EA042959B1 - BROAD SPECTRUM ANTIBODIES AND METHODS OF THEIR APPLICATION - Google Patents

Description

I. Application for Federal Funding

The present invention was made with government support under grant AI100148 from the National Institutes of Health (NIH). The State of the United States has certain rights to the present invention.

II. The field of technology to which the invention belongs

The field of the invention is broad-spectrum neutralizing anti-HIV-1 antibodies (bNab) and methods for their use.

III. State of the art

Human immunodeficiency virus (HIV) is a lentivirus (family Retroviridae) that infects humans. If left untreated, HIV infection will eventually lead to the development of acquired immunodeficiency syndrome (AIDS), a condition in which a progressive deficiency of the immune system allows life-threatening opportunistic infections (such as toxoplasmosis) and cancers (such as Kaposi's sarcoma) to thrive. , which ultimately leads to the death of the infected person. Without treatment, the median survival time after primary infection is only 9 to 11 years. HIV/AIDS is a major global health crisis that the CDC considers a pandemic, with about 37 million people infected with HIV as of 2014, which resulted in about 1.2 million deaths that same year.

HIV has several subtypes, including HIV-1 and HIV-2. HIV-1 is more virulent, more infectious, and the predominant form worldwide, while HIV-2 is less virulent, less infectious, and currently only found in West Africa. HIV-1 is the cause of most HIV infections and therefore represents a more clinically relevant target. HIV-1 includes several subgroups, including group M, group N, group O, and group P. Group M, which means the main one, includes approximately 90% of HIV infections and is subdivided into other subtypes, A-K, which are sometimes subdivided more on additional subtypes (A1, A2, etc.). The exceptional diversity of HIV-1, caused by its high mutation rate, makes it difficult to effectively treat HIV. To combat this problem, researchers have attempted to develop so-called broad-spectrum neutralizing antibodies (bNAbs), so designated due to their ability to neutralize multiple HIV viral strains, such as multiple HIV-1 viral strains, such as those disclosed in US 2014/0328862. included in the present description in its entirety by reference. A subset of people infected with HIV-1 generate bNAbs that typically develop over 1-3 years, during which circulating viral strains and antibodies co-evolve. However, these bNabs are generally unable to neutralize coexisting autologous viruses due to antibody-mediated selection against susceptible viral strains. Accordingly, there is an urgent need for novel broad spectrum anti-HIV neutralizing antibodies that exhibit a high level of activity against HIV-1 infection.

IV. The essence of the invention

The present invention relates to a number of neutralizing effective broad-spectrum anti-HIV-1 antibodies and to methods of their use. Accordingly, in some embodiments, the present invention includes an anti-HIV-1 antibody. In some embodiments, the anti-HIV-1 antibody is a broad-spectrum anti-HIV-1 neutralizing antibody (bNab) or an antigen-binding portion thereof. In some embodiments, the anti-HIV-1 antibody comprises NC37 and antigen-binding portions thereof. In some embodiments, the anti-HIV-1 antibody comprises BG1 and antigen-binding portions thereof. In some embodiments, the anti-HIV-1 antibody comprises BG18 and antigen-binding portions thereof. In some embodiments, the invention includes the BG18 variant and antigen-binding portions thereof. In some embodiments, the BG18 variant includes 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.

In some embodiments, the anti-HIV-1 antibody or antigen-binding portions thereof comprise a heavy chain variable region. In some embodiments, the heavy chain variable region comprises one of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, and 89. In some embodiments, the heavy chain variable region has at least 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 with 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

- 1 042959

SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81 and 89 have conservative substitutions.

In some embodiments, the anti-HIV-1 antibody or antigen-binding portions thereof comprise a light chain variable region. In some embodiments, the light chain variable region comprises one of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, and 93. In some embodiments, the light chain variable region has at least 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.

In some embodiments, the anti-HIV-1 antibody, or antigen-binding portion thereof, comprises at least one complementarity determining region (CDR) in the heavy chain variable region. In some embodiments, 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 at least 97%, at least 98%, or at least 99% homology with 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, 6668, 74-76, 82-84 and 90-92 have conservative substitutions.

In some embodiments, the anti-HIV-1 antibody, or antigen-binding portion thereof, comprises at least one complementarity determining region (CDR) in the light chain variable region. In some embodiments, 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 with 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.

In some embodiments, the implementation of the present invention relates to kits containing the first antibody. In some embodiments, the first antibody is an anti-HIV1 antibody, or an antigen-binding portion thereof. In some embodiments, the anti-HIV-1 antibody, or antigen-binding portion thereof, comprises a broad-spectrum anti-HIV-1 neutralizing antibody (bNab). In some embodiments, the broad spectrum anti-HIV-1 neutralizing antibody is any anti-HIV-1 antibody of 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 kits contain a second antibody. In some embodiments, the second antibody comprises an anti-HIV-1 antibody, or an 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 associated with a substrate. In some embodiments, the second antibody, or antigen-binding portion thereof, is associated with 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 and the second antibody or antigen-binding portion is detectably labeled. In some embodiments, the implementation of the detectable label contains a reporter molecule. In some embodiments, the implementation of the reporter molecule contains a fluorescent molecule. In some embodiments, the implementation of the reporter contains a radioactive label. In other embodiments, the detectable label contains an enzyme. In some embodiments, the kits include a substrate for the enzyme. In some embodiments, the addition of a substrate to the enzyme results in the generation of a detectable signal. In some embodiments, the detection signal includes a colored soluble product. In some embodiments, the implementation of the radioactive label includes I-125. In some embodiments, the implementation of the enzyme includes horseradish peroxidase. In some embodiments, the implementation of the substrate for the enzyme includes TMB. In some embodiments, the kits are capable of detecting HIV-1 or antigenic fragments thereof in a sample. In some embodiments, the kits provide a quantitative determination of HIV-1 or its antigenic fragments present in a sample.

In some embodiments, the kits further comprise an anti-HIV agent. In some embodiments, the anti-HIV agent is selected from the group consisting of non-nucleoside inhibitors.

- 2 042959 reverse transcriptase inhibitors (NNRTIs), nucleoside reverse transcriptase inhibitors (NRTIs), protease inhibitors (PIs), fusion inhibitors, CCRS antagonists/entry inhibitors, integrase strand transfer inhibitors (INSTIs) and combinations thereof. In some embodiments, the anti-HIV agent is in unit dosage form. In some embodiments, the anti-HIV-1 antibody, or antigen-binding portion thereof, is in a unit dosage form. In some embodiments, the unit dosage form is an injectable unit dosage form. In some embodiments, the anti-HIV agent is in the same container as the anti-HIV-1 antibody or antigen-binding portion thereof. In some embodiments, the anti-HIV agent is in a separate container. In some embodiments, the kits further comprise one or more additional anti-HIV-1 antibodies or antigen-binding portions thereof. In some embodiments, one or more additional anti-HIV-1 antibodies, or antigen-binding portions thereof, comprise any of the anti-HIV-1 antibodies of the present invention. In some embodiments, one or more additional anti-HIV-1 antibodies or antigen-binding portions thereof are selected from the group consisting of NC37, NC133, NC102, AC40, AC41, AC72, and combinations thereof. In some embodiments, the kits contain instructions for use. In some embodiments, the kits contain one or more pharmaceutically acceptable carriers or preservatives.

In some embodiments, the present invention relates to a method for detecting HIV-1, or an antigenic fragment thereof, present in a sample. In some embodiments, the implementation of the method includes obtaining a sample containing HIV-1 or its antigenic fragment. 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 an anti-HIV-1 antibody or antigen-binding portion thereof to HIV or an antigenic fragment thereof. In some embodiments, the implementation of the method includes the quantitative determination of HIV-1 or its antigenic fragments 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, in accordance with 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, detection of the presence of a specific binding is performed by an immunoassay. In some embodiments, detection of the presence of a specific binding is performed by a competitive immunoassay.

In some embodiments, the present invention relates to a method for diagnosing an individual as having an HMP-1 infection. In some embodiments, the method includes identifying an individual who has or is suspected of having HIV-1 infection. In some embodiments, the method comprises obtaining from an individual 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 anti-HIV-1 antibody, or antigen-binding portion thereof, is an anti-HIV-1 antibody, or antigen-binding portion thereof, in accordance with any aspect of the present invention. In some embodiments, the method includes detecting the presence of specific binding of an anti-HIV-1 antibody or antigen-binding portion thereof to HIV or an antigenic fragment thereof. In some embodiments, the method includes diagnosing an individual as HIV-1 infected. In some embodiments, the individual is diagnosed as having 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, detection of the presence of a specific binding is performed by an immunoassay. In some embodiments, detection of the presence of a specific binding is performed by a competitive immunoassay.

In some embodiments, the present invention relates to a method of treating an HIV-1 infection in an individual in need thereof. In some embodiments, a method for treating an HIV-1 infection comprises administering to an individual having or suspected of having an HIV-1 infection at least one anti-HIV-1 antibody, or an antigen-binding portion thereof. In some embodiments, at least one anti-HIV-1 antibody or antigen-binding antibody thereof

- 3 042959 The main part contains a broad-spectrum anti-HIV-1 neutralizing antibody (bNab) or an antigen-binding part thereof. In some embodiments, the implementation of at least one anti-HIV-1 antibody or antigennegative part includes any anti-HIV-1 antibody or antigennegative part in accordance with the present invention. In some embodiments, the implementation of the method includes prior to the stage of introduction of the stage of identifying the individual as HIV-1 infected. 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 broad-spectrum neutralizing anti-HIV-1 antibody (bNab) or antigen-binding portion thereof. In some embodiments, the implementation of at least one anti-HIV-1 antibody or antigennegative part includes any anti-HIV-1 antibody or antigennegative part in accordance with 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 method further comprises administering an anti-HIV agent. In some embodiments, the anti-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, CCRS antagonists/entry inhibitors, integrase strand transfer inhibitors ( INSTI) and combinations thereof. In some embodiments, the anti-HIV-1 antibody or antigen-binding portion thereof may be co-administered with additional anti-HIV-1 antibody or antibodies or antigen-binding portion(s) thereof and/or an anti-HIV agent or anti-HIV agents. In other embodiments, the anti-HIV antibody or antigen-binding portion thereof is administered prior to the administration of additional anti-HIV-1 antibody or antibodies or antigen-binding portion(s) thereof and/or the anti-HIV agent or anti-HIV agents. In other embodiments, the anti-HIV-1 antibody or antigen-binding portion thereof is administered following the administration of additional anti-HIV-1 antibody or antibodies or antigen-binding portion(s) thereof and/or the anti-HIV agent or anti-HIV agents. In other embodiments, the anti-HIV-1 antibody or antigen-binding portion thereof may be administered between additional anti-HIV-1 antibodies or antigen-binding portion(s) thereof and/or the anti-HIV agent or anti-HIV agents.

In another embodiment, the present invention relates to a passive vaccine. In some embodiments, the implementation of the vaccine contains at least one anti-HIV-1 antibody or antigennegative part. In some embodiments, the anti-HIV-1 antibody, or antigen-binding portion thereof, comprises a broad-spectrum anti-HIV-1 neutralizing antibody (bNab), or antigen-binding portion thereof. In some embodiments, the anti-HIV-1 antibody, or antigen-binding portion thereof, comprises a broad-spectrum anti-HIV-1 neutralizing antibody, or antigen-binding portion thereof, in accordance with 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 for preventing HIV-1 infection in an individual in need thereof. In some embodiments, the method includes administering to an individual a passive vaccine composition in accordance with any aspect of the present invention.

In some embodiments, the present invention relates to a nucleic acid. In some embodiments, the nucleic acid comprises 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 acid comprises 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 acid has conservative substitutions. In some embodiments, the implementation of the nucleic acid contains a nucleotide sequence encoding one or more CDRs of anti-HIV-1 antibodies 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 9496. In some embodiments, the nucleic acid has conservative substitutions.

In some embodiments, the present invention provides a vector or vector system comprising one or more nucleic acid sequences encoding the heavy and/or light chains and/or CDRs of one or more 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 heavy chain variable region and a nucleic acid sequence encoding a light chain variable region. In some embodiments, the nucleic acid sequence encoding the heavy chain is in the same vector as the nucleic acid sequence encoding the light chain. In some embodiments, the nucleic acid sequence encoding the heavy chain variable region is located in a different vector than the vector that contains the nucleic acid sequence encoding the light chain variable 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 vectors. In some embodiments, the phage vector is a γ flag. In some embodiments, the vector or vectors are a cosmid or cosmids. In some embodiments, the vector or vector system is a recombinant chromosome or recombinant chromosomes. In some embodiments, the vector system is a combination of different vectors. In some embodiments, expression of different nucleic acid sequences may be co-expressed. In other embodiments, expression of different nucleic acid sequences can be independently induced. In another embodiment, the present invention relates to a vector or system of vectors 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 according to the present invention.

V. Brief Description of Drawings

In FIG. 1A, 1B, 1C, 1D, 1E, 1F show the repertoire of anti-HIV-1 antibodies obtained from donor EB354. Fig. 1A: Viral load values measured in 2002-2015. The graph shows the time points when neutralizing antibodies were isolated. The point in time of plasma collection is marked with arrows pointing down. Fig. 1B: Heat map showing IC ₅₀ values obtained from TZM-bl assay for purified serum IgG at 4 time points (indicated by arrows in (1A)) tested against a panel of HIV-1 strains. All neutralization assays were performed in duplicate. Fig. 1C: Upper panels: IgG+ memory B cells pre-enriched with CD19 were stained with CD19 and three non-native HIV-1 baits: 2CC core, gp140 _YU2 folded trimer, and gp14092u _G37 . ₈ +gp140 _CZA7901 2 (gp140 A+C) trimers, and one native bait BG505.SOSIPAviB. Populations in square boxes represent cells obtained by sorting single cells, and the numbers indicate the percentage of CD19+/primer+ cells relative to the total number of IgG+ cells. Lower panels: The pie charts represent the total number of Ig sequences amplified from cells obtained by single cell sorting from the corresponding FACS plot shown above. The number in the middle of the charts indicates the total amount of antibodies. Empty slices are sequences presented in a single copy that do not have clonally related species. Sections represent antibody clones and are proportional to the number of sequences in each clone. Clones marked with an asterisk are clones that have demonstrated tier 2 neutralization and are variants NC37, BG18 and BG1. Fig. 1D: top panel: heat map showing neutralization efficiency based on IC ₅₀ values obtained in TZM-bl assay, monoclonal antibodies NC37, BG1 and BG18. Lower panel: Neutralization efficiency based on IC ₅₀ values obtained by TZMbl analysis of polyclonal IgG donor from 2014 sample and NC37, BG1 and BG18 to YU2 _WT , YU2 _N280Y , YU2N _N332K and YU2N _N160K . All TZM-bl assays were performed in duplicate. Fig. 1E: Heat map of neutralization efficiency, on IC ₅₀ values obtained from the TZM-bl assay, of NC37, BG1, BG18 and 1:1:1 combination antibodies (comb) tested against a panel of 120 level 2 HIV-1 pseudoviruses. Mean the geometric value of all neutralized viruses and the percentage of coverage are shown in the bottom panel. Neutralization assays were performed in duplicate. Fig. 1F: coverage curve based on values from (1E).

In FIG. 2A, 2B, 2C, 2D, 2E, 2F, 2G show the sequence and structural analysis of bNab BG18. Fig. 2A: Amino acid sequence alignment of heavy chains 10-1074, PGT121 and BG18. The asterisks represent the gaps that occur during the alignment process. Arrows indicate positions in the framework at which all three antibodies are mutated relative to their respective germline genes. CDR H1, H2 and H3 are indicated. Fig. 2B: Light chain sequence alignment as in (A) for 10-1074, PGT121 and BG18. Fig. 2C: left panel: V _H domain strip chart

- 5 042959 (dark grey) and V _L (light grey) Fab BG18 shown at 1.3 A resolution. Middle panel: BG18 variable region surface representation. Right panel: The CDRH3 conformation is stabilized by hydrogen bonds (shown in dotted lines) within the loop. Interacting residues are shown as rods, the CDRH3 residue and adjacent CDRH1 are shown as strip charts. Fig. 2D: left panel: strip charts showing the overlay of the BG18 (purple), PGT121 (green) and 10-1074 (blue) variable domains. CDRH3 BG18 in red, CDRH3 loops PGT121 and 10-1074 in maroon. Right panel: V _L BG18 (light pink) and PGT121 (light green) are shown after V _H overlay. CDRL2 _BG18 does not have an ordered structure in the crystal and is indicated by a dotted line. 10-1074 is closely related to PGT121 and is not shown for clarity. Fig. 2E: Representation of BG18 and PGT121 surfaces. Fig. 2F: EM structure of a single particle (transparent gray density) BG505 SOSIP.664 associated with Fab BG18 and Fab 179NC75. Coordinates for BG505 SOSIP.664, Fab BG18 and model for Fab 179NC75 (Fab CH103: PDB 4JAM) were fitted into EM density as described in Example 1 below. Average values of 2D classes are given. Fig. 2G: Comparison of BG505 binding angle of Fab BG18 versus Fab 10-1074, PGT122 (PDB 5FYJ), PGT135 (PDB 4JM2) and PGT128 (PDB 5ACO). The densities for Fabs 179NC75 have been subtracted from the BG18-179NC75-BG505 map to facilitate comparisons.

In FIG. 3A, 3B, 3C, 3D, 3E show autologous viruses present in plasma from an EB354 donor. Fig. 3A: Maximum likelihood phylogenetic tree of env gene sequences derived from a single genome from EB354 donor samples taken in 2006, 2010, 2013 and 2014. A red asterisk indicates phylogenetic branches with a bootstrap score of >90%. The three main groups of sequences are arbitrarily named clusters A, B, and C. The three triangles indicate env sequences that lack an N-linked glycosylation site. The squares highlighted with dotted lines indicate the VOC of the culture; the number in brackets represents the number of env viral sequences in the VOC. Fig. 3B: Scatterplots depicting pairwise nucleotide sequence diversity for plasma env sequences obtained in 2010, 2013 and 2014. Each point represents a genetic difference in a pairwise comparison of two sequences at a given point in time. P-values were determined using a Z-test based on two-sample U-statistics and indicate ***p<0.0005. Fig. 3C: Heatmaps showing IC ₅₀ values obtained by TZM-bl analysis for BG18, BG1, NC37, and IgG purified at the same time point as viral env genes to autologous pseudoviruses using donor EB354 env sequences . Stars denote pseudoviruses that are resistant to all three autologous bNAbs. The 3BNC117 antibody (described in US 2014/0328862) served as a control. The circles on the left correspond to the time points at which the sequence indicated in (3A) was received. Neutralization assays were run in duplicate and repeated at least twice. Gray squares indicate that the analysis was not performed. Fig. 3D: Pie charts represent the sensitivity of autologous pseudoviruses, obtained using donor EB354 env sequences, to each of the three autologous bNAbs at the different time points analyzed. The number in the middle of the circle means the number of pseudoviruses env tested, and the different slices are proportional to the number of pseudoviruses. bNAbs are indicated on the left, and the time points at which the samples were obtained are indicated above the graphs. The bars below the circles show when antibody transcripts were detected by PCR. Fig. 3E: IC ₅₀ values obtained by TZM-bl assay for BG18, BG1, NC37 and control bNAb 3BNC117 to VOCs derived from EB354 donor CD35 T cells in 2014 and 2015.

In FIG. 4A, 4B, 4C, 4D, 4E show HIV treatment of _YU 2 infected mice. Fig. 4A: Viral load values in four (BG18) and five (NC37) HIV _YU2 -infected hu mice before and after BG18 (upper panel) or NC37 (lower panel) monotherapy. The shaded gray area on the graph indicates the time period during which BG18 (top) or NC37 (bottom) was administered. Bold lines indicate geometric mean values in each treatment experiment. Fig. 4B: Amino acid sequence alignment of gp120 from viruses cloned 21 days after treatment with BG18 (top) or NC37 (bottom). Each horizontal bar represents the sequence of one gp120 clone aligned with HIV-1 _YU2 . Amino acid substitutions are indicated as ticks. The identifier (ID) of the mouse from which the sequence was obtained is indicated on the vertical columns. An enlarged view of the regions, shown in individual squares, is shown on the right side of each panel. Fig. 4C: Pie charts showing recurrent mutations for BG18 (top) and NC37 (bottom) in gp120 relative to the wild-type HIV-1 _YU2 sequence. The different cuts are proportional to the number of sequences that carry the mutations. The number in the center of the circle indicates the total number of cloned sequences. A white cut in NC37 indicates the absence of any recurring mutations. Fig. 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 (open icons). T3-T7 mice received the BG18+NC37+BG1 combination (solid symbols). The shaded area indicates the time period during which the bNAb combination was administered. The geometric mean values are shown by bold lines,

- 6 042959 the dotted line refers to the untreated group and the solid lines to the treated group. Fig.

4E: Log10 difference in viral load compared to day 0 (before antibody administration). An asterisk indicates an outlier value.

In FIG. 5A, 5B show the results of a competitive ELISA. Each of the neutralizing antibodies (equal amounts) was analyzed by ELISA for binding to BG505 SOSIP.664 in the presence of increasing amounts of different competing antibodies. The black line indicates the binding of the biotinylated antibody in the absence of competition. Fig. 5A: BG18, fig. 5V: BG1.

In FIG. 6A, 6B, 6C, 6D are sequence comparisons between BG18, PGT121 and 101074. FIG. 6A: Dendrogram of the heavy chain sequences of PGT121, 10-1074, and nine BG18 clonal variants and their predicted germlines. Fig. 6B: heavy chain CDR alignment. Positions common to PGT121/10-1074 and BG18 variants are highlighted. Fig. 6C: same as (6A) but for light chains. Fig. 6D: Light chain CDR alignment. Positions common to PGT121/101074 and BG18 variants are highlighted.

In FIG. 7 shows the electrostatic potential of BG18. Electrostatic surface potentials Fab were calculated using the APBS program, and the surface representation was obtained using UCSF Chimerac shading. The predicted binding interfaces are shown with a dotted black line. Exemplary areas of recognition on the surfaces of Fab PGT121 and 10-1074 gp120 glycans attached to Asn137gp120, Asn156gp120 and Asn332gp120 are indicated by black triangles.

In FIG. 8A, 8B, 8C show the structure of the NC37 Fab-gp120 complex. Fig. 8A: Crystal structure (at 2.7 A resolution) of the NC37 Fab-gp120 complex superimposed on the structure of the 8ab134 Fab-gp120 complex (PDB 4RX4) shows a similar gp120 binding orientation in the two antibodies. Fig. 8B: A close-up of the Fab-gp120 interface shows the increased CDRH3 of the NC37 compared to the 8ANC134 CDRH3. Fig. 8C: Side view of the BG505 trimer surface structure (PDB 4TVP) with the predicted epitopes for NC37, 8ANC134 and NIH45-46 shown on one trimer protomer. The longer CDRH3 NC37 suggests a larger contact area on the adjacent protomer (light grey) of the trimer.

In FIG. 9A, 9B, 9C, 9D, 9E, 9F show binding of the BG18 antibody to HIV-1 synthetic V3 glycopeptides. Biotin-labeled V3 glycopeptides were immobilized on a neutravidin chip, and IgG BG8 was used as an analyte. Fig. 9A: Interaction between BG18 and the JR-FL mini-V3 glycopeptide carrying the Man9GlcNAc2 glycan in N301. Weak binding is observed. Fig. 9B: Interaction between BG18 and the JR-FL mini-V3 glycopeptide carrying the Man9GlcNAc2 glycan in N332. Weak binding is observed. Fig. 9C: Interaction between BG18 IgG and JR-FL mini-V3 glycopeptide carrying the 2-antennary N-glycan complex type in N301. Linking is not observed. Fig. 9D: Interaction between BG18 IgG and the JR-FL mini-V3 glycopeptide carrying the 2-antennary N-glycan complex type in N332. Linking is not observed. Fig. 9E: Interaction between BG18 IgG and A244 mini-V3 glycopeptide carrying the Man9lGcNAc2 glycan in N334. Weak binding is observed. Fig. 9F: Interaction between BG18 IgG and CAP45 mini-V3 glycopeptide carrying the Man9GlcNAc2 glycan in N334. Weak binding is observed.

In FIG. 10A, 10B, 10C show the sequencing of a single virus genome obtained from the plasma of an EB354 donor. Fig. 10A: The pie charts indicate the total number of env sequences amplified by PCR from a single genome for each sample collection time point. The total number of sequences obtained at each time point is shown in the center of the pie chart, and the different slices are proportional to the number of sequences. Fig. 10B: Maximum likelihood phylogenetic tree of env gene sequences isolated from a single genome from EB354 donor samples collected in 2006, 2010, 2013 and 2014. An asterisk denotes phylogenetic branches with a bootstrap score of >90%. The table on the right shows the values of TSH ₅₀ and IC ₅₀ obtained in TZM-bl analysis, for BG18, BG1, NC37 and control antibody 3BNC117 to autologous pseudoviruses using the env sequences of donor EB354. Fig. 10C: Comparison of TSS ₅₀ values for pseudoviruses using EB354 donor env that were found to be resistant to all three bNAbs, in contrast to pseudoviruses using EB354 donor env sequences that were found to be susceptible to at least one bNAb. P-value (t-test)=0.0016.

In FIG. 11A, 11B, 11C show the results of treatment with BG8 antibody of HIV _YU 2 infected hu mice. Fig. 11A: Viral load values in four hu mice before and after BG8 administration. The shaded gray area on the graph indicates the time period during which the antibody was administered. Bold lines represent geometric mean values. Fig. 11B: Amino acid sequence alignment of gp120 from viruses cloned 21 days post-treatment. Each horizontal gray bar represents the sequence of a single gp120 clone aligned to HIV-1 _YU 2. Amino acid substitutions are indicated by black checkmarks. The identifier (ID) of the mouse from which the sequence was obtained is indicated on the vertical black bars. An enlarged view of the areas shown in individual squares is shown on the right side of each panel. Fig. 11C: Pie charts showing recurrent mutations in gp120 relative to sequence

- 7 042959

HIV-1 _YU2 wild-type after BG8 therapy. The number in the center of the circle indicates the total number of cloned sequences; slices represent the most stably mutated regions in gp120 and are proportional to the number of sequences that carry mutations.

In FIG. 12A, 12B show viral loads in HIV _YU2 -infected, antibody-treated mice. Viral load values in HIV _YU2 -infected humanized mice before and after BG18 (FIG. 12A) and NC37 (FIG. 12B) monotherapy. The shaded area on the graph indicates the time period during which the antibody was administered. Each graph shows the results of two independent experiments, indicated by circles (one experiment) and squares (second experiment). Filled and unfilled forms denote treated and control mice, respectively. The dotted and solid thick lines show the mean values for control and treated mice, respectively.

VI. Detailed description of the invention

A. Definitions.

It is known in the art that an anti-HIV-1 antibody can take one of numerous forms as described herein. Antibodies are partly determined by the antigens to which they bind, thus an anti-HIV-1 antibody is any antibody that specifically binds to at least one epitope found in the human immunodeficiency virus 1 (HIV-1) viral envelope, such as in gp120 and/or gp140 described in this application. It is known in the art that an antibody is a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or an antigen-binding portion thereof. The heavy chain consists of the heavy chain variable region (VH) and the heavy chain constant region (CH1, CH2 and CH3). The light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The variable regions of both heavy and light chains include framework regions (FWRs) and complementarity determining regions (CDRs). The four FWR regions are relatively conserved, while the CDR regions (CDR1, CDR2 and CDR3) are hypervariable regions and are located in the direction from the NH ₂ -terminus to the COOH-terminus as follows: FWR1, CDR1, FWR2, CdR2, FWR3, CDr3, FWR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen, while the constant region(s), depending on the isotype, can mediate the binding of the immunoglobulin to host tissues or factors. It is known in the art that it is possible to manipulate monoclonal and other antibodies and use recombinant DNA technology techniques to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such methods may include introducing DNA encoding an immunoglobulin variable region or CDRs of an antibody into constant regions or constant regions plus framework regions of another immunoglobulin.

As used herein, the term antibody (Ab) is used in its broadest sense and specifically can include any immunoglobulin, whether natural or partially or fully synthesized, including, without limitation, monoclonal antibodies, polyclonal antibodies, polyspecific antibodies (e.g., bispecific antibodies and polyreactive antibodies) and antibody fragments. Thus, the term antibody, as used herein in any context, includes, without limitation, any specific binding element, class, and/or isotype of an immunoglobulin (e.g., IgG1, IgG2a, IgG2b, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE) and a biologically relevant fragment or specific binding element thereof, including, without limitation, Fab, F(ab') ₂ , scFv (single chain or related entity) and (scFv) ₂ .

Used in this application, the term antibody fragments may include fragments of antibodies, which are obtained by methods well known and available to specialists in this field, described in this application. Therefore, in addition to the above definition of antibody, the term antibody can further encompass any polypeptide or protein containing a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody. They may be obtained from natural sources or may be synthesized in whole or in part. Examples of antibody fragments include, without limitation, 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 antigens. One skilled in the art will appreciate that a Fab consists of one constant and one variable domain each of an antibody heavy and light chain.

As used herein, the term monoclonal antibody or mAb may refer to an antibody derived from a population of substantially homogeneous antibodies, ie. the individual antibodies that make up a population are identical except for possible naturally occurring mutations that may be present in minor amounts.

Monoclonal antibodies may include chimeric antibodies, humanized antibodies, recombinant antibodies, and human antibodies. Methods for making monoclonal antibodies are well known in the art, including some specific examples discussed below, including hybridoma technology, production of recombinant antibodies such as phage display or yeast display technology, production of transgenic mice, and cultures of single B -cells. Techniques that involve the amplification of heavy and/or light chain antibody genes (e.g., plasmids or cosmids) by PCR (or a similar method) either in vitro or in bacterial and/or mammalian and/or yeast systems are included in the scope of the present invention.

As used herein, the terms conservative sequence modifications or conservative substitutions may refer to amino acid modifications to the target epitope of the invention that do not significantly affect or alter the binding characteristics of anti-HIV-1 antibodies to the epitope(s). Such conservative modifications include amino acid substitutions, additions and deletions. Conservative amino acid substitutions are substitutions 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 are 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), non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine, phenylalanine , tryptophan, histidine). Thus, one or more amino acid residues in the target epitope to which the anti-HIV-1 antibodies of the invention specifically bind, for example epitopes on the envelope of the HIV-1 virus, for example epitopes on gp120 and/or gp140, can be replaced with other amino acid residues from the same family of side chains, and the antibodies of the present invention can be tested against the target epitope, for example, using functional assays described in this application or otherwise known in the art.

The term biological sample refers to a sample obtained from an organism (eg, a patient) or from components (eg, cells) of an organism. The sample may be any biological tissue, cell(s), or fluid. The sample may be a clinical sample, which is a sample obtained from a subject, such as a human patient. Such samples include, without limitation, saliva, sputum, blood, blood cells (e.g., white blood cells), bodily fluids, lavage, pancreatic juices, gastric juices, secretions, CSF, amniotic lymphatic fluid, plasma, semen, bone marrow, and tissue biopsy samples or punch biopsy samples, urine, stool, peritoneal fluid and pleural fluid, or cells thereof, and any combinations thereof. Biological specimens may also include tissue sections, such as frozen sections taken for histological purposes. A biological sample may also be referred to as a patient sample. The biological sample may also include a substantially purified or isolated protein, a membrane preparation, or a cell culture.

The term bispecific antibody refers to artificial immunoglobulin constructs consisting of fragments from two different monoclonal antibodies that bind to two different antigens. There are several different types of bispecific antibodies, including, without limitation, trifunctional antibodies and chemically linked Fabs. Anti-HIV-1 antibodies of the present invention may include bispecific antibodies and fragments of one or more different anti-HIV-1 antibodies, including one or more different anti-HIV-1 antibodies of the present invention, for example, BG18 and BG1 or one or more anti-HIV-1 antibodies. HIV-1 antibodies of the present invention and known anti-HIV-1 antibodies such as BG18 and 3BNC117 (described in US 2014/0328862) or BG18 and VRC01 described in US 8,637,036. Bispecific bNabs for HIV and methods for their preparation and use are described in PCT/US16/64713, incorporated herein by reference.

As used herein, the terms effective amount or therapeutically effective amount may refer to an amount of a compound or agent that is capable of providing a medically desired result in a subject being treated. The method of treatment can be performed in vivo or ex vivo, alone or in combination with other drugs or therapy. A therapeutically effective amount may be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular drug or route of administration.

The terms specific binding, selective binding, selectively binds, and specifically binds may refer to the binding of an antibody to an epitope on a predetermined antigen, but not to other antigens. Typically, an antibody will bind with an equilibrium dissociation constant (KD) of less than about 10 ^-6 M, for example, less than about 10 ^-7 M, 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M, or even lower, as determined by for example, by equilibrium dialysis or surface plasmon resonance (SPR) in a BIACORE® 2000 surface plasmon resonance instrument using a predetermined antigen, such as an epitope on the envelope of the HIV-1 virus, such as gp120, as the analyte, and an antibody as ligand, or by Scatchard analysis of antibody binding to antigen-positive cells, and (ii) binds to a predetermined antigen with an affinity that is at least twice its binding affinity for a non-specific antigen (e.g., BSA, casein) other than from a predetermined antigen or a closely related antigen.

As used herein, the term homology may refer to the existence of a common structure between two compositions. The term homology in the context of proteins can refer to the amount (eg, expressed as a percentage) of overlap between two or more amino acid and/or peptide sequences. In the context of nucleic acids, the term may refer to the amount (eg, expressed as a percentage) of overlap between two or more nucleic acid sequences. As used herein, the percentage (%) of homology between two sequences is equivalent to the percentage of identity between the two sequences. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions/total number of positions x100), taking into account the number of gaps and the length of each gap that must be entered to optimally align the two sequences. Sequence comparison and determination of percent identity between two sequences can be performed using a mathematical algorithm. Such homology is well represented in the art through local alignment tools and/or algorithms, such as BLAST and/or BLAST 2.0, and may include pairwise alignment methods, multiple sequence alignment methods, structural alignment methods, and/or phylogenetic analysis methods.

As used herein, the terms co-administration, co-administration, and in combination with may refer to the administration of at least two agents or therapies to a subject. In some embodiments, the simultaneous administration of two or more agents/therapies is simultaneous. In other embodiments, the first agent/therapy is administered prior to the second agent/therapy. Those skilled in the art will appreciate that the formulations and/or routes of administration of the various agents/therapies employed may vary. For example, the anti-HIV-1 antibodies of the present invention may be co-administered with each other or may be co-administered with other antibodies, e.g., other broad spectrum neutralizing antibodies such as, for example, the broad spectrum anti-HIV neutralizing antibodies disclosed in US 2014/0328862, such as 3BNC117 or VRC01 as described in US Pat. Co-administration may or may not occur simultaneously, for example, one or more anti-HIV-1 antibodies of the present invention may be administered before or after another anti-HIV-1 antibody of the present invention (or other anti-HIV antibody or other anti-HIV antibodies). agents) or may be administered at the same or substantially the same time. One of skill in the art will appreciate that co-administration is not intended to be limited in time as it is intended to describe administration with other compounds and/or therapies as part of a patient's treatment regimen.

As used herein, the term anti-HIV agent may refer to a therapy for the treatment of HIV that does not itself include anti-HIV-1 antibodies. For example, non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleoside reverse transcriptase inhibitors (NRTIs), protease inhibitors (PIs), fusion inhibitors, CCRS antagonists/entry inhibitors, integrase transfer inhibitors (INSTIs), and combinations thereof, are all anti-HIV agents, as they are non-antibody based therapies that can be administered in conjunction with one or more anti-HIV-1 antibodies of the present invention and/or additional anti-HIV-1 antibodies. One of ordinary skill in the art will appreciate that this list should not be considered exhaustive, and as modern advances and additional processing techniques become available, they are also within the scope of this definition.

As used herein, the term carriers may include pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to the cell or mammal exposed to the substances at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffer solution. Examples of physiologically acceptable carriers include, without limitation, buffers such as phosphate, citrate and other organic acids; antioxidants, including, without limitation, ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins such as, without limitation, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as, without limitation, polyvinylpyrrolidone; amino acids such as, without limitation, glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including, without limitation, glucose, mannose, or dextrins; chelating agents such as, without limitation, EDTA; sugar alcohols such as, without limitation, mannitol or sorbitol; salt-forming counterions such as, without

- 10 042959 restrictions, sodium; and/or nonionic surfactants such as, without limitation,

Twin; polyethylene glycol (PEG) and PLURONICS.

The term treatment of a disease refers to the implementation of a protocol, which may include the administration of one or more drugs to a patient (human or otherwise) to relieve the signs or symptoms of the disease. Relief can occur before the onset of signs or symptoms of the disease, as well as after they appear. Thus, treatment includes prevention of the disease. The term prophylaxis refers to prophylactic and/or preventive measures, the purpose of which is to prevent or delay the development of the target pathological condition or disorder. For example, in the case of infection with the immunodeficiency virus 1 (HIV-1), prophylaxis may occur in a situation of prior treatment to prevent or block infection with HIV-1, such as passive vaccination. Such prophylaxis also occurs in the case of latent HIV-1 infection, for example, in those people who are seropositive without showing any symptoms (i.e. without progressing to the onset of AIDS symptoms), while preventing the development of active infection and / or clearing the patient of said HIV-1 infection. In addition, treatment does not necessarily lead to complete relief of signs or symptoms, a cure and, in particular, it includes protocols that have only a minimal but positive effect on the patient.

As used herein, the term epitope can refer to the region of an antigen to which an antibody or T cell binds, such as the envelope region of the HIV-1 virus, including but not limited to a glycoprotein, such as gp120 or a region on a glycoprotein. Antigen refers to a substance that causes an immunological reaction or binds to the products of this reaction.

The terms purified or isolated antibody, peptide, polypeptide or protein refer to a peptide, polypeptide or protein as used herein and may refer to a peptide, polypeptide or protein that has been separated from other proteins, lipids and nucleic acids with which it is bound in natural conditions. The polypeptide/protein may be at least 10% (i.e., any percentage from 10 to 100%, such as 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, and 99%) of calculated on the dry weight of the purified drug. Purity can be measured by any suitable standard method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. The isolated polypeptide/protein (eg, anti-HIV-1 antibodies) described in the invention can be obtained by recombinant DNA methods.

B. Broad-spectrum anti-HIV-1 neutralizing antibodies.

The present invention relates to broad spectrum anti-HIV-1 neutralizing antibodies (bNab) 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, without limitation, their complementarity determining regions (CDRs), as well as their derivatives and variants, 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 strong therapeutic efficacy when used either alone or in combination with each other or with other bNabs or other HIV treatments as discussed herein above.

Each of BG18, NC37 and BG1 has different non-overlapping epitopes. NC37 recognizes CD4bs. NC37 and its clonal variants show characteristics of bNAbs derived from both V _H1-2 and V _H1-46 ; however, according to the results of the structural analysis, it is assumed that NC37 recognizes Env as type V _H1-46 . While not wishing to be bound by any theory, it is hypothesized that the long CDRH3 of NC37 makes contact with an adjacent protomer on the trimer, thereby recognizing a trimer quaternary epitope whose core overlaps with CD4bs.

BG1 binds to the V1V2 Env region, another common bNAb target that occurs during natural infection. BG1 is the first antibody in this class (bNab) isolated from a donor infected with type (phylogenetic branch) B. Its activity is similar to members of the VRC26 antibody family, such as described by N.A. Doria-Rose et al., Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies. Nature 509, 55-62 (2014), but it is less effective than bNAb PG9/16 and PGDM1400. Like other V1V2 bNAbs isolated to date, BG1 has a long, tyrosine-rich CDRH3.

BG18, the most potent of BG18, NC37, and BG1, targets the Asn332 _gp120 -centered glycan site at the base of the V3 loop. This is a heavily glycosylated region that includes carbohydrates at positions Asn332 _gp120 , Asn301 _gp120 , Asn386 _gp120 , Asn392 _gp120 , Asn137 _gp120 , Asn156 _gp120 . A number of monoclonal bNAbs have been isolated that bind to both protein and carbohydrate components at this site, including PGT121-124 described in LM Walker et al., Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature 477, 466-470 (2011), and 10-1074 described in 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 109, E3268-3277 (2012). All of these bNAbs have been isolated from a type A infected donor and can be subdivided into antibodies that

- 11 042959 bind exclusively to the Asn332 _gp1 2o glycan (10-1074 and PGT124), and those that also bind to surrounding glycans in the V3 glycan region (PGT121-123). BG18 and its clonal variants are similar to 10-1074 in that they involve Asn332 _gp120 , but BG18 is more potent than published anti-V3 bNAbs and was first isolated from a type B infected donor.

BG18 differs from previously characterized members of this class in its remarkably characteristic orientation of the CDRH3 and light chain domain. In addition, BG18 differs from other members of this group of antibodies in that it has a shorter CDRH3 and does not contain insertions or deletions, although, without wishing to be bound by any theory, it can be assumed that BG18-like antibodies are more easily generated. In the absence of the high resolution structure of the BG18-Env complex, it is difficult to predict how the shorter BG18 CDRH3 interacts with Env. Structural studies of Env complexes with Fab PGT122 and 10-1074 show that they approach the gp120 protomers in the Env trimer at approximately the same angles, approximately perpendicular. The EM structure of BG18-Env, although it does not have sufficient resolution to more accurately determine the type of interactions, nevertheless demonstrates that BG18 approaches Env at a different angle, which is shifted by 40° to the gp120 promoter relative to the approach angles of 10-1074 and PGT122 . Clonal variants and derivatives of BG18, including 354BG8, 354BG18, 354BG42, 354BG33, 354BG129, 354BG188, 354BG411, and 354BG426, as well as their full length heavy and light chain complementarity determining region (CDR) sequences, are shown in Table 1 below. 1. The dendrogram of BG18 variants is shown in FIG. 6 and discussed in Example 1 below. BG1 clonal variants and derivatives, including BG1, BG22, and BG47, along with their full length heavy and light chain complementarity determining regions (CDR) sequences, are shown in Table 1 below. 2.

Name SEQ ID NO: Subsequence 354BG8IgH 1 EVOLRESGPRLVKPSETLSLSCDVFGDSRPSDHSWTWVROPPG KALEWIGDVHYNGDNTYNPSLRGRVKIDVDRSTHRFSLTLKSL TAADT GIY FCARNVIRVFGVISLGEWFHYGMDVWGPGTAVIVS S CDR1 2 GDSRPSDHS CDR2 3 VHYNGDN CDR3 4 NVIRVFGVISLGEWFHYGMDV 354BG8 IgL 5 SSELTOAPSVSVSPGOTATIACSGPPLASRYTYWYRQKPGOAP VLIIFRPRQFPSGVSGRFSASKSGTTATLTIRDVQVEDEGDYY CQSSDTSDSYKMFGGGTTL TVL CDR1 6 PLASRY CDR2 7 RPR CDR3 8 QSSDTSDSYKM 354BG18 IgH 9 S CDR1 10 NDSRPSDHS CDR2 eleven IHYNGAT CDR3 12 NAIRIYGVVALGEWFHYGMDV 354BG18 IgL 13 SSELTQPPSVSVSPGQTARITCSGAPLTSRFTYWYRQKPGQAP VLIISRSSQRSSGWSGRFSASWSGTTVTLTIRGVQADDEADYY CQSSDTSDSYKMFGGGTKLTVL CDR1 14 PLTSRF CDR2 15 RSS CDR3 16 QSSDTSDSYKM

- 12 042959

354BG42 IgH 17 EVOLRESGPGLVKPSETLSLSCDVFGDSRPSDHSWTWVROPPG KALEWIGDVHYNGDTTYNPSLRGRVKIDVDRSTHRFSLTLNSL TAADT GIY FCARNVIRVFGVISLGEWFHYGMDVWGOGTAVTVS S CDR1 18 GDSRPSDHS CDR2 19 VHYNGDT CDR3 20 NVIRVFGVISLGEWFHYGMDV 354BG42 IgL 21 SSELTOAPSVSVSPGOTATIACSGPPLASRYTYWYROKPGOAP VLIIFRORQFPSGVSGRFSASKSGTTATLTIRDVQVEDEGDYY CQSSDTSDSYKMFGGGTTL TVL CDR1 22 PLASRY CDR2 23 RPR CDR3 24 QSSDTSDSYKM 354BG33 IgH 25 OVOLRESGPGLVKPSETLSLTCTVSNDSRPSDHSWTWVRQSPG KALEWIGDIHYNGATTYNPSLRSRVRIELDQSIPRFSLKMTSM TAADTGMYYCARNAIRIYGVVALGEWFHYGMDVWGOGTAVTVS S CDR1 26 NDSRPSDHS CDR2 27 IHYNGAT CDR3 28 NAIRIYGVVALGEWFHYGMDV 354BG33 IgL 29 SSELTQPPSVSVSPGOTAKITCSGAALTSRFTYWYRQKPGOAP VLIISRTSQRSSGWSGRFSASWSGTTVTLTIRGVQADDEGDYY CQSSDTSDSYKMFGGGTKLTVL CDR1 thirty ALTSRF CDR2 31 RTS CDR3 32 QSSDTSDSYKM 354BG129 IgH 33 P CDR1 34 GDSRPSDHS CDR2 35 IHYGGDI CDR3 36 NVIRVFGVIALGEWFHYGMDV 354BG129 IgL 37 SSELTQTPSVTVSPGETARIACSGPPLASRYCYWYRQKPGQAP VL11FRDRQFSS GMS GRFAS SHSGTTVTLTIRDVRVE DEADYY

- 13 042959

COSSPINPSYKMFGGGTKVTVL CDRl 38 PLAS-RY CDR2 39 RPR CDR3 40 QSSPINPSYKM 354BG188 IgH 41 EVQLRESGPGLVKPSGNMALTCTISGPSRPSPHSWTWVRQSPG KTLEWIGPIHYGGPITYNPSLRSRVKLEVPTSSNRFFLKMTSL TVADT GIY FCARNVIRVFGVIALGEWFHYGMPVWGOGTAITVS P CDRl 42 GDSRPSDHS CDR2 43 IHYGGDI CDR3 44 NVIRVFGVIALGEWFHYGMPV 354BG188 IgL 45 SSELTOTASVTVSPGETARIACSGPPLASRYCYWYRQKPGOAP VLIIFRpRQFSSGISGRFSSSQSGTTVTLTIRDVRVEDEADYY CQSSPTSPSFKMFGGGTKLTVL CDRl 46 PLASRY CDR2 47 RPR CDR3 48 QSSPTSPSFKM 354BG411 IgH 49 OVOLRESGPGLVKPSGNMALTCTISGPSRPSPHSWTWVROSPG KALEWIGPIHYGGPITYNPSLRSRVELEVPRSTNRFFLKMTSL SVAPTGMYFCARNVIRVFGVIALGEWFHYGMPVWGOGTAITVS P CDRl 50 GPSRPSPHS CDR2 51 IHYGGPI CDR3 52 NVIRVFGVIALGEWFHYGMPV 354BG411 IgL 53 SSELTOAPSVTVSPGPTARIACSGPPLATRYCYWYRQKSGOAP VL11FRPRQFSSGVSGRFSSSQSGSTVTLTIRPVRVE PEAPYY CQSSPTSPSYKMFGGGTKLTVL CDRl 54 PLATRY CDR2 55 RPR CDR3 56 QSSPTSPSYKM 354BG419 IgH 57 OVOLRESGPGLVKPSETLSLSCPVFGPSRPSPHSWTWVROPPG KALEWIGPIHYNGPKTYNPSLRGRVKIPVPRSTHRFSLTLNSL TAAP T GMY FCARNVIRVFGVISLGEWFHYGMPVWGPGTAVTVS S CDRl 58 GPSRPSPHS

- 14 042959

CDR2 59 IHYNGDK CDR3 60 NVIRVFGVISLGEWFHYGMDV 354BG419 IgL 61 SSELTOAPSVSVSPGOTARIACSGPPLASRYTYWYROKPGOAP VLIIFRDRQFPSGVSGRFSASKSGTTGTLTIRDVQAEDEGDYY COSSDTSDSYKMFGGGTTL TVL CDR1 62 PLASRY CDR2 63 RPR CDR3 64 QSSDTSDSYKM 354BG426 IgH 65 OVOLRESGPGLVKPSGNMALTCTISGDSRPSDHSWTWVRQSPG KALEWIGDIHYGGDITYNPSLRSRVKLEVDTSSNRFFLKMTSL TVADT GIY FCARNVIRVFGVIALGEWFHYGMDVWGOGTAITVS P CDR1 66 GDSRPSDHS CDR2 67 IHYGGDI CDR3 68 NVIRVFGVIALGEWFHYGMDV 354BG426 IgL 69 SSELTOAPSVTLSPGETARIACSGPPLASRYCYWYRQKPGOAP VLIIFRDRQFSSGISGRFSSSQSGTTVTLTIRDVRVEDEADYY CQSSDNSDSFKMFGGGTKLTVL CDR1 70 PLASRY CDR2 71 RPR CDR3 72 QSSPNSPSFKM

Table 1. BG18 variant sequences.

Name SEQ ID NO: Subsequence BGlIgH 73 AEOLVESGGGLVPPGRSLRLSCSASGFYFPDYAMAWVROAPGO GLOWVGFMRGWAYGGSAQFAAFAVGKFAISRDDGRNWYLDVK NP T FE D T GVY FCAREQRNKDYRYGQEGFGYSYGMDVWGRGTTV WST CPR1 74 GFYFPDYA CPR2 75 MRGWAYGGSA CPR3 76 EQRNKDYRYGQEGFGYSYGMDV BG1 IgL 77 DIHMTQSPVSLSASVGDRVTITCRASHFIANYVNWYQQKPGKA PTTLIFESSTLORGVPSRFSAYGDGTEFTLSINTLQPEDFASY ICOQSHSPPVTFGAGTRVDQK CPR1 78 HFIANY

- 15 042959

CDR2 79 ESS CDR3 80 QQSHSPPVT BG22 1yN 81 EERLVESGGGLVPPRSLRLSCSAFDFYFPDYAMAWVRQAPGK GLE WIG FIRGWAYGQAAQ YGKS AS GRMTIS RDDS RRWYL DIK S PIEE DT GAY FCAREQRGGDGRYSGDGFGYPYGMDVWGRGTMV TVSA CDR1 82 DFYFPDYA CDR2 83 IRGWAYGQAA CDR3 84 EQRGGDGRYSGDGFGYPYGMDV BG22 IgL 85 DILMTQSPVSLSASIGERITITCRASHFIANYVNWYQQRPGKA PKLLIFQSWTLNRGIPSRFSGYGDGTEFTLSISALQSEDFGTY ICQQSHSPPLSFGGGTRVDQT CDR1 86 HFIANY CDR2 87 QSW CDR3 88 QQSHSPPLS BG47 IgH 89 EERLVESGGGLVPPGRSLRLSCSAFDFYFPDYAMAWVRQAPGR ALE WIG FIRGWAYGQSAQ YGKS AS GRMT IS RDDS RRWYL DIK S P T HE D T GVY FCAREQRGANGRYGGDGFGYSYGMDVWGRGTMV SVSA CDR1 90 DFYFPDYA CDR2 91 IRGWAYGQSA CDR3 92 EQRGANGRYGGDGFGYSYGMDV BG47 IgL 93 DIQMTQSPFTLSASVGERVTITCRASHFIANYVNWYQQRPGRA PKLLIFESSTLNRGVPSRFSGSGDGTEFTLSISALQSEDFATY ICQQSHSPPVSFGGGTRVDQT CDR1 94 HFIANY CDR2 95 ESS CDR3 96 QQSHSPPVS

Table 2. BG1 variant sequences.

One skilled in the art will appreciate that the complementarity determining regions (CDRs) of most antibodies, such as NC37, BG1, including all BG1 variants, and BG18, including all BG18 variants, largely determine the biological activity of an antibody by forming a paratope on the antibody, wherein CDRH3 has the most specific character. Accordingly, 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. The person skilled in the art will also understand that the antibodies of the present invention may contain conservative substitutions, including in the CDR, and still remain within the scope of the invention. Accordingly, some embodiments of the present invention relate to antibodies and their antigennegative parts, expressing the CDRs listed in table. 1 and 2.

For example, some embodiments of the present invention relate to antibodies, or antigen-binding portions thereof, having a heavy chain comprising one or more of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81 and 89 or having a certain degree of homology with one or more of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81 and 89, for example at least 75, 80, 85, 90, 95, 96, 97, 98, 99% and any range in between. 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 present 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 certain degree of homology with one or more of SEQ ID NOs: 55, 13, 21, 20, 37, 45, 53, 61, 69, 77, 85 and 93, for example, at least 75, 80, 85, 90, 95, 96, 97, 98, 99% and any range in between. One or more of SEQ ID NOs: 5, 13, 21, 20, 37, 45, 53, 61, 69, 77, 85 and 93 may have conservative substitutions.

IgG heavy chains typically range in size from about 400 to about 600 amino acids, more typically from 450 to about 550 amino acids. The variable region of each heavy chain, regardless of antibody class, is typically about 100-140 amino acids in length, most typically about 110-16 042959

130 amino acids. Light chains are typically about 210-220 amino acids in length, more typically 211217. The light chain variable region is typically about 100-120 amino acids in length.

Some embodiments of the present invention relate to antibodies, or antigen-binding portions thereof, having one or more heavy chain CDRs comprising 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 or having a certain degree of homology with 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, e.g. at least 75, 80, 85 , 90, 95, 96, 97, 98, 99% and any range in between. One or more of SEQ ID NOs: 2-4, 10-12, 18-20, 26-28, 34-36, 4244, 50-52, 58-60, 66-68, 74-76, 82-84 and 90-92 may have conservative substitutions.

Some embodiments of the present invention relate to antibodies, or antigen-binding portions thereof, having one or more light chain CDRs comprising 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 or having a certain degree of homology with 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, e.g. at least 75, 80, 85 , 90, 95, 96, 97, 98, 99% and any range in between. One or more of SEQ ID NOs: 6-8, 14-16, 22-24, 30-32, 38-40, 4648, 54-56, 62-64, 70-72, 78-80, 86-88 and 94-96 may have conservative substitutions.

In addition, the antibodies of the present invention may be composed of antigen-binding portions, such as CDRs, from different individual bNabs, including, but not limited to, the bNab of the present invention. By way of example only, the engineered derivative may contain the light chain of one of NC37, BG1, or BG18, including any variants, and the heavy chain of another bNab, including one of NC37, BG1, or BG18, including any variants. Alternatively, the engineered derivative may contain the light chain of one BG18 variant and the heavy chain of another BG18 variant. One skilled in the art will appreciate that methods for producing recombinant antibodies allow for a significant number of recombinant antibodies, each of which is clearly within the scope of the present invention. Accordingly, the bNab of the present invention can be produced by recombinant DNA techniques such as those described in US Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be easily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of mouse antibodies). Once isolated, the DNA can be placed into expression vectors that are then transfected into host cells, such as monkey COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to allow the synthesis of monoclonal antibodies in recombinant host cells. The DNA can also be modified, for example, by covalently attaching all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. Such a non-immunoglobulin polypeptide may be replaced by the constant domains of an antibody of the invention or may be replaced by the variable domains of a single antigen-binding site of an antibody of the invention to create a chimeric bivalent antibody.

Once generated, recombinantly or otherwise, anti-HIV-1 antibodies can be purified or isolated such that the antibodies are substantially free of, for example, no serum, supernatant or other cell cultures or related materials. Methods for purifying antibodies are known in the art and may include selective enrichment or specific isolation techniques. For example, affinity purification may be used, such as purification based on class-specific affinity. Fractionation techniques can also be used initially to facilitate the isolation of a subset of protein samples that include immunoglobulins. Typically, antigen-specific affinity is used to isolate antibodies that bind to an antigen. The antibodies can then be combined with a carrier, buffer, diluent, diluent and/or preservatives, etc., which serve to create pharmaceutically acceptable anti-HIV-1 antibody compositions.

The antibodies may be monovalent antibodies. Methods for making 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 typically truncated at any point in the Fc region to prevent crosslinking of the heavy chain. Alternatively, the corresponding cysteine residues are replaced with another amino acid residue or removed to prevent crosslinking. In vitro methods are also suitable for preparing monovalent antibodies. Cleavage of antibodies to obtain their fragments, in particular fragments of Fab, can be performed by conventional methods known in the art.

Human monoclonal antibodies can be generated by various methods 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)]. For the production of human monoclonal antibodies, the methods described by Cole et al. are also available. and Boerner et al. (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)]. 042959 Alternatively, human antibodies can be obtained by introducing human immunoglobulin loci into transgenic animals such as mice in which endogenous immunoglobulin genes have been partially or completely inactivated. in humans, including gene rearrangement, antibody assembly and repertoire.This approach is described, for example, in US Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 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).

Some embodiments of the invention relate to vectors and vector systems containing nucleotide sequences that encode variable regions, including, without limitation, the CDRs of anti-HIV-1 antibodies of the present invention, for example, NC37, BG1 and BG18, including any variants, and their antigen-binding parts. The vector may be, for example, but not necessarily, a plasmid; other recombinant vectors are known in the art and may include, for example, phage vectors such as the A phage vector, other viral vectors such as a non-replicating adenovirus vector, cosmids and/or artificial chromosomes. The vector system may or may not be separately induced, i.e. may have different promoter and/or repressor elements. Common to most engineered vectors are origins of replication, multicloning sites, and selectable markers, provided that the vector (including vector systems such as multiple plasmids) contains such a system and is considered to be within the scope of the present invention. One skilled in the art will understand that it is possible to obtain nucleic acid sequences from a given peptide sequence and clone them into a selection system. Accordingly, some embodiments of the present invention relate to a vector or system of vectors 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 a vector or system of vectors 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 anti-HIV-1 antibodies of the present invention, for example a nucleotide sequence or 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, 3840, 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 heavy/light chain and/or CDR-encoding nucleotides may have conservative substitutions, and the CDR-encoding nucleotides, regardless of such conservative substitutions, may have a degree of homology (e.g., at least 75, 80, 85, 90, 95, 96, 97, 98 or 99%) with these nucleotide sequences. The vector or vector system of the present invention may be introduced into one or more cells (ie, the cells are transformed with the vector), and such methods are known in the art.

C. Pharmaceutical formulations and delivery.

One of the embodiments of the present invention relates to pharmaceutical compositions containing at least one anti-HIV-1 antibody or antigennegative part of the present invention, as well as methods of using them to treat a patient in need thereof. The patient may have latent or active HIV-1 infection. The anti-HIV-1 antibodies or antigen-binding portions thereof used in these compositions and methods can be any of the anti-HIV-1 antibodies or antigen-binding portions thereof of the present invention, but such anti-HIV antibodies or antigen-binding portions thereof which contain BG18 are particularly useful. or variants thereof, including recombinant variants and/or engineered derivatives thereof.

A pharmaceutically acceptable composition of the anti-HIV-1 antibody or antigen-binding portion thereof suitable for administration to a patient may comprise an effective amount of the anti-HIV-1 antibody or antibodies or antigen-binding portions thereof in a formulation that retains biological activity while providing maximum storage stability. within an acceptable temperature range. Pharmaceutical compositions may also include, depending on the intended formulation, pharmaceutically acceptable diluents, pharmaceutically acceptable carriers and/or pharmaceutically acceptable excipients, or any such carrier vehicle commonly used in the preparation of pharmaceutical compositions for administration to animals or humans. The diluent is chosen so that it does not affect the biological activity of the combination. Examples of such diluents are distilled water, phosphate buffered saline, Ringer's solutions, dextrose solution and Hank's solution. The amount of excipient that is used in a pharmaceutical composition or composition of the present invention is an amount that serves to evenly distribute the antibody throughout the composition so that it can be evenly dispersed upon delivery to a subject in need. It can serve to dilute the antibody to a concentration that provides the desired beneficial palliative or curative results, when

- 18 042959 thereby minimizing any side effects that may occur at too high a concentration. It may also have a preservative effect. Thus, higher levels of excipient can be used for antibodies having high physiological activity. On the other hand, for any active ingredient(s) that exhibits lower physiological activity, a smaller amount of excipient will be used.

The pharmaceutically acceptable composition may be in liquid or solid form. The solid formulation is usually, but not necessarily, lyophilized and reconstituted prior to administration for single or multiple dosing. Formulations should not be exposed to extreme temperatures or pH to avoid thermal denaturation. Thus, it is important to prepare the composition of the antibodies of the present invention in a biologically significant pH range. A buffered solution is often needed to maintain the proper pH range during storage, especially for liquid formulations stored for longer periods from preparation to administration. Typically, liquid and solid formulations require storage at lower temperatures (typically 2-8°C) to maintain stability for longer periods of time. Prepared antibody compositions, especially liquid formulations, may contain a bacteriostat to prevent or minimize proteolysis during storage, including, but not limited to, effective concentrations (typically <1% w/v) of benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben and/or propylparaben. Bacteriostat may be contraindicated in some patients. Therefore, the lyophilized composition can be reconstituted in solution containing or not containing such a component. Additional components may be added to the buffered liquid or solid antibody composition, including, but not limited to, sugars as a cryoprotectant (including, but not limited to, polyhydroxylated hydrocarbons such as sorbitol, mannitol, glycerol, and dulcitol, and/or disaccharides such as sucrose, lactose, maltose or trehalose) and, in some cases, the corresponding salt (including, without limitation, NaCl, KCl or LiCl). Such antibody formulations, especially liquid formulations intended for long-term storage, are prepared with a useful range of total osmolarity both to ensure stability at 2-8°C or higher for an extended period of time, and to obtain a formulation suitable for parenteral injection. For example, but not necessarily, an effective range of total osmolarity (total number of molecules in solution) may be from about 200 mOs/L to about 800 mOs/L. Obviously, the amount of a ciroprotectant, such as sucrose or sorbitol, will depend on the amount of salt in the composition so that the total osmolarity of the solution remains in the appropriate range. Therefore, the salt-free formulation may, but need not, contain from about 5% to about 25% sucrose.

Alternatively, the salt-free sorbitol-based composition may, but need not, contain sorbitol in the range of from about 3% to about 12%. Salt-free formulations may require an increased range of the appropriate cryoprotectant to maintain effective osmolarity levels. These compositions may also contain a divalent cation (including, but not necessarily, MgCl ₂ , CaCl ₂ and MnCl ₂ ); and a non-ionic surfactant 32 (including, but not limited to, polysorbate-80 (Tween 80®), polysorbate-60 (Tween 60®), polysorbate-40 (Tween 40®), and polysorbate-20 (Tween 20®), polyoxyethylene alkyl ethers, including but not limited to Brij 58®, Brij 35®, as well as others such as Triton X-100®, Triton X 114®, NP40®, Span 85 and a number of non-ionic surfactants Pluronic series (for example, Pluronic 121)). Any combination of such components, including the optional inclusion of a bacteriostat, may be useful in filling the antibody formulations of the present invention. The anti-HIV-1 antibodies or antigen-binding portions thereof of the present invention may also be a chemical derivative, which is an antibody containing additional chemical moieties not normally part of an immunoglobulin molecule (eg, pegylation). Such moieties can improve solubility, increase half-life, improve absorption, and so on. main molecule. Alternatively, such fragments may reduce the undesirable side effects of the main molecule or reduce the toxicity of the main molecule.

For in vivo treatment, a patient is administered or provided with a pharmaceutical composition comprising at least one anti-HIV antibody or antigen-binding portion thereof of the present invention. The anti-HIV antibodies, or antigen-binding portions thereof, of the invention, when used for in vivo therapy, are administered to the patient in therapeutically effective amounts (i.e., amounts that eliminate or reduce the overall viral load, as described in Example 1 below). Antibodies are administered to the human patient by known methods, such as intravenous administration, for example, as a bolus or by continuous infusion over a period of time, intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical routes of administration, or by inhalation . The antibodies, or antigen-binding portions thereof, may be administered parenterally, if possible, to the target site of the cells, or intravenously. In some embodiments, the antibody is administered intravenously or subcutaneously. The therapeutic compositions of the invention may be administered to a patient or subject systemically, parenterally, or topically. The above parameters for assessing successful treatment and improvement of the disease are easily measurable using routine procedures familiar to the physician.

For parenteral administration, anti-HIV-1 antibodies, or antigen-binding portions thereof, may be formulated into an injectable dosage form (solution, suspension, emulsion) in combination with a pharmaceutically acceptable parenteral carrier. Examples of such carriers include, without limitation, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous carriers include, without limitation, fixed oils and ethyl oleate. Liposomes can be used as carriers. The carrier may contain minor amounts of additives, such as substances which increase isotonicity and chemical stability, such as, for example, buffers and preservatives.

The anti-HIV-1 antibodies or antigen-binding portions thereof of the present invention may be administered to the host by any route, strategy and/or combination available in the art, in amounts sufficient to provide therapeutic treatment for HIV-1. These compositions can be delivered to the individual by various routes of administration known in the art, in particular by parenteral administration, as well as, without limitation, non-parenteral routes of administration, such as intravenous (IV), intramuscular (IM) or subcutaneous (SC) administration, moreover, intravenous administration is the norm in the field of therapeutic administration of antibodies. These compositions can be administered as single or multiple doses (ie, administration of the antibody in doses spaced apart in time, maintaining the sterility of the composition during the treatment regimen).

The dose and dosing regimen depend on a variety of factors easily determined by the physician, such as the nature of the infection, for example, its therapeutic index, the patient's condition and medical history. Typically, a therapeutically effective amount of the antibody is administered to the patient. In some embodiments, the amount of antibody administered is in the range of about 0.001 mg/kg to about 100 mg/kg of the patient's body weight, and any range in between. Depending on the type and severity of the infection, from about 0.1 to about 50 mg/kg body weight (e.g., about 0.1-15 mg/kg/dose) of antibody is the initial dose administered to the patient, either, for example, in as one or more separate injections, or as a continuous infusion. Anti-HIV-1 antibodies can be delivered at relatively low volumetric rates, for example, but not necessarily, from about 0.001 ml/day to 10 ml/day, to minimize damage or injury to tissue near the site of release of the composition. In the case of a low dose, the composition can be released at a rate, depending on the specific biological agent(s), for example, from about 0.01 µg/h or 0.1 µg/h, 0.25 µg/h, 1 µg/h to , typically about 200 μg/hr, or the drug may be delivered at a low volume rate, e.g., a volume rate of about 0.001 ml/day to about 1 ml/day, e.g., 0.01 micrograms per day to about 20 milligrams per day. day. The dose will depend on a number of factors such as the potency, bioavailability and toxicity of the active ingredient used (eg, anti-HIV-1 antibody or antigen-binding portions thereof) and the requirements of the subject. The progress of this therapy is easily tracked using conventional methods and assays and is based on criteria known to the physician or others skilled in the art. The above parameters for assessing the success of treatment and improvement in disease are easily measurable using routine procedures familiar to the physician.

Specific embodiments for the anti-HIV-1 antibody delivery medium of the present invention include PLGA microspheres as discussed herein and as known in the art, as well as non-degradable polymer-based carriers containing poly(ethylene vinyl acetate); PEVAc). In addition, a review of controlled release and localized delivery of antibody-based therapeutic products can be found in Grainger, et al., 2004, Expert Opin. Biol. Ther. 4(7): 1029-1044). Suitable microcapsules to encapsulate the antibody may also include hydroxymethylcellulose or gelatin microcapsules and polymethyl methacrylate microcapsules prepared by coacervation or interfacial polymerization techniques. See PCT Publication WO 99/24061 entitled Process for the Preparation of IGF-1 Extended Release Formulations wherein the protein is encapsulated in PLGA microspheres, which document is incorporated herein in its entirety by reference. In addition, microemulsions or colloidal drug delivery systems such as liposomes and albumin microspheres can also be used. Other preferred sustained release formulations use a bioadhesive to hold the antibody at the injection site. As noted above, a sustained release formulation may contain a biodegradable polymer in which the antibody is placed and which may provide a sustained release. Non-injectable devices may be referred to herein as an implant, a pharmaceutical depot implant, a depot implant, a non-injectable depot, or may be referred to by a similar term. Conventional depot implants may include, without limitation, solid biodegradable and non-biodegradable polymeric devices (such as elongated polymeric or coaxial rod-like devices) as well as numerous pumping systems also known in the art. Injectable devices are classified into bolus injections (release and dissipation of the drug after injection) and storage or depot injections, which provide a storage reservoir at the injection site, allowing gradual release of the biological agent over time. A depot implant can be surgically placed at the point of delivery to provide an adequate reservoir for sustained release of the antibody over time. Such a device is capable of carrying the drug composition in such amounts as are therapeutically or prophylactically necessary for treatment over a preselected period of time. The depot implant can also protect the drug from degradation by bodily processes (such as proteases) for the duration of treatment. As known in the art, the term sustained release refers to the gradual (continuous or intermittent) release of such an agent from the block polymer matrix over an extended period of time. Regardless of the specific device, the sustained release formulation provides localized, biologically effective concentrations of the antibody. Sustained release of the biological agent(s) may occur over one day, several days, a week or more; but most likely within a month or more, or up to about six months, depending on the composition. Natural or synthetic polymers known in the art may be useful as a depot implant due to characteristics such as versatile degradation kinetics, safety and biocompatibility. These copolymers can be manipulated to modify the pharmacokinetics of the active ingredient, protect the agent from enzymatic attack, and allow them to degrade over time at the site of attachment or injection. One skilled in the art will appreciate that there are many ideas in the art for manipulating the properties of these copolymers, including the appropriate manufacturing process, the catalysts used, and the final molecular weight of the sustained release depot implant or depot injection. Natural polymers include, without limitation, proteins (eg, collagen, albumin, or gelatin); polysaccharides (cellulose, starch, alginates, chitin, chitosan, cyclodextrin, dextran, hyaluronic acid) and lipids. Biodegradable synthetic polymers may include, without limitation, various polyesters, copolymers of L-glutamic acid and gamma-ethyl-E-glutamate (Sidman et al., 1983, Biopolymers 22:547-556), polylactides ([PLA]; US patent US 3,773,919 and EP 058481), polylactate polyglycolate (PLGA) such as polylactide-co-glycolide (see, for example, US Pat. ), polyorthoesters, polyaspirins, polyphosphagenes, vinylpyrrolidone, polyvinyl alcohol (PVA), PVA-g-PLGA, PEGT-PBT copolymer (polyactive), methacrylates, poly(Nisopropylacrylamide), PEO-PPO-PEO (Pluronics), PEO-PPO copolymers -PAA, PLGA-PEO-PLGA, polyorthoethers (POE), or any combination thereof, as described above (see, for example, US patent No. 6991454 and US patent No. 20050187631, each of which is included in this description in its entirety in the form references), hydrogels (see, for example, Langer et al., 1981, J. Biomed. Mater. Res. 15: 167277; Langer, 1982, Chem. Tech. 12:98-105, non-degradable ethylene vinyl acetate (e.g., ethylene vinyl acetate discs and poly(ethylene-vinyl acetate)), degradable lactic acid-glycolic acid copolymers such as Lupron Depot™, poly-D-(-)-3-hydroxy-butyric acid (EP 133988), hyaluronic acid gels (see, for example, US Pat. No. 4,636,524), alginic acid suspensions, polyorthoesters (POE), and the like. Polylactide (PLA) and its glycolide copolymers (PLGA) have been well known in the art since the commercialization of Lupron Depot™ approved in 1989 as the first sustained release parenteral formulation using PLA polymers. Additional examples of products that use PLA and PLGA as excipients to achieve sustained release of the active ingredient include Amidox (PLA; periodontal disease), Nutropin Depot (PLGA; with hGH), and Trelstar Depot (PLGA; prostate cancer). Other synthetic polymers include, without limitation, poly(c-caprolactone), poly-3-hydroxybutyrate, poly(β-malic acid), and poly(dioxanone)]; polyanhydrides, polyurethane (see WO2005/013936), polyamides, cyclodextrans, polyorthoethers, n-vinyl alcohol, polyethylene oxide/polyethylene terephthalate, polyphosphate, polyphosphonate, polyorthoester, polycyanoacrylate, polyethylene glycol, polydihydropyran and polyacetal.

Non-biodegradable devices include, without limitation, various cellulose derivatives (carboxymethyl cellulose, cellulose acetate, cellulose acetate propionate, ethyl cellulose, hydroxypropyl methyl cellulose), silicon-based implants (polydimethylsiloxane), acrylic polymers, (polymethacrylate, polymethyl methacrylate, polyhydroxy(ethyl methacrylate)), and polyethylene-co(vinyl acetate), poloxamer, polyvinylpyrrolidone, poloxamine, polypropylene, polyamide, polyacetal, polyester, poly(ethylene-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, without limitation, 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 pa

- 21 042959 US tarpaulin No. 6,953,593; 6946146; 6656508; 6541033; and 6,451,346, the contents of each of which are incorporated herein by reference. The dosage form must be capable of carrying the dosage composition in such quantities and concentrations as are therapeutically necessary for treatment for a preselected period of time, and must provide sufficient protection of the composition from degradation by processes occurring in the body during the entire period of treatment. For example, the dosage form may be surrounded by an outer shell made of a material that has protective properties against degradation due to metabolic processes and risk, such as leakage, cracking, breaking or deformation. This can prevent the contents of the dosage form from being released in an uncontrolled manner by stresses that it may be subjected to during use, such as physical forces acting on the drug release device as a result of normal articulation and other movements of the subject, or, for example, in convective devices. delivery of medicinal substances, physical forces associated with the pressure created in the reservoir. The drug reservoir or other means for storing or retaining the drug should also be made of a material that avoids unintended reactions with the active agent formulation and should preferably be biocompatible (e.g., when a dosage form is implanted, the formulation should not substantially be reactive with the subject's body or body fluids). Typically, anti-HIV-1 antibodies are administered to an individual over a period of at least 12 hours, at least a week, and most likely via a drug delivery implant for at least 10, 20, 30, 100 days, or at least 4 months, or at least 6 months, or at least 12 months, or at least 24 months, if necessary.

In some embodiments, the anti-HIV-1 antibodies or antigen-binding portions thereof of the present invention may be administered in combination with one or more additional HIV-1 treatments, such as in combination with one or more anti-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 their antigennegative parts, including, without limitation, additional anti-HIV-1 antibodies or their antigennegative parts of the present invention. As shown in Example 1 below, administration of NC17, BG1 and BG18 in a 1:1:1 ratio was effective in reducing viral load in vivo. While not wishing to be bound by theory, the data indicate that monoclonal bNAbs and very low levels of neutralization-sensitive viruses coexist in the EB354 donor, suggesting that the antibodies promote control initiation in this individual. According to another embodiment, the present invention provides a passive vaccine or pharmaceutical compositions comprising at least one anti-HIV-1 antibody or antigen-binding portions thereof of the invention and a pharmaceutically acceptable carrier. In one embodiment, the vaccine or pharmaceutical compositions are compositions comprising at least one antibody described herein and a pharmaceutically acceptable excipient. The vaccine may include a plurality of antibodies having the characteristics described herein, in any combination, and may additionally include other anti-HIV-1 antibodies or antigen-binding portions thereof. The passive vaccine may contain one or more pharmaceutically acceptable preservatives, carriers and/or excipients that are known in the art. In other embodiments, the present invention provides an active vaccine or pharmaceutical compositions containing HIV-1 or an antigenic fragment thereof. If HIV-1 is used, it may be attenuated. It can be killed by heat. If antigenic fragments are used, it may be preferable, although not mandatory, to use the gp120 glycoprotein, although fragments of the gp120 glycoprotein may also be used.

The compositions of the present invention may be used in an immunogenic composition for immunizing an animal. Such an immunogenic composition according to the invention can be used to prepare a vaccine. Preferably, a prophylactic and/or therapeutic vaccine is prepared. Thus, within the scope of the present invention is an immunogenic or vaccine composition that contains a pharmaceutically acceptable carrier and an effective amount of an antigen as described above. The carriers used in the composition may be selected based on the mode and route of administration and standard pharmaceutical practice. The composition may also contain an adjuvant. Examples of the adjuvant include cholera toxin, heat-labile Escherichia coli enterotoxin, liposome, unmethylated DNA (CpG), or any other innate immunostimulatory 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, polyurethane polyols, polyanions, peptides, oil emulsions, snail limpet hemocyanin and dinitro

- 22 042959 phenol.

The vaccine composition may be administered to the subject per se or as a pharmaceutical or therapeutic composition. The compositions of the invention and the adjuvant may be prepared by conventional mixing, dissolving, granulating, drageeing, pulverizing, emulsifying, encapsulating, trapping, or lyophilizing processes. Pharmaceutical compositions can be prepared in the usual way using one or more physiologically acceptable carriers, diluents, excipients or excipients that facilitate processing of the antigens of the invention for use in pharmaceutically acceptable preparations. The appropriate drug depends on the chosen route of administration.

For injection, vaccine preparations may be prepared in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, phosphate buffered saline, or any other physiological saline buffer. The solution may contain agents suitable for formulating formulations such as suspending, stabilizing and/or dispersing agents. Alternatively, the composition may be in the form of a powder which is reconstituted with a suitable vehicle, eg sterile, pyrogen-free water, before use.

The amount of composition administered depends, for example, on the specific antigen in the composition, whether the adjuvant is administered along with the antigen, the type of co-administration of the adjuvant, the method and frequency of administration, and the desired effect (e.g., protection or treatment), which can be determined by a person skilled in the art. . Determining the effective amount of the vaccine composition for administration is within the skill of the art, especially in light of the detailed description provided herein. The effective dose may be initially estimated from the results of in vitro assays. For example, the dose may be prepared using animal models to achieve the induction of an immune response by methods well known in the art. A person skilled in the art can easily optimize administration for all animal species based on the results described in this application. The amount and dosing interval can be adjusted individually. For example, when used as a vaccine, the vaccine formulations of the invention may be administered in 1 to 3 doses over 1-36 weeks. Vaccination can continue indefinitely depending on the circumstances, especially with passive vaccination. It is preferred to administer 1 or 2 doses at intervals of about 3 weeks to about 4 months, and thereafter periodic boosters may be given. For individual animals, alternative protocols may be appropriate. A suitable dose is the amount of the vaccine composition which, when administered as described above, is capable of inducing an immune response in the immunized animal sufficient to protect the animal from infection for at least 4-12 months. Typically, the amount of antigen present in a dose ranges from about 1 μg to about 100 mg per kg of host weight, typically from about 10 μg to about 1 mg, and preferably from about 100 μg to about 1 μg. A suitable dosage range will vary depending on the route of administration and the size of the subject, but is usually from about 0.1 ml to about 5 ml. Additional booster injections may be given as needed.

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 contain an anti-HIV-1 antibody or an antigen-binding portion thereof of the present invention and various reagents, e.g., reagents that help detect binding of an anti-HIV-1 antibody to an epitope present on HIV-1, e.g. gp120 and/or gp140 or their antigenic fragment.

The kits may be in vitro assays such as immunoassays, e.g. enzyme immunoassays (EIA), enzyme immunosorbent assay (ELISA), ELISPOT (enzymatic immunoassay), radioimmunoassays (RIA), immunofluorescence, and other assays known in the art, including , without limitation, Western blot analysis and/or immunoprecipitation methods. The in vitro assays may be competitive or indirect, such as a sandwich assay, or may be an antibody capture method. For example, in direct ELISA, a buffered antigen solution, such as a sample containing HIV-1 or an antigenic fragment thereof, or a biological sample containing or suspected of having HIV-1, is added to a well of a microtiter plate, such as a 96-well plate. A solution of a non-reactive protein, such as bovine serum albumin or casein, is then added to the well. Add an anti-HIV-1 antibody or antigen-binding portions thereof conjugated to an enzyme, a reporter molecule, e.g. conjugated to horseradish peroxidase, although not necessarily to this particular enzyme, as there are other commonly used enzymes, including alkaline phosphatase or β-D-galactosidase , other enzymes are also possible and are included in the scope of the invention. An enzyme substrate is then added, resulting in a detectable signal. For example, the addition of TMB to horseradish peroxidase results in a colored product, in which case the ELISA is a colorimetric assay. ELISA methods can be carried out in the format of a qualitative or quantitative analysis. Qualitative results give a simple positive or negative result in relation to the sample.

- 23 042959 positive result (yes or no). The gap between a positive and negative result is determined by the analyst and may be statistical. Sandwich ELISA is usually performed according to the following protocol. The anti-HIV-1 capture antibody, or antigen-binding portions thereof, is bound (ie, 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, after which the sample is captured by anti-HIV-1 antibodies. The substrate is then washed to remove unbound antigen. A second anti-HIV-1 antibody, or antigen-binding portions thereof, is added that binds to a different epitope on HIV-1, such as other epitopes on gp120 or other antigens, such as gp140. The second anti-HIV-1 antibody, or antigen-binding portions thereof, is coupled to a reporter molecule, such as an enzyme, although the reporter molecule can be any molecule that results in a detectable signal. The plate may be washed a second time and, in cases where the reporter molecule is an enzyme, a substrate such as TMB may be added which results in a detectable signal (also colorimetric analysis). The third type of general ELISA is the competitive ELISA. In these embodiments, an unlabeled anti-HIV-1 antibody, or antigen-binding portions thereof, is incubated in the presence of an antigen-containing sample, which is then added to an antigen-coated well. The plate is washed to remove unbound antibodies. A secondary antibody is added that is specific for the primary antibody, eg a secondary antibody specific for anti-HIV-1 antibodies. The secondary antibody is bound to a reporter molecule as described above, such as an enzyme (or any other molecule that can result in a detectable signal). Some competitive ELISA methods use labeled antigens rather than labeled antibodies; the smaller the amount of antigen in the sample, the more labeled antigen is retained and the stronger the detectable signal.

Other forms of in vitro assays include radioimmunoassay (RIA). Typically, a known amount of antigen is bound to a radioactive label, such as I-125, although other labels, such as ⁹⁹ Tc, are also used, which is then mixed with a known amount of antibodies specific for that antigen, such as an anti-HIV-1 antibody or its antigen-binding parts. A sample containing an unknown amount of antigen is then added (eg, a biological sample that contains or is suspected to contain HIV-1 or an antigenic fragment thereof). This method is a direct competitive specific binding method; as the concentration of unlabeled antigen increases, the binding between anti-HIV-1 antibodies and the labeled standard decreases, which can be measured directly by measuring radioactivity. Other assays are known and one of skill in the art would be able to use them.

In some embodiments, the invention relates to a method for detecting HIV-1 or an antigenic fragment thereof in a sample. Any of the assays described herein or other assays known in the art can be used in such methods. Some of the assays described herein quantify an antigen present in a sample, and accordingly, in some embodiments, the present invention relates to methods for quantifying HIV-1 or antigenic fragments thereof present in a sample, such as a biological sample. Assays containing anti-HIV-1 antibodies or antigen-binding portions of the present invention may or may not be used for diagnostic purposes.

Accordingly, in some embodiments, the invention provides methods for using the anti-HIV-1 antibodies or antigen-binding portions thereof of the present invention for diagnosis. Due to the specificity of the anti-HIV-1 antibodies of the present invention or their antigen-binding portions, immunoassays containing the anti-HIV-1 antibodies or antigen-binding portions of the present invention may be sufficient to diagnose an individual as having active or latent HIV-1 infection. The antibodies or antigen-binding portions thereof are not to be limited to any particular epitopes, provided that the antibodies or antigen-binding portions used are specific for HIV-1. For example, in a sandwich assay, a first anti-HIV-1 antibody or antigen-binding portions thereof may bind to a first epitope, such as gp120, and a second anti-HIV-1 antibody, or antigen-binding portions thereof (bound to a reporter molecule) may bind to a second epitope. , which may or may not be present on the same antigen. Accordingly, kits and methods used to detect or quantify HIV-1 or an antigenic fragment thereof may comprise BG18, NC37, and BG1, including their antigen-binding portions, and are contemplated within the scope of the present invention, or any of BG18, NC37, and BG1, including their antigen-binding portions; and any other anti-HIV-1 antibody, provided it is suitable for inclusion in such kits/methods. It is understood that anti-HIV-1 antibodies, or antigen-binding portions thereof, used for the purpose of detecting and/or quantifying HIV-1 present in a sample, or even for diagnostic purposes, do not need to have a broad-spectrum neutralizing ability against HIV1 in order to be able to their use for such purposes.

- 24 042959

E. Equivalents.

Where a range of values is indicated, it is to be understood that the scope of the invention includes every intermediate value, to the nearest tenth of one of the lower limit, unless the context clearly implies otherwise, between the upper and lower limits of that range and any another specified value or an intermediate value within the specified range. The upper and lower limits of these smaller ranges, which may independently be included in the smaller ranges, are also within the scope of the invention, subject to any specifically excluded limit within the specified range. Where the specified range includes one or both of the limits, ranges excluding both limits of the included limits are also included within the scope of the invention.

Unless otherwise indicated, all technical and scientific terms used in the present description have the same meanings as they are commonly understood by specialists in the field of technology to which the present invention pertains. Although any methods and materials similar or equivalent to those described in this application can also be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications mentioned in the present description are incorporated herein in their entirety by reference.

As used herein and in the appended claims, the singular forms include references to the plural, unless the context clearly indicates otherwise.

The term roughly refers to a range of values that would not be considered by one of ordinary skill in the art to be substantially different from baseline values. For example, a term roughly can refer to a value that is between 20, 15, 10, 9, 8, 7.6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, or 0 .01% of the declared value, as well as a value that is within the specified values.

The publications cited in this application are provided solely for their disclosure prior to the filing date of the present invention. Nothing herein should be construed as an admission that the present invention does not give any right to backdate such disclosure based on an earlier invention. In addition, publication dates shown may differ from actual publication dates, which may require independent confirmation.

Each of the applications and patents cited in this specification, as well as each document or reference, patent or non-patent literature cited in each of the applications and patents (including during the litigation of each patent granted; documents cited in the application) and each of the PCT and other country applications or patents corresponding to and/or claiming priority with respect to any of those applications and patents, and each of the documents cited or referenced in each of the documents cited in the application are hereby expressly incorporated herein description in their entirety by reference. In a more general sense, documents or references are cited in the present description or in the list of references before the claims; or in the text itself; and each of those documents or references (references cited in this document) and each document or reference cited in each of the references cited in this document (including specifications, instructions, etc. of any manufacturer), hereby expressly form is incorporated herein by reference.

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

VII. Examples

1. Coexistence of effective broad-spectrum anti-HIV-1 neutralizing antibodies and antibody-sensitive viruses in a viremia controller.

A. Methods.

HIV-1 infected subject EB354.

Purified IgG from donor EB354 ranked in the top 1% in terms of breadth and neutralization efficiency in a cohort of 394 HIV-1 infected long-term nonprogressors. Donor EB354 was diagnosed with HIV-1 group B in 1986. He received didanosine and stavudine treatment between 1995 and 1998, but has not received antiretroviral therapy since. He has been under routine medical supervision since 2002 (Table 3: HLA A*01:01, 24:02, B*27:05, 57:01, C*01:02, 06:02). Most individuals rapidly develop strain-specific antibodies shortly after infection. This response is associated with the selection of resistant viral variants, which in some cases cause the formation of bNAb. However, detailed screening individuals differ from EB354 in that bNAb development is associated with rapid selection of autologous plasma viruses resistant to coexisting bNAbs. EB354 is important because susceptible and resistant viral strains coexist with bNAbs, and resistant strains are unable to induce high levels of viremia, either because they are in some way partially effective or controlled by CD8+ T cells. Thus, in this individual, bNAb-susceptible plasma viruses were unable to escape immune pressure, resulting in a bNAb:virus equilibrium in which the virus persists but is unable to induce high levels of viremia. The EB354 is unusual in that it is both an elite controller and an elite converter. The Elite Controllers of HIV-1 Are Infections

- 25 042959 individuals who maintain a low viral load for many years. These individuals are much less likely to transmit the virus and demonstrate long-term AIDS-free survival. HLA B57*01 and/or B27*05 alleles were found in 85% of elite HIV-1 controllers. These alleles are associated with increased cytotoxic activity of CD8 T cells. Compared to viraemic professors (i.e. patients with a high viral load), elite controllers are less likely to develop bNAb. Regardless of robust CD8+ T cell responses that are able to partially control infection, the level of HIV-1 replication in EB354 was sufficient to generate bNAb and affinity maturation. As discussed herein, bNAbs that confer serological neutralizing activity in the EB354 donor organism recognize multiple non-overlapping epitopes.

Date of diagnosis 1986 HLA A101.2402 B2705.5701 cwl02,602 Date of visit Viral load CD4abs CD8abs 4/24/2002 310 957 1406 8/7/2002 653 554 12/3/2002 2520 983 1787 3/11/2004 1035 1323 1634 6/18/2004 171 1231 1753 8/20/2004 467 10/7/2004 182 789 2/28/2005 1048 1227 8/25/2005 < 400 1080 11/17/2005 1665 1551 8/3/2006 < 400 10/24/2006 < 400 744 1132 11/15/2006 206 814 4/6/2007 < 400 564 11/28/2007 < 400 781 7/17/2008 149 815 3/17/2009 107 734 2/19/2010 170 900 1933 8/10/2010 65 1096 1706 7/13/2011 380 795 1512 3/28/2013 414 1127 1809 9/18/2013 547 674 1152 7/21/2014 454 697 1321 7/17/2015 461 605 1224

Table 3. Clinical parameters of the EB354 donor.

B cell sorting and antibody isolation.

Sorting of single bait+CD19+IgG+B cells from EB354 donor PBMC was performed as described by JF 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). Memory B cells were pre-enriched with anti-CD19 magnetic particles (MACS) and stained using four different baits: 2CC gp120 core protein as bait, gp140 _YU2 , 1:1 mixture of gp140 _92UG37 . ₈ (type A) + gp140 _CZA79012 (type B) and BG505 SOSIP.664. Reserve (rescue) primers were used to amplify both heavy chains and IgA genes, and conventional primers were used for the IgK chain. All PCR products were sequenced and analyzed for Ig gene usage, CDR3 and the number of somatic VH/VL hypermutations (IgBLAST and IMGT). Purified, digested PCR products were cloned into human IgY1, Igk, or Igλ expression vectors as 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 immunology- 26 042959 cal methods 329, 112-124 (2008), and produced by transient transfection of expression plasmids

IgH, IgK and IgL into exponentially growing HEK 293-6E cells as described in F. Klein et al., Enhanced

HIV-1 immunotherapy by commonly occurring antibodies that target virus escape variants. The Journal of experimental medicine 211, 2361-2372 (2014).

The study of neutralization.

HIV-1 neutralization was assessed using a luciferase-based TZM.bl cell assay. Briefly, envelope pseudoviruses were incubated with five-fold serial dilutions of individual antibodies and applied to TZM.bl cells that carried the luciferase reporter gene. After 48 hours, the cells were lysed and the luminescence was measured. The IC ₅₀ and IC ₈₀ values reflect the concentrations of individual antibodies that cause a reduction in luminescence in relative units (RLU) of 50% and 80%, respectively. Activity spectrum curves were plotted using the Antibody Database computational tool.

Crystallization, X-ray data collection and structure determination.

BG18 Fab crystals with the potential N-linked glycosylation site at position 26 of the heavy chain removed by mutation of Asn26HC in Gln showed superior size and morphology compared to wild-type BG18 Fab crystals, thus structure determination was performed using Fab BG18 _N26 q. Fab BG18 _N26 q crystals (space group P21; a=46.12 A, b=71.04 A, c=69.54 A; β=98.48°; 1 molecule per asymmetric unit); prepared by combining 0.2 μl of a 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% (v/v) PEG 8000 at 20° C. was cryoprotected in the mother liquor supplemented with 20% (v/v) ethylene glycol and flash-cooled in liquid nitrogen. Fab NC102 crystals (space group P212121; a=60.2 A, b=82.5 A, c=117.2 A; 1 molecule per asymmetric unit) were prepared by vapor phase diffusion in sessile drops with a reservoir of 0.1 M sodium acetate trihydrate, pH 4.6, and 2.0 M ammonium sulfate using 250 nl drops with a protein:reservoir ratio of 1.5:1. The crystals were cryoprotected in a 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 A, b=67.4 A, c=210.4 A; 1 complex per asymmetric unit) were prepared by vapor phase diffusion in 250 nl sessile drops by protein incubation and a reservoir (comprising 0.1 M HEPES, pH 7.5 and 20% (w/v) PEG 10,000) at a protein:reservoir ratio of 1.5:1. The crystals were cryoprotected in a mother liquor supplemented with 20% (w/v) PEG 400 and flash-cooled in liquid nitrogen.

X-ray diffraction data were collected on the 12-2 beam line of a Stanford synchrotron light source equipped with a Pilatus 6M pixel detector (Dectris). XDS was used for indexing, integrating and scaling data. BG18 _n26 Q Fab crystals were diffracted to 1.5 A and structure determined by molecular replacement using V _H V _L 101074 Fab assays (PDB code 4FQ2) with CDR and CH1CL loops removed. The structure was refined using an iterative approach with the Phenix program and manual model building in the Coot graphics program. The final model Fab BG18 _N26 q (R _work =18.0%, R _free =18.9%) contained 98.3%, 1.7%, and 0% residues in the preferred, allowed, and forbidden regions of the Ramachandran site, respectively. Fab NC102 was diffracted to 1.6 A and structure determined by molecular substitution using NIH45-46 (PDB code 3U7W) with V _H V _L with CDR and CH1CL loops removed as study models. The final Fab NC102 model (R _work =18.0%, R _free =20.0%) contained 98, 2 and 0% residues in the preferred, allowed and forbidden areas of the Ramachandran site, respectively. The structure of the NC37-93TH057 gp120 complex was obtained by molecular replacement using the V _H V _L , C _H 1C _L NC102 models and the truncated gp120 core (from PDB 3U7Y) as study models. The final model of the NC37-93TH057 complex (R _work =21.0%, R _free =26.0%) contained 96, 4, and 0% residues in the preferred, allowed, and forbidden regions of the Ramachandran site, respectively. The results of statistics on data collection and refinement are presented in Table. 4.

354BG18 Fab 354NC37+93TH05 7 354NC102 Fab

- 27 042959

Range permissions (A) 35.44-1.3 (1.346-1.3) 38.6-2.7 (2.82.7) 38.91-1.60 (1.66-1.60) Spatial th group R21 vg^^! Unit cell parameters a, b, c (A) 46, 12, 71, 04, 69.54 63, 60, 67.41, 210.42 60.22, 82.50, 117.21 a, b, g (°) 90, 98.48, 90 90, 90, 90 90, 90, 90 Reflections measured 634239 (60770) 172153 (17052) 515918 (48175) Independent reflections 106792 (10428) 25596 (2517) 77546 (7649) multiplicity 5.9 (5.7) 6.7 (6.8) 6.7 (6.3) Completeness (%) 0.98 (0.98) 1.00 (1.00) 1.00 (1.00) Meaning I/sigma (I) 14.48 (0.86) 14.80 (2.73) 11.12 (1.68) B-factor Wilson (A ² ) 14.92 41, 63 15, 84 R merge 0.06152 (1.772) 0.1272 (0.7028) 0.108 (1.031) SS1/2 0.999 (0.318) 0.996 (0.832) 0.997 (0.996) SS* 1 (0.694) 0.999 (0.953) R work 0.180 0.21 0.18 free 0.189 0.26 0.2 Number of atoms 3616 6161 4159 macromolecules 3167 5924 3402 ligands 235 25 solvent 449 2 732 protein residue 432 781 448 RMS (communications) (A) 0.009 0.005 0.007 RMS (Angles) (°) 1 0.96 1.145 Clashscore (overlap index) 6, 17 13.99 3.41 The average value of the B-factor (A ² ) 22.89 36, 55 20.37 macromolecules 21.3 35, 77 17.77 ligands 56, 03 34.45 solvent 34.17 34.8 31.96

Table 4. Results of statistics on data collection and statistics refinement. The statistics for the highest resolution shell are shown in brackets.

The structure of the BG505 SOSIP.664-BG18-179NC75 complex was obtained by cryoelectron tomography/subtomogram averaging up to ~40 A and used as a reference structure to obtain the EM structure of a single particle from negatively stained samples. Purified BG505 SOSIP.664-BG18-179NC75 complexes were diluted to a concentration of 10 μg/ml in TBS immediately before

- 28 042959 by adding 3 µl to immersion ultra-thin film C on perforated carbon film support, 400 mesh, Cu-grids (Ted Pella, Inc.). Mesh samples were 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 equipped with a Gatan Ultrascan 2kx2k CCD camera. Images were obtained with a time exposure of 0.5 s at a nominal magnification of 42000x with a defocus of 1 μm, with a resolution of 2.5 A per pixel. A total of 25639 particles were selected by particle swarm using the EMAN2.1 program, and CTF correction was performed using the EMAN2.1 program. Initial averaging over 2D classes, without reference, was performed using the RELION program, and all particles were sorted into 250 classes. 9827 particles with good class averages were selected, and the particles were further sorted by 3D classification using the RELION program, after which 7925 particles were selected for refinement. To obtain a reference structure for 3D classification and refinement, data were collected from an independent sample of the BG505 SOSIP.664-BG18-179NC75 complex using cryoelectron tomography, and the structure obtained from averaged 40 A subtomograms was obtained by 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):p. 1379-90. The structure averaged over subtomograms was filtered with a low pass filter up to 80 A to be used as a reference structure for single particle reconstruction. The resolution of the final single particle reconstruction was ~25 A calculated using the RELION program and the FSC gold standard with a cutoff of 0.143 recommended for evaluating the resolution for single particle EM reconstructions.

Fitting to an EM density map.

EM structures were visualized using the USCF Chimera program. Coordinates from crystal structures were fitted into sub-tomogram-averaged or negatively stained single particle EM structures using the Fit in map utility in UCSF Chimera with the following parameters: real-time correlation/mean update, use simulated atom map, 25 A resolution First, the structure BG505 SOSIP.664 (PDB 4TVP) was inscribed into the density, and then the coordinates for Fab BG18 and Fab CH103 (PDB 4JAM; as a model for Fab 179NC75) separately into the corresponding densities. The C _H 1-C _L Fab domain regions were of low density.

Protein production and purification for structure determination studies.

6x-His-labeled Fabs BG18, BG18 _n26 q, NC102 and NC37 were expressed by transient transfection in HEK293-6E cells and purified from transfected cell supernatants by Ni ²⁺ -NTA affinity chromatography (GE Healthcare) and Superdex 200 size exclusion chromatography. 16/60 (SEC) (GE Healthcare). The truncated, His-tagged, 93TH057 gp120 core protein was produced in baculovirus-infected insect cells and purified as described by Ni ²⁺ affinity chromatography (GE Healthcare) (Diskin et al. Science 2011; Diskin et al. NSMB 2010). Prior to crystallization testing, the purified NC37 and 93TH057 proteins were co-incubated (at a 2:1 molar ratio of Fab to gp120 core) and treated with 5 kU of endoglycosidase H per mg of gp120 protein for 3 h at 25°C. The endoglycosidase H-treated complex was purified by size exclusion chromatography on a Superdex 200 10/300 GL column (GE Healthcare).

Soluble BG505 SOSIP.664 trimers for EM studies were constructed as described by Sanders, R.W., 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. el003618. HEK293-6E cells treated with 5 μM kifunensin (Sigma) were co-transfected with plasmids encoding BG505 SOSIP.664 and soluble furin in a 4:1 ratio. The BG505 SOSIP.664 protein was harvested from cell supernatants using a 2G12 immunoaffinity column prepared by covalently linking the 2G12 IgG monomer to an NHS-activated sepharose column (GE Healthcare). The protein was eluted with 3M MgCl2 followed by an immediate buffer exchange with trisbuffered saline (TBS), pH 7.4 (50 mM NaCl). The trimers were further purified on a Mono Q 5/50 GL column (GE Healthcare) and then on a Superose 6 10/300 SEC column (GE Healthcare).

autologous viruses.

For single genome sequencing (SGS) of HIV-1 env genes, 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 with Superscript III reverse transcriptase using the envB3out HIV-1 specific primer:

envB5out: 5'-TAGAGCCCTGGAAGCATCCAGGAAG-3' (SEQ ID NO: 97);

envB3out: 5'-TTGCTACTTGTGATTTGCTCCATGT-3' (SEQ ID NO: 98);

envB5in: 5'-TTAGGCATCTCCTATGGCAGGAAGAAG-3' (SEQ ID NO: 99);

envB3in: 5'-GTCTCGAGATACTGCTCCCACCC-3' (SEQ ID NO: 100).

The first PCR cycle was carried out in a volume of 20 µl containing 1x High Fidelity buffer, 2 mM MgSO ₄ , 0.2 mM dNTP and 0.5 U. High Fidelity Platinum Taq and 0.2 µM each of envB5out and envB3out primers. A second round of PCR was performed using 1 μl of PCR 1 and 0.2 μM envB5in and envB3in primers.

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PCR conditions were the same as for PCR-1 except for 45 cycles and an elevated annealing temperature of 58°C. PCR-2 products were tested using 1% 96-well E-gels (Invitrogen). From the bands obtained by PCR with an amplification efficiency of less than 30%, libraries were obtained, which were sequenced using the Nextera DNA Sample Preparation Kit (Illumina). CMV-Env expression cassettes were generated according to the protocol described in J. L. Kirchherr et al., High throughput functional analysis of HIV-1 env genes without cloning. J Virol Methods 143, 104-111 (2007). 500 ng of CMV-env were co-transfected with pSG3Aenv in 6-well plates into 293T cells and the supernatant was collected 48 hours later. All plasmid sequences were verified prior to expression. Supernatants were tested for neutralization using TZM-bl analysis.

TZM-bl assays for glycol mutant viruses Env.

Pseudoviruses were generated by transfection of 293T cells (ATCC) with a HIV-1 Env expression plasmid and an Env-deficient genomic backbone plasmid named pSG3AEnv 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):p. 10108-25. Pseudoviruses were harvested 72 hours after transfection for use in neutralization assays. Neutralizing activity was assessed in a single run assay of pseudovirus replication and TZM-B1 target cells as described by Li et al. higher. Briefly, TZM-bl cells were seeded in a flat-bottomed 96-well plate. Pseudovirus was added to this plate and pre-incubated with serial dilutions of antibodies for 1 hour at 37°C. 72 hours after infection, luciferase reporter gene expression was quantified after lysis and addition of Bright-Glo™ luciferase substrate (Promega). To determine the values of IC ₅₀ received curves dose-response method of non-linear regression.

Synthesis of glycopeptides V3 HIV-1.

V3 glycopeptides derived from several strains of HIV-1 were synthesized using a chemoenzymatic method, which consists of automated solid-phase peptide synthesis of the GlcNAc-peptide precursor and subsequent enzymatic transglycosylation of the GlcNAc peptide to produce target glycopeptides, following the procedures described in Amin, MN, et al. , Synthetic glycopeptides reveal the glycan specificity of HIV-neutralizing antibodies. Nat Chem Biol, 2013. 9(8):p. 521-6. Interactions between biotinylated synthetic glycopeptides and IgG antibodies BG18 and BG8 were evaluated by SPR using a BIAcore T200 system (GE Healthcare) at 25°C. Biotinylated glycopeptides were immobilized on neutravidin-coated CM5 sensor chips in HBS-P buffer (10 mM HEPES, 150 mM NaCl, P20 surfactant 0.05% v/v, pH 7.4) until a response of 200 units was reached. (RU). BG18 (or BG8) was injected at a 2-fold serial dilution starting at 4 μM in BS-P buffer at a flow rate of 40 μl/min for 180 seconds. Buffer BS-P was then injected for 1210 seconds at a flow rate of 40 μl/min to ensure dissociation. Regeneration was performed by injecting 3 M MgCl ₂ at a flow rate of 50 μl/min for 3 minutes followed by an injection of HBS-P buffer at a flow rate of 50 μl/min for 5 minutes.

In vivo mouse model.

Humanized mice NOD Rag1'/'I12rg ^nu (NOD. Cg-Rag1 ^tm1Mom Il2rgtm ^1Wjl /SzJ) (The Jackson Laboratory) were injected subcutaneously (sc) with 1 mg of each antibody twice a week for 3 weeks, with a total number of antibody injections equal to six. Control hu mice were generated using human cells from the same donor and infected with BH4-1YU2 but not challenged with antibodies. Plasma virus load was measured weekly. The gp120 sequences from mice with increased viral load 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).

ELISA methods.

High binding 96-well ELISA plates (Costar) were coated overnight with 5 μg/ml of purified 2CC core, gp120.YU2 folded trimer (wild type or mutants) or gp140.YU2 in PBS. After washing 6 times with PBS + 0.05% Tween 20, the plates were blocked for 2 h with 2% BSA, 1 μM EDTA and 0.05% Tween-PBS (blocking buffer), and then incubated for 1 h with IgG, which was added in the form of seven serial dilutions 1:4 in PBS with an initial concentration of 4 µg/ml. After additional washing, the plates were stained by incubation with goat HRP-conjugated anti-human IgG (Jackson ImmunoResearch) (at 0.8 μg/ml in blocking buffer) for 1 h followed by the addition of horseradish peroxidase (HRP) chromogenic substrate (ABTS solution); Invitrogen). For competitive ELISA, plates were coated with 0.5 µg/mL BG505.SOSIP.664, washed, blocked for 2 h with blocking buffer, and then incubated for 1 h with seven serial 1:4 dilutions of IgG in PBS starting at 32 μg/ml in the presence of a biotinylated antibody at a constant concentration of 4 μg/ml. The plates were then stained using HRP-conjugated streptavidin (Jackson ImmunoReseach) (at a concentration of 1 μg/ml in blocking buffer).

For ELISA using BG505 SOSIP.664 trimers labeled with epitope D7324 (BG505

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SOSIP.664-D7324) plates were coated overnight with 5 μg/ml of D7324 antibody as described in Sanders, R.W., 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 and then washed and incubated with 500 ng/ml trimer. After additional washing, IgG was added for 1 h in the form of seven serial 1:4 dilutions in PBS with an initial concentration of 4 μg/ml. The final product was obtained by incubation with goat HRP-conjugated antibodies against human IgG, as described above. All experiments were performed at least 3 times.

Generation of CMV-Env viruses.

CMV-env expression cassettes were generated according to an established protocol (52). Briefly, the CMV promoter was amplified from the pcDNA 3.1D/V5-His-TOPO expression vector with the following primers:

CMVenv: 5'-AGTAATCAATTACGGGGTCATTAGTTCAT-3' (SEQ ID NO: 101) and

CMVenv1A 5'-CATAGGAGATGCCTAAGCCGGTGGAGCCTCTGCTTATATAGACCTC-3' (SEQ ID NO: 102).

The PCR product was purified using a Macherey-Nagel PCR kit and gel cleanser. 1 µl of the product from the first PCR cycle 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 volume of 20 µl containing 1x High Fidelity buffer, 2 mM MgSO ₄ , 0.2 mM dNTP, 0.5 units High Fidelity Platinum Taq and 0.2 µM of each primer. Cycle conditions were as follows: 94° C., 2 min; (94°C, 15 s; 55°C, 30 s; 68°C, 4 min) x35; 68°C, 10 min. The presence of env was confirmed by analysis on a 0.7% agarose gel and the product was purified using the Macherey-Nagel Gel purification kit and PCR. Then, 10 ng of shell and 0.5 ng of CMV were subjected to overlap PCR using primers CMVenv and Rev19 in triplicate. Total reaction volume 6 was 50 µl containing 1x High Fidelity Buffer, 0.2 µM MgSO4, 0.2 mM dNTP, 1 U. High Fidelity Platinum Taq and 0.4 µM of each primer. PCR was performed at 94°C, 2 min; (94°C, 30 s; 60°C, 30 s; 68°C, 4 min) x25; 68°C, 10 min. 500 ng of CMV-env were co-transfected with pSG3Aenv in 6-well plates into 293T cells and the supernatant was collected 48 hours later. Supernatants were tested for neutralization using the TZM-bl assay as described above.

Virus Culture (VOA).

Virus from an EB354 donor was obtained by co-culture of a patient's peripheral blood mononuclear cells (PBMCs) with healthy donor PBMCs as described in van't Wout et al., Isolation and propagation of HIV-1 on peripheral blood mononuclear cells. Nat Protoc, 2008. 3(3):p. 363-70. Healthy donor PBMCs were obtained from patients by leukapheresis according to Rockefeller University study protocol MNU-0628. Healthy donor PBMCs were prestimulated at a density of 5x106 cells per ml in IMDM containing 10% FBS, 1% penicillin-streptomycin and 1 μg/ml PHA for 2-3 days at 37° C. at 5% CO ₂ . Then 6x106 stimulated donor PBMCs were transferred into IMDM containing 10% FBS, 1% penicillin-streptomycin, 10 IU/ml IL-2 and 5 μg/ml polybrene and co-cultured with 5-10x106 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 containing more than 1 ng p24 per ml supernatant were frozen and stored at -80°C. Determination of tissue culture infectious dose 50 (TCID ₅₀ ) and subsequent susceptibility testing of autologous viruses to various broad spectrum neutralizing antibodies and autologous serum IgG was performed using a TZM-bl neutralization assay according to the protocols described above. All neutralization assays were performed in duplicate.

HIV-1 _YU2 envelope mutants.

Single, double and triple mutations were introduced into the wild-type HIV-1 _YU2 envelope using the QuikChange multiple site-directed mutagenesis kit according to the manufacturer's specifications (Agilent Technologies).

Analysis of the evolution of the virus.

Env nucleotide sequence alignments were performed using the ClustalW program (version 2.11) or by manual alignment using Geneious sequence analysis software (version 8.1.6). For phylogenetic analysis and diversity calculations, areas that could not be uniquely aligned were removed. Evolutionary model classes for phylogenetic analysis were selected by the maximum likelihood method using the jModelTest program. Phylogenetic trees were obtained using the maximum likelihood method using the PhyML program (version 3) with a joint assessment of the values of the model and phylogeny parameters. Pairwise comparisons of genetic diversity between samples were performed using the two-sample U statistical test in the DIVEIN web tool.

Bioinformatics processing of enV sequences using the MiSeq system.

Sequence adapters were removed using Cutadapt v1.8.3. Assembly reading

- 31 042959 for each virus was performed in three stages. First, de novo assembly was done with Spades v3.6.1, producing long contig files. Then contig longer than 255 b.p. aligned with the reference HIV envelope sequence and generated a consensus sequence using the Geneious 8 program. Finally, the reads were realigned with the consensus sequence to close the gaps and a final consensus was obtained. Sequences with double peaks (consensus identity with cut-off for any residue <75%) were excluded from subsequent analysis.

Statistical analysis.

Statistical differences were analyzed using 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.

B. Results.

Several BNAbs.

Purified IgG from the EB354 donor sample ranked in the top 1% in terms of breadth and neutralization efficiency in a cohort of 394 long-term non-progressors infected with HIV-1. This donor was diagnosed with HIV-1 type B in 1986. EB354 was treated with didanosine and stavudine between 1995 and 1998 but has not received antiretroviral therapy since. In 2002, the viral load in EB354 was <400 copies/ml, and the CD4 and CD8 counts were 954 and 1046 cells/mm ³ , respectively. Between 2002 and 2006, EB354 had three documented peaks of viremia (FIG. 1A). Serological neutralizing activity and spectrum breadth were first assessed in 2006 when the viral load was <400 copies/ml (FIGS. 1A and 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, 06:02 (Table 3).

To isolate antibodies that are responsible for serological activity in this subject, single cell selection was performed using four different HIV-1 baits; core 2CC, gp140 _YU2 / 1:1 mix gp140 _92UG37 . ₈ (type A) + gp140 _CZA79012 (type C) and BG505 SOSIP.664 (Fig. 1C). The total number of isolated paired heavy and light chains was 241, of which 152 antibodies formed 22 different clones. Antibodies from 3 clones showed level 2 neutralizing activity (FIG. 1C and Table 13). Compared with serum IgG in the period 2010-2015. antibodies belonging to this clone, exemplified by the BG18 antibody (FIG. 1C), accounted for most of the serological activity (FIG. 1D). Antibodies belonging to two other neutralizing clones, exemplified by the NC37 antibody and the BG1 antibody (FIG. 1C), were less effective but complemented the activity of BG18 (FIG. 1D).

To map antibody binding sites in 3 neutralizing clones, TZM-bl assays were performed using HIV-1 _YU2 variants carrying epitope-specific point mutations in Env. Given the fact that polyclonal IgG showed no measurable change in susceptibility to the mutants, the BG18 antibody was sensitive to YU2 _N332K (glycan-V3), the BG1 antibody to YU2 _N160K (V1V2) and the NC37 antibody to YU2 _n280y (CD4b) (Fig. .1D). According to the results of neutralization, the binding of BG18 in the ELISA method was competitively inhibited by PGT121 and 10-1074; and BG1 binding decreased in the presence of PGT145 (FIG. 5).

BG18 neutralized 64% of viruses in a panel of 118 viruses (FIG. 1E, Table 5) with a geometric mean IC _{50 of} 0.03 µg/mL and was comparable in broadband to but more effective than PGT121 or 10-1074 ( Fig. 1F, tables 5-10). Antibodies NC37 and BG1 had geometric mean IC ₅₀ values of 0.3 μg/ml and 0.67 μg/ml and a latitude of 33 and 37%, respectively. (FIGS. 1E and 1F, Tables 6 and 7). A 1:1:1 mixture of three bNAbs neutralized 81% of the viruses in a panel of 118 viruses with a geometric mean IC ₅₀ of 0.130 μg/ml indicating an additive effect (FIG. 1E, Table 11).

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BG18 Virus ID Group 1С ₅₀ !From ₈ o 6535.3 IN 0.001 0.005 QH0692.42 IN 0.013 0.042 SC422661.8 IN 0.005 0.021 PVO. 4 V 0.003 0.013 TRO.11 V 0.001 0.002 ASYU. 0.2 9 V 0.001 0.003 RHPA4259.7 V 1.437 >30 THRO4156.18 V >30 >30 REJO4541.67 V >30 >30 TRJO4551.58 V 3.341 26,938 WITO4160.33 V 0.018 0.250 CAAN5342.A2 V 0.008 0.040 WEAU_dl5_410_787 In (T/F) 0.003 0.012 1006_ll_C3_1601 In (T/F) 0.001 0.004 1054_07_TC4_1499 In (T/F) 0.005 0.022 1056_1O_TA11_1826 In (T/F) 0.007 0.045 1012_ll_TC21_3257 In (T/F) 0.003 0.012 6240_08_TA5_4622 In (T/F) 0.003 0.010 6244_13_B5_4576 In (T/F) 0.011 0.049 62357_14_D3_4589 In (T/F) 3,008 >30 SC05_8Cll_2344 In (T/F) 0.003 0.016 Dul56.12 WITH 0.003 0.010 Dul72.17 WITH >30 >30 Du422.1 WITH 0.009 0.057 ZM197M.PB7 WITH >30 >30 ZM214M.PL15 WITH >30 >30 ZM233M.PB6 WITH 2.866 29.805 ZM249M.PL1 WITH >30 >30

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ZM53M.PB12 C >30 >30 ZM109F.PB4 C >30 >30 ZM135M.PL10a C 0.029 0.161 CAP45.2.00.G3 C >30 >30 CAP210.2.00.E8 C 3.469 10.748 HIV-001428-2.42 C 0.004 0.021 HIV-0013095-2.11 C 10.516 29.966 HIV-16055-2.3 C >30 >30 HIV-16845-2.22 C 0.019 0.090 CelO86_B2 C(T/F) >30 >30 CeO393_C3 C(T/F) >30 >30 Cell76_A3 C(T/F) 0.004 0.012 Ce2010_F5 C(T/F) >30 >30 CeO682_E4 C(T/F) >30 >30 Cell72_Hl C(T/F) 0.002 0.007 Ce2060_G9 C(T/F) >30 >30 Ce703010054_2A2 C(T/F) >30 >30 BF1266.431a C(T/F) >30 >30 246F CIG C(T/F) 0.034 0.257 249M Bio C(T/F) >30 >30 ZM247vl(Rev-) C(T/F) 1.454 8.725 7030102001E5(Rev-) C(T/F) 0.002 0.008 1394C9G1(Rev-) C(T/F) 0.029 0.350 Ce704809221_1B3 C(T/F) 4,394 >30 CNE19 BC >30 >30 CNE2 0 BC 0.002 0.006 CNE21 BC 0.002 0.007 CNE17 BC 0.020 0.109 CNE3 0 BC 0.019 0.059 CNE52 BC 0.012 0.045 CNE53 BC 0.003 0.011 CNE58 BC 0.024 0.073

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MS208.Al A >30 >30 Q23.17 A 0.006 0.020 Q461.e2 A >30 >30 Q769.d22 A >30 >30 Q259.d2.17 A >30 >30 Q842.dl2 A 13.986 >30 3415.vl.cl A 0.023 0.087 3365.v2.c2 A 0.013 0.037 191955_A11 A(T/F) >30 >30 191084 B7-19 A(T/F) 0.011 0.043 9004SS_A3_4 A(T/F) 0.005 0.056 T257-31 CRF02_AG >30 >30 928-28 CRF02_AG 0.761 3,934 263-8 CRF02_AG 1.380 7.548 T250-4 CRF02_AG 0.001 0.006 T251-18 CRF02_AG 0.086 0.328 T278-50 CRF02_AG 2.947 12.875 T255-34 CRF02_AG >30 >30 211-9 CRF02_AG 1.299 5,529 235-47 CRF02_AG 0.005 0.017 620345.cOl CRF01_AE >30 >30 CNE8 CRF01_AE >30 >30 C1080.C03 CRF01_AE 5.369 >30 R2184.C04 CRF01_AE >30 >30 R1166.C01 CRF01_AE >30 >30 R3265.C06 CRF01_AE >30 >30 C2101.cOl CRF01_AE >30 >30 C3347.C11 CRF01_AE >30 >30 C4118.c09 CRF01_AE >30 >30 CNE5 CRF01_AE >30 >30 BJOX009000.02.4 CRF01_AE 24.376 >30

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BJOX015000.11.5 CRF01_AE (T/F) >30 >30 BJOX010000.06.2 CRF01_AE (T/F) >30 >30 BJOX025000.01.1 CRF01_AE (T/F) >30 >30 BJOX028000.10.3 CRF01_AE (T/F) 0.474 14.851 Х1193_с1 G 0.005 0.022 Р0402_с2_11 G 0.005 0.029 Х1254_сЗ G 0.004 0.012 X2088_s9 G 0.004 0.009 Х2131_С1_В5 G 0.002 0.008 Р1981_С5_3 G 0.006 0.019 X1632_S2_B10 G >30 >30 3016.v5.c45 D >30 >30 А07412М1.vrcl2 D 0.081 1.715 231965.c01 D >30 >30 231966.s02 D >30 >30 191821_E6_1 D(T/F) 14.076 >30 3817.v2.c59 CD 0.219 1.059 6480.v4.c25 CD 0.016 0.116 6952. vl.с20 CD 0.005 0.011 6811,v7,s18 CD 0.001 0.006 89-E1_2_25 CD >30 >30 3301.vl.с24 AC 0.025 0.082 6041.v3.c23 AC >30 >30 6540.v4. cl AC 28.197 >30 6545.v4. cl AC >30 >30 0815.v3.c3 ACD 0.160 5,964 3103.v3.cl0 ACD 0.002 0.005 BG505/T332N A <0.0001 0.002 YU2_WT IN 0.033 0.198 YU2_N276D IN 0.012 0.036 YU2_N332K IN >30 >30 YU2_N160K IN 0.028 0.150 YU2_N280Y IN 0.005 0.016

Table 5. IC ₅₀ and IC ₈₀ values in TZM-bl assay for BG18.

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NC37 Virus ID Group 1C _5O 1C _8O 6535.3 IN >30 >30 QH0692.42 IN 0.582 2.054 SC422661.8 IN 0.071 0.321 PVO. 4 V 0.412 2.825 TRO.11 V 0.337 1.761 ASYU. 0.29 V >30 >30 RHPA4259.7 V 0.345 2.289 THRO4156.18 V >30 >30 REJO4541.67 V 0.104 0.304 TRJO4551.58 V 0.143 1,615 WITO4160.33 V 0.733 17.779 CAAN5342.A2 V 0.911 3,694 WEAU_dl5_410_787 In (T/F) 0.037 0.133 1006_ll_C3_1601 In (T/F) 0.054 0.207 1054_07_TC4_1499 In (T/F) >30 >30 1056_1O_TA11_1826 In (T/F) 1.048 5,510 1012_11_TC21_3257 In (T/F) 0.099 0.638 6240_08_TA5_4622 In (T/F) 1.236 6, 388 6244_13_B5_4576 In (T/F) 5,574 >30 62357_14_D3_4589 In (T/F) 1,860 29.422 SC05_8Cll_2344 In (T/F) 0.202 0.714 Dul56.12 WITH >30 >30 Dul72.17 WITH >30 >30 Du422.1 WITH >30 >30

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ZM197M.PB7 C 3,098 17.368 ZM214M.PL15 C >30 >30 ZM233M.PB6 C >30 >30 ZM249M.PL1 C >30 >30 ZM53M.PB12 C >30 >30 ZM109F.PB4 C >30 >30 ZM135M.PL10a C >30 >30 CAP45.2.00.G3 C >30 >30 CAP210.2.00.E8 C >30 >30 HIV-001428-2.42 C 0.124 2.275 HIV-0013095-2.11 C >30 >30 HIV-16055-2.3 C >30 >30 HIV-16845-2.22 C >30 >30 CelO86_B2 C(T/F) 2.322 20.996 CeO393_C3 C(T/F) >30 >30 Cell76_A3 C(T/F) >30 >30 Ce2010_F5 C(T/F) >30 >30 CeO682_E4 C(T/F) >30 >30 Cell72_Hl C(T/F) >30 >30 Ce2060_G9 C(T/F) >30 >30 Ce703010054_2A2 C(T/F) >30 >30 BF1266.431a C(T/F) >30 >30 246F CIG C(T/F) >30 >30 249M Bio C(T/F) >30 >30 ZM247vl(Rev-) C(T/F) >30 >30 7030102001E5(Rev-) C(T/F) >30 >30 1394C9G1(Rev-) C(T/F) >30 >30 Ce704809221_1B3 C(T/F) >30 >30 CNE19 BC >30 >30 CNE2 0 BC >30 >30 CNE21 BC >30 >30 CNE17 BC >30 >30

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CNE3 0 BC 0.915 11.813 CNE52 BC >30 >30 CNE53 BC >30 >30 CNE58 BC >30 >30 MS208.Al A >30 >30 Q23.17 A >30 >30 Q461.e2 A >30 >30 Q769.d22 A 0.242 1.574 Q259.d2.17 A >30 >30 Q842.dl2 A 0.255 1.821 3415.vl.cl A 0.183 0.654 3365.v2.c2 A >30 >30 191955_A11 A(T/F) >30 >30 191084 B7-19 A(T/F) 0.064 0.208 9004SS_A3_4 A(T/F) >30 >30 T257-31 CRF02_AG >30 >30 928-28 CRF02_AG >30 >30 263-8 CRF02_AG 0.269 1.523 T250-4 CRF02_AG >30 >30 T251-18 CRF02_AG 1.835 7.857 T278-50 CRF02_AG >30 >30 T255-34 CRF02_AG >30 >30 211-9 CRF02_AG >30 >30 235-47 CRF02_AG 14.369 >30 620345.cOl CRF01_AE >30 >30 CNE8 CRF01_AE >30 >30 C1080.C03 CRF01_AE >30 >30 R2184.c04 CRF01_AE 0.025 0.082 R1166.C01 CRF01_AE >30 >30 R3265.C06 CRF01_AE >30 >30 C2101.C01 CRF01_AE >30 >30

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С3347.С11 CRF01_AE >30 >30 С4118.с09 CRF01_AE >30 >30 CNE5 CRF01_AE >30 >30 BJOX009000.02.4 CRF01_AE >30 >30 BJOX015000.11.5 CRF01_AE (T/F) >30 >30 BJOX010000.06.2 CRF01_AE (T/F) >30 >30 BJOX025000.01.1 CRF01_AE (T/F) >30 >30 BJOX028000.10.3 CRF01_AE (T/F) >30 >30 Х1193_с1 G >30 >30 Р0402_с2_11 G >30 >30 Х1254_сЗ G >30 >30 X2088_s9 G >30 >30 Х2131_С1_В5 G >30 >30 Р1981_С5_3 G >30 >30 X1632_S2_B10 G >30 >30 3016.v5.c45 D 23.592 >30 А07412М1.vrcl2 D >30 >30 231965.c01 D >30 >30 231966.s02 D >30 >30 191821_E6_1 D(T/F) 9,963 >30 3817.v2.c59 CD >30 >30 6480.v4.c25 CD 3,060 >30 6952.vl.c20 CD 0.104 0.477 6811.v7.cl8 CD >30 >30 89-E1_2_25 CD 17.963 >30 3301.vl.c24 AC >30 >30 6041.v3.c23 AC 0.018 0.054 6540.v4. cl AC >30 >30 6545.v4.cl AC >30 >30 0815.v3.c3 ACD 0.022 0.082 3103.v3.cl0 ACD >30 >30 BG505/T332N A YU2_WT IN 0.12 YU2_N276D IN NT YU2_N332K IN 0.05 YU2_N160K IN 0.05 YU2_N280Y IN >30

Table 6. IC ₅₀ and IC ₈₀ values in TZM-bl analysis for NC37.

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Bgl Virus ID Group 1С ₅₀ 1C ₈ about 6535.3 IN 0.322 1.130 QH0692.42 V >30 >30 SC422661.8 V >30 >30 PVO. 4 V >30 >30 TRO.11 V >30 >30 AGIO.0.29 V 0.012 0.043 RHPA4259.7 V >30 >30 THRO4156.18 V >30 >30 REJO4541.67 V 0.342 1,662 TRJO4551.58 V >30 >30 WITO4160.33 V 0.002 0.014 CAAN5342.A2 V >30 >30 WEAU_dl5_410_787 In (T/F) >30 >30 1006_ll_C3_1601 In (T/F) >30 >30 1054_07_TC4_1499 In (T/F) >30 >30 1056_1O_TA11_1826 In (T/F) 5,428 29.924 1012_ll_TC21_3257 In (T/F) >30 >30 6240_08_TA5_4622 In (T/F) >30 >30 6244_13_B5_4576 In (T/F) >30 >30 62357_14_D3_4589 In (T/F) 0.638 2.889

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SC05_8Cll_2344 In (T/F) >30 >30 Dul56.12 C 1.525 6, 031 Dul72.17 C >30 >30 Du422.1 C 1,804 13.708 ZM197M.PB7 C >30 >30 ZM214M.PL15 C >30 >30 ZM233M.PB6 C >30 >30 ZM249M.PL1 C 1.223 6.795 ZM53M.PB12 C >30 >30 ZM109F.PB4 C >30 >30 ZM135M.PL10a C >30 >30 CAP45.2.00.G3 C 0.149 0.383 CAP210.2.00.E8 C >30 >30 HIV-001428-2.42 C 1,094 3, 124 HIV-0013095-2.11 C >30 >30 HIV-16055-2.3 C 4.296 16,939 HIV-16845-2.22 C >30 >30 CelO86_B2 C(T/F) >30 >30 CeO393_C3 C(T/F) >30 >30 Cell76_A3 C(T/F) 0.031 0.165 Ce2010_F5 C(T/F) >30 >30 CeO682_E4 C(T/F) >30 >30 Cell72_Hl C(T/F) >30 >30 Ce2060_G9 C(T/F) >30 >30 Ce703010054_2A2 C(T/F) >30 >30 BF1266.431a C(T/F) >30 >30 246F CIG C(T/F) >30 >30 249M Bio C(T/F) 1,473 5,445 ZM247vl(Rev-) C(T/F) >30 >30 7030102001E5(Rev-) C(T/F) >30 >30 1394C9G1(Rev-) C(T/F) 1.279 7.249 Ce704809221_lB3 C(T/F) 3.251 18.455

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CNE19 BC 1.267 10.883 CNE2 0 BC 4.264 >30 CNE21 BC >30 >30 CNE17 BC >30 >30 CNE3 0 BC >30 >30 CNE52 BC 0.975 5,008 CNE53 BC 1.318 7.470 CNE58 BC >30 >30 MS208.Al A 1.478 6,543 Q23.17 A 0.008 0.016 Q461.e2 A >30 >30 Q769.d22 A >30 >30 Q259.d2.17 A >30 >30 Q842.dl2 A 1.395 4.297 3415.vl.cl A 14.412 >30 3365.v2.c2 A 1.161 4.272 191955_A11 A(T/F) 0.008 0.018 191084 B7-19 A(T/F) >30 >30 9004SS_A3_4 A(T/F) >30 >30 T257-31 CRF02_AG >30 >30 928-28 CRF02_AG >30 >30 263-8 CRF02_AG >30 >30 T250-4 CRF02_AG 3,921 >30 T251-18 CRF02_AG 3.591 17.977 T278-50 CRF02_AG 19.313 >30 T255-34 CRF02_AG 11,589 >30 211-9 CRF02_AG 2.852 12.939 235-47 CRF02_AG 1.275 4.384 620345.cOl CRF01_AE 0.626 4.229 CNE8 CRF01_AE >30 >30 C1080.C03 CRF01_AE 0.002 0.005

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R2184.c04 CRF01_AE >30 >30 R1166.c01 CRF01_AE >30 >30 R3265.c06 CRF01_AE 0.611 3.438 C2101.cOl CRF01_AE >30 >30 C3347.C11 CRF01_AE >30 >30 C4118.c09 CRF01_AE 22.788 >30 CNE5 CRF01_AE 0.351 1.277 BJOX009000.02.4 CRF01_AE >30 >30 BJOX015000.11.5 CRF01_AE (T/F) >30 >30 BJOX010000.06.2 CRF01_AE (T/F) >30 >30 BJOX025000.01.1 CRF01_AE (T/F) >30 >30 BJOX028000.10.3 CRF01_AE (T/F) >30 >30 X1193_cl G 0.138 0.463 P0402_c2_ll G >30 >30 X1254_c3 G 0.048 0.086 X2088_c9 G >30 >30 X2131_C1_B5 G 0.116 0.313 P1981_C5_3 G >30 >30 X1632_S2_B10 G 0.079 0.250 3016.v5.c45 D >30 >30 A07412M1.vrcl2 D >30 >30 231965.cOl D >30 >30 231966.c02 D >30 >30 191821_E6_1 D(T/F) >30 >30 3817.v2.c59 CD 11.147 >30 6480.v4.c25 CD >30 >30 6952.vl.c20 CD >30 >30 6811.v7.cl8 CD >30 >30 89-Fl_2_25 CD >30 >30

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3301. vl.с24 AC >30 >30 6041.v3.c23 AC 6, 745 16.641 6540.v4.cl AC >30 >30 6545.v4.cl AC >30 >30 0815.v3.c3 ACD >30 >30 3103.v3.cl0 ACD >30 >30 BG505/T332N A 0.034 0.125 YU2_WT IN 15.816 >30 YU2_N276D IN 18.765 >30 YU2_N332K IN 19.434 >30 YU2_N160K IN >30 >30 YU2_N280Y IN 8.998 23.023

Table 7. Values of 1C ₅ o and 1Cg0 in TZM-bl analysis for BG1.

BG8 Virus ID Group 1C ₅₀ !From ₈ o 6535.3 IN 0.007 0.022 QH0692.42 IN 0.106 0.383 SC422661.8 IN 0.058 0.368 PVO. 4 V 0.072 0.252 TRO.11 V 0, OH 0.039 AGIO.0.29 V 0.015 0.043 RHPA4259.7 V 0.023 0.083 THRO4156.18 V >10 >10 REJO4541.67 V >10 >10 TRJO4551.58 V 0.057 0.218 WITO4160.33 V 0.111 0.828 CAAN5342.A2 V 0.005 0.019 WEAU_dl5_410_787 In (T/F) 0.031 0.112 1006_11_SZ_1601 In (T/F) 0.002 0.006 1054_07_TS4_1499 In (T/F) 0.048 0.222 1056_10_TA11_1826 In (T/F) 0.038 0.168

-45042959

1012_11_TS21_3257 In (T/F) 0.011 0.047 6240_08_TA5_4622 In (T/F) 0.038 0.131 6244_13_B5_4576 In (T/F) 0.091 0.380 62357_14_D3_4589 In (T/F) >10 >10 SC05_8Cll_2344 In (T/F) 0.017 0.048 Dul56.12 C 0.010 0.036 Dul72.17 C 0.049 0.194 Du422.1 C 0.047 0.113 ZM197M.PB7 C >10 >10 ZM214M.PL15 C 0.415 2,970 ZM233M.PB6 C 0.058 0.371 ZM249M.PL1 C >10 >10 ZM53M.PB12 C >10 >10 ZM109F.PB4 C >10 >10 ZM135M.PL10a C 0.061 0.263 CAP45.2.00.G3 C >10 >10 CAP210.2.00.E8 C >10 >10 HIV-001428-2.42 C 0.035 0.126 HIV-0013095-2.11 C >10 >10 HIV-16055-2.3 C >10 >10 HIV-16845-2.22 C 0.727 3,930 CelO86_B2 C(T/F) >10 >10 CeO393_C3 C(T/F) >10 >10 Cell76_A3 C(T/F) 0.022 0.070 Ce2010_F5 C(T/F) >10 >10 CeO682_E4 C(T/F) >10 >10 Cell72_Hl C(T/F) 0.019 0.083 Ce2060_G9 C(T/F) >10 >10 Ce703010054_2A2 C(T/F) >10 >10 BF1266.431a C(T/F) >10 >10 246F CIG C(T/F) 0.035 0.119 249M Bio C(T/F) >10 >10

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ZM247vl(Rev-) C(T/F) 0.042 0.146 7030102001E5(Rev-) C(T/F) 0.009 0.019 1394C9G1(Rev-) C(T/F) 0.031 0.093 Ce704809221_1B3 C(T/F) 0.084 0.395 CNE19 BC 0.075 >10 CNE2 0 BC 0.002 0.005 CNE21 BC 0.047 0.124 CNE17 BC 1.180 4,853 CNE3 0 BC 0.261 0.678 CNE52 BC 1.991 9.763 CNE53 BC 0.013 0.058 CNE58 BC 0.165 0.476 MS208.Al A >10 >10 Q23.17 A 0.005 0.015 Q461.e2 A >10 >10 Q769.d22 A >10 >10 Q259.d2.17 A >10 >10 Q842.dl2 A >10 >10 3415.vl.cl A >10 >10 3365.v2.c2 A 0.057 0.200 191955_A11 A(T/F) >10 >10 191084 B7-19 A(T/F) 0.026 0.108 9004SS_A3_4 A(T/F) 0.012 0.032 T257-31 CRF02_AG >10 >10 928-28 CRF02_AG 0.616 2.396 263-8 CRF02_AG 0.416 5.714 T250-4 CRF02_AG 0.002 0.005 T251-18 CRF02_AG 0.399 2.292 T278-50 CRF02_AG 1.713 >10 T255-34 CRF02_AG >10 >10 211-9 CRF02_AG 0.065 0.403

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235-47 CRF02_AG 0.058 0.224 620345.c01 CRF01_AE >10 >10 CNE8 CRF01_AE >10 >10 С1080.с03 CRF01_AE >10 >10 R2184.c04 CRF01_AE >10 >10 R1166.C01 CRF01_AE >10 >10 R3265.C06 CRF01_AE >10 >10 С2101.с01 CRF01_AE >10 >10 C3347 .cP CRF01_AE >10 >10 С4118.с09 CRF01_AE >10 >10 CNE5 CRF01_AE >10 >10 BJOX009000.02.4 CRF01_AE >10 >10 BJOX015000.11.5 CRF01_AE (T/F) >10 >10 BJOX010000.06.2 CRF01_AE (T/F) >10 >10 BJOX025000.01.1 CRF01_AE (T/F) >10 >10 BJOX028000.10.3 CRF01_AE (T/F) >10 >10 Х1193_с1 G 0.070 0.207 Р0402_с2_11 G 0.008 0.024 Х1254_сЗ G 0.063 0.239 X2088_s9 G 0.003 0.010 Х2131_С1_В5 G 0.014 0.056 Р1981_С5_3 G 0.004 0.013 X1632_S2_B10 G >10 >10 3016.v5.с45 D >10 >10 А07412М1.vrcl2 D 0.011 0.037 231965.c01 D >10 >10 231966.s02 D >10 >10 191821_E6_1 D(T/F) >10 >10 3817.v2.с59 CD 0.706 4.492 6480.v4.c25 CD 0.006 0.023 6952. vl.с20 CD 0.023 0.086 6811.v7.c18 CD 0.003 0.009 89-Fl_2_25 CD >10 >10 3301.vl.с24 AC 0.018 0.049 6041.v3.c23 AC >10 >10 6540.v4.cl AC >10 >10 6545.v4.cl AC >10 >10 0815.v3.c3 ACD 0.043 0.197 3103.v3.cl0 ACD 0.024 0.061

Table 8. IC ₅₀ and IC8o values in TZM-bl analysis for BG8.

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10-1074 Virus ID Group 1C ₅₀ 1C ₈ about 6535.3 IN 0.007 0.022 QH0692.42 V 0.106 0.383 SC422661.8 V 0.058 0.368 PVO. 4 V 0.072 0.252 TRO.11 V 0.011 0.039 ASYU. 0.2 9 V 0.015 0.043 RHPA4259.7 V 0.023 0.083 THRO4156.18 V >10 >10 REJO4541.67 V >10 >10 TRJO4551.58 V 0.057 0.218 WITO4160.33 V 0.111 0.828 CAAN5342.A2 V 0.005 0.019 WEAU_dl5_410_787 In (T/F) 0.031 0.112 1006_ll_C3_1601 In (T/F) 0.002 0.006 1054_07_TC4_1499 In (T/F) 0.048 0.222 1056_1O_TA11_1826 In (T/F) 0.038 0.168 1012_ll_TC21_3257 In (T/F) 0.011 0.047 6240_08_TA5_4622 In (T/F) 0.038 0.131 6244_13_B5_4576 In (T/F) 0.091 0.380

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62357_14_D3_4589 In (T/F) >10 >10 SC05_8Cll_2344 In (T/F) 0.017 0.048 Dul56.12 C 0.010 0.036 Dul72.17 C 0.049 0.194 Du422.1 C 0.047 0.113 ZM197M.PB7 C >10 >10 ZM214M.PL15 C 0.415 2,970 ZM233M.PB6 C 0.058 0.371 ZM249M.PL1 C >10 >10 ZM53M.PB12 C >10 >10 ZM109F.PB4 C >10 >10 ZM135M.PL10a C 0.061 0.263 CAP45.2.00.G3 C >10 >10 CAP210.2.00.E8 C >10 >10 HIV-001428-2.42 C 0.035 0.126 HIV-0013095-2.11 C >10 >10 HIV-16055-2.3 C >10 >10 HIV-16845-2.22 C 0.727 3,930 CelO86_B2 C(T/F) >10 >10 CeO393_C3 C(T/F) >10 >10 Cell76_A3 C(T/F) 0.022 0.070 Ce2010_F5 C(T/F) >10 >10 CeO682_E4 C(T/F) >10 >10 Cell72_Hl C(T/F) 0.019 0.083 Ce2060_G9 C(T/F) >10 >10 Ce703010054_2A2 C(T/F) >10 >10 BF1266.431a C(T/F) >10 >10 246F CIG C(T/F) 0.035 0.119 249M Bio C(T/F) >10 >10 ZM247vl(Rev-) C(T/F) 0.042 0.146 7030102001E5(Rev-) C(T/F) 0.009 0.019 1394C9G1(Rev-) C(T/F) 0.031 0.093

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Ce704809221_1B3 C(T/F) 0.084 0.395 CNE19 BC 0.075 >10 CNE2 0 BC 0.002 0.005 CNE21 BC 0.047 0.124 CNE17 BC 1.180 4,853 CNE3 0 BC 0.261 0.678 CNE52 BC 1.991 9.763 CNE53 BC 0.013 0.058 CNE58 BC 0.165 0.476 MS208.Al A >10 >10 Q23.17 A 0.005 0.015 Q461.e2 A >10 >10 Q769.d22 A >10 >10 Q259.d2.17 A >10 >10 Q842.dl2 A >10 >10 3415.vl.cl A >10 >10 3365.v2.c2 A 0.057 0.200 191955_A11 A(T/F) >10 >10 191084 B7-19 A(T/F) 0.026 0.108 9004SS_A3_4 A(T/F) 0.012 0.032 T257-31 CRF02_AG >10 >10 928-28 CRF02_AG 0.616 2.396 263-8 CRF02_AG 0.416 5.714 T250-4 CRF02_AG 0.002 0.005 T251-18 CRF02_AG 0.399 2.292 T278-50 CRF02_AG 1.713 >10 T255-34 CRF02_AG >10 >10 211-9 CRF02_AG 0.065 0.403 235-47 CRF02_AG 0.058 0.224 620345.cOl CRF01_AE >10 >10

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CNE8 CRF01_AE >10 >10 С1080.С03 CRF01_AE >10 >10 R2184.c04 CRF01_AE >10 >10 R1166.C01 CRF01_AE >10 >10 R32 65.co 6 CRF01_AE >10 >10 С2101.сО1 CRF01_AE >10 >10 С3347.С11 CRF01_AE >10 >10 С4118.с09 CRF01_AE >10 >10 CNE5 CRF01_AE >10 >10 BJOX009000.02.4 CRF01_AE >10 >10 BJOX015000.11.5 CRF01_AE (T/F) >10 >10 BJ0XO1OOOO.06.2 CRF01_AE (T/F) >10 >10 BJOX025000.01.1 CRF01_AE (T/F) >10 >10 BJOX028000.10.3 CRF01_AE (T/F) >10 >10 Х1193_с1 G 0.070 0.207 Р0402_с2_11 G 0.008 0.024 Х1254_сЗ G 0.063 0.239 X2088_s9 G 0.003 0.010 Х2131_С1_В5 G 0.014 0.056 Р1981_С5_3 G 0.004 0.013 X1632_S2_B10 G >10 >10 3016.v5.с45 D >10 >10 А07412М1.vrcl2 D 0.011 0.037 231965.cOl D >10 >10 231966.s02 D >10 >10 191821_E6_1 D(T/F) >10 >10 3817.v2.с59 CD 0.706 4.492 6480.v4.c25 CD 0.006 0.023 6952.vl.с20 CD 0.023 0.086 6811.v7.c18 CD 0.003 0.009 89-Fl_2_25 CD >10 >10 3301.vl.с24 AC 0.018 0.049 6041.v3.c23 AC >10 >10 6540.v4. cl AC >10 >10 6545.v4.cl AC >10 >10 0815.v3.c3 ACD 0.043 0.197 3103.v3.cl0 ACD 0.024 0.061

Table 9. IC ₅₀ and IC ₈₀ values in TZM-bl analysis for 10-1074.

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PGT121 Virus ID Group 1C _5O ICso 6535.3 IN 0.003 0.010 QH0692.42 IN 0.498 8.507 SC422661.8 IN 0.079 0.373 PVO. 4 V 0.094 0.450 TRO.11 V 0, OH 0.037 AGIO.0.29 V 0.019 0.060 RHPA4259.7 V 0.013 0.046 THRO4156.18 V >10 >10 REJO4541.67 V >10 >10 TRJO4551.58 V 0.802 >10 WITO4160.33 V 0.301 3,200 CAAN5342.A2 V 0.008 0.026 WEAU_dl5_410_787 In (T/F) 0.017 0.062 1006_ll_C3_1601 In (T/F) 0.002 0.008 1054_07_TC4_1499 In (T/F) 0.041 0.299 1056_1O_TA11_1826 In (T/F) 0.034 0.148 1012_H_TC21_3257 In (T/F) 0.008 0.034 6240_08_TA5_4622 In (T/F) 0.068 0.231 6244_13_B5_4576 In (T/F) 0.172 1.471 62357_14_D3_4589 In (T/F) 6.215 >10 SC05_8CH_2344 In (T/F) 0.030 0.108

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Dul56.12 C 0.006 0.019 Dul72.17 C 0.062 0.531 Du422.1 C 0.017 0.088 ZM197M.PB7 C >10 >10 ZM214M.PL15 C 0.288 2.456 ZM233M.PB6 C 2.809 >10 ZM249M.PL1 C >10 >10 ZM53M.PB12 C 0.001 0.005 ZM109F.PB4 C 8.924 >10 ZM135M.PL10a C 0.730 5, 727 CAP45.2.00.G3 C 1,300 >10 CAP210.2.00.E8 C >10 >10 HIV-001428-2.42 C 0.020 0.126 HIV-0013095-2.11 C >10 >10 HIV-16055-2.3 C 0.772 7.986 HIV-16845-2.22 C 8.512 >10 CelO86_B2 C(T/F) 0.003 0.016 CeO393_C3 C(T/F) >10 >10 Cell76_A3 C(T/F) 0.022 0.063 Ce2010_F5 C(T/F) >10 >10 CeO682_E4 C(T/F) >10 >10 Cell72_Hl C(T/F) 0.007 0.030 Ce2060_G9 C(T/F) >10 >10 Ce703010054_2A2 C(T/F) >10 >10 BF1266.431a C(T/F) >10 >10 246F CIG C(T/F) 0.082 0.363 249M Bio C(T/F) >10 >10 ZM247vl(Rev-) C(T/F) 0.036 0.123 7030102001E5(Rev-) C(T/F) 0.016 0.042 1394C9G1(Rev-) C(T/F) 0.823 6, 227 Ce704809221_1B3 C(T/F) 0.043 0.285 CNE19 BC 0.012 0.083

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CNE2 0 BC 0.003 0.006 CNE21 BC 0.009 0.031 CNE17 BC 3,962 >10 CNE3 0 BC 0.088 0.288 CNE52 BC 4.914 >10 CNE53 BC 0.014 0.062 CNE58 BC >10 >10 MS208.Al A >10 >10 Q23.17 A 0.005 0.017 Q461.e2 A >10 >10 Q769.d22 A >10 >10 Q259.d2.17 A 3,071 >10 Q842.dl2 A 0.010 0.037 3415.vl.cl A >10 >10 3365.v2.c2 A 0.243 2.133 191955_A11 A(T/F) >10 >10 191084 B7-19 A(T/F) 0.038 0.118 9004SS_A3_4 A(T/F) 0.006 0.016 T257-31 CRF02_AG >10 >10 928-28 CRF02_AG >10 >10 263-8 CRF02_AG 0.647 4,680 T250-4 CRF02_AG 0.003 0.009 T251-18 CRF02_AG 5, 183 >10 T278-50 CRF02_AG >10 >10 T255-34 CRF02_AG 2,631 >10 211-9 CRF02_AG 0.866 6,631 235-47 CRF02_AG 0.259 1,601 620345.cOl CRF01_AE >10 >10 CNE8 CRF01_AE >10 >10 C1080.C03 CRF01_AE >10 >10 R2184.c04 CRF01_AE >10 >10

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R1166.C01 CRF01_AE >10 >10 R3265.C06 CRF01_AE >10 >10 С2101.с01 CRF01_AE >10 >10 С3347.С11 CRF01_AE >10 >10 С4118.с09 CRF01_AE >10 >10 CNE5 CRF01_AE >10 >10 BJOX009000.02.4 CRF01_AE 9,001 >10 BJOX015000.11.5 CRF01_AE (T/F) >10 >10 BJOX010000.06.2 CRF01_AE (T/F) >10 >10 BJOX025000.01.1 CRF01_AE (T/F) >10 >10 BJOX028000.10.3 CRF01_AE (T/F) >10 >10 Х1193_с1 G 0.022 0.088 Р0402_с2_11 G 0.007 0.022 Х1254_сЗ G 0.029 0.085 X2088_s9 G 0.007 0.022 Х2131_С1_В5 G 0.011 0.037 Р1981_С5_3 G 0.002 0.009 X1632_S2_B10 G >10 >10 3016.v5.c45 D >10 >10 А07412М1.vrcl2 D 0.034 0.255 231965.c01 D >10 >10 231966.s02 D >10 >10 191821_E6_1 D(T/F) >10 >10 3817.v2.с59 CD >10 >10 6480.v4.c25 CD 0.008 0.029 6952. vl.с20 CD 0.049 0.348 6811.v7.c18 CD 0.002 0.007 89-Fl_2_25 CD >10 >10 3301.vl.c24 AC 0.013 0.041 6041.v3.c23 AC >10 >10 654 0.v4. cl AC >10 >10 6545.v4.cl AC >10 >10 0815.v3.c3 ACD 0.017 0.085 3103.v3.cl0 ACD 0.024 0.073

Table 10. IC ₅₀ and IC ₈₀ values in TZM-bl assay for PGT121.

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Titers in TZM.bl cells (µg/ml)

NC37+BG1+BG18

Virus ID Group 1С ₅₀ !From ₈ o 6535.3 IN 0.006 0.030 QH0692.42 IN 0.033 0.130 SC422661.8 IN 0.024 0.089 PVO. 4 IN 0.012 0.050 TRO.11 V 0.004 0.015 ASYU. 0.2 9 V 0.006 0.016 RHPA4259.7 V 0.210 1,669 THRO4156.18 V >50 >50 REJO4541.67 V 0.199 0.696 TRJO4551.58 V 0.938 7.063 WITO4160.33 V 0.021 0.075 CAAN5342 .A2 V 0.036 0.162 YU2.DG V 0.052 0.147 WEAU_dl5_410_787 in (T/F) 0.022 0.074 1006_11_SZ_1601 In (T/F) 0.005 0.013 1054_07_TC4_1499 In (T/F) 0.022 0.122 1056_10_TA11_1826 In (T/F) 0.031 0.266 1012_11_TC21_3257 In (T/F) 0.012 0.055 6240_08_TA5_4622 In (T/F) 0.009 0.028 6244_13_B5_4576 In (T/F) 0.045 0.216 62357_14_D3_4589 In (T/F) 0.840 5,585 SC05_8Cll_2344 In (T/F) 0.045 0.083 Dul56.12 WITH 0.018 0.050 Dul72.17 WITH >50 >50

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Du422.1 C 0.076 0.347 ZM197M.PB7 C 4.123 >50 ZM214M.PL15 C >50 >50 ZM233M.PB6 C 7.814 >50 ZM249M.PL1 C 2.916 17.336 ZM53M.PB12 C >50 >50 ZM109F.PB4 C >50 >50 ZM135M.PL10a C 6, 018 31.451 CAP45.2.00.G3 C 0.251 1.097 CAP210.2.00.E8 C 19.144 >50 HIV-001428-2.42 C 0.029 0.075 HIV-0013095-2.11 C 31.333 >50 HIV-16055-2.3 C 12.372 43.320 HIV-16845-2.22 C 0.092 0.316 CelO86_B2 C(T/F) 2.088 13.998 CeO393_C3 C(T/F) >50 >50 Cell76_A3 C(T/F) 0.013 0.030 Ce2010_F5 C(T/F) >50 >50 CeO682_E4 C(T/F) >50 >50 Cell72_Hl C(T/F) 0.017 0.047 Ce2060_G9 C(T/F) >50 >50 Ce703010054_2A2 C(T/F) >50 >50 BF1266.431a C(T/F) 45.589 >50 246F CIG C(T/F) 0.172 1.220 249M Bio C(T/F) 4.422 17.159 ZM247vl(Rev-) C(T/F) 6.417 >50 7030102001E5(Rev-) C(T/F) 0.011 0.035 1394C9G1(Rev-) C(T/F) 0.078 1.405 Ce704809221_1B3 C(T/F) 10.937 >50 CNE19 BC 1.576 17.656 CNE2 0 BC 0.008 0.024 CNE21 BC 0.009 0.022 CNE17 BC 0.195 0.709 CNE3 0 BC 0.067 0.183

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CNE52 BC 0.077 0.199 CNE53 BC 0.015 0.036 CNE58 BC 0.055 0.206 MS208.Al A 2.393 17.933 Q23.17 A 0.009 0.024 Q461.e2 A >50 >50 Q769.d22 A 0.364 2.151 Q259.d2.17 A >50 >50 Q842.dl2 A 0.405 1.371 0260.v5.c36 A 0.018 0.055 3415.vl. cl A 0.039 0.187 3365.v2.c2 A 0.020 0.049 191955_AH A(T/F) 0.830 4.335 191084 B7-19 A(T/F) 0.029 0.071 9004SS_A3_4 A(T/F) 0.027 0.090 T257-31 CRF02_AG >50 >50 928-28 CRF02_AG 1.919 7.522 263-8 CRF02_AG 0.544 3.759 T250-4 CRF02_AG 0.005 0.029 T251-18 CRF02_AG 0.133 0.639 T278-50 CRF02_AG 9,069 44.006 T255-34 CRF02_AG 37.020 >50 211-9 CRF02_AG 1.976 18.953 235-47 CRF02_AG 0.013 0.046 620345.cOl CRF01_AE 1.325 8.233 CNE8 CRF01_AE >50 >50 C1080.C03 CRF01_AE 0.012 0.042 R2184.c04 CRF01_AE 0.084 0.376 RH66.c01 CRF01_AE >50 >50 C2101.cOl CRF01_AE >50 >50 C3347.C11 CRF01_AE >50 >50 C4118.c09 CRF01_AE >50 >50 CNE5 CRF01_AE 1.465 6,578 BJOX009000.02.4 CRF01_AE >50 >50

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BJOX015000.11.5 CRF01_AE (T/F) >50 >50 BJOX010000.06.2 CRF01_AE (T/F) >50 >50 BJOX025000.01.1 CRF01_AE (T/F) >50 >50 BJOX028000.10.3 CRF01_AE (T/F) >50 >50 Х1193_с1 G 0.036 0.130 Р0402_с2_11 G 0.023 0.088 Х1254_сЗ G 0.016 0.038 X2088_s9 G 0.007 0.021 Х2131_С1_В5 G 0.016 0.036 Р1981_С5_3 G 0.022 0.063 X1632_S2_B10 G 0.275 0.986 3016.v5.c45 D 8.359 >50 А07412М1.vrcl2 D 0.189 2.455 231965.c01 D >50 >50 231966.s02 D >50 >50 191821_E6_1 D(T/F) >50 >50 3817.v2.с59 CD 0.582 2,883 6480.v4.c25 CD 0.108 0.476 6952.vl.с20 CD 0.011 0.027 6811.v7.c18 CD 0.009 0.035 89-Fl_2_25 CD 5, 379 >50 3301.vl.с24 AC 0.074 0.260 6041.v3.c23 AC 0.085 0.233 654 0.v4. cl AC >50 >50 6545.v4.cl AC >50 >50 0815.v3.c3 ACD 0.071 0.321 3103.v3.cl0 ACD 0.009 0.017

Table 11. IC ₅₀ and IC ₈₀ values in TZM-bl assay for a 1:1:1 combination of NC37, BG1 and BG18 to a panel of 118 viruses.

BG18.

At the amino acid level, BG18 is 63% and 62% identical to heavy chains 10-1074 and PGT121, respectively, both of which are derived from the germline precursor VH4-59. BG18 is derived from a closely related (90.8% identity) germline VH4-4 gene (FIG. 6A). An alignment of the complementarity determining regions (CDRs) of PGT121, 10-1074 and BG18 heavy chains and their clonal variants showed a high degree of identity between CDRH1 and CDRH2. The CDRH3 of BG18 is three residues shorter than that of PGT121/10-1074 (21 versus 24 residues; Fig. 2A and Fig. 6B). In addition, BG18 has 6 of the 7 mutations in the framework regions that are common to the PGT121 family (FIG. 2A), but unlike members of PGT121/10-1074 does not contain any insertions or deletions.

BG18 contains the VL2-25 segment of the VL gene and has only 46 and 50% amino acid sequence identity with the VL3-21 segment of the gene contained in PGT121 and 10-1074, respectively (Fig. 2B, Fig. 6C and Fig. 6D). In addition, the light chains of BG18 and its variants have slightly more mutations than 10-1074 and PGT121 but, like the heavy chains, do not contain any insertions or deletions. Thus, BG18 and its clonal variants have heavy chains similar to those of previously isolated PGT121/10-1074 class antibodies, but different light chains.

The 1.3 A resolution crystal structure of Fab BG18 showed that the loops of the CDRs formed a compact binding site, except for CDRH3, which protruded ˜11 A beyond the other CDRs as an apex consisting predominantly of hydrophobic residues (Fig. 2C). Instead of fully unfolding, CDRH3 folds back into a conformation stabilized by hydrogen bonds between the Gly100aHC, Val100bHC, Val100cHC, Gly100eHC, and Glu100fHC residues in CDRH3 (Fig. 2C). CDRL2 has a disordered structure, suggesting that it can assume multiple conformations in an unbound antibody. An overlay of the unrelated structures of BG18, PGT121 (PDB 4FQ1) and 10-1074 Fab (PDB 4FQ2) showed that their V _H domains are highly identical, according to the alignment results, with the exception of CDRH3, which is located above the VH domain in BG18, but tilts markedly over V _L in Fab structures PGT121 and 10-1074 (FIG. 2D). IN

- 60 042959 consistent with low levels of BG18 and PGT121 light chain sequence identities (FIG. 2B, FIG. 6C, and FIG. 6D), aligned V _L domains (3.1 A standard deviations were found by aligning 93 or 92 Ca atoms when comparing light chains in superpositions of BG18-PGT121 and BG18-10-1074, respectively) showed differences in the orientations of all CDRL loops (Fig. 2D). Despite these differences, a gap between CDRH2 and CDRH3 in PGT121 and 10-1074 is also visible in BG18 (Fig. 2E). The residues lining this cleft interact with the complex-type N-glycan in the glycosylated Fab structure of PGT121 and interact with the N-glycan attached to Asn137 _gp1 2o in the structure of the PGT122-BG505 SOSIP complex (PDB 4TVP). BG18 has a second gap between CDRH3 and CDRL1/CDRL3, which cannot be formed in PGT121 and its clonal relatives due to the location of CDRH3 in these antibodies above the light chain (Fig. 2E). In addition to these structural differences, the electrostatic surface potential of the V _H -V _L domains of BG18 shows a different distribution of positively charged regions compared to the surface potentials of PGT121 and 10-1074 (FIG. 7). In sum, these results are consistent with differences in Env recognition by BG18 compared to recognition by PGT121-related bNAbs.

To study how BG18 recognizes Env, a single particle of BG505 SOSIP.664 trimer complexed with Fabs from BG18 and CD4bs bNAb 179NC75 was generated, at ~25 A resolution, by electron microscopy (EM) after negative staining. Inscribing coordinates from the FG BG18 crystal structure into the EM map (Fig. 2F) allowed comparison of approach angles and potential Env interactions between BG18 and other Asn332 _gp120 -V3 bNAb (Fig. 2G). Similar to PGT122 (a variant of PGT121) and 10-1074, BG18 CDRH3 is predicted to interact with the Asn332 _gpl20 glycan and the 324GDIR327 gp120 motif. However, while PGT122 and 10-1074 show similar angles of approach when binding to Env, BG18 binds to the Env trimer at a different angle, shifted 34-41° from PGT122 and PGT124 (FIG. 2G).

BG18 was evaluated for neutralization against a panel of HIV-1 pseudoviruses with deletions of specific N-linked glycosylation sites. The results showed that none of the glycans at the base of the V3 loop, with the exception of Asn332 _gp120 , had an effect on the neutralizing activity (Table 12). Consistent with these data, BG18 binds preferentially to synthetic V3 glycopeptides containing oligomannose N-glycans at the Asn332 position of _gp120 rather than to glycopeptides containing complex-type N-glycans attached to this position or to other potential N-linked glycosylation sites at the Asn332 position. gp120 (Fig. 9). These data suggest that the recognition properties of BG18 are more similar to 10-1074 than to PGT121.

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BG8 BG18 PGT121 12А12 BG505 (T332N) 0.007 0.001 0.012 0.030 N137A 0.003 0.001 0.002 0.027 N156A 0.008 0.001 0.007 0.024 N2 95A 0.009 0.001 0.014 0.044 N301A 0.004 0.001 0, he 0.027 N339A 0.007 0.001 0.016 ND N3 8 6A 0.004 0.001 0.015 0.030 N392A 0.005 0.001 0.006 0.012 BG8 BG18 PGT121 12 Al 2 JR-CSF 0.009 0.001 0, he 0.233 N2 95A 0.006 0.001 0.016 0.203 N301A 0.006 0.001 0, he 0.010 N332A > 50 > 50 > 50 0.240 N339A 0.010 0.001 0.007 0.028 N362A 0.008 0.001 0.018 0.267 N3 8 6A 0.012 0.001 0.024 0.332 N392A 0.014 0.002 0.017 0.230 G324A 0.005 0.001 0.044 0.236 D325A 0.005 0.001 0.079 0.203 I326A 0.005 0.001 0.024 0.278 R327A 0.005 0.001 0.018 0.131 Q328A 0.023 0.001 0.057 0.038 NZZOA 0.050 0.001 0.017 0.288 BG8 BG18 PGT121 12 Al 2 92BR020 0.015 0.001 0.004 0.050 N136A 0.005 0.001 0.002 0.027 N156A 0, OH 0.001 0.003 0.035 N301A 0.009 0.001 0.003 0, OH N332A > 50 > 50 0.080 0.039 N3 8 6A 0.012 0.003 0.006 0.055 N392A 0.023 0.001 0.007 0.105 D325A 0.006 0.001 0.006 0.050 I326A 0.006 0.001 0.002 0, OH R327A 0, OH 0.001 0.001 0.022 Q328A 0.018 0.001 0.002 0.028 NZZOA 0.018 0.001 0.003 0.073 N136A+N332A > 50 > 50 > 1 0.025 N156A+N332A > 50 > 50 > 1 0.023 N301A+N332A > 50 > 50 > 1 0.007 N13 6A+N15 6A+N3 01A 0.001 0.001 0.001 0.013

Table 12. IC ₅₀ and IC ₈₀ values in TZM-bl assay of BG18, BG8 PGT121 and control mAb 12A12 against selected V3 envelope mutants.

NC37.

Crystal structure of Fab NC37 (FIG. 1C) at 2.7 A resolution bound to core gp120 confirmed that NC37 recognizes CD4bs (FIGS. 8A and 8B). Based on sequence alone, it was initially difficult to determine whether VH NC37 was derived from the VH1-2 or VH1-46 germline gene because some members of the NC37 family were aligned to V _H 1-2 while others were aligned to VH1-46 (Table 13). However, structure comparison showed that NC37 adopts the same orientation and binding angle as the 8ANC131/134 antibodies derived from VH1-46 (Fig.

- 62 042959

8A and 8B). As a consequence, NC37 uses the light chain to bind to Asn280gp ₁₂₀ of loop D, while heavy chains are used to bind to Asn280 _gp120 in antibodies derived from VH1-2, such as NIH45-46. CDRH3 NC37 extends towards the internal domain of gp120, forming contacts between the Tyr100G _HC residue in CDRH3 and the residues Lys97 _gp120 and Glu102 _gp120 of the gp120 internal domain. Overlay of the NC37 Fab-gp120 NC37 complex onto the SOSIP trimer structure suggests that the NC37 CDRH3 binds to an adjacent protomer in the Env trimer (Fig. 8C).

NC37 was isolated in 2010, while BG1 and BG18 were isolated from PBMCs collected in 2014. Available PBMCs were examined in 2010 and 2013 using PCR primers specific for BG1 and BG18. While BG1 transcripts were found at both time points, BG18 was first discovered in 2013. This indicates that BG18 emerged between 2010 and 2013, while BG1 and NC37 appeared earlier.

autologous neutralization.

To study the effect of three bNAbs on autologous viruses, individual env genes from circulating plasma viruses were subjected to single genome sequencing (SGS) from five different time periods during 2006-2015. (2006, 2010, 2013, 2014 and 2015). Only 37 functional transcripts were recovered within the reading frame, partly due to the viral load, which was below 400 copies/ml at all time points. Moreover, all sequences recovered since 2015 were non-functional (Fig. 10). The 37 functional sequences formed three clusters - Cluster A, containing one 2006 sequence that differed by ~15% from the rest of the sequences, Cluster B, the largest cluster, containing sequences from all four time points, and Cluster C, containing 3 sequences, all 2014 d (Fig. 3A). An increase in viral diversity was observed between 2013 and 2014, consistent with the emergence of a new sequence cluster in 2014 (Fig. 3B).

All three sequences in cluster C contained mutations altering the N-linked glycosylation site in Asn332 _gp120 / while the other 34 sequences had an intact N-linked glycosylation site in Asn332 _gp120 (FIG. 3A). In addition, all three sequences in cluster C had an Asp282 _gp120 residue that was not found in the other 34 sequences. 94.4% of all HIV-1 sequences in the Los Alamos database contain Lys at position 282 _{of gp120} , and Asp at this position is associated with resistance to CD4bs antibodies.

To determine the sensitivity of functional Env to 3 autologous bNAbs, pseudoviruses were generated and tested by TZM-bl assay. Of the 35 successfully created pseudoviruses, only 4 were resistant to all 3 bNAbs. These were Env from cluster C found in plasma in 2014 and one from 2013 (Fig. 3C and Fig. 10). The remaining 31 pseudoviruses (88.5% of all pseudoviruses) were susceptible to at least 1 of the 3 bNAbs. In addition, 2006, 2010, and 2014 pseudoviruses were tested for susceptibility to parallel serum IgG isolated at the same time point as the viral env genes (IgG from 2013 plasma was not available for this analysis). The results showed that the parallel isolated IgG had a neutralizing effect on 19 of the 22 viruses tested (FIG. 2C and FIG. 10).

Viruses sensitive to BG18 and NC37, as well as viruses sensitive to competitive IgG, were detected at all analyzed time points, including the time points at which BG18 and NC37 were isolated (Fig. 3D). In contrast, most strains, with the exception of one 2006 virus, were found to be resistant to BG1. This suggests that most of the autologous viruses in the EB354 donor eluded BG1 but failed to elude BG18 and NC37 antibodies, indicating the breadth and efficacy of BG18 and NC37.

Although it was not possible to obtain viral sequences from 2015 plasma samples, it was possible to obtain virus from two of five EB354 CD4+ virus propagation cultures (VOC). According to p24 ELISA results, in both cases it took more than five weeks for the cultures to become positive for the virus. The SGS sequences from both 2015 virus propagation cultures are clustered adjacent to cluster C sequences and do not contain an N-linked glycosylation site in Asn332 _gp120 (Fig. 3A). In addition, all sequences showed the presence of Asp282 _gp120 . Consistent with these data, 2015 culture supernatants were resistant to all three bNAbs in TZM-bl assays (Fig. 3E).

In vivo efficacy of BG18, NC37 and BG1.

To determine whether BG18 or NC37 are capable of independently selective pressure on HIV-1 in vivo, humanized mice (hu mice) were infected with HIV-1 _YU2 and the established infection was treated. BG1 showed low activity against HIV-1 _YU2 (IC ₅₀ =15.8 μg/ml) and was therefore only used in subsequent combination treatment experiments and not in monotherapy experiments. Similar to 10-1074, administration of BG18 or BG8, a less potent but still effective clonal variant, was associated with a rapid decrease (mean 1.5 log ₁₀ ) in viral load followed by rebound viremia (Fig. 4A, Fig. 11 and Fig. . 12). All viral sequences causing the rebound reaction contained a mutation that altered the N-linked glycosylation site in _gp120 Asn332 (FIGS. 4B and 4C). NC37 monotherapy also temporarily suppressed viremia, but the magnitude

- 63 042959 reduction in viral load was less pronounced than with BG18, on average 0.5 log10 (Fig. 4A and Fig. 12). This indicates that BG18 and NC37 are effective in treating HIV, with BG18 having the most pronounced effects. Most of the Env sequences of the NC37 rebound virus showed the R456K mutation, which is associated with resistance to α anti-CD4bs antibodies (FIGS. 4B and 4C). This further indicates that BG18 and NC37 allow selection of BH4-1 _yu2 antibody resistant variants in vivo from hu mice.

Antibody combinations can control HIV-1 _YU2 infection in hu mice. To determine if the combination of BG18, NC37 and BG1 could control viremia, five HIV-1 _YU2 -infected hu mice were treated with a 1:1:1 combination of three bNAbs (BG18, NC37 and BG1). Shortly after administration of the three bNAbs, the viral load decreased in all animals by an average of 1.74 Log ₁₀ , and surprisingly, 4/5 mice showed a virtually undetectable viral load after 3 weeks (FIGS. 4D and 4E). Viremia recovered in only one mouse, T6, and this did not occur until 3 weeks after cessation of therapy. The remaining 4 mice continued to show virtually undetectable viremia even 4 weeks after the end of therapy (FIGS. 4D and 4E). This indicates that whether NC37, BG1 and BG18 are used alone, but especially in combination, NC37, BG1 and especially BG18 are effective for long-term suppression and neutralization of HIV-1 in vivo.

ANTIBODY CLOSER Shii VH CLOSER Shii VL SOMATIC MUTATIONS IN V (nt)% AFTER DEPARTMENT CDRH3 vh VL NC37 1-2/1- 46 KZ-20 28.8 27.9 DNFGTRPVPGRGYYYGMDV (SEQ ID NO: 105) NC133 1-46 KZ-20 28 23.2 DNRGDRNVPGRGYYYGMDV (SEQ ID NO: 106) NC102 1-2 KZ-20 27.1 22.2 DNFGTRPVPGRGYYYGMDV (SEQ ID NO: 107) AC40 1-46 KZ-20 33.5 21.1 DNFGE SHKGYTYGMDA (SEQ ID NO: 108) AC41 1-46 KZ-20 30.1 21.5 DNFGE SHKGYTYGMDA (SEQ ID NO: 109) AC72 1-46 KZ-20 29.8 20.8 DNFGE SHKGYTYGMDA (SEQ ID NO: 110) BG8 4-4 L3-25 25.4 20.5 NVIRVFGVISLGEWFHYGMDV (SEQ ID NO: 4) BG18 4-4 L3-25 21.2 17.7 NAIRIYGWALGEWFHYGMDV (SEQ ID NO: 12) BG33 4-4 L3-25 25 18.4 NVIRVFGVISLGEWFHYGMDV (SEQ ID NO: 28) BG42 4-4 L3-25 23.7 19.4 NVIRVFGVISLGEWFHYGMDV (SEQ ID NO: 20) BG1 3-4 9 K1-39 27.2 19.9 EQRNKDYRYGQEGFGYSYGMDV (SEQ ID NO: 76) BG22 3-4 9 K1-39 26.8 23.4 EQRGGDGRYSGDGFGYPYGMDV (SEQ ID NO: 84) BG47 3-4 9 K1-39 27.1 22 EQRGANGRYGGDGFGYSYGMDV (SEQ ID NO: 92)

Table 13. Neutralizing clones. Genetic characterization of 13 antibodies, representative variants from each of the three clones (blue, magenta and green), which showed neutralizing activity. V _H , VL and mutation rate are indicated, as well as the sequence of CDRH3 (according to IMGT).

The above examples and description of the preferred embodiments should be considered as an illustration and not as a limitation of the present invention defined by the claims. It should be understood that numerous variations and combinations of the above features may be used without departing from the present invention as set forth in the claims. Such changes are not intended to be a departure from the scope of the invention, and all such variations are included within the scope of the following claims. All references cited in the present description are incorporated in their entirety by reference.

Claims (20)

1. An isolated anti-HIV-1 antibody or antigen-binding portion thereof, containing: i) a heavy chain variable region that contains CDRH1, CDRH2 and CDRH3, where CDRH1, CDRH2 and CDRH contain the respective sequences of SEQ ID NOs: 10-12, and ii) a light chain variable region that contains CDRL1, CDRL2 and CDRL3, where CDRL1 , CDRL2 and CDRL3 contain the respective sequences of SEQ ID NOs: 14-16. 2. An isolated anti-HIV-1 antibody according to claim 1, or an antigen-binding portion thereof, wherein the heavy chain variable region and the light chain variable region comprise the respective sequences of SEQ ID NOS: 9 and 13. 3. An isolated anti-HIV-1 antibody according to any one of claims 1, 2, or an antigen-binding portion thereof, wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody. 4. An isolated anti-HIV-1 antibody according to any one of claims 1 to 3, or an antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof is recombinantly produced. 5. A bispecific antibody comprising an isolated anti-HIV-1 antibody, or an antigen-binding portion thereof, according to any one of claims 1-4. 6. An isolated nucleic acid containing a sequence encoding an anti-HIV-1 antibody according to any one of claims 1 to 4, or an antigen-binding portion thereof. 7. An isolated nucleic acid containing a sequence encoding a bispecific antibody according to claim 5 or an antigen-binding portion thereof. 8. A vector containing a nucleic acid according to claim 6 or 7. 9. Cultured cell containing the vector according to claim 8. 10. A pharmaceutical composition comprising (i) an anti-HIV-1 antibody or an antigen-binding portion thereof according to any one of claims 1 to 5 or a bispecific antibody according to claim 5 and (ii) a pharmaceutically acceptable carrier. 11. The pharmaceutical composition according to claim 10, wherein the CDRH1, CDRH2, CDRH3, and CDRL1, CDRL2, and CDRL3 anti-HIV-1 antibodies comprise the respective sequences of SEQ ID NOS: 10-12 and 14-16. 12. The pharmaceutical composition of claim 11, wherein the heavy chain variable region and light chain variable region of the anti-HIV-1 antibody comprise the respective sequences of SEQ ID NOS: 9 and 13. 13. Pharmaceutical composition according to claim 10, further comprising a second anti-HIV-1 antibody or an antigen-binding portion thereof. 14. A method for treating an HIV-1 infection, comprising identifying a subject in need thereof and administering to said subject a therapeutic agent containing a therapeutically effective amount of an anti-HIV-1 antibody or an antigen-binding antibody thereof according to any one of claims 1 to 4 or a bispecific antibody according to 5 or a pharmaceutical composition according to any one of claims 10-13. 15. The method of claim 14, further comprising administering a second therapeutic agent. 16. The method of claim 15, 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. 17. A vaccine composition comprising an anti-HIV-1 antibody or an antigen-binding portion thereof according to any one of claims 1 to 4 or a bispecific antibody according to claim 5. 18. The vaccine composition according to claim 17, wherein the CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2 and CDRL3 anti-HIV-1 antibodies comprise the respective sequences of SEQ ID NOS: 10-12 and 14-16. 19. The vaccine composition of claim 18, wherein the heavy chain variable region and the light chain variable region comprise the respective sequences of SEQ ID NOS: 9 and 13. 20. A method for preventing HIV-1 infection in a subject in need thereof, comprising administering to said subject a vaccine composition according to any one of claims 17-19.