w / v) PEG 8,000 at 20 °C, cryoprotecting in mother liquor supplemented with 20% (v / v) ethylene glycol, and flash-cooling in liquid nitrogen. Crystals of NC102Fab (space group P212121; a = 60.2 Å, b = 82.5 Å, c = 117.2 Å; 1 molecule per asymmetric unit) were prepared by vapor diffusion in sitting drops using 250 nL droplets in 0.1 M sodium acetate trihydrate pH 4.6 and a reservoir of 2.0 M ammonium sulfate at a protein:reservoir ratio of 1.5:1. Crystals were cryoprotected in mother liquor supplemented with 25% (v / v) ethylene glycol and flash-cooled in liquid nitrogen. Crystals of the NC37-93TH057 complex (space group P212121; a = 63.6 Å, b = 67.4 Å, c = 210.4 Å; one complex per asymmetric unit) were prepared by vapor diffusion in 250 nL sitting drops by incubating the protein with a reservoir (containing 0.1 M HEPES pH 7.5 and 20% (w / v) PEG 10,000) at a protein:reservoir ratio of 1.5:1. Crystals were cryoprotected in mother liquor supplemented with 20% (w / v) PEG 400 and flash-cooled in liquid nitrogen.

[0086] X-ray diffraction data were collected at the Stanford Synchrotron Radiation Lightsource beamline 12-2 equipped with a Pilatus 6M pixel detector (Dectris). XDS was used for data indexing, integration, and scaling. BG18 N26QThe Fab crystal diffracted to 1.5 Å, and the search model was 10-1074 Fab (PDB code 4FQ2) V with the CDR loops removed. H V L and C H 1C L The structure was solved by molecular replacement using BG18. The structure was refined using an iterative approach of manual model building and refinement in Phenix and Coot. N26Q Fab's final model (R work =18.0%, R free = 18.9%) had 98.3%, 1.7%, and 0% of residues in the favored, allowed, and disallowed regions of the Ramachandran plot, respectively. NC102 Fab diffracted to 1.6 Å and was modeled as V with the CDR loops removed. H V L A model of NIH45-46 (PDB code 3U7W) with C H 1C L The structure was elucidated by molecular replacement using the final model of NC102 Fab (R work =18.0%, R free = 20.0%) had 98%, 2%, and 0% of residues in the favored, allowed, and disallowed regions of the Ramachandran plot, respectively. H V L , C H 1C L The NC37-93TH057 gp120 complex structure was solved by molecular replacement using a search model of the NC37-93TH057 gp120 core (from PDB 3U7Y). The final model of the NC37-93TH057 complex (R work =21.0%, R free = 26.0%) had 96%, 4%, and 0% of residues in the favored, allowed, and disallowed regions of the Ramachandran plot, respectively. Data collection and refinement statistics are shown in Table 4.

[0087] [Table 4]

[0088] The structure of the BG505 SOSIP.664-BG18-179NC75 complex was solved by cryoelectron tomography / subtomogram averaging to a resolution of approximately 40 Å and used as a reference structure for solving single-particle EM structures from negatively stained samples. The purified BG505 SOSIP.664-BG18-179NC75 complex was diluted to 10 μg / mL in TBS and immediately added 3 μL to a glow-discharged ultrathin C film on a holey carbon support film (400 mesh, Cu grid (Ted Pella, Inc.)). The sample on the grid was crosslinked using glutaraldehyde vapor and then stained with 3% uranyl acetate. Data were collected using an FEI Tecnai T12 transmission electron microscope operating at 120 keV with a Gatan Ultrascan 2k x 2k CCD. Images were acquired using a 0.5-second exposure time at 42,000x nominal magnification with a 1 μm defocus resulting in 2.5 Å per pixel. A total of 25,639 particles were picked using swarm picking in EMAN 2.1, and CTF correction was performed using EMAN 2.1. An initial reference-free 2D class averaging was performed using RELION to classify all particles into 250 classes. 9,827 particles corresponding to good class averages were selected and further classified using 3D classification in RELION, after which 7,925 particles were selected for refinement. To obtain a reference structure for 3D classification and refinement, data were collected from an independent BG505 SOSIP.664-BG18-179NC75 complex sample by cryo-electron tomography. A 40 Å subtomogram-averaged structure was obtained using the method described by Scharf, L. et al., Broadly Neutralizing Antibody 8ANC195 Recognizes Closed and Open States of HIV-1 Env. Cell, 2015. 162(6):1379-90. The subtomogram-averaged structure was low-pass filtered to 80 Å for use as a reference structure for single-particle reconstruction. The resolution of the final single-particle reconstruction was calculated (approximately 25 Å) using RELION and the gold-standard FSC cutoff of 0.143, recommended for estimating the resolution of single-particle EM reconstructions.

[0089] Fitting the EM density map EM structures were visualized using UCSF Chimera. Coordinates from the crystal structure were fitted to subtomogram-averaged or negative-stain single-particle EM structures using the fit in map utility within UCSF Chimera with the following options: real-time correlation / average update, use of atom-simulated map, and 25 Å resolution. The BG505 SOSIP.664 structure (PDB 4TVP) was first fitted to the density, and then the corresponding densities for BG18 Fab and CH103 Fab (PDB 4JAM; as models for 179NC75 Fab) were individually fitted. The C of the Fab was H 1-C L The domain region exhibited low density.

[0090] Protein production and purification for structure determination studies 6×-His-tagged BG18, BG18 by transient transfection N26Q, NC102, and NC37 Fabs were expressed in HEK293-6E cells and purified from transfected cell supernatants using Ni2+-NTA affinity chromatography (GE Healthcare) and Superdex 200 16 / 60 size exclusion chromatography (SEC) (GE Healthcare). Truncated His-tagged 93TH057 gp120 core protein was produced in baculovirus-infected insect cells and purified using Ni2+-NTA affinity chromatography (GE Healthcare). 2+ Purification was performed using affinity chromatography (GE Healthcare) as described (Diskin et al., Science 2011; Diskin et al., NSMB 2010). Prior to crystallization studies, purified NC37 and 93TH057 proteins were co-incubated (2:1 molar ratio of Fab to gp120 core) and treated with 5 kU of endoglycosidase H per mg of gp120 protein at 25°C for 3 h. The endoglycosidase H-treated complex was purified by size-exclusion chromatography using a Superdex 200 10 / 300 GL column (GE Healthcare).

[0091] Soluble BG505 SOSIP.664 trimers for EM studies were constructed as described in Sanders, RW et al., "A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies." PLoS Pathog, 2013.9(9):p.e1003618. HEK293-6E cells treated with 5 μM kifunensine (Sigma) were cotransfected with plasmids encoding BG505 SOSIP.664 and soluble furin at a 4:1 ratio. BG505 SOSIP.664 protein was recovered from cell supernatants using a 2G12 immunoaffinity column constructed by covalently coupling 2G12 IgG monomer to an NHS-activated Sepharose column (GE Healthcare). The protein was eluted with 3 M MgCl, followed by immediate buffer exchange into Tris-buffered saline (TBS) pH 7.4 (50 mM NaCl). The trimer was further purified using Mono Q 5 / 50 GL (GE Healthcare) followed by Superose 6 10 / 300 SEC (GE Healthcare).

[0092] Autologous virus For single-genome sequencing (SGS), HIV-1 env gene HIV-1-RNA was extracted from patient plasma using the Qiagen MinElute Virus Spin kit according to the manufacturer's instructions. The extracted RNA was subjected to Env-specific cDNA synthesis using SuperScript III Reverse Transcriptase and the HIV-1-specific primer envB3out.

[0093] envB5out: 5'-TAGAGCCCTGGAAGCATCCAGGAAG-3' (SEQ ID NO: 97) envB3out: 5'-TTGCTACTTGTGATTGCTCCATGT-3' (SEQ ID NO: 98) envB5in: 5'-TTAGGCATCTCCTATGGCAGGAAGAAG-3' (SEQ ID NO: 99) envB3in: 5'-GTCTCGAGATACTGCTCCCACCC-3' (SEQ ID NO: 100).

[0094] The first round of PCR was performed in a volume of 20 μL containing 1× High Fidelity buffer, 2 mM MgSO4, 0.2 mM dNTPs, and 0.5 units of High Fidelity Platinum Taq using 0.2 μM each of primers envB5out and envB3out. The second round of PCR was performed using 1 μL of PCR-1 and 0.2 μM of primers envB5in and envB3in. PCR conditions were the same as PCR-1 except for 45 cycles and an increased annealing temperature of 58°C. PCR-2 products were confirmed using 1% 96-well E-Gels (Invitrogen). Bands from PCR with amplification efficiencies less than 30% were subjected to library preparation and sequenced using the Illumina Nextera DNA Sample Preparation Kit (Illumina). The CMV-Env expression cassette was prepared according to the protocol described in JL Kirchherr et al., "High throughput functional analysis of HIV-1 env genes without cloning." J Virol Methods 143, 104-111 (2007). 293T cells were cotransfected with 500 ng of CMV-env and pSG3Δenv in a 6-well plate, and the supernatant was harvested 48 hours later. Both plasmids were sequence-confirmed before expression. The supernatant was subjected to neutralization testing using the TZM-bl assay.

[0095] TZM-bl assay of Env glycol mutant viruses Pseudoviruses were generated by transfecting 293T cells (ATCC) with an HIV-1 Env expression plasmid and an Env-deficient genomic backbone plasmid called pSG3ΔEnv, as described in Li, M. et al., Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies. J Virol, 2005. 79(16):10108-25. Pseudoviruses were harvested 72 hours posttransfection for use in neutralization assays. Neutralization activity was assessed using a single-round replicative pseudovirus assay with TZM-Bl target cells, as described in Li et al., supra. Briefly, TZM-Bl cells were seeded into 96-well flat-bottom plates. Pseudoviruses were added to the plates and preincubated with serial dilutions of antibody at 37°C for 1 hour. At 72 hours post-infection, luciferase reporter gene expression was quantified upon lysis and addition of Bright-Glo™ luciferase substrate (Promega). IC 50 To determine values, dose-response curves were fitted by nonlinear regression.

[0096] Synthesis of HIV-1 V3 glycopeptides Following the procedure described in Amin, MN et al., "Synthetic glycopeptides reveal the glycan specificity of HIV-neutralizing antibodies," Nat Chem Biol, 2013.9(8):521-6, we achieved the synthesis of V3 glycopeptides derived from several HIV-1 strains using a chemoenzymatic method consisting of automated solid-phase peptide synthesis of GlcNAc-peptide precursors and subsequent enzymatic transglycosylation of the GlcNAc-peptides to generate the target glycopeptides. The interaction of biotinylated synthetic glycopeptides with BG18 and BG8 IgG antibodies was assessed by SPR using a BIAcore T200 system (GE Healthcare) at 25°C. Biotinylated glycopeptides were immobilized on a neutravidin-coated CM5 sensor chip in HBS-P buffer (10 mM HEPES, 150 mM NaCl, P20 surfactant 0.05% v / v, pH 7.4) until 200 response units (RU) were achieved. BG18 (or BG8) was injected in BS-P buffer at a flow rate of 40 μL / min at concentrations starting at 4 μM and serially diluted two-fold. Dissociation was then performed by injecting BS-P buffer at a flow rate of 40 μL / min for 1210 seconds. Regeneration was performed by injecting 3M MgCl2 at a flow rate of 50 μL / min for 3 minutes, followed by injecting HBS-P buffer at a flow rate of 50 μL / min for 5 minutes.

[0097] In vivo mouse model Humanized NOD Rag1 was administered subcutaneously (sc) at 1 mg of each antibody twice a week for 3 weeks. - / - Il2rg null (NOD.Cg-Rag1 tm1Mom Il2rg tm1Wjl Control hu mice were reconstituted with human cells from the same donor and were infected with HIV-1. YU2Mice were infected with HIV but not treated with antibodies. Plasma viral load was measured weekly. gp120 sequences from mice carrying rebound virus were obtained as described in F. Klein et al., HIV therapy by a combination of broadly neutralizing antibodies in humanized mice. Nature 492, 118-122 (2012).

[0098] ELISA High-binding 96-well ELISA plates (Costar) were coated overnight with 5 μg / mL purified 2CC core, gp120.YU2 (wild-type or mutant), or gp140.YU2 fold-on trimer in PBS. After six washes with PBS + 0.05% Tween 20, the plates were blocked for 2 hours with 2% BSA, 1 μM EDTA, and 0.05% Tween-PBS ("blocking buffer") and then incubated for 1 hour with IgG, added as seven serial 1:4 dilutions in PBS starting at an initial concentration of 4 μg / mL. After further washes, the plates were developed by incubation for 1 hour with goat HRP-conjugated anti-human IgG antibody (Jackson ImmunoResearch) (0.8 μg / mL in blocking buffer), followed by incubation with horseradish peroxidase (HRP) chromogenic substrate (ABTS solution; Invitrogen). For competitive ELISA, plates were coated with 0.5 μg / mL BG505.SOSIP.664, washed, and blocked with blocking buffer for 2 hours. Plates were rocked and then incubated for 1 hour in the presence of biotinylated antibody at a constant concentration of 4 μg / mL with IgG added as seven serial 1:4 dilutions in PBS from an initial concentration of 32 μg / mL. Plates were then developed with HRP-conjugated streptavidin (Jackson ImmunoResearch) at 1 μg / mL in blocking buffer.

[0099] For ELISAs using the BG505 SOSIP.664 trimer with the D7324 epitope tag (BG505 SOSIP.664-D7324), plates were coated overnight with 5 μg / mL of D7324 antibody, washed, and incubated with 500 ng / mL of trimer, as described by Sanders, RW et al., "A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies." PLoS Pathog, 2013.9(9):p.e1003618. After further washing, IgG was added over 1 hour in seven serial 1:4 dilutions in PBS, starting at an initial concentration of 4 μg / mL. The endpoint was generated by incubation with a goat HRP-conjugated anti-human IgG antibody, as described above. All experiments were performed at least three times.

[0100] Preparation of CMV-Env virus The CMV-env expression cassette was generated according to established protocols ( 52 ). Briefly, the CMV promoter was amplified from the pcDNA 3.1D / V5-His-TOPO expression vector using the following primers: CMVenv: 5'-AGTAATCAATTACGGGGTCATTAGTTCAT-3' (SEQ ID NO: 101) and CMVenv1A 5'-CATAGGAGATGCCTAAGCCGGTGGAGCTCTGCTTATATAGACCTC-3' (SEQ ID NO: 102).

[0101] The PCR product was purified using a Macherey-Nagel PCR and Gel Purification kit. 1 μl of the first round PCR product was amplified using the following primers: env1ATOPO 5'-CACCGGCTTAGGCATCTCCTATGGCAGGAAGAA-3' (SEQ ID NO: 103) and Rev19 5'-ACTTTTTGACCACTTGCCACCCAT-3' (SEQ ID NO: 104) (in a 20 μL volume containing 1× High Fidelity buffer, 2 mM MgSO4, 0.2 mM dNTPs, 0.5 units of High Fidelity Platinum Taq, and 0.2 μM of each primer). Cycling conditions were 94°C, 2 min; (94°C, 15 s; 55°C, 30 s; 68°C, 4 min) × 35; 68°C, 10 min. The presence of env was confirmed by analysis on a 0.7% agarose gel, and the product was purified using a Macherey-Nagel Gel and PCR Purification kit. Then, 10 ng of envelope and 0.5 ng of CMV were subjected to overlap PCR in triplicate using primers CMVenv and Rev19. The total reaction volume was 50 μL, containing 1× High Fidelity buffer, 0.2 μM MgSO, 0.2 mM dNTPs, 1 unit of High Fidelity Platinum Taq, and 0.4 μM of each primer. PCR was performed at 94°C for 2 min; (94°C for 30 s; 60°C for 30 s; 68°C for 4 min) × 25; and 68°C for 10 min. 293T cells were cotransfected with 500 ng of CMV-env and pSG3Δenv in a 6-well plate, and supernatants were harvested 48 h later. Supernatants were subjected to neutralization assays with TZM-bl as described above.

[0102] Virus of Association (VOA) Virus from donor EB354 was obtained by co-culturing peripheral blood mononuclear cells (PBMCs) from the patient with PBMCs from a healthy donor, as described in van 't Wout et al., "Isolation and propagation of HIV-1 on peripheral blood mononuclear cells." Nat Protoc, 2008. 3(3):363-70. Healthy donor PBMCs were obtained from the patient by leukapheresis under Rockefeller University protocol MNU-0628. 5 × 10 cells per mL were cultured in IMDM containing 10% FBS, 1% penicillin-streptomycin, and 1 μg / mL PHA. 6 Healthy donor PBMCs were pre-stimulated for 2-3 days at 37°C and 5% CO at a cell density of 6 x 10 stimulated donor PBMCs. 6 Transfer the cells to IMDM containing 10% FBS, 1% penicillin-streptomycin, 10 IU / mL IL-2, and 5 µg / mL polybrene and culture at 5–10 × 10 6 The cells were co-cultured with EB354 PBMCs at 37°C and 5% CO2. The medium was changed weekly, and the presence of p24 in the culture supernatant was quantified using the Lenti-X p24 Rapid Titer Kit (Clontech). Cultures with greater than 1 ng of p24 per mL of supernatant were frozen and stored at -80°C. A TZM-bl neutralization assay was used according to the protocol described above to determine the tissue culture infectious dose 50 (TCID 50 We determined the sensitivity of the autologous virus to a range of broadly neutralizing antibodies and autologous serum IgG. All neutralization assays were performed in duplicate.

[0103] HIV-1 YU2 Envelope mutants Wild-type HIV-1 was transfected using the QuikChange (multi-)site-directed mutagenesis kit according to the manufacturer's specifications (Agilent Technologies). YU2 Single, double and triple mutations were introduced into the envelope.

[0104] Analysis of viral evolution Alignments of env nucleotide sequences were generated using ClustalW (version 2.11) or by manual alignment using Geneious (version 8.1.6) sequence analysis software. Regions that could not be unambiguously aligned were removed for phylogenetic analysis and diversity calculations. jModelTest was used to select evolutionary model classes for maximum likelihood phylogenetic analysis. PhyML (version 3) was used with model parameter values ​​and combined phylogenetic estimates to generate maximum likelihood molecular phylogenetic trees. Pairwise genetic diversity between samples was compared using the two-sample U-statistic test in the DIVEIN webtool.

[0105] Bioinformatic processing of MiSeq env sequences Sequence adapters were removed using Cutadapt v1.8.3. Read assembly for each virus was performed in three steps. First, de novo assembly was performed using Spades v3.6.1 to generate long contig files. Subsequently, contigs longer than 255 bp were aligned to the HIV envelope reference sequence, and a consensus sequence was generated using Geneious 8. Finally, reads were realigned to the consensus sequence to fill gaps and generate the final consensus. Sequences with double peaks (cutoff consensus identity for any residue <75%) were omitted from downstream analysis.

[0106] statistical analysis Statistical differences were analyzed by the Mann-Whitney test. GraphPad Prism software was used for analysis, and data were considered significant at *p≦0.05, **p≦0.01, and ***p≦0.001.

[0107] B. Results Multiple bNAbs We purified IgG from donor EB354, who ranked in the top 1% for neutralizing broadness and potency within a cohort of 394 HIV-1-infected long-term nonprogressors. This donor was diagnosed with HIV-1 clade B in 1986. EB354 was treated with didanosine and stavudine between 1995 and 1998 but has not received antiretroviral therapy since. In 2002, EB354's viral load was <400 copies / mL, and his CD4 and CD8 counts were 954 and 1046 cells / mm, respectively. 3 EB354 had three documented peaks in viremia between 2002 and 2006 (Fig. 1A). Serological neutralization potency and broad spectrum were first assessed in 2006 when the viral load was <400 copies / ml (Fig. 1A and Fig. 1B and Table 3). Neutralizing activity increased until 2010 and has remained broad and potent since then (Fig. 1B). HLA typing revealed HLA A*01:01, 24:02, B*27:05, 57:01, C*01:02, and 06:02 (Table 3).

[0108] To isolate antibodies that account for this serological activity, single cells were screened using four HIV-1 baits: 2CC core, gp140 YU2 , gp140 92UG37.8 (Clade A)+gp140 CZA79012 A 1:1 mixture of BG505 (clade C) and BG505 SOSIP.664 (Figure 1C). A total of 241 pairs of heavy and light chains were isolated, of which 152 antibodies formed 22 distinct clones. Antibodies from three clones exhibited tier 2 neutralizing activity (Figure 1C and Table 13). When compared to serum IgG obtained between 2010 and 2015, antibodies belonging to the clone exemplified by antibody BG18 (Figure 1C) reproduced most of the serological activity (Figure 1D). Antibodies belonging to two other neutralizing clones exemplified by antibody NC37 and antibody BG1 (Figure 1C) complemented the activity of BG18, albeit less potently (Figure 1D).

[0109] To map the antibody binding sites in the three neutralizing clones, we analyzed HIV-1 carrying epitope-specific point mutations in Env. YU2 The TZM-bl assay was performed with the mutants. Polyclonal IgG showed no measurable change in sensitivity to the mutants, whereas antibody BG18 showed no measurable change in sensitivity to the YU2 mutants. N332K (Glycan-V3) and antibody BG1 is sensitive to YU2 N160K (V1V2), and antibody NC37 is sensitive to YU2 N280Y (CD4bs) (Fig. 1D). Consistent with the neutralization results, BG18 binding by ELISA was competitively inhibited by PGT121 and 10-1074. BG1 binding was reduced by PGT145 (Fig. 5).

[0110] BG18 inhibited 64% of the viruses in the 118 virus population (Figure 1E, Table 5) with a geometric mean IC of 0.03 μg / ml. 50 Antibodies NC37 and BG1 neutralized IgG1 with similar broad spectrum but more potent than either PGT121 or 10-1074 (Figure 1F, Tables 5-10). Antibodies NC37 and BG1 neutralized IgG1 with geometric mean IC50 of 0.3 μg / mL and 0.67 μg / mL, respectively. 50 and broad spectrum of 33% and 37% (Figures 1E and 1F, Tables 6 and 7). A 1:1:1 mixture of the three bNAbs inhibited 81% of the viruses in the 118 virus population with a geometric mean IC of 0.130 μg / mL. 50 and showed an additive effect (Fig. 1E, Table 11).

[0111] [Table 5-1]

[0112] [Table 5-2]

[0113] [Table 5-3]

[0114]

Table 5-4

[0115]

Table 6-1

[0116]

Table 6-2

[0117]

Table 6-3

[0118]

Table 6-4

[0119]

Table 7-1

[0120]

Table 7-2

[0121]

Table 7-3

[0122]

Table 7-4

[0123]

Table 8-1

[0124]

Table 8-2

[0125]

Table 8-3

[0126]

Table 8-4

[0127]

Table 9-1

[0128]

Table 9-2

[0129]

Table 9-3

[0130]

Table 9-4

[0131]

Table 10-1

[0132]

Table 10-2

[0133]

Table 10-3

[0134]

Table 10-4

[0135] [Table 11-1]

[0136] [Table 11-2]

[0137] [Table 11-3]

[0138] [Table 11-4]

[0139] BG18 At the amino acid level, BG18 has both V H It is 63% and 62% identical to the heavy chains of 10-1074 and PGT121, respectively, which arise from the 4-59 germline precursor. BG18 is a member of the closely related (90.8% identity) germline gene V H The heavy chain complementarity-determining regions (CDRs) of PGT121, 10-1074, and BG18 and their clonal variants show a high degree of similarity between CDRH1 and CDRH2. The CDRH3 of BG18 is three residues shorter than that of PGT121 / 10-1074 (21 vs. 24 residues; Figures 2A and 6B). Furthermore, BG18 shares six of the seven mutations within the framework region common to the PGT121 family (Figure 2A), but unlike members of PGT121 / 10-1074, does not contain any insertions or deletions.

[0140] BG18 is a VL gene segment V L 2-25 and V used by PGT121 and 10-1074, respectively LIt is related to the 3-21 gene segment at the amino acid sequence level with only 46% and 50% identity (Figures 2B, 6C, and 6D). Furthermore, the light chains of BG18 and its variants are slightly more mutated than those of 10-1074 and PGT121, but do not contain insertions or deletions as the heavy chains. Thus, BG18 and its clonal variants display similar heavy chains but different light chains to the previously isolated PGT121 / 10-1074 class of antibodies.

[0141] The 1.3 Å resolution crystal structure of BG18 Fab revealed that the CDR loops form a compact binding site, exhibiting a tip composed primarily of hydrophobic residues, except for CDRH3, which protrudes approximately 11 Å beyond the other CDRs (Fig. 2C). Instead of being fully extended, CDRH3 residue Gly100a HC , Val100b HC , Val100c HC , Gly100e HC and Glu100f HC The CDRL2 folds back on itself in a conformation stabilized by hydrogen bonds between the V and V domains (Figure 2C). The structure of CDRL2 is disordered, suggesting that CDRL2 can adopt multiple conformations in the unbound antibody. Superposition of the unbound BG18, PGT121 (PDB 4FQ1), and 10-1074 Fab (PDB 4FQ2) structures reveals that their V domains are distinct. H The domain is BG18 V H Located on the V domain of the PGT121 and 10-1074 Fab structures L The results revealed that the aligned Vs were closely aligned, with the exception of CDRH3, which was significantly more tilted than BG18 (Figure 2D). Consistent with the low sequence identity for the BG18 and PGT121 light chains (Figures 2B, 6C, and 6D), the aligned Vs LThe superposition of the domains (BG18-PGT121 and BG18-10-1074, respectively, found a root-mean-square deviation of 3.1 Å for superimposing 93 or 92 Cα atoms in the light chain comparison) showed differences in the orientation of all CDRL loops (Figure 2D). Despite these differences, a cleft between CDRH2 and CDRH3 is also visible in BG18, PGT121, and 10-1074 (Figure 2E). Residues lining this cleft interact with complex-type N-glycans in the glycosylated PGT121 Fab structure and Asn137 in the PGT122-BG505 SOSIP complex structure (PDB 4TVP). gp120 BG18 exhibits a second cleft between CDRH3 and CDRL1 / CDRL3, which cannot form in PGT121 and its clonal relatives due to the location of CDRH3 on the light chain of these antibodies (Figure 2E). In addition to these structural differences, BG18 V H -V L The electrostatic surface potential of the domains shows a different distribution of positively charged patches compared to the surface potential of PGT121 and 10-1074 (Figure 7). Taken together, these results are consistent with differences in Env recognition of BG18 compared to recognition by PGT121-related bNAbs.

[0142] To address how BG18 recognizes Env, we solved a negative-stain single-particle electron microscopy (EM) structure at approximately 25 Å resolution of the BG505 SOSIP.664 trimer in complex with BG18-derived Fab and the CD4bs bNAb 179NC75. Coordinate fitting from the BG18 Fab crystal structure to the EM map (Figure 2F) revealed the approach angle and the position of Asn332 in BG18 and other Fabs. gp120 This allowed for a comparison of the potential Env interactions of BG18 with the V3 bNAb (Figure 2G). Similar to PGT122 (a variant of PGT121) and 10-1074, the CDRH3 of BG18 is located at Asn332. gp120 glycans, and gp120 324 GDIR 327However, while PGT122 and 10-1074 exhibit similar approach angles for binding Env, BG18 contacts the Env trimer at a different angle, shifted by 34–41° relative to PGT122 and PGT124 (Fig. 2G).

[0143] BG18 was evaluated for neutralization against a panel of HIV-1 pseudoviruses lacking specific N-linked glycosylation sites. gp120 The results showed that none of the glycans at the base of the V3 loop affected neutralizing activity, except for BG18 at position Asn332 (Table 12). Consistent with these data, BG18 had a glycan at position Asn332. gp120 BG18 preferentially bound to a synthetic V3 glycopeptide containing an oligomannose N-glycan at 10-1074, but not to glycopeptides containing complex N-glycans at this position or to other potential N-linked glycosylation sites within gp120 (Fig. 9). These data suggest that BG18 recognition properties are more similar to those of 10-1074 than to those of PGT121.

[0144] [Table 12-1]

[0145] [Table 12-2]

[0146] NC37 The 2.7 Å resolution crystal structure of NC37 Fab bound to the gp120 core (Fig. 1C) demonstrated that NC37 recognizes CD4bs (Fig. 8A and 8B). Some members of the NC37 family bind to V H They lined up in 1-2, and the other members were V. H 1-46, so based on sequence alone, V H V H 1-2 or V HIt was initially difficult to determine whether NC37 was derived from the V1-46 germline gene (Table 13). However, structural comparisons revealed that NC37 was derived from the V1-46 germline gene. H The results revealed that NC37 adopts a similar orientation and binding angle to the 1-46-derived antibody 8ANC131 / 134 (Figures 8A and 8B). As a result, NC37 uses its light chain to bind to loop D residue Asn280. gp120 contact with V H 1-2 derived antibodies, such as NIH45-46, use their heavy chains to target Asn280 gp120 The CDRH3 of NC37 extends toward the gp120 inner domain, contacting CDRH3 residue Tyr100G. HC and gp120 inner domain residue Lys97 gp120 and Glu102 gp120 Superimposition of the NC37 Fab-gp120 complex onto the SOSIP trimer structure suggests that the NC37 CDRH3 contacts the adjacent promoter within the Env trimer (Fig. 8C).

[0147] NC37 was isolated in 2010, whereas BG1 and BG18 were isolated from PBMCs collected in 2014. Using PCR primers specific for BG1 and BG18, we examined available PBMCs from 2010 and 2013. BG1 transcripts were detected at both time points, whereas BG18 was first detected in 2013. This indicates that BG18 emerged between 2010 and 2013, whereas BG1 and NC37 were expressed earlier.

[0148] Autologous Neutralization To examine the effects of the three bNAbs on autologous virus, we cloned individual env genes from circulating plasma viruses obtained by single-genome sequencing (SGS) at five different time points between 2006 and 2015 (2006, 2010, 2013, 2014, and 2015). Only 37 functional in-frame transcripts were recovered, partly due to viral loads below 400 copies / mL at all time points. Furthermore, all sequences recovered from 2015 were nonfunctional (Figure 10). The 37 functional sequences formed three clusters: Cluster A, which contained one sequence obtained in 2006 and differed by approximately 15% from the remaining sequences; Cluster B, the largest cluster containing sequences from all four time points; and Cluster C, which contained three sequences, all obtained in 2014 (Figure 3A). Consistent with the new cluster of sequences emerging in 2014, there was an increase in viral diversity between 2013 and 2014 (Fig. 3B).

[0149] All three sequences in cluster C contain Asn332 gp120 34 sequences contained a mutation that altered the N-linked glycosylation site at Asn332, whereas the other 34 sequences contained a mutation that altered the N-linked glycosylation site at Asn332. gp120 In addition, all three sequences in cluster C contained a complete N-linked glycosylation site at Asp282, which was not found in the other 34 sequences. gp120 It contained 282 residues. gp120 This position harbors a Lys in 94.4% of all HIV-1 sequences in the Los Alamos database, and an Asp at this position is associated with resistance to CD4bs antibodies.

[0150] To determine the sensitivity of functional Envs to the three autologous bNAbs, pseudoviruses were generated and tested in the TZM-bl assay. Of the 35 successfully generated pseudoviruses, only four were resistant to all three bNAbs. These were Envs from cluster C found in plasma obtained in 2014 and plasma obtained in 2013 (Figure 3C and Figure S10). The remaining 31 pseudoviruses (88.5% of all pseudoviruses) were sensitive to at least one of the three bNAbs. Furthermore, pseudoviruses obtained in 2006, 2010, and 2014 were tested for their sensitivity to contemporaneous serum IgG isolated from the same time point as the viral Env gene (plasma IgG obtained in 2013 was unavailable for this assay). Results showed that 19 of the 22 viruses tested were still neutralized by contemporaneous IgG (Figure 3C and Figure S10).

[0151] BG18- and NC37-sensitive viruses, as well as viruses sensitive to simultaneous IgG, were found at every time point analyzed, including the time when BG18 and NC37 were isolated (Figure 3D). In contrast, most strains were resistant to BG1, except for one virus obtained in 2006. This suggests that the majority of donor EB354's autologous virus escaped BG1 but was unable to escape BG18 and NC37 antibodies, thus demonstrating the broad spectrum and potency of BG18 and NC37.

[0152] Although it was not possible to obtain viral sequences from plasma samples obtained in 2015, it was possible to grow virus from two of the five EB354 CD4+ viral expansion cultures (VOCs). In both cases, it took more than five weeks for the cultures to become positive for virus, as measured by p24 ELISA. SGS sequences from both 2015 expansion cultures clustered closely with sequences from cluster C, with a mutation at Asn332. gp120 Both sequences lacked N-linked glycosylation sites (Fig. 3A).gp120 Consistent with these findings, culture supernatants obtained in 2015 were resistant to all three bNAbs in the TZM-bl assay (Figure 3E).

[0153] In vivo efficacy of BG18, NC37 and BG1 To determine whether BG18 or NC37 can independently exert selective pressure on HIV-1 in vivo, humanized mice (hu mice) were cultured with HIV-1 YU2 BG1 was used to treat established HIV-1 infections. YU2 showed low activity against 50 = 15.8 μg / mL), which was used only in subsequent combination treatment experiments and not in monotherapy experiments. Similar to 10-1074, administration of the less potent but still effective clonal variants BG18 or BG8 resulted in a rapid reduction in viral load (average 1.5 log 10 ), and were associated with subsequent rebound viremia (Fig. 4A, Fig. 11, and Fig. 12). All rebound viral sequences contained a residue at Asn332 gp120 NC37 monotherapy also transiently suppressed viremia, but the magnitude of the reduction in viral load was less pronounced than with BG18, averaging 0.5 log 10 The results were consistent with those of the NC37-rebound virus (Figures 4A and 4C). This indicates that BG18 and NC37 are effective in treating HIV, with BG18 having the most pronounced effect. The majority of NC37-rebound virus Env sequences showed the R456K mutation, which is associated with resistance to CD4bs antibodies (Figures 4B and 4C). This further indicates that BG18 and NC37 inhibit HIV-1 in vivo in human mice. YU2 This demonstrates that antibody-resistant mutants can be selected.

[0154] By combining antibodies, HIV-1 in mice was YU2To determine whether the combination of BG18, NC37, and BG1 can suppress viremia, a 1:1:1 combination of three bNAbs (BG18, NC37, and BG1) was used to test HIV-1 YU2 Five infected hu mice were treated. Immediately after administration of the three bNAbs, the viral load across all animals averaged 1.74 Log 10 Strikingly, 4 / 5 mice showed virtually undetectable viral loads after 3 weeks (Figures 4D and 4E). Viremia rebounded in only one mouse, T6, only 3 weeks after treatment was discontinued. The remaining four mice continued to show remarkably suppressed or undetectable viremia even 4 weeks after treatment was discontinued (Figures 4D and 4E). This demonstrates that NC37, BG1, and especially BG18, whether applied individually or especially in combination, are potent in sustained suppression and neutralization of HIV-1 in vivo.

[0155] [Table 13]

[0156] The foregoing examples and description of preferred embodiments should be construed as illustrative, not limiting, of the invention as defined by the claims. As will be readily understood, numerous variations and combinations of the features described above can be utilized without departing from the invention as set forth in the claims. Such variations are not to be considered a departure from the scope of the invention, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated by reference in their entirety.

Claims

1. three heavy chain complementarity determining regions (CDRH1, CDRH2 and CDRH3) and three light chain complementarity determining regions (CDRL1, CDRL2 and CDRL3); An isolated anti-HIV-1 antibody or antigen-binding portion thereof, wherein the CDRH1, CDRH2, and CDRH3 comprise the sequences of SEQ ID NOs: 74 to 76, respectively, and the CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences of SEQ ID NOs: 78 to 80, respectively.

2. 2. The isolated anti-HIV-1 antibody or antigen-binding portion thereof of claim 1, wherein the anti-HIV-1 antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, and the heavy chain and the light chain comprise the amino acid sequences of SEQ ID NOs: 73 and 77, respectively.

3. The isolated anti-HIV-1 antibody or antigen-binding portion thereof of claim 1 or 2, wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

4. The isolated anti-HIV-1 antibody or antigen-binding portion thereof of any one of claims 1 to 3, wherein the antibody or antigen-binding portion thereof is recombinantly produced.

5. A bispecific antibody comprising the isolated anti-HIV-1 antibody or antigen-binding portion thereof according to any one of claims 1 to 4.

6. An isolated nucleic acid comprising a sequence encoding the anti-HIV-1 antibody or antigen-binding portion thereof of any one of claims 1 to 4, or the bispecific antibody of claim 5.

7. A vector comprising the nucleic acid of claim 6.

8. A cultured cell comprising the vector of claim 7.

9. A pharmaceutical composition comprising: (i) the anti-HIV-1 antibody or antigen-binding portion thereof according to any one of claims 1 to 4, or the bispecific antibody according to claim 5; and (ii) a pharmaceutically acceptable carrier.

10. 10. The pharmaceutical composition of claim 9, wherein the anti-HIV-1 antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, and the heavy chain and the light chain comprise the amino acid sequences of SEQ ID NOs: 77 and 73, respectively.

11. The pharmaceutical composition of claim 9 or 10, further comprising a second anti-HIV-1 antibody or an antigen-binding portion thereof.

12. Use of a first therapeutic agent comprising a therapeutically effective amount of the anti-HIV-1 antibody or antigen-binding portion thereof of any one of claims 1 to 4, or the bispecific antibody of claim 5, or the pharmaceutical composition of any one of claims 9 to 11, in the manufacture of a medicament for treating HIV-1 infection.

13. 13. The use of claim 12, wherein the treatment further comprises administering a second therapeutic agent.

14. 14. The use of claim 13, wherein the second therapeutic agent is selected from the group consisting of a non-nucleoside reverse transcriptase inhibitor, a protease inhibitor, an entry or fusion inhibitor, an integrase inhibitor, and a second anti-HIV-1 antibody or antigen-binding portion thereof.

15. A vaccine composition comprising the anti-HIV-1 antibody or antigen-binding portion thereof according to any one of claims 1 to 4, or the bispecific antibody according to claim 5.

16. 16. The vaccine composition of claim 15, wherein the anti-HIV-1 antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, and the heavy chain and the light chain comprise the amino acid sequences of SEQ ID NOs: 73 and 77, respectively.

17. Use of a vaccine composition according to claim 15 or 16 in the manufacture of a medicament for preventing HIV-1 infection.

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