EA046696B1 - BISPECIFIC ANTI-HIV ANTIBODY AND ITS APPLICATION - Google Patents

Description

Cross reference to related applications

This application claims priority under 35 U.S.C. § 119(e) based on U.S. Provisional Patent Application No. 61/715,642, filed October 18, 2012, which is incorporated herein in its entirety.

Government interests

The invention disclosed herein was made, at least in part, with government support under Grant No. P01 AI081677 awarded by the National Institutes of Health. Accordingly, the US Government owns certain rights to the present invention.

Field of technology to which the invention relates

The present invention relates to effective broad-spectrum antibodies against the human immunodeficiency virus (HIV).

State of the art

HIV causes acquired immunodeficiency syndrome (AIDS), a condition in humans characterized by a range of clinical features, including wasting syndromes, central nervous system degeneration, and profound immunosuppression that leads to life-threatening opportunistic infections and malignancies. Since its discovery in 1981, HIV type 1 (HIV-1) has killed at least 25 million people worldwide. It is predicted that 20-60 million people will be infected over the next two decades, despite an annual decline of 2.5% in HIV infections. There is a need for therapeutic agents and methods for treating or suppressing HIV infection.

Broad-spectrum neutralizing IgG antibodies are found in the serum of some HIV-infected people. However, little is known about the specificity and activity of such antibodies, despite their possible importance for the development of effective vaccines. In animal models, passive transfer of neutralizing antibodies may contribute to protection against viral infection. Neutralizing antibody responses can also develop in HIV-infected individuals, but the detailed composition of the serological response has not yet been fully elucidated.

The essence of the invention

The present invention relates to new categories of broad-spectrum neutralizing anti-HIV antibodies. The consensus amino acid sequences of the antibody heavy and light chains are listed below and shown in Figures 3a and 3b:

QVQLQESGPGLVKPSETLSLTCSSVSGX1SX2X3DX4YWSWIRQSPGKGLEWIGYVHDS

GDTNYNPSLKSRVX5X6SLDTSKNQVSLKLX7X8VTAADSAX9YYCARAX10HGX11RIYGIV

AFGEX12FTYFYMDVWGKGTTVTVSS (SEQ ID NO :1)

SX1VRPQPPSLSVAPGETARIX2CGEX3SLGSRAVQWYQQRPGQAPSLIIYNNQDRPS

GIPERFSGSPDX4X5FGTTATLTITX6VEAGDEADYYCHIWDSRX7PTX8WVFGGGTTLTVL (SEQ ID NO:2)

In SEQ ID NO: 1 or 2, each X can be any amino acid residue or the absence of an amino acid. Preferably, each of X may be a residue at a corresponding position of clonal variants 10-259, 10-303, 10-410, 10-847, 10-996, 101074, 10-1121, 10-1130, 10-1146, 10- 1341 and 10-1369, which are shown in figures 3a and 3b, and an artificially modified version of the antibody 10-1074, 10-1074GM.

Accordingly, one aspect of the present invention features an isolated anti-HIV antibody, or antigen binding portion thereof, having at least one complementarity determining region (CDR) having a sequence selected from the group consisting of the sequences of SEQ ID NO:33-38, provided that that the antibody is not PGT-121, 122 or 123. SEQ ID NO:33-38 refers to the sequences CDR heavy chain (CDRH) 1-3 and CDR light chain (CDRL) 1-3 according to the Kabat system, as shown in fig. 3a and 3b. In one embodiment, the CDR may comprise a sequence selected from the group consisting of sequences SEQ ID NO:39-104, i.e., Kabat CDR sequences, which are shown in table. 1 below. Alternatively, the CDR may contain a sequence selected from the CDR sequences of the corresponding antibodies according to the IMGT system, which are shown in table. 1 below.

In one embodiment, the isolated anti-HIV antibody or antigen binding portion thereof comprises a heavy chain variable region that includes CDRH 1, CDRH 2 and CDRH 3, wherein CDRH 1, CDRH 2 and CDRH 3 contain the corresponding sequences of SEQ ID NO:33 -35. CDRH 1, CDRH 2 and CDRH 3 may also contain corresponding sequences of the CDRH set selected from the group consisting of sequences SEQ ID NO:39-41, SEQ ID NO:45-47, SEQ ID NO:51-53, SEQ ID NO : 57-59, SEQ ID NO: 63-65, SEQ ID NO: 69-71, SEQ ID NO:75-77, SEQ ID NO:81-83, SEQ ID NO:87-89, SEQ ID NO: 93 -95, SEQ ID NO: 99-101 and SEQ ID NO: 131-133. Alternatives

- 1 046696 but the CDRH may contain corresponding sequences selected from sequences corresponding to the CDR sequences of antibodies according to the IMGT system, which are shown in table. 1 below.

In another embodiment, the isolated anti-HIV antibody or antigen binding portion thereof comprises a light chain variable region that includes CDRL 1, CDRL 2 and CDRL 3, wherein CDRL 1, CDRL 2 and CDRL 3 contain the corresponding sequences of SEQ ID NO:36- 38. For example, CDRL 1, CDRL 2, and CDRL 3 may comprise corresponding sequences from the CDRL set selected from the group consisting of SEQ ID NO:42-44, SEQ ID NO:48-50, SEQ ID NO:54-56, SEQ ID NO: 60-62, SEQ ID NO: 66-68, SEQ ID NO:72-74, SEQ ID NO:78-80, SEQ ID NO:84-86, SEQ ID NO: 90-92, SEQ ID NO : 96-98, SEQ ID NO: 102-104 and SEQ ID NO: 134-136. Alternatively, the CDRLs may contain corresponding sequences selected from the corresponding antibody CDR sequences according to the IMGT system, which are shown in table. 1 below.

In a further embodiment, the above isolated anti-HIV antibody or antigen binding portion thereof comprises (i) a heavy chain variable region that includes CDRH 1, CDRH 2, and CDRH 3, and (ii) a light chain variable region that includes CDRL 1, CDRL 2 and CDRL 3. CDRH 1, CDRH 2, CDRH 3, CDRL 1, CDRL 2 and CDRL 3 may contain corresponding sequences from a set of CDRs selected from the group consisting of the sequences SEQ ID NO:39-44, SEQ ID NO: 45-50, SEQ ID NO: 51-56, SEQ ID NO: 57-62, SEQ ID NO: 63-68, SEQ ID NO: 69-74, SEQ ID NO:75-79, SEQ ID NO:81- 86, SEQ ID NO: 87-92, SEQ ID NO: 93-98, SEQ ID NO: 99-104 and SEQ ID NO: 131136. Alternatively, CDRH and CDRL may contain corresponding sequences selected from sequences corresponding to antibody CDR sequences according to IMGT system, which are shown in table. 1 below.

In a further embodiment, the isolated anti-HIV antibody or antigen binding portion thereof comprises one or both of (i) a heavy chain having the consensus amino acid sequence of SEQ ID NO: 1, and (ii) a light chain having the consensus amino acid sequence of SEQ ID NO: 1 : 2. The heavy chain may comprise a sequence selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 129, and the light chain may comprise a sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and 130. For example, the heavy chain and light chain may contain the corresponding SEQ ID sequences NO:3-4, SEQ ID NO: 5-6, SEQ ID NO:7-8, SEQ ID NO: 910, SEQ ID NO: 11-12, SEQ ID NO: 13-14, SEQ ID NO: 15- 16, SEQ ID NO: 17-18, SEQ ID NO: 19-20, SEQ ID NO: 21-22, SEQ ID NO: 23-24 and 129-130.

In a preferred embodiment, the isolated anti-HIV antibody is an antibody selected from the group consisting of antibodies 10-259, 10-303, 10-410, 10-847, 10-996, 10-1074, 10-1074GM, 10 -1121, 10-1130, 10-1146, 10-1341 and 10-1369. Their respective heavy chain variable regions, light chain variable regions, CDRH 1-3 and CDRL 1-3 are shown in FIG. 3a and 3b. In a more preferred embodiment, the isolated anti-HIV antibody is a 10-1074-like antibody, i.e., an antibody selected from the group consisting of 10-847, 10-996, 10-1074, 101074GM, 10-1146 and 10- 1341. An antibody from this group is more effective in neutralizing modern viruses than PGT121. The antibody discussed above may be a human antibody, a humanized antibody, or a chimeric antibody.

In a second aspect, the invention provides an isolated nucleic acid having a sequence encoding a CDR, heavy chain variable region or light chain variable region of the above-discussed anti-HIV antibody or an antigen binding portion thereof. Another distinctive feature of the invention is a vector containing a nucleic acid and a cultured cell containing the vector.

The nucleic acid, vector, and cultured cell can be used in a method for producing an anti-HIV antibody or fragment thereof. The method includes, among others, the stages of: obtaining the above-mentioned cultured cell; culturing the cell in a medium under conditions allowing expression of the polypeptide encoded by the vector and assembly of the antibody or fragment thereof, and purifying the antibody or fragment from the cultured cell or cell medium.

In a third aspect, the invention is characterized by a pharmaceutical composition comprising (i) at least one anti-HIV antibody as defined above or an antigen-binding portion thereof and (ii) a pharmaceutically acceptable carrier.

In a fourth aspect, the invention relates to a method for preventing or treating HIV infection or an HIV-related disease. The method includes, among others, the steps of: identifying a patient in need of such prophylaxis or treatment, and administering to said patient a first therapeutic agent containing a therapeutically effective amount of at least one anti-HIV antibody as defined above, or an antigen-binding portion thereof. . The method may further include administering a second therapeutic agent, such as an antiviral agent.

In a fifth aspect, the invention provides a kit comprising a pharmaceutically acceptable unit dosage form comprising a pharmaceutically effective amount of at least

- 2 046696 at least one isolated anti-HIV antibody specified above, or an antigen-binding portion thereof, and a pharmaceutically acceptable unit dosage form containing a pharmaceutically effective amount of an anti-HIV agent. The two pharmaceutically acceptable unit dosage forms may not necessarily be in the form of one pharmaceutically acceptable unit dosage form. An example of an anti-HIV agent may be an agent selected from the group consisting of a non-nucleoside reverse transcriptase inhibitor, a protease inhibitor, an entry or fusion inhibitor, and an integrase inhibitor.

In a sixth aspect, the invention relates to a kit for diagnosing, prognosticating or monitoring the treatment of HIV infection in an individual. The kit contains one or more detection reagents that specifically bind to neutralizing anti-HIV antibodies in a biological sample obtained from an individual. The kit may additionally contain reagents for PCR or mass spectrometry.

A detailed description of one or more embodiments of the invention is provided below. Other features, purposes and advantages of the invention will be clear from the description and claims.

Brief description of drawings

In fig. Figure 1 shows the neutralizing activity of PGT121-like and 10-1074-like variants. (A) Two-dimensional color map comparing the neutralization efficiencies of PGT121-like and 101074-like antibodies in the TZM-b1 assay. Darker color = more effective neutralization; white = no neutralization. (B) Correlation between the average IC80 value against 9 viruses (y-axis) and the apparent KD values for binding to gp120 and gp140 (x-axis). (C) Graph comparing the breadth and neutralization efficiency of antibodies PGT121, 10-996, and 10-1074 in the TZMb1 assay against an expanded panel of 119 viruses. The y-axis shows the cumulative frequency of IC50 values up to the concentration shown on the x-axis. The spider plot (top left) shows the frequency distribution of neutralized viruses according to HIV-1 clades. (D) Scatter plot showing molar neutralization ratios (MNR; ratio of IC50 concentrations of Fab and IgG). Horizontal bars represent average IC50s for all viruses. (E) Bar graph comparing the neutralization efficiencies of PGT121 (dark gray) and 10-1074 (light gray) against viruses isolated from historical (historical) and modern (modern) seroconverters. ns - not significant; **, p<0.005. The fold difference between the median IC50 values for neutralization of modern viruses by PGT121 and 10-1074 is indicated.

In fig. Figure 2 shows the binding and neutralization activities of mutant antibodies PGT12GM and 10-1074GM. (A) Bar graphs comparing apparent KD values for binding of antibodies 10-1074, PGT121, PGT121GM, and 10-1074GM to gp120 and gp140. Error bars show SEM of KD values obtained from three independent experiments. The fold differences between the KD values of wild-type and glycomutant antibodies are shown. (B) Bar graphs comparing glycan binding (Figure 7A) by antibodies PGT121 and 10-1074 and glycan binding by mutant antibodies (PGT121GM and 10-1074GM). Numerical estimates of binding were given as fluorescence intensity (average of duplicate spots) for probes distributed at 5 fmol per spot. (C) Coverage plot comparing the breadth and neutralization efficiencies of antibodies PGT121, PGT121GM, 10-1074, and 10-1074GM in the TZM-bl assay against a panel of 40 viruses.

In fig. 3 depicts: sequence alignments of clonal variants PGT121 and 101074. (A) Amino acid alignment of the heavy chains (IgH) of PGT121-like and 10-1074-like antibodies and the probable germline (GL) VH for all clonal variants. Amino acid numbering based on crystal structures, framework (FWR) and complementarity determining regions (CDR) according to the Kabat system (J. Exp. Med. 132(2): 211-250) and IMGT (Nucleic Acids Res. 37 (Database issue) ): D1006-1012). Color shading shows acidic (red), basic (blue) amino acids, and tyrosine (green). (B) Same as in A, but for light chains (IgL).

Fig. 4 shows: Binding affinity of clonal variants PGT121 and 10-1074. (A) Binding affinity of the PGT121 IgG antibody variants with YU-2 gp140 and gp120 ligands, measured using surface plasmon resonance (SPR). M, mol/l; sec, seconds; RU, response units; /, no binding detected. A chi2 (χ2) value < 10 indicates that the 1:1 binding model used to fit the curves adequately described the experimental data. Equilibrium and kinetic constants were considered to be apparent constants, taking into account avidity effects resulting from bivalent IgG binding. (B) Dot plots showing the rate constants of association (ka) and dissociation (kd) for PGT 121-like (blue shading) and 10-1074-like (green shading) antibodies. (C) Linear regression plots comparing the ka and kd values of IgG antibodies for their binding to gp120 and gp140 (x-axis) against their neutralization efficiencies (mean IC80 values) of the 9 viruses shown in Table. 4 (y-axis).

In fig. Figure 5 shows the binding of PGT121 variants to gp120 core proteins, the gp120GD324-5AA mutant, and gp120v3 linear peptides. (A) ELISA-based assays of binding of PGT121-like and 10-1074-like antibodies to gp120core HXB2 and 2CC core proteins compared to intact gp120 YU-2. The x-axis shows the antibody concentration (M) required to obtain

- 3,046,696 ELISA values (OD405 nm) shown on the y-axis. Anti-CD4bs antibody VRC01 (Science 329(5993): 856861), anti-V3 loop antibody 10-188 (PLoS One 6(9): e24078) and HIV non-reactive antibody mGO53 (Science 301(5638): 1374- 1377) were used as controls. (B) Same as (A) but for binding to the g120GD324-5AA mutant protein, (C) Bar graph comparing the ELISA reactivities of PGT121- and 10-1074-like antibodies and control antibodies (positive control, 10-188, 1-79, 2-59 and 2-1261 (Nature 458(7238): 636-640), and negative control, mGO53) against gp120v3-C3 overlapping peptides. The y-axis shows the ELISA values (OD405 nm) obtained when testing IgG antibodies at a concentration of 2 μg/ml. The amino acid sequences of individual peptides are shown at the bottom right. All experiments were performed at least in duplicate. Typical data shown.

In fig. 6 shows the binding of PGT121 to gp120 glycosylation mutants and deglycosylated gp120. (A) ELISA-based binding assays of antibody variants PGT121 and 10-1074 to gp120, gp120NNT301-303AAA, gp120N332A, and gp120N332A/NNT301-303AAA. The x-axis shows the antibody concentration (M) required to obtain the ELISA values (OD405 nm) indicated on the y-axis. Black dotted and solid lines show the average reactivity against the four antigens of positive (10-188) and negative (mGO53) control antibodies. (B) Silver-stained SDS-PAGE gel comparing untreated gp120 (WT), PNGase F-digested, and EndoH gp120s. L, protein ladder. (C) Same as (A), but comparison between untreated and PNGase-treated F gp120. (D) Same as (A), but comparison between untreated and EndoH-treated gp120. All experiments were performed at least in duplicate.

In fig. Figure 7 shows the binding of clonal variants PGT121 and 10-1074 to glycans. (A) Monosaccharide sequences of a set of N-glycan probes used in a glycan microarray assay to probe PGT121-like and 10-1074-like antibodies for direct binding to N-glycans. DH stands for the lipid tag 1,2-dihexadecyl-sn-glycero-3phosphoethanolamine (DHPE), to which N-glycans were conjugated via reductive amination. The key features to note are (i) the PGT121 group antibodies bound the single antenna N-glycan probe 10 (N2), with the galactose-terminated antenna binding in a 1-3 linkage to the mannose core, but did not bind the isomeric N-glycan probe 11 (denoted N4) with an antenna connected by a 1-6 bond to the core mannose; (ii) the presence of a galactose-terminated 1-6-linked antenna, as in dual-antenna probe 13 (NA2), allowed binding as in the presence of a2-6-linked (but not a2-3-linked) sialic acid; (iii) dual antenna probe 12 (NGA2), which lacks galactose and ends with N -acetylglucosamine, did not bind. (B) Bar graphs comparing glycan binding by P6T121-like, 10-1074-like, and germline variant (GL) antibodies. 10-188, an antibody against the V3 loop, was used as a negative control. A numerical estimate of binding was obtained as fluorescence intensity (average of duplicate spots) for probes distributed at 2 fmol (white) and 5 fmol per spot (gray).

In fig. Figure 8 shows antibody binding and neutralization activity against high-mannose-only gp120 and viruses. (A) Silver-stained SDS-PAGE gel compares gp12 0 YU-2 produced in cells treated with kifunensin (gp120kif) and gp120 produced in untreated cells (WT, wild type). L, protein ladder. (B) ELISA comparison of PGT binding 121-like (blue labels) and 10-1074-like (green labels) antibodies to gp120 YU-2 (gp120 WT) and gp120kif. The x-axis shows the antibody concentration (M) required to obtain the ELISA values (OD405 nm) shown on the y-axis. (C) Neutralization curves for PGT121 evaluated against selected P6T121-sensitive/10-1074-resistant pseudoviruses generated in the presence (kif virus) or absence (WT virus) of kifunensin. The dotted horizontal line indicates 50% neutralization, from which the IC50 value can be derived from the antibody concentration on the x-axis. Experiments were carried out in triplicate. Error bars indicate SD for triplicate measurements. (D) Bar graphs comparing the neutralizing activity of selected antibodies against YU-2 and PVO.4 pseudoviruses generated in HEK 2 93S GnTI −/− cells (67GnT Virus −/−) or in wild-type cells (WT virus). The y-axis shows the mean IC50 values (μg/mL) for neutralization of the viruses shown on the x-axis. Error bars show SEM of IC50 values obtained from two independent experiments.

In fig. 9 shows activity in neutralizing PGT121, 10-996 and 10-1074. (A) Graph comparing the neutralization efficiencies of PGT121, 10-996, and 10-74 against viruses of the indicated HIV-1 clades (determined using the TZM-bl assay and a panel of 119 pseudoviruses). The x-axis shows the antibody concentration (μg/mL) required to achieve 50% neutralization (IC50). The y-axis shows the cumulative frequency of IC50 values up to the concentration shown on the x-axis. (B) Graph comparing the breadth and neutralization efficiencies for antibodies PGT121, 10-996, and 10-1074 against an expanded panel of 119 viruses that were measured in the TZM-bl neutralization assay. The y-axis shows the cumulative frequency of IC80 values up to the concentration shown on the x-axis. (C) The graphs show the neutralization curves of selected viruses by PGT121 and 10-1074. Dotted

- 4 046696 the horizontal line indicates 50% neutralization from which the IC50 value can be derived from the antibody concentration shown on the x-axis. Experiments were carried out in triplicate. Error bars indicate SD for triplicate measurements.

In fig. 10 depicts neutralization activity against historical viruses compared to modern clade B viruses. Scatter plots compare neutralization efficiencies against clade B viruses isolated from historical (historical) and modern (modern) seroconverters for selected bN-Abs. Horizontal bars represent median IC50s for all viruses in a patient. Differences between groups were assessed using the Mann-Whitney test, ns - not significant.

In fig. Figure 11 shows neutralization of two tropical SHIV R5s using a panel of 11 broad-spectrum anti-HIV-1 mAbs. Calculated IC50 values for neutralization of SHIVAD8EO (A) and SHIVDH12-V3AD8 (B).

In fig. Figure 12 shows: Correlation of plasma concentrations of passively administered neutralizing mAbs with viral infection after challenge of macaques with two different SHIV R5. Filled circles indicate protected (non-challenged) monkeys; open circles indicate infected animals.

In fig. 13 shows: plasma concentration of bN-Ab. The mAb concentration was determined by measuring the neutralizing activity in plasma samples. (A) ID50 values measured in neutralization assays of TZM.bl 10-1074 and 3BNC117 against HIV-1 strains that are sensitive to one but not the other bN Ab (i.e., HIV-1 strain X2088_9 (susceptible to 10-1074); HIV-1 strain Q769_d22 (sensitive to 3BNC117). (B) Neutralizing activities of plasma obtained before the introduction of antibodies (preP), but with a pulse of 0.01, 0.1, 1, 10 and 100 μg. /ml antibodies 10-1074 (blue) or 3BNC117 (green) Neutralizing activity was reported as plasma ID50 titers (left columns) and converted to antibody concentrations (right columns) based on the measured ID50 values reported in case (A). (C) ID50 titers (left columns) and bN-Ab concentrations (right columns) measured in the indicated macaque plasma samples before (pre-immunization) and after (day) bN-Ab administration.

Detailed Description of the Invention

The present invention is based, at least in part, on the unexpected discovery of a new category of broad-spectrum neutralizing antibodies (bN-Abs) against HIV that can recognize carbohydrate-dependent epitopes, including complex-type N-glycan, on gp120.

Antibodies are essential for the success of most vaccines, and HIV antibodies appear to be only a correlate of prevention in the recent RV144 HIV vaccine trial. Some HIV-1-infected patients developed broad-spectrum neutralizing serologic activity against the gp160 spike virus 2 to 4 years after infection, but such antibodies generally did not protect infected individuals because autologous viruses escaped due to mutation. However, broad neutralizing activity imposes selection pressure on the virus, and passive transfer of broadly neutralizing antibodies (bN-Abs) to macaques protects against SHIV infection. It has therefore been suggested that vaccines that induce such antibodies may protect people from HIV infection.

The development of methods for cloning antibodies in single cells has shown that bN Abs target different epitopes on the HIV-1 gp160 spike. The most effective anti-HIV-1 bN Abs recognize the CD4 binding site (CD4bs) (Science 333(6049): 1633-1637; Nature 477(7365): 466-470; Science 334(6060): 12891293) and carbohydrate-dependent epitopes associated with variable loops (Nature 477(7365):466-470; Science 326(5950):285-289; Science 334(6059):1097-1103; Nature 480(7377):336-343), including V1 loops /V2 (PG9/PG16) (Science 326(5950): 285-289) and V3 (PGT) (Nature 477(7365): 466-470). Less is known about carbohydrate-dependent epitopes because the antibodies studied to date are either unique examples or representatives of small clonal families.

To better understand the neutralizing antibody response to HIV-1 and the epitope targeted by PGT antibodies, we isolated members of a large clonal family that predominates in the secondary immune response in the form of gp160-specific IgG from a patient infected with clade A viruses, which produced PGT121. As described herein, PGT121 antibodies are divided into two groups, P6T121-like and 10-1074-like antibodies, according to sequence, binding affinity, neutralizing activity and recognition of carbohydrates and the V3 loop. 10-1074 and related members of the family produce unusually effective neutralization, including broad reactivity against newly transmitted viruses. In contrast to previously characterized carbohydrate-dependent bN-Abs, PGT121 binds to complex-type N-glycans rather than high-mannose N-glycans in glycan microarray experiments. The crystal structures of PGT121 and 10-1074, compared with the structures of their germline precursor and the structure of PGT121 bound to a complex-type N-glycan, explain their different properties.

In one example, assays were performed to isolate B cell clones encoding the antibody PGT121, which is unique among glycan-dependent bN Abs in recognizing N

- 5,046,696 complex-type glycans rather than high-mannose N-glycans. PGT121 clones are divided into PGT121- and 10-1074-like groups, differing in sequence, binding affinity, carbohydrate recognition and neutralizing activity. Group 10-1074 has remarkable potency and a broad spectrum of activity, despite the lack of detectable binding to protein-free glycans. The crystal structures of the unliganded PGT121, 101074, and its germline precursor indicate that this differential carbohydrate recognition maps to the recess between CDRH2 and CDRH 3, which was occupied by a complex-type N-glycan in the separate PGT121 structure. The exchange of glycan-contacting residues between PGT121 and 10-1074 confirmed the importance of such residues for neutralization activity. HIV envelopes have different ratios of high-mannose N-glycans and complex-type N-glycans, so the findings, including the first structural characterization of complex-type N-glycan recognition by an HIV bN antibody, are critical to understanding how antibodies and ultimately, vaccines can achieve broad neutralizing activity.

The term antibody (Ab) as used herein includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies and polyreactive antibodies) and antibody fragments. Thus, the term antibody, as used in any context herein, is intended to include, without limitation, any specifically binding member of a class and/or isotype of immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM); and a biologically suitable fragment thereof or a specific binding representative thereof, including but not limited to Fab, F(ab')2, Fv and scFv (single chain or linked unit). It is known in the art that an antibody is a glycoprotein having at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. The heavy chain consists of a heavy chain variable region (VH) and a 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 heavy and light chain variable regions contain 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 distributed from the KH2-conduct to the COOH-terminus as follows: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, and FWR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen, while depending on the isotype, the constant region(s) may mediate binding of the immunoglobulin to host tissues or factors.

Also included within the definition of antibody as used herein are chimeric antibodies, humanized antibodies and recombinant antibodies, human antibodies generated in a transgenic non-human animal, as well as antibodies selected from libraries using enrichment techniques available to one skilled in the art.

The term variable refers to the fact that some regions of the variable (V) domains vary greatly in sequence among antibodies. The V domain mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the 110 amino acid length of the variable regions. In fact, V regions are composed of relatively invariant regions called framework regions (FRs) of 15–30 amino acids in length, separated by shorter regions of extreme variability called hypervariable regions, each 9–12 amino acids in length. Each of the variable regions of the native heavy and light chains contains four FRs, generally adopting a beta-sheet conformation, linked by three hypervariable regions that form loops that link and in some cases form part of the beta-sheet structure. The hypervariable regions on each chain are held together in close proximity by FR and, with hypervariable regions from the other chain, contribute to the formation of the antigen-binding region of antibodies (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

The term hypervariable region as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region typically contains amino acid residues from the complementarity determining region (CDR).

The term monoclonal antibody as used herein refers to an antibody derived from a population of essentially homogeneous antibodies, i.e. the individual antibodies that make up the population are identical, except for possible naturally occurring mutations that may be present in minor quantities. The term polyclonal antibody refers to drugs that include different antibodies directed against different determinants (epitopes).

Monoclonal antibodies according to the present invention include chimeric antibodies in which part of the heavy and/or light chain is identical or homologous to the corresponding sequences

- 6 046696 sequences in antibodies obtained from a particular species, or belonging to a particular class or subclass of antibodies, the remainder of the chain(s) being identical or homologous to the corresponding sequences in antibodies obtained from another species or belonging to a different class or subclass of antibodies, as well as fragments of such antibodies, provided that they exhibit the desired biological activity (see, for example, US Pat. No. 4816567 and Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984) ). The described invention provides antigen binding variable region sequences derived from human antibodies. Accordingly, chimeric antibodies of primary interest according to the present invention include antibodies having one or more human antigen binding sequences (eg, CDRs) and containing one or more sequences derived from a non-human animal antibody, for example, an FR or C- sequence areas. In addition, chimeric antibodies included in the present invention are antibodies containing an antigen binding sequence of a human variable region of one class or subclass of antibodies and another sequence, such as an FR sequence or C region, derived from another class or subclass of antibodies.

A humanized antibody is generally considered to be a human antibody that has one or more amino acid residues introduced into it from a non-human animal source. Such non-human amino acid residues are often referred to as imported residues, which are typically taken from the imported variable region. Humanization can be accomplished following the description of Winter et al. (See, for example, Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al. ., Science, 239: 1534-1536 (1988)), replacing the imported hypervariable region sequences with the corresponding human antibody sequences. Accordingly, such humanized antibodies are chimeric antibodies (see, for example, US Pat. No. 4,816,567) in which significantly less than the intact human variable region is replaced by the corresponding sequence of a non-human species.

An antibody fragment contains a portion of an intact antibody, such as an antigen binding or variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments; diantibodies; linear antibodies (see, for example, US patent No. 5641870; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and polyspecific antibodies formed from antibody fragments.

Fv means the minimum fragment of an antibody that contains the complete antigen recognition and antigen binding site. Such a fragment contains a dimer of one heavy chain variable region and one light chain variable region in close non-covalent association. As a result of the folding of these two domains, six hypervariable loops (three loops from each of the H and L chains) arise, which provide amino acid residues for antigen binding and give the antibody specificity in binding the antigen. However, even a single variable region (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, although with lower affinity than the full binding region.

Single chain Fv (sFv or scFv) refers to antibody fragments that contain the antibody VH and VL domains linked in a single polypeptide chain. The sFv polypeptide may further comprise a polypeptide linker between the VH and VL domains, which provides the ability of the sFv to form the required structure for antigen binding. For a review regarding sFv, see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, below.

The term diabodies refers to small antibody fragments obtained by constructing sFv fragments with short linkers (approximately 5-10 residues) between the VH and VL domains, so that interchain but not intrachain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites. Bispecific diabodies are heterodimers consisting of two crossover sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described more fully, for example, in EP 404097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

Domain antibodies (dAbs), which can be produced in fully human form, are the smallest known antigen-binding antibody fragments, ranging from about 11 kDa to about 15 kDa. Dab are the stable variable regions of the heavy and light chains of immunoglobulins (VH and VL, respectively). They are highly expressed in microbial cell culture, exhibit suitable biophysical properties including, for example, but not limited to, solubility and temperature stability, and are well suited for in vitro selection and affinity maturation, such as in phage display. Dab are biologically active as monomers and, due to their small size and inherent stability, can be formulated into larger molecules to create longer lasting drugs.

- 7 046696 no half-life in serum or other pharmacological activities. Examples of such techniques have been described, for example, in W09425591 for antibodies derived from camelid heavy chain Ig, and also in US20030130496, which describes the isolation of single domain fully human antibodies from phage libraries.

Fv and sFv are the only species with intact antigen-binding regions that lack constant regions. Thus, they are suitable for reduced non-specific binding during in vivo use. sFv fusion proteins can be engineered to produce a fusion with an effector protein at either the amino or carboxyl terminus of the sFv. See, for example, Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a linear antibody, as described, for example, in US Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

In some embodiments, the antibodies of the invention are bispecific or polyspecific. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Examples of bispecific antibodies can bind to two different epitopes of the same antigen. Other antibodies may combine a first antigen-binding site with a binding site for a second antigen. Alternatively, the anti-HIV arm can be combined with an arm that binds to an initiation molecule on a leukocyte, such as a T cell receptor molecule (eg, CD3) or Fc receptors in the case of IgG (Fc gamma R), such as Fc- gamma RI (CD64), Fc-gamma RII (CD32) and Fc-gamma RIII (CD 16), in order to focus and localize cellular defense mechanisms on the infected cell. Bispecific antibodies can also be used to localize cytotoxic agents to infected cells. Bispecific antibodies can be produced as full-length antibodies or antibody fragments (eg, bispecific F(ab')2 antibodies). For example, WO 96/16673 discloses a bispecific anti-ErbB2/anti-Fc gamma RIII antibody, and US Pat. No. 5,837,234 discloses a bispecific anti-ErbB2/anti-Ec gamma RI antibody. For example, a bispecific anti-ErbB2/Fc alpha antibody is described in WO98/02463; US Pat. No. 5,821,337 describes a bispecific anti-ErbB2/anti-CD3 antibody. See also, for example, Mouquet et al., Polyreactivity Increases The Apparent Affinity Of Anti-HIV Antibodies By Heteroligation. Nature. 467, 591-5 (2010), and Mouquet et al., Enhanced HIV-1 neutralization by antibody heteroligation Proc Natl Acad Sci USA. 2012 Jan 17;109(3):875-80.

Methods for producing bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, the two chains having different specificities (see, for example, Millstein et al., Nature, 305: 537-539 (1983)). Similar methods are described, for example, in WO 93/08829, Traunecker et al., EMBO J., 10: 3655-3659 (1991), and also see Mouquet et al., Enhanced HIV-1 neutralization by antibody heteroligation Proc Natl Acad Sci USA. 2012 Jan 17;109(3):87 5-80.

Alternatively, antibody variable regions with the desired binding specificities (antibody-antigen binding sites) are fused to immunoglobulin constant domain sequences. The fusion is carried out with an Ig heavy chain constant domain containing at least part of the hinge region, CH2 and CH3 regions. In some embodiments, a first heavy chain constant region (CH1) containing a site required for light chain binding is present in at least one of the fusions. DNAs encoding immunoglobulin heavy chain and, optionally, immunoglobulin light chain fusions are inserted into separate expression vectors and cotransfected into a suitable host cell. This provides greater flexibility in adjusting the relative ratios of the three polypeptide fragments in those embodiments where unequal ratios of the three polypeptide chains used in the construct provide optimal yield of the desired bispecific antibody. However, it is possible to incorporate coding sequences for two or all three polypeptide chains into a single expression vector when expressing at least two polypeptide chains in equal ratios produces high yields or when the ratios do not significantly affect the yield of the desired combination of chains.

Methods for creating bispecific antibodies from antibody fragments are also described in the literature. For example, bispecific antibodies can be produced using chemical coupling. For example, Brennan et al. (Science, 229: 81 (1985)) describe a method in which intact antibodies are proteolytically cleaved to create F(ab')2 fragments. The resulting fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent dithiols and prevent the formation of intermolecular disulfide. The generated Fab fragments are then converted into thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then converted back to a Fab'-thiol by reduction using mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form a bispecific antibody. The resulting bispecific antibodies can be used as agents for selective immobilization of enzymes.

The present invention contemplates other modifications of antibodies. For example, the antibody may be associated with one of a variety of non-protein polymers, such as polyethylene glycol, poly

- 8 046696 propylene glycol, polyoxyalkylenes or copolymers of polyethylene glycol and polypropylene glycol. The antibody can also be enclosed in microcapsules prepared, for example, by coacervation or interfacial polymerization methods (e.g., hydroxymethylcellulose microcapsules or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such methods are described, for example, in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).

Typically, antibodies according to the described invention are produced recombinantly using vectors and methods available in the art. Human antibodies can also be generated by in vitro activated B cells (see, for example, US Pat. Nos. 5,567,610 and 5,229,275). General molecular genetics and genetic engineering techniques applicable to the present invention are described in current publications Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185 , edited by D. Goeddel, 1991. Academic Press, San Diego, CA), Guide to Protein Purification in Methods in Enzymology (M.P. Deutschcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R.I. Freshney. 1987. Liss, Inc. New York, NY), and Gene Transfer and Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.). Reagents, cloning vectors, and genetic manipulation kits are available from commercial suppliers such as BioRad, Stratagene, Invitrogen, ClonTech, and Sigma Aldrich Co.

Human antibodies can also be produced in transgenic animals (eg, mice), which are capable of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice has been described to result in complete inhibition of endogenous antibody production. Transfer of a number of human germline immunoglobulin genes into such germline mutant mice will result in the production of human antibodies upon antigen stimulation. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno., 7:33 (1993); US Patents No. 5545806, 5569825, 5591669 (all GenPharm), US Patent No. 5545807 and WO 97/17852. Such animals can be genetically engineered to produce human antibodies containing a polypeptide according to the described invention.

Various methods for producing antibody fragments have been developed. Traditionally, such fragments were obtained by proteolytic cleavage of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al., Science, 229:81 (1985) ). However, such fragments can now be directly produced by recombinant host cells. Fab, Fv and ScFv antibody fragments can be expressed and secreted from E. coli, thereby allowing large quantities of such fragments to be easily produced. Fab'-SH fragments can be directly recovered from E. coli cells and chemically coupled to form F(ab')2 fragments (see, for example, Carter et al., Bio/Technology 10: 163167 (1992)). According to another method, F (ab')2 fragments can be isolated directly from a culture of recombinant host cells. Fab and F(ab')2 fragments with extended in vivo half-lives containing salvage receptor binding epitope residues are described in US Pat. No. 5,869,046. Other methods for producing antibody fragments will be known to those skilled in the art.

Other methods that are known in the art for selecting antibody fragments from libraries using enrichment techniques include, but are not limited to, phage display, ribosomal display (Hanes and Pluckthun, 1997, Proc. Nat. Acad. Sci. 94: 4937- 4942), bacterial display (Georgiou, et al., 1997, Nature Biotechnology 15: 29-34) and/or yeast display (Kieke, et al., 1997, Protein Engineering 10: 1303-1310) can be used as an alternative to previously described method for selecting single-chain antibodies.

Single chain antibodies are selected from a library of single chain antibodies produced directly using filamentous phage based techniques. The phage display technique is known in the art (see Cambridge Antibody Technology (CAT)) and is described in US Pat. Nos. 6521404, 6544731, 6555313 , 6582915, 6593081, and other US patents or applications that are based on the priority of GB 9206318, filed May 24, 1992; see also Vaughn, et al. 1996, Nature Biotechnology 14: 309-314). Single-chain antibodies can also be designed and constructed using available DNA recombination techniques, such as DNA amplification techniques (eg, PCR), or possibly using the corresponding hybridoma cDNA as a template.

Antibody variants are also included within the scope of the invention. Thus, the sequence variants listed in the application are also included within the scope of the invention. Additional antibody sequence variants having improved affinity can be generated using methods known in the art and are included within the scope of the invention. For example, amino acid substitutions can be used to produce antibodies with further improved affinity.

- 9 046696

Alternatively, codon optimization of the nucleotide sequence can be used to improve translation efficiency in expression systems for antibody production.

The sequences of such antibody variants will have 70% or greater (ie, 80%, 85%, 90%, 95%, 97%, 98%, 99% or greater) sequence identity with the sequences reported in the application. Such sequence identity is calculated relative to the full length of the reference sequence (ie, the sequence specified in the application). Percentage identities reported herein were determined using BLAST, version 2.1.3, using the default parameters defined by NCBI (the National Center for Biotechnology Information; www.ncbi.nlm.nih.gov/) [Blosum matrix 62 ; penalty for opening a space = 11 and penalty for extending a space = 1]. For example, the present invention provides peptide sequences that contain at least about 5, 10, 15, 20, 30, 40, 50, 75, 100, 150 or more contiguous peptides of one or more sequences disclosed herein description, as well as all intermediate lengths in between. As used herein, the term intermediate lengths is intended to describe any length between the specified values, such as 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, etc. ; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.

The present invention relates to antibodies, either alone or in combination with other antibodies, such as, but not limited to, VRC01, anti-V3 loop, CD4bs and CD4i antibodies, as well as PG9/PG16-like antibodies, which have broad neutralizing activity in serum.

In another embodiment, the present invention provides methods for preparing and administering an HIV antibody composition that is suitable for administration to a human patient or non-human primate having or at risk of HIV infection, in an amount and in a schedule sufficient to induce a protective immune response against HIV or reduce the amount of HIV virus in a person.

In another embodiment, the present invention provides a vaccine comprising at least one antibody of the invention and a pharmaceutically acceptable carrier. In one embodiment, the vaccine is a vaccine comprising at least one antibody described herein and a pharmaceutically acceptable carrier. The vaccine may contain a plurality of antibodies having the characteristics described herein, in any combination, and may additionally contain HIV neutralizing antibodies that are known in the art.

It should be understood that the compositions may have one or a combination of the antibodies described herein, which may be the same or different, to prophylactically or therapeutically treat the progression of various subtypes of HIV infection after vaccination. Such combinations can be selected according to the required immunity. When an antibody is administered to an animal or human, it can be combined with one or more pharmaceutically acceptable carriers, excipients or adjuvants known to one of ordinary skill in the art. The combination may further include broad spectrum neutralizing antibodies known in the art, including, but not limited to, VRC01, b12, anti-V3 loop, CD4bs and CD4i antibodies, as well as PG9/PG16-like antibodies.

In addition, with regard to determining the effective level in a patient for the treatment of HIV, suitable animal models in particular are available and have been widely used to evaluate in vivo anti-HIV efficacy in various gene therapy protocols (Sarver et al. (1993b) ), higher). Such models include mice, monkeys and cats. Even though such animals are not naturally susceptible to HIV disease, chimeric mouse models (e.g., SCID, bg/nu/xid, NOD/SCID, SCID-hu, immunocompetent SCID-hu, BALB/c bone-destroyed brain) reconstructed using human peripheral blood mononuclear cells (PBMCs), lymph nodes, fetal liver/thymus, or other tissues can be infected with a lentiviral vector or HIV and used as models of HIV pathogenesis. Likewise, the simian immunodeficiency virus (VII)/monkey model can be used, as well as the feline immunodeficiency virus (IIV)/cat model. The pharmaceutical composition may contain other pharmaceutical agents together with the vector according to the invention in case of use for the therapeutic treatment of AIDS. Such other pharmaceutical agents can be used in their traditional manner (ie, as agents for the treatment of HIV infection).

In another embodiment, the present invention provides an antibody pharmaceutical composition comprising an effective amount of an isolated HIV antibody or affinity matured variant that provides a choice of prophylactic or therapeutic treatment to reduce infection by the HIV virus. The antibody-based pharmaceutical composition of the present invention can be prepared by several methods known in the art (for example, see McGoff and Scher, 2000, Solution Formulation of Proteins/Peptides: In McNally, E.J., ed. Protein Formulation and Delivery. New York , NY: Marcel Dekker; pp. 139-158; Akers and Defilippis, 2000, Peptides and Proteins as Parenteral Solutions.

- 10 046696

Peptides and Proteins. Philadelphia, PA: Talyor and Francis; pp. 145-177; Akers, et al., 2002, Pharm. Biotechnol. 14:47-127).

A pharmaceutically acceptable composition suitable for administration to a patient will contain an effective amount of antibody in a formulation that will retain biological activity while also providing maximum stability during storage within an acceptable temperature range. Pharmaceutical compositions may also contain, depending on the desired preparation, pharmaceutically acceptable diluents, pharmaceutically acceptable carriers and/or pharmaceutically acceptable excipients or any such excipients commonly used for 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, physiological phosphate-buffered saline, Ringer's solution, dextrose solution and Hanks solution. The amount of excipient that is useful in the pharmaceutical composition or preparation according to the present invention is an amount that serves to evenly distribute the antibody in the composition so that it is evenly distributed when it is delivered to the body of an individual. It can serve to dilute the antibody to a concentration that provides the desired beneficial palliative or curative results while minimizing any adverse side effects that may arise from too high a concentration. It may also act as a preservative. Thus, in the case of an antibody with high physiological activity, a larger amount of excipient can be used. On the other hand, for any active ingredient(s) that exhibit lower physiological activity, less excipient will be used.

The antibodies described above and antibody compositions or vaccine compositions containing at least one or a combination of antibodies described herein can be administered for the prophylactic and therapeutic treatment of infection with the HIV virus.

The present invention also provides isolated polypeptides containing novel light chain and heavy chain amino acid sequences, as well as heavy and light chain consensus sequences SEQ ID NO: 1 and 2, which are indicated in FIG. 3.

In other related embodiments, the invention provides polypeptide variants that encode the amino acid sequences of HIV antibodies listed in FIG. 3; heavy and light chain consensus sequences SEQ ID NO: 1 and 2. Such polypeptide variants are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher sequence identity compared to a polypeptide sequence of the present invention, as determined using the methods described herein (eg, BLAST analysis using standard parameters). One skilled in the art will appreciate that such values may be adjusted accordingly to determine the appropriate identity of the encoded proteins, taking into account amino acid similarities and the like.

The term polypeptide is used in its usual meaning, i.e., as a sequence of amino acids. Polypeptides are not limited to a specific product length. Peptides, oligopeptides and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically stated otherwise. Such a term also includes modifications of the polypeptide after expression, for example, glycosylation, acetylation, phosphorylation and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. The polypeptide may be a complete protein or a subsequence thereof. Polypeptides of particular interest in the context of the present invention are amino acid subsequences containing CDR, VH and VL, capable of binding an antigen or an HIV-infected cell.

A variant polypeptide, as used herein, is a polypeptide that generally differs from the polypeptide specifically disclosed herein by one or more substitutions, deletions, additions and/or insertions. Such variants may occur naturally or may be created synthetically, for example, by modifying one or more of the polypeptide sequences of the invention described above and assessing one or more biological activities of the polypeptide as described herein and/or using a variety of methods well known. in this area.

For example, some amino acids can be replaced by other amino acids in the structure of a protein without significantly losing its ability to bind other polypeptides (eg, antigens) or cells. Since the binding ability and nature of the protein determine the biological functional activity of the protein, certain amino acid sequence substitutions can be made in the protein sequence, and thus in the underlying DNA coding sequence, to produce a protein with similar properties. Thus, it is contemplated that various changes may be made to the peptide sequences of the claimed compositions or the corresponding DNA sequences that encode said peptides without

- 11 046696 significant loss of their biological benefit or activity.

In many cases, a polypeptide variant will contain one or more conservative substitutions. A conservative substitution is a substitution in which an amino acid is replaced with another amino acid that has similar properties such that one skilled in the art of peptide chemistry would expect that the secondary structure and hydropathic nature of the polypeptide will not be significantly changed.

Amino acid substitutions are generally based on the relative similarity of amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, and the like. Examples of substitutions that take into account the above characteristics are well known to those skilled in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

Homology or sequence identity refers to the percentage of residues in a polynucleotide or polypeptide sequence variant that are identical to the invariant sequence after sequence alignment and the introduction of gaps as necessary to achieve maximum percentage homology. In specific embodiments, the polynucleotide and polypeptide variants have at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98% or at least about 99% homology of the polynucleotides or polypeptides with the polynucleotide or polypeptide described in this publication.

Such polypeptide sequence variants will have 70% or greater (ie, 80%, 85%, 90%, 95%, 97%, 98%, 99% or greater) sequence identity to the sequences reported in the application. In additional embodiments, the described invention relates to polypeptide fragments having varying lengths of contiguous stretches of amino acid sequences disclosed herein. For example, the present invention provides peptide sequences that contain at least about 5, 10, 15, 20, 30, 40, 50, 75, 100, 150 or more contiguous peptides of one or more sequences disclosed herein description, as well as all intermediate lengths in between.

The invention also relates to nucleic acid sequences encoding part or all of the light and heavy chains of the described antibodies according to the invention and fragments thereof. Due to the degeneracy of the genetic code, there will be variants of such sequences that encode the same amino acid sequences.

The present invention also provides isolated nucleic acid sequences encoding the HIV antibody heavy and light chain polypeptides shown in FIG. 3, and the heavy and light chain consensus sequences of SEQ ID NO: 1 and 2.

In other related embodiments, the disclosed invention provides polynucleotide variants that encode the HIV antibody heavy and light chain peptide sequences set forth in FIG. 3; heavy and light chain consensus sequences SEQ ID NO: 1 and 2. Such polynucleotide variants have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity to the polynucleotide sequence of the present invention, as determined using the methods described herein publications (for example, BLAST analysis using standard parameters). One skilled in the art will appreciate that such values can be adjusted accordingly to determine the respective identities of the proteins encoded by the two nucleotide sequences, taking into account codon degeneracy, amino acid similarity, reading frame location, and the like.

The terms nucleic acid and polynucleotide are used interchangeably herein to refer to single- or double-stranded RNA, DNA, or mixed polymers. Polynucleotides may comprise genomic sequences, extragenomic and plasmid sequences, and smaller engineered gene regions that express or can be adapted to express polypeptides.

An isolated nucleic acid is a nucleic acid that is substantially separated from other genomic DNA sequences, as well as proteins or complexes, such as ribosomes and polymerases, that naturally accompany the native sequence. The term covers a nucleic acid sequence that has been extracted from its natural environment and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues synthesized biologically in heterologous systems. Substantially pure nucleic acid includes isolated forms of nucleic acid. Accordingly, it refers to the nucleic acid that is originally isolated and does not exclude genes or sequences artificially added to the isolated nucleic acid later.

The term polynucleotide variant, as used herein, is a polynucleotide that is typically different from a polynucleotide, particularly those disclosed in

- 12 046696 worthwhile description, one or more substitutions, divisions, additions and/or insertions. Such variants may occur naturally or may be created by synthesis, for example, by modifying one or more polynucleotide sequences of the invention and assessing one or more biological activities of the encoded polypeptide, as described herein and/or using any of a number of methods , well known in the field.

Modifications can be made to the structure of the polynucleotides of the invention and still provide a functional molecule that encodes a variant or derivative of a polypeptide with the desired characteristics. When it is desired to change the amino acid sequence of a polypeptide to create an equivalent or even an improved variant or portion of the polypeptide of the invention, one of ordinary skill in the art can usually change one or more codons of the DNA coding sequence.

Typically, polynucleotide variants contain one or more substitutions, additions, deletions and/or insertions such that the immunogenic binding properties of the polypeptide encoded by the polynucleotide variant are not significantly reduced compared to the polypeptide encoded by the polynucleotide sequence specifically identified herein.

In additional embodiments, the described invention relates to polynucleotide fragments containing varying lengths of contiguous stretches of sequence identical or complementary to one or more sequences described herein. For example, the present invention provides polynucleotides that contain at least about 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, or 1000 or more contiguous nucleotides of the same or more than one sequence described in this publication, all intermediate lengths and any length between the specified values are also included, for example, 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; and including all integers in the ranges 200-500; 500-1000.

In another embodiment, the invention provides polynucleotide compositions that are capable of hybridizing under moderate to high stringency conditions with a polynucleotide sequence of the present invention, or a fragment or complementary sequence thereof. Hybridization methods are well known in the field of molecular biology. For purposes of illustration, suitable moderate stringency conditions for testing hybridization of a polynucleotide of the present invention with other polynucleotides include a prewash in a solution of 5x SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization at 50-6°C, 5xSSC, overnight; subsequent washing twice at 65°C for 20 minutes with 2x, 0.5x and 0.2x SSC containing 0.1% SDS. One skilled in the art will appreciate that the stringency of hybridization can be easily manipulated, for example, by changing the salt content of the hybridization solution and/or the temperature at which hybridization is performed. For example, in another embodiment, suitable high stringency hybridization conditions include those described above, except that the hybridization temperature is increased, for example, to 60-65°C or 65-70°C.

In some embodiments, the polypeptide encoded by the polynucleotide variant or fragment has the same binding specificity (ie, specifically or preferentially binds to the same HIV epitope or strain) as the polypeptide encoded by the native polynucleotide. In some embodiments, the described polynucleotides, polynucleotide variants, fragments, and hybridizing sequences encode polypeptides that have a level of binding activity that is at least about 50%, at least about 70%, and at least about 90% of the level for the polypeptide sequence. specifically mentioned in the present description.

The polynucleotides of the invention, or fragments thereof, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding regions, and the like, so that they the total length can vary greatly. A nucleic acid fragment of almost any length can be used. For example, exemplary stretches of polynucleotides having a total length of about 10,000, about 5,000, about 3,000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs, and the like (including all lengths therein) are included in many embodiments implementation of the present invention.

Also included within the scope of the invention are vectors, such as expression vectors, containing a nucleic acid sequence according to the invention. Cells transformed with such vectors are also included within the scope of the invention.

The present invention also relates to vectors and host cells containing a nucleic acid of the invention, as well as recombinant methods for producing a polypeptide of the invention. Vectors of the invention include vectors capable of replicating in any cell type

- 13 046696 or an organism, including, for example, plasmids, phage, cosmids and minichromosomes. In some embodiments, vectors containing a polynucleotide according to the described invention are vectors suitable for propagation or replication of a polynucleotide, or vectors suitable for expressing a polypeptide according to the described invention. Such vectors are known in the art and are commercially available.

The vector includes shuttle vectors and expression vectors. Typically, the plasmid design will also include an origin of replication (eg, ColE1 origin of replication) and a selectable marker (eg, ampicillin or tetracycline resistance) for replication and selection, respectively, of plasmids in bacteria. An expression vector refers to a vector that contains the necessary regulatory sequences or regulatory elements for the expression of antibodies, including an antibody fragment of the invention, in bacterial or eukaryotic cells.

As used herein, the term cell can mean any cell, including, but not limited to, a cell of a eukaryotic multicellular species (eg, as opposed to unicellular yeast), such as, but not limited to, a mammalian cell or a human cell. A cell may be a single entity or may be part of a larger group of cells. Such a larger group of cells may include, for example, a cell culture (either mixed or pure), a tissue (for example, endothelial, epithelial, mucosal or other tissue), an organ (for example, lung, liver, muscle and other organs), an organ system (eg, circulatory system, respiratory system, digestive system, urinary system, nervous system, integumentary system, or other organ system) or organism (eg, bird, mammal, or the like).

The polynucleotides of the invention can be synthesized as a whole or in parts that are then combined and inserted into a vector using conventional molecular and cell biology techniques, including, for example, subcloning the polynucleotide into a linearized vector using suitable restriction sites and restriction enzymes. Polynucleotides according to the described invention are amplified in a polymerase chain reaction using oligonucleotide primers complementary to each of the polynucleotide strands. Such primers also include restriction enzyme cleavage sites to facilitate subcloning into the vector. Components of a replicable vector typically include, without limitation, one or more of the following components: a signal sequence, an origin of replication, and one or more marker or selectable genes.

To express a polypeptide of the invention, nucleotide sequences encoding the polypeptide or functional equivalents can be inserted into a suitable expression vector, ie, a vector that contains the necessary elements for transcription and translation of the inserted coding sequence. Methods well known to those skilled in the art can be used to construct expression vectors containing sequences encoding the polypeptide of interest and suitable transcriptional and translational regulatory elements. Such methods include in vitro DNA recombination methods, synthesis methods, and in vivo genetic recombination. Such methods are described, for example, in Sambrook, J., et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.

The present invention also relates to kits used to perform diagnostic and prognostic assays using antibodies, polypeptides and nucleic acids according to the present invention. The kits of the present invention include a suitable container containing the HIV antibody, polypeptide or nucleic acid of the invention in either labeled or unlabeled form. In addition, when the antibody, polypeptide or nucleic acid is supplied in a labeled form suitable for indirect binding assay, the kit further contains reagents for performing the appropriate indirect assay. For example, the kit may include one or more suitable containers containing enzyme substrates or derivatizing agents, depending on the nature of the label. Control samples and/or instructions may also be included. The present invention also relates to kits for detecting the presence of HIV antibodies or a nucleotide sequence for an HIV antibody according to the present invention in a biological sample using PCR or mass spectrometry.

A label as used herein refers to a registered compound or composition that is conjugated directly or indirectly to an antibody so as to create a labeled antibody. The label may also be conjugated to a polypeptide sequence and/or nucleic acid sequence disclosed herein. The label may be detected as such (eg, radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze a chemical change in the compound or substrate composition that can be detected. Antibodies and polypeptides according to the described invention can also be modified to include an epitope tag, for example, for purification or diagnostic use. Suitable means of detection include the use of labels such as, but not limited to, radionuclides, enzymes, coenzymes, fluorescent substances, chemiluminescent substances, chromogens, enzyme substrates or cofactors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes and the like.

According to another embodiment, the present invention relates to diagnostic methods.

Diagnostic methods typically involve bringing a biological sample obtained from a patient, such as, for example, blood, serum, saliva, urine, sputum, cell smear, or tissue biopsy, into contact with an HIV antibody and determining whether the antibody preferentially binds to the sample. which is compared with a control sample or a predetermined cutoff value, thereby determining the presence of the HIV virus.

According to another embodiment, the present invention relates to methods for detecting the presence of HIV antibodies according to the present invention in a biological sample obtained from a patient. Detection methods typically involve obtaining a biological sample from a patient, such as, for example, blood, serum, saliva, urine, sputum, cell smear, or tissue biopsy, and isolating HIV antibodies or fragments thereof or nucleic acids that encode the HIV antibody, and analysis of the presence of HIV antibodies in a biological sample. The present invention also relates to methods for detecting the nucleotide sequence of an HIV antibody in a cell. The nucleotide sequence of an HIV antibody can also be detected using the primers described in this publication. The presence of an HIV antibody in a biological sample from a patient can be determined using known recombination techniques and/or using a mass spectrometer.

In another embodiment, the present invention relates to a method for detecting in a biological sample an HIV antibody comprising a heavy chain that contains a highly conserved consensus sequence and a light chain that contains a highly conserved consensus sequence, comprising obtaining a biological sample containing immunoglobulins from a mammalian individual, isolating HIV -antibodies from the specified sample and identification of highly conserved heavy chain and light chain consensus sequences. The biological sample may be blood, serum, saliva, urine, sputum, cell smear, or tissue biopsy. Amino acid sequences can be determined by methods known in the art, including, for example, PCR and mass spectrometry.

The term assessment includes any form of measurement and involves determining whether an item is present or absent. The terms definition, measurement, assessment, evaluation and analysis are used interchangeably and include quantitative and qualitative definitions. Evaluation can be relative or absolute. Assessing presence involves determining the quantity of what is present and/or determining whether it is present or absent. As used herein, the terms definition, measurement and evaluation and analysis are used interchangeably, and the terms include both quantitative and qualitative definitions.

II. A method to reduce viral replication.

In addition, methods are provided to reduce increases in HIV viral titer, viral replication, viral proliferation, or the amount of HIV viral protein in an individual. In another aspect, the method includes administering to an individual an amount of an HIV antibody effective to reduce increases in HIV titer, viral replication, or amounts of HIV protein of one or more strains or isolates of HIV in the individual.

In another embodiment, the present invention provides a method of reducing viral replication or spread of HIV infection into additional host cells or tissues, comprising contacting a mammalian cell with an antibody or portion thereof that binds an antigenic epitope on gp120.

III. Method of treatment.

In another embodiment, the present invention provides a method of treating a mammal infected with a virus, such as HIV, comprising administering to said mammal a pharmaceutical composition containing the HIV antibodies disclosed herein. In one embodiment, a method of treating a mammal infected with HIV includes administering to said mammal a pharmaceutical composition that contains an antibody of the present invention or a fragment thereof. The compositions of the invention may contain more than one antibody having the characteristics described (eg, a plurality or pool of antibodies). The composition may also contain other HIV neutralizing antibodies that are known in the art, such as, but not limited to, VRC01, PG9 and b12.

Passive immunization has proven to be an effective and safe technique for the prevention and treatment of viral diseases (see, for example, Keller et al., Clin. Microbiol. Rev. 13:602-14 (2000); Casadevall, Nat. Biotechnol. 20: 114 (2002) ; Shibata et al., Nat. Med. 5:204-10 (1999); Passive immunization using human monoclonal antibodies provides an immediate treatment modality for acute HIV prevention and treatment.

Individuals at risk of developing HIV-related diseases or disorders include patients who have been in contact with an infected person or who have been exposed to HIV.

- 15 046696 I get HIV in some other way. Administration of a prophylactic agent may be administered prior to the onset of symptoms characteristic of an HIV-related disease or disorder in order to prevent the disease or disorder, or alternatively slow its progression.

In the case of in vivo treatment of sick humans or sick non-human animals, the patient is administered or given a pharmaceutical preparation containing an HIV antibody according to the invention. When used for in vivo therapy, antibodies of the invention are administered to a patient in therapeutically effective amounts (ie, amounts that eliminate or reduce the patient's viral load). Antibodies are administered to a sick person by known routes, such as intravenous administration, for example, as a bolus or by continuous infusion over a period of time, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, ocular, topical or inhalation routes. Antibodies can be administered parenterally, where possible, to the target cell site, or intravenously. In some embodiments, the antibody is administered by intravenous or subcutaneous administration. The therapeutic compositions of the invention may be administered to a patient or individual systemically, parenterally or topically. The above parameters for assessing successful treatment and improvement in the condition of the disease can be easily measured by conventional methods known to the attending physician.

In the case of parenteral administration, antibodies can be obtained in a standard dosage injection form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral excipient. Examples of such vehicles include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous excipients include, but are not limited to, fixed oils and ethyl oleate. Liposomes can be used as carriers. the filler may contain minor amounts of additives, such as substances that increase isotonicity and chemical stability, such as, for example, buffers and preservatives. Antibodies can be formulated in such excipients at concentrations ranging from about 1 mg/ml to 10 mg/ml.

The dose and dosage schedule depend on many factors easily determined by the attending physician, such as, for example, the nature of the infection, its therapeutic index, the patient and the patient's medical history. In general, a therapeutically effective amount of antibody is administered to the patient. In some embodiments, the amount of antibody administered ranges from about 0.1 mg/kg to about 50 mg/kg of the patient's body weight. Depending on the type and severity of the infection, the initial dose of antibody selected to be administered to the patient is from about 0.1 mg/kg to about 50 mg/kg body weight (e.g., about 0.1 to 15 mg/kg/dose), administered using either one or more separate administrations or by continuous infusion. The success of such therapy is easily monitored by conventional methods and tests and based on criteria known to the attending physician or other specialist in the field. The above parameters for assessing successful treatment and improvement in the condition of the disease can be easily measured by conventional methods known to the attending physician.

Other therapeutic regimens can be combined with the administration of the HIV antibody according to the present invention.

Combination administration includes co-administration using separate drugs or a single pharmaceutical drug and sequential administration in any order, preferably there is a period of time when both (or all) active agents simultaneously exhibit their biological activities. Such combination therapy may result in a synergistic therapeutic effect. The above parameters for assessing successful treatment and improvement in the condition of the disease can be easily measured by conventional methods known to the attending physician.

The terms treatment process or treatment or relief refer to both therapeutic treatment and prophylactic or preventive measures; the goal being to prevent or slow down (reduce) the target pathological condition or disorder. Patients in need of treatment are those who already have the disorder, as well as those who are predisposed to developing the disorder, or those in whom the disorder must be prevented. An individual or mammal is successfully treated for an infection if, after receiving a therapeutic amount of antibody according to the methods of the present invention, the patient has an observable and/or measurable decrease or absence of one or more of the following: a decrease in the number of infected cells or the absence of infected cells; reduction in the overall percentage of cells that are infected; and/or relief, to some extent, of one or more symptoms associated with a particular infection; reducing morbidity and mortality and improving quality of life. The above parameters for assessing successful treatment and improvement in the condition of the disease can be easily measured by conventional methods known to the attending physician.

The term therapeutically effective amount refers to an amount of an antibody or drug that is effective in treating a disease or disorder in an individual or mammal.

Administration in combination with one or more additional therapeutic agents

- 16 046696 includes simultaneous (coinciding in time) and sequential administration in any order.

Carriers as used herein include pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to the cell or mammal exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is a pH buffered aqueous solution. Examples of physiologically acceptable carriers include, but are not limited to, buffers such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins such as, but not limited to, serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as, but not limited to, polyvinylpyrrolidone; amino acids such as, but not limited to, glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including, 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, but not limited to, sodium; and/or nonionic surfactants such as, but not limited to, Tween, polyethylene glycol (PEG) and Pluronics.

Where ranges are specified, it is understood that every intervening value up to the lower limit of tenths of a unit, unless the context clearly dictates otherwise, between the upper and lower limit of the range and any other specified or intervening value within that specified range is included in the scope inventions. The upper and lower limits of such smaller ranges, which may be independently included within the smaller ranges, are also within the scope of the invention, and any specifically excluded limit within the specified range is permitted. In the event that the specified range includes one or both limits, ranges excluding either one or both of such included limits are also covered by the invention.

Example 1.

This example describes the materials and methods used in Examples 2-5 described below.

HIV antibodies were cloned and produced after capturing gp140-specific individual B cells as described previously (Mouquet, H. et al. PLoS One 6, e24078 (2011); Tiller, T. et al. J Immunol Methods 329, 112-24 ( 2008); and Scheid, J.F. et al. Nature 458, 636-40 (2009)). Glycomutant antibodies PGT121GM and 10-1074GM were created by replacing residues 10-1074 at HC positions 32, 53, 54, 58, 97, 1001 with residues PGT121 and vice versa. The binding properties of anti-gp140 antibodies to HIV Env envelope proteins were analyzed in ELISA, SPR, and glycan microarray assays as described (Scheid, J.F. et al. Science 333, 1633-7 (2011); Walker, L.M. et al. Nature 477, 466-70 (2011); and Mouquet, H. et al. PLoS One 6, e24078 (2011). Neutralization was assessed using (i) a luciferase-based assay in TZM.bl cells and (ii) a PBMC-based assay using infection with major HIV-1 variants as described previously (Li, M. et al. J Virol 79, 10108- 25 (2005); Euler, Z. et al. Journal of virology 85, 7236-45 (2011); The structures of the Fab fragments of PGT121 (unliganded and liganded), 10-1074 and GL were obtained by molecular replacement with resolutions up to 2.8 Å, 2.3 Å, 1.8 Å and 2.4 Å, respectively. .

Single B cell RT-PCR and Ig gene assays.

Single cell sorting of gp140+CD19+IgG+ B cells from PBMCs from patient 10 (pt10; referred to as patient 17 in Nature 477(7365): 466-470), cDNA synthesis, and nested PCR amplification of Ig genes were performed in a previous study (PLoS One 6(9) :e24078). Ig/_ genes. expressed by clonal PGT121 variants were PCR amplified using a forward primer (L-V/3-21*02: 5′ CTGGACCGTTCTCCTCCTCG 3′) upstream of the leader region to avoid the potentially mutated region ( 31 ). All PCR products were sequenced and analyzed for the Ig gene used, CDR3 analysis, and the number of somatic VH/Vk hypermutations (IgBLAST; http://www.ncbi.nlm.nih.gov/igblast and IMGT®; http://www.imgt .org). Multiple sequence alignments were performed using MacVector (v.12.5.0) with the ClustalW analysis function (default parameters) and used to generate dendrograms by neighbor pairing (using best tree selection mode and using outgroups for rooting). Alternatively, dendrograms were generated using the UPGMA method (using best tree selection mode).

Germline progenitor gene (GL) regions of PGT121-like and 10-1074-like antibodies were identified using IgBLAST (http://www.ncbi.nlm.nih.gov/igblast) and IMGT®/V-QUEST (http: //www.imgt.org/IMGT_vquest/share/textes/) as VH4-59*01, JH6*03, VL3-21*02 and JL3*02. (These gene regions are among the most commonly used in the human antibody repertoire (PLoS One 6(8) : e22365; Immunogenetics 64(5) : 337-350). To construct a representative GL precursor sequence, we aligned the IgH and IgL sequences 10-996 (antibodies containing the least somatic hypermutations) with GL sequences using IgBLAST (http://www.ncbi.nlm.nih.gov/igblast) The IgH GL sequence was constructed by replacing the sep regions.

- 17 046696 of the VH and JH genes with their GL analogues and using the sequence 10-996 for the CDRH 3 region, including the nucleotides of the N region and the DH gene region. The IgL GL sequence was assembled from the sequences of the VL3-21*02 and JL3*02 gene regions.

Cloning and production of antibodies.

Purified digested PCR products were cloned into vectors expressing Igy1 or Iq/. human (J. Immunol. Methods 329(1-2): 112-124). The vectors containing the IgH and IgX genes were then sequenced and compared with the original sequences of the PCR products. PGT121 and 10-303 shared the same IgA gene, and had one amino acid difference at position 2 of the IgH gene (Fig. 4); Therefore, to produce IgG PGT121, we used the IgX gene, 10-303, and the IgH gene PGT121, formed by introducing a single substitution (V2M) into the IgH gene 10-303 by site-directed mutagenesis (QuikChange site-directed mutagenesis kit; Stratagene). To generate His-tagged Fabs, the VH genes PGT121 and 10-1074 were subcloned into the 6xHis-IgCeee1 expression vector, derived from modification of the present inventors' standard Igy1 vector (Science 301(5638): 1374-1377) to encode the CH1 domain of IgG1 , followed by the label 6x-1 lis. IgH DNA fragments encoding mutant antibodies PGT121GM (S32Y, K53D, S54R, N58T, H97R, T1001Y) and 10-1074GM (Y32S, D53K, R54S, T58N, R97H, Y1001T) were obtained as a synthetic minigene (IDT) and subcloned into Igy1 expression vectors.

The heavy chain sequence of 10-1074GM is shown below, with the mutations highlighted. The light chain sequence of 10-1074GM is the same as for 10-1074.

QVQLQESGPGLVKPSETLSVTCSVSGDSMNNSYWTWIRQSPGKGLEWIGYISKSESAN

YNPSLNSRWISRDSKNQLSLKLNSVPADTAVYYCATARHGQRIYGWSFGEFFTYYSMDVWG KGTTVTVSS

Antibodies and Fab fragments were generated by transient transfection of IgH and IgL expressing plasmids into exponentially growing HEK 293T cells (ATCC, CRL-11268) using the polyethylenimine (PEI) precipitation method (PLoS One 6(9) : e24078). IgG antibodies were affinity purified using Protein G Sepharose beads (GE Healthcare) according to the manufacturer's instructions. Fab fragments were affinity purified using cobalt-containing HisPur™ resin (Thermo scientific) as described below.

HIV-1 Env proteins.

Alanine mutations were introduced into the pYU-2 gp120 vector (a gift from J. Sodroski, Harvard Medical School) at positions 301-303 (Asn-Asn-Thr), 324-325 (Gly-Asp) and 332 (Asn) (amino acid numbering HXBc2) , using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. The same method was used to create double glycan mutants by introducing single alanine mutations into the gp120N332A pYU-2 vector in each PNGS located between Asn262gp120 and Asn406gp120. Site-specific mutations were confirmed by DNA sequencing.

Expression vectors encoding proteins gp140 YU-2 (Journal of virology 74(12): 5716-5725), gp120 YU-2, HXB2c gp120core (Nature 393(6686): 648-659), HXB2c 2CCcore (PLoS Pathog. 5( 5): e1000445) and YU-2 gp120 mutant proteins were used to transfect HEK 293T cells. To produce YU-2 high-mannose-only gp120 protein (gp120kif), 25 μM kifunensine (Enzo Life Sciences) was added at the time of transfection. Culture supernatants were collected and concentrated using centrifugation-based filter devices (Vivacell 100, Sartorius Stedim Biotech Gmbh), which provided buffer exchange of the samples with a buffer containing 10 mM imidazole, 50 mM sodium phosphate, 300 mM sodium chloride; pH 7.4. Proteins were purified by affinity chromatography using HisPur™ cobalt resin (Thermo scientific) according to the manufacturer's instructions.

For deglycosylation reactions, 50 μg of gp120 YU-2 produced by HEK 293T cells in PBS was digested overnight at 37°C using 200 units of PNGase F (New England Biolabs) or 10,000 units of Endo-Hf (New England Biolabs) in appropriate reaction buffers without denaturing agents. After buffer exchange to PBS, glycosidase-treated gp120 (200 ng) was examined on SDS-PAGE using a 4-12% NuPAGE gel (Invitrogen), followed by silver staining (Pierce Silver Stain Kit) using centrifugal filter devices (Amicon® Ultra, Millipore). , Thermo Scientific).

ELISA.

96-well high binding ELISA plates (Costar) were coated overnight with 100 ng/well of purified gp120 in PBS. After washing, the plates were blocked for 2 hours using 2% BSA, 1 μM EDTA, 0.05% Tween-PBS (blocking buffer) and then incubated for 2 hours with IgG at a concentration of 26.7 nM (or 427.2 nM in the case of ELISA using the double glycan mutant gp120 YU-2) and 7 serial dilutions 1:4 in PBS. After washing, plates were developed by incubating with HRP-conjugated goat anti-human IgG (Jackson ImmunoReseach) (at a concentration of 0.8 μg/ml in blocking buffer) for 1 hour and adding HRP chromogenic substrate (ABTS solution, Invitrogen) ( PLoS One 6(9): e24078). Antibody binding to you

- 18046696 gpl2 0V3 brane overlapping peptides were tested using a previously described peptide ELISA method.

For competitive ELISA, gp120-coated plates were blocked for 2 h with blocking buffer and then incubated for 2 h with biotinylated antibodies (at a concentration of 26.6 nM for PGT121, 0.21 nM for 10-1074, 0.43 nM in the case of 10-996 and 1.67 nM in the case of 10-1369) in solutions of competitor antibodies serially diluted 1:2 in PBS (IgG concentration range is from 5.2 to 667 nM). Plates were developed as described above using HRP-conjugated streptavidin (Jackson ImmunoReseach) (at a concentration of 0.8 μg/ml in blocking buffer). All experiments were performed at least in duplicate.

Glycan microarray analysis.

Microarrays were created using robotic printing of lipid-linked glycan probes (neoglycolipids) onto nitrocellulose-coated glass slides (Methods Mol. Biol. 808: 117-136) at two levels (2 and 5 fmol/spot) in duplicate. Binding assays were performed using microarrays containing 15 neoglycolipidomes derived from high-mannose and complex-type N-glycans. Probe sequences are shown in FIG. 7A. Briefly, antibodies were tested at a concentration of 50 μg/ml and binding was detected using biotylated anti-human IgG (Vector) followed by AlexaFluor 647-labeled streptavidin (Molecular Probes).

Surface plasmon resonance.

Experiments were performed using Biacore T100 (Biacore, Inc) (Nature 467(7315): 591-595). Briefly, YU-2 gp140 and gp120 proteins were coupled with primary amines on CM5 chips (Biacore, Inc.) at a binding density of 300 response units. Anti-gp120-IgG and germline (GL) precursor were injected onto the flow cells at 1 μM and 10 μM, respectively, with a flow rate of 35 μL/min and a 3-min association phase and a 5-min dissociation phase. The sensor surface was regenerated using a 30-second injection of 10 mM glycine-HCl, pH 2.5, at a flow rate of 50 μL/min. Dissociation constants (kd (sec-1)), association (1<;ι(Μ-1 sec-1) and binding (KD (M) or KA (M-1)) were calculated from kinetic analyzes after background subtraction using 1:1 binding model without mass reflectance (RI) correction (Biacore T100 computer program) Binding constants for bivalent IgGs calculated using the 1:1 binding model are referred to as apparent affinities in the text to emphasize that the KD values include. potential avidity effects. Neutralization assays.

Viral neutralization was assessed using a luciferase-based assay in TZM.bl cells (J. Virol. 79(16): 10108-10125). The HIV-1 pseudoviruses tested contained mainly tier-2 and tier-3 viruses (Journal of virology 84(3) : 1439-1452) (Tables 4 and 5). High-mannose-only pseudoviruses were produced in wild-type cells treated with 25 μM kyfunesin (Enzo Life Sciences) (Fig. 8C) or in HEK 293S GnTI −/− cells (Fig. 8D). Nonlinear regression analysis was used to calculate the concentrations at which half-maximal inhibition was observed (IC50 values). Neutralizing activities were also assessed in a previously characterized PBMC-based assay using infection with major HIV-1 variants (n=95) isolated from clade B-infected donors with known seroconversion dates from either 1985 to 1989 (historical seroconverters, n=14 ), or from 2003 to 2006 (modern seroconverters, n=21) (Journal of virology 5(14): 7236–7245; Nat. Med. 16(9): 995–997). The neutralizing activity of each antibody was calculated using the computer program GraphPad (v5.0b) as the area under the best-fit curve, which corresponds to the proportion of viruses neutralized above IC50 values ranging from 0.001 to 50 μg/ml. Relative area under the curve (RAUC) values were obtained by normalizing all AUC values relative to the largest value (obtained using 10-1074).

Statistical analyses.

Statistical analyzes were performed using the computer program GraphPad Prism (v5.0b). Neutralization efficiencies in the TZM-bl assay against a selected panel of 9 virus strains compared with apparent antibody binding affinities for gp120 and gp140 were analyzed using Spearman's correlation coefficient. The Mann-Whitney test was used to compare: (i) the gp120/gp140 affinities of antibodies classified in the PGT121 or 101074 group, and (ii) neutralization activity against viruses isolated from historical and modern seroconverters.

Crystallization and structure determination.

6x-His-tagged Fabs PGT121, 10-1074 and 10-996GL were expressed for crystallization. Fabs were purified from supernatants of transiently transfected HEK 2936E cells sequentially by Ni2+-NTA affinity chromatography (Qiagen) and size exclusion chromatography on Superdex200 10/300 (GE Healthcare). Crystals of unliganded PGT121 Fab, PGT121 IgG were isolated from supernatants of transiently transfected HEK 293-bE cells by protein A affinity chromatography (Pierce), and Fab fragments were prepared by papain digestion of IgG and subsequent purification using excl.

- 19 046696 sion chromatography by size on

Superdex200 10/300 (GE Healthcare).

Purified Fabs were concentrated to 8-20 mg/ml (unliganded PGT121, 8 mg/ml; 101074 and GL, 20 mg/ml) in PBS buffer. Ligand-bound PGT121 Fab crystals were prepared from a protein sample (final concentration: 15 mg/mL) that was mixed with a 3-fold molar excess of NA2 glycan and incubated at 20°C for 2 hours. Screening for crystallization conditions was performed at 20°C using a Mosquito® robotic crystallization device (TTP labs) in 400 nL droplets using a 1:1 ratio of protein to reservoir solution. Crystals of unliganded PGT121 Fab (P212121; a=56.8, b=74.7, c=114.9 A) were prepared in 24% PEG 4000, 0.1 M Tris-HCl, pH 8.5, 10 mM CuCl2, and crystals of ligand-bound PGT121 Fab (P212121; a=67.8, b=67.8, c=94.1 A) were grown in 17% PEG 10000, 0.1 M bis-Tris, pH 5, 5, 0.1 M CH3COOHNH4. Crystals of 10-1074 Fab (P21; a=61.4, b=40.3, c=84.5 A; β=95.39°) were obtained in 25% PEG 3350, 0.1 M bis-Tris, pH 5.5, 0.2 M NaCl, and GL Fab crystals (P21; a=54.9, b=344.7, c=55.2 A; β=91.95°) were grown in 20% PEG 3350, 0.24 M sodium malonate, pH 7.0, 10 mM MnCl2. Crystals were cryoprotected by soaking in a mother liquor containing 20% glycerol (unliganded and liganded PGT121 Fab) or 20% ethylene glycol (10-1074 Fab and GL Fab) and then flash cooled in liquid nitrogen.

Diffraction data were acquired using the 12-2 synchrotron radiation beam (wavelength = 1.029 A) from the Stanford Synchrotron Light Source (SSRL) on a 6-megapixel Pilatus 6M detector (Dectris). Data was indexed, integrated and scaled using XDS. Using data obtained for unliganded PGT121 Fab crystals, the authors used Phenix to find a solution for molecular substitution in the case of one Fab per asymmetric unit (H and L chains for the heavy and light chain, respectively) using two search models, domains CH-CL Fab PGT128 (PDB code 3PV3) and VH-VL domains 2F5 (PDB code 3IDJ) after excluding residues in the CDRH 3 and CDRL 3 loops. We then used the unliganded structure of PGT121 as a search model to find solutions for molecular replacement in the case of ligand-bound Fabs PGT121 (one Fab per asymmetric cell), Fab 10-1074 (one Fab per asymmetric cell), and GL (four Fabs per asymmetric cell).

Iterative refinement (including non-crystallographic symmetry constraints for GL) was performed using Phenix and manually fitting models to produce an electron density map using the Coot program. Atomic models were refined with a resolution of up to 3.0 A in the case of Fab PGT121 (Rwork=21.6%; Rfree=26.4%), with a resolution of 1.9 A in the case of Fab 10-1074 (Rwork=18.7%; Rfree =22.3%), with a resolution of 2.4 A in the case of four Fab GL molecules (Rwork = 19.4%; Rfree = 23.7%) and with a resolution of 2.4 A in the case of the ligand-bound Fab PGT121 (Rwork =20.1%; Rfree=24.9%). The atomic model of Fab PGT121 contains 95.2%, 4.9% and 0.0% of residues in the preferred, allowed and forbidden regions of the Ramachandran map, respectively (Fab 10-1074: 98.8%, 0.9%, 0.2 %; Fab GL: 96.0%, 3.8%, 0.23%; ligand-bound Fab PGT121: 96.7%, 3.1%, 0.2%). PyMOL was used for molecular visualization and to generate figures depicting Fab structures. Buried surface area calculations were performed using Areaimol (CCP4 software package) and a 1.4 A probe.

Fab structures were aligned using Super script in PyMOL. Pairwise Ca alignments were performed using PDBeFold.

Example 2. Prevalence and diversity of clonotype PGT121.

Memory B cells for gp140-specific IgG were isolated from an African donor infected with clade A virus using gp140 YU-2 trimmers as bait. Eighty-seven matching immunoglobulin heavy (IgH) and light (IgL) chain genes corresponding to 23 unique clone families were identified. The anti-gp140 IgH repertoire was dominated by one clonal family, representing ~28% of all expanded B cell clones. This B cell family corresponded to the same clone as PGT121-123 (Nature 477(7365): 466-470) and contained 38 members, 29 of which were unique variants at the nucleotide level (Table 3). Based on the IgH nucleotide sequence, the PGT121 family is divided into two groups: a P6T121-like group containing PGT121-123 and 9 closely related variants, and a second group, 10-1074-like, containing 20 members. Although traditional primers designed by the inventors (J. Immunol. Methods 329(1-2):112-124; Science 301(5638):1374-1377) did not amplify IgL genes expressed by PGT121 B cell clones due to nucleotide deletions in the region encoding framework region 1, 24 of the 38 Ig/.. genes were obtained using new Iqλ-specific primers designed to amplify highly somatically mutated genes (Table 3). Consistent with the high levels of hypermutation in the IgH genes (on average 18.2% of VH genes), the amplified Ig/ genes had a high level of mutations (on average 18.2% of V/ gene) and carried nucleotide deletions in framework region 1 (FWR1) (12-21 nucleotides) and a 9-nt long insertion in framework region 3 (FWR3) (Fig. 3B and Table 3).

Sequence alignments of three PGT antibodies (PGT-121, -122 and -123), eleven clonal variants PGT121 and 10-1074 (10-259, 10-303, 10-410, 10-847, 10-996, 10-1074 , 10-1121, 10-1130,

- 20 046696

10-1146, 10-1341, 10-1369 and 10-1074GM), probable germline (GL) and consensus sequences are shown in FIG. 3(a) and 3(b). The sequences of the corresponding heavy chain variable regions, light chain variable regions, heavy chain CDRs and light chain CDRs according to the IMGT and CABT systems are shown in table. 1 below. The assigned sequence identification numbers in the case of sequences according to CAVT systems are indicated in Table. 2 below:

IgH sequences.

Table 1

IMGT FWRl CDRl FWR2 CDR2 FWR3 CDR3 FWR4 10-1369 QVQLQESGPGLVKPLETLSLTCN VS GAFIADHY WSWIRLPLGKG PEWIGY VEDSGDI NYNPSLKNRVHLSLDKSTNQVSLKLM AVTAGDSALYYC ATTKHGRRIYGWAFGE WFTYFYMDV WGRGTTVTVSS 10-259 QVHLQESGEGLVKPSHTLSLTCN GTLVHDNI WSWMRQPLGKQ PEWIGY VRDSGDT NINPSLKSKVHLSLDKSNNLVSLRL ¹ - ¹ AVTAADSATYYC AT TKHGKRIIGIVAENE WFTYFYMDV WGKGTTVTVSS 10-303 QVQLQESGPGLVKPSETLSLTCS GASISDSY WSWIRRSPGKG LEWIGY VHKSGDT NYSPSLKSRVNLSLDTSKNQVSLSLV AATAADSGKYYC ARTL HGRRIYGIVAENE WFTYFYMDV WGNGTQVTVSS 10-410 QVQLQESGPGLVKPPETLSLTCS GASVNDAY WSWIRQSPGKR PEWVGY VHHSGDT NYNPSLKRRVTFSLDTAKNEVSLKLV ALTAADSAVYFC AS HGKRIYGIVALGE LFTYFYMDV WGKGTTVTVSS 10-1130 QVQLGESGPGLVKPPETLSLTCS GASINDAY WSWIRQSPGKR PEWVGY VHHSGDT NYNPSLKRRVTFSLDTAKNEVSLKLV DLTAADSAVYFC ARALHGKRIYGIVALGE LFTYFYMDV WGKGTTVTVSS 10-1121 QVQLQESGPGLVKPPETLSLTCS GASINDAY WSWIRQSPGKR PEWVGY VHHSGDT NYNPSLKRRVSFSLDTAKNEVSLKLV DLTAADSAIYFC ARALHGKRIYGIVALGE LFTYFYMDV WGKGTTVTVSS 10-1146 QVQLVESGPGLVTP3ETLSLTCT NGSVSGRF WSWIRQSPGRG LEWIGY FSDTDRS EYSPLSLRSRLTLSLDASRNQLSLKLK SVTAADSATYYC ARAQ QC-KRI YG I VS FGE FFYYYYMDA WGKGTAVTVSS 10-996 QVQLQESGPGLVKPSETLSLTCS NGSVSGRF WSWIRQSPGRG LEWIGY FSDTEKS NYNPSLRSRLTLSVDASKNQLSLKLN SVTAADSATYYC ARTQQGKRIYGWSFGE FFHYYYMDA WGKGTAVTVSS G.L. QVQLQESGPGLVKPSETLSLTCT VS GGSISSYY WSWIRQPPGKG LEWIGY IYYSGST NYNPSLKSRVTISVDTSKNQFSLKLS SVTAADTAVYYC ARTQGC-KRIYGWSFGD YYYYYYMDV WGKGTTVTVSS 10-1341 QVQLQESGPGLVKPSETLSVTCS GDSMNNYY WTWIRQSPGKG LEWIGY ISDRESA TYNPSLNSRWISRDTSTNQLSLKLN SVTPADTAVYYC ATARRC-QRIYGWSFGE FFYYYSMDV WGRGTTVTVSS 10-847 QVQLQESGPGLVKPSETLSVTCS GDSMNNYY WTWIRQSPGKG LEWIGY ISDRASA TYNPSLNSRWISRDTSKNQLSLKLN SVTPADTAVYYC ATARRC-QRIYGWSFGE FFYYYSMDV WGKGTTVTVSS 10-1074 QVQLQESGPGLVKPSETLSVTCS VS GDSMNNYY WTWIRQSPGKG LEWIGY ISDRESA TYNPSLNSRWISRDTSKNQLSLKLN SVTPADTAVYYC ATARRC-QRIYGWSFGE FFYYYSMDV WGKGTTVTVSS 101074GM QVQLQESGPGLVKPSETLSVTCS VS GDSMNNSY WTWIRQSPGKG LEWIGY ISKSFSA NYNPSLNSRWTSRDTSKNQLSLKLN SVTPADTAVYYC ATARHC-QRTYGWSFGE FFTYYSMDV WGKGTTVTVSS RABAT FWRl CDRl FWR2 CDR2 FWR3 CDR3 FWR4 10-1369 QVQLQESGPGLVKPLHTLSLTCN VSGAFTA DHYWS WIRLPLGKGPE WIG YVEDSGDINY NPSLKN RVHLSLDKSTNQVSLKLMAVTAGDSA LYYCAT TKHGRRIYGWAFGEWF TYFYMDV WGRGTTVTVSS 10-259 QVHLQESGPGLVKPSHTLSLTCN VSGTLVR DNYWS WMRQPLGKQPE WIG YVEDSGDTNY NPSLKS RVHLSLDKSNNLVSLRLTAVTAADSA TYYCAT TKHGRRIYGIVAFNEWF TYFYMDV WGKGTTVTVSS 10-303 QVQLQESGPGLVKPSETLSLTCS DSYWS WIRRSPGKGLE WIG YVHKSGDTNY SPSLKS RVNL S L D T SKNQVS L S LVAATAAD S G KYYCAR TLHGRRIYGIVAFNEWF TYFYMDV WGNGTQVTVSS 10-410 QVQLQESGPGLVKPPETLSLTCS VSGASVN DAYWS WIRQSPGKRPE WVG YVEESGDTNY NPSLKR RVTFSLDTAKNEVSLKLVALTAADSA VYFCAR AL HGKRIY GIVAL GE L F TYFYMDV WGKGTTVTVSS 10-1130 QVQLQESGPGLVKPPETLSLTCS VSGASIN DAYWS WIRQSPGKRPE WVG YVEESGDTNY NPSLKR RVTFSLDTAKNEVSLKLVDLTAADSA VYFCAR AL HGKRIY GIVAL GE L F TYFYMDV WGKGTTVTVSS 10-1121 QVQLQESGPGLVKPPETLSLTCS VSGASIN DAYWS WIRQSPGKRPE WVG YVEESGDTNY NPSLKR RVSFSLDTAKNEVSLKLVDLTAADSA IYFCAR AL HGKRIY GIVAL GE L F TYFYMDV WGKGTTVTVSS 10-1146 QVQLVESGPGLVTP3ETL3LTCT VSNGSVS GRFWS WIRQSPGRGLE WIG YFSDTDRSEY SPSLRS RLTLSLDASRNQLSLKLKSVTAADSA TYYCAR AQQC-KRIYGIVSFGEFF YYYYMDA WGKGTAVTVSS 10-996 QVQLQESGPGLVKPSETLSLTCS VSNGSVS GRFWS WIRQSPGRGLE WIG YFSDTEKSNY NPSLRS RLTLSVDASKNQLSLKLNSVTAADSA TYYCAR TQQC-KRIYGWSFGEFF EYYYMDA WGKGTAVTVSS G.L. QVQLQESGPGLVKPSETLSLTCT SYYWS WIRQPPGKGLE WIG YIYYSGSTNY NPSTKS RVTISVDT SKNQ F S LKL S SVTAAD TA VYYCAR TQQGKRIYGWSFGDYYYYYYMDV WGKGTTVTVSS 10-1341 QVQLQESGPGLVKPSETLSVTCS VSGDSMN NYYWT WIRQSPGKGLE WIG YISDRESATY NPSTiNS RWISRDTSTNQLSLKLNSVTPADTA VYYCAT ARRGQRIYGWSFGEFF YYYSMDV WGRGTTVTVSS 10-847 QVQLQESGPGLVKPSETLSVTCS VSGDSMN NYYWT WIRQSPGKGLE WIG YISDRASATY NPSLNS RWISRDTSKNQLSLKLNSVTPADTA VYYCAT ARRGQRIYGWSFGEFF YYYSMDV WGKGTTVTVSS 10-1074 QVQLQESGPGLVKPSETLSVTCS VSGDSMN NYYWT WIRQSPGKGLE WIG YISDRESATY NPSLNS RWI SRDTSKNQLSLKLNSVTPADTA VYYCAT ARRGQRIYGWSFGEFF YYYSMDV WGKGTTVTVSS ΙΟ- Ι 07 4GM QVQLQESGPGLVKPSETLSVTCS VSGDSMN NSIWT WIRQSPGKGLE WIG IISKSESANI NPSLNS RWI SRDTSKNQLSLKLNSVTPADTA VYYCAT ARHGQRI IGVVSFGEh'F TYYSMDV WGKGTTVTVSS

IgL sequences.

IMGT FWRl CDRl FWR2 CDR2 FWR3 CDR3 FWR4 G.L. SYVLIQPPSVSVAPGQTARITCG NIGSKS VHWYQQKPGQA PVLWY DDS DRPSGIPERFSGSNSGNTATLTISRV eagdeadyyc QVWDSSSDHPWV FGGGTKLTVL 10-1369 SSMSVSPGETAKITCGEK SIGSRA VQWYQKKPGQP PSLIIY NNQ DRPSGVPERFSASPDIEFGTTAILTI TNVEAGDEADYYC HIYDARRPTNWV FDRGTTLTTVL 10-259 SSMSVSPGETAKISCGKE SIGSRA VQWYQQKSGQP PSLIIY NNQ DRPSGVPERFSATPDFGAGTTAILTI TNVEADDEADYYC HIYDARGGTNWV FDRGATLTVL 10-303 SDISVAPGETARISCGEK SLGSRA VQWYQHRAGQA PSLIIY NNQ DRPSGIPERFSGSPDSPFGTTAILTI TSVEAGDEADYYC HIWDSRVPTKWV FGGGTTLTVL 10-1121 SFVSVAPGQTARITCGEE SLGSRS VIWYQQRPGQA PSLIMY NNH DRPSGIPERFSGSPGSTFGTTATLTI TSVEAGDEADYYC HIWDSRRPTNWV FGEGTTLTVL 10-410 SFVSVAPGQTARITCGEE SLGSRS VIWYQQRPGQA PSLIIY NNN DRPSGIPERFSGSPGSTFGTTATLTI TSVEAGDEADYYC HIWDSRRPTNWV FGEGTTLTVL 10-1130 SFVSVAPGQTARITCGEE SLGSRS VIWYQQRPGQA PSLIIY NNN DRPSGIPERFSGSPGSTFGTTATLTI TSVEAGDEADYYC HIWDSRRPTNWV FGEGTTLTVL 10-847 SYVRPLSVALGETASISCGRQ ALGSRA VQWYQHRPGQA PILLIY NNQ DRPSGIPERFSGTPDINFGTRATLTISGVEAGDEADYYC HMWDSRSGFSWS FGGATRLTVL 10-1074 SYVRPLSVALGETARISCGRQ ALGSRA VQWYQHRPGQA PILLIY NNQ DRPSGIPERFSGTPDINFGTRATLTISGVEAGDEADYYC HMWDSRSGFSWS FGGATRLTVL 10-1341 SYVRPTiSVATiGETART SCGRQ ALGSRA VQWYQHRPGQA PILLIY NNQ DRPSGIPERFSGTPDTNFGTRATTiTT SGVEAGDEADYYC HMWDSRSGFSWS FGGATRLTVL 10-996 SSLPLSVAPGATAKIACGEK SFASRA VQWYQQKPGQA PVLIIY NNQ DRPAGVSERFSGTPDVGFGSTATLTI SRVEAGDEADYYC HKWDSRSPLSWV FGGGTQLTVL 10-1146 SSLPLSLAPGATAKIPCGEK SRGSRA VQWYQQKPGQA PTLIIY NNQ DRPAGVSERYSGNPDVAIGVTATTLTI SRVEAGDEAEYYC HYWDRSRSPISWV FGGWTQLTVL KABAT FWRl CDRl FWR2 CDR2 FWR3 CDR3 FWR4 G.L. SYVLIQPPSVSVAPGQTARITC GGNNIGKSSVH WYQQKPGQAPV DDSDRPS GIPERFSGSNSGNTATLTISRVEAGD EADYYC QVWDSSSDHPWV FGGGTKLTVL 10-1369 SSMSVSPGETAKITC GEKSIGSRAVQ WYQKKPGQPPS NNQDRPS GVPERFSASPDIEFGTTATLTIINVE AGDEADYYC HIYDARRPTNWV FDRGTTLTTVL 10-259 SSMSVSPGETAKISC GKESIGSRAVQ WYQQKSGQPPS LIIY NNQDRPS GVPERFSATPDFGAGTTATLTIINVE ADDEADYYC HIYDARGGTNWV FDRGATLTVL 10-303 SDISVAPGETARISC GEKSLGSRAVQ WYQHRAGQAPS LIIY NNQDRPS GIPERFSGSPDSPFGTTATLTIISVE AGDEADYYC HIWDSRVPTKWV FGGGTTLTVL

10-1121 SFVSVAPGQTARITC GEESLGSRSVI WYQQRPGQAPS LIMY NNHDRPS GIPERFSGSPGSTFGTTATLTITSVE AGDEADYYC HIWDSRRPTNWV FGEGTTLTVL 10-410 SFVSVAPGQTARITC GEESLGSRSVI WYQQRPGQAPS LIIY NNNDRPS GIPERFSGSPGSTFGTTATLTITSVE AGDEADYYC HIWDSRPT1VW FGEGTTLTVL 10-1130 SFVSVAPGQTARITC GEESLGSRSVI WYQQRPGQAPS LIIY NNNDRPS GIPERFSGSPGSTFGTTATLTITSVE AGDEADYYC HIWDSRRPTNWV FGEGTTLTVL 10-847 SYVRPLSVALGETASISC GRQALGSRAVQ WYQHRPGQAPI LLIY NNQDRPS GIPERFSGTPDINFGTRATLTISGVE AGDEADYYC HMWDSRSGFSWS FGGATRLTVL 10-1074 S Y VRP L SVAL GE TARISO GRQALGSRAVQ WYQHRPGQAPI LLIY NNQDRPS GIPERFSGTPDINFGTRATLTISGVE AGDEADYYC HMWDSRSGFSWS FGGATRLTVL 10-1341 SUVRPL SVALGE TARISC GRQALGSRAVQ WYQHRPGQAPI LLIY NNQDRPS GIPERFSGTPDINFGTRATLTISGVE AGDEADYYC HMWDSRSGFSWS FGGATRLTVL 10-996 SSLPLSVAPGATAKIAC GEKSFASRAVQ WYQQKPGQAPV NNQDRPA GVSERFSGTPDVGFGSTATLTISRVE AGDEADYYC HKWDSRSPLSWV FGGGTQLTVL 10-1146 SSLPLSLAPGATAKIPC GEKSRGSRAVQ WYQQKPGQAPT LIIY NNQDRPA GVSERYSGNPDVAIGVTATTLTISRVE AGDEAEYYC HYWDRSRSPISWV FGGWTQLTVL

table 2

Name SEQ ID NO Variable region CDR 1-3 Heavy chain (H) Light chain (L) H L consensus SEQ ID NO 1 SEQ ID NO: 2 SEQ ID NO: 33-35 SEQ ID NO: 36-38 10-259 SEQ ID NO 3 SEQ ID NO: 4 SEQ ID NO:39-41 SEQ ID NO: 42-44 10-303 SEQ ID NO 5 SEQ ID NO: 6 SEQ ID NO: 45-47 SEQ ID NO: 48-50 10-410 SEQ ID NO 7 SEQ ID NO: 8 SEQ ID NO: 51-53 SEQ ID NO ; 54-56 10-847 SEQ ID NO 9 SEQ ID NO: 10 SEQ ID NO: 57-59 SEQ ID NO: 60-62 10-996 SEQ ID NO eleven SEQ ID NO: 12 SEQ ID NO: 63-65 SEQ ID NO: 66-68 10-1074 SEQ ID NO 13 SEQ ID NO: 14 SEQ ID NO: 69-71 SEQ ID NOs: 72-74 10-1121 SEQ ID NO 15 SEQ ID NO: 16 SEQ ID NO: 75-77 SEQ ID NO: 78-80 10-1130 SEQ ID NO 17 SEQ ID NO: 18 SEQ ID NO: 81-83 SEQ ID NO: 84-86 10-1146 SEQ ID NO 19 SEQ ID NO: 20 SEQ ID NO: 87-89 SEQ ID NO: 90-92 10-1341 SEQ ID NO 21 SEQ ID NO: 22 SEQ ID NO: 93-95 SEQ ID NO: 96-98 10-1369 SEQ ID NO 23 SEQ ID NO; 24 SEQ ID NO ; 99-101 SEQ ID NO: 102-104 PGT-121 SEQ ID NO 25 SEQ ID NO: 26 SEQ ID NO: 105-107 SEQ ID NO: 108-110 PGT-122 SEQ ID NO 27 SEQ ID NO: 28 SEQ ID NO: 111-113 SEQ ID NO: 114-116 PGT-123 SEQ ID NO 29 SEQ ID NO: 30 SEQ ID NO: 117-119 SEQ ID NO: 120-122 G.L. SEQ ID NO 31 SEQ ID NO: 32 SEQ ID NO: 123-125 SEQ ID NO: 126-128 10-1074GM SEQ ID NO 129 SEQ ID NO: 130 SEQ ID NO: 131-133 SEQ ID NO: 134-136

Eleven new unique variants were expressed (Table 3) and shown to bind to YU-2 gp120 and gp140 using ELISA and surface plasmon resonance (SPR). Unless otherwise stated, the gp120 and gp140 proteins for these and other experiments were expressed in mammalian cells, which can bind either the complex type or the high mannose type N-glycan to PNGS. The levels of reactivity to gp120 differ for antibodies belonging to the PGT121 and 10-1074 groups, the latter group having higher apparent affinities (Fig. 3A), mainly due to slower dissociation from the gp120/gp140 complex in the case of 10-1074. 1074-related antibodies (Fig. 4B).

Example 3. Epitopes PGT121 and 10-1074.

Asn332gp120 near the V3 stem loop has been reported to be important for the binding and neutralization of viruses by PGT121 (Nature 477(7365): 466-470), so we investigated the role of V3 in antigen recognition by P6T121-like and 10-1074-like antibodies. ELISA was performed using gp120 core proteins HXB2, which lack the V1-V3 loops (gp120core) or retain part of V3 (2CC-cogs), and using the mutant gp120 YU-2 protein carrying a double alanine substitution in the V3 stem (gp120GD324-5AA) . Antibodies tested exhibited reduced reactivity against variants lacking the V3 loop and gp120GD324-5AA compared to intact YU-2 gp120, with binding of group 10-1074 antibodies most affected (Fig. 5A and B). The results obtained indicate that protein determinants near the V3 loop are involved in recognition by both groups of antibodies. None of the antibodies bound to the overlapping peptides spanning V3, suggesting that the target epitopes are discontinuous and/or require a special conformation that is not achieved with the isolated peptides (Fig. 5C).

Asn332gp120 (Asn337gp120 in the earlier numbering (J. Proteome Res 7(4): 1660-1674)) is the N-terminal residue of a potential N-glycosylation site (PNGS), defined as the sequence Asn-X-Ser/Thr. To determine whether Asn332gp120 and/or its N-linked glycan are required for gp120 reactivity of the new PGT121- and 10-1074-group antibodies, we tested their binding to YU-2 gp120N332A in an ELISA. The N332A substitution reduced the binding of PGT121 and all new antibody variants, while their reactivity against a gp120 mutant lacking an adjacent glycosylation site (gp120NNT301-3AAA mutant) was not affected. To determine whether PNGS in addition to Asn332gp120 PNGS affects recognition by new antibodies, we constructed a series of 11 double glycan mutants in which the N332A mutation in YU-2 gp120 was combined with a mutation in PNGS located between Asn262gp120 and Asn406gp120. All PGT121-like and 10-1074-like antibodies bound to each of the double glycan mutants with comparable affinities as in the case of gp120N332A.

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To compare the overall glycan recognition of PGT121- and 10-1074-like antibodies, we examined their binding to YU-2 gp120 treated with PNGase F, which cleaves both complex-type and high-mannose N-glycans. . Because gp120 cannot be completely enzymatically deglycosylated unless it is denatured, treatment with PNGase F resulted in partial deglycosylation of native folded gp120 (Fig. 6). However, the reactivities of the two groups of antibodies differed in that partial deglycosylation of gp120 by PNGase F reduced the binding activity of all PGT121-like antibodies but did not reduce the activity of any of the 10-1074-like antibodies (Fig. 6C). Similar experiments performed using YU-2 gp120 treated with Endo H, which cleaves high-mannose N-glycans but does not cleave complex-type N-glycans, revealed a greater effect on binding of 101074-like antibodies than PGT121-like antibodies ( Fig. 6D).

The N-glycan microarray showed that six of the seven PGT121-like antibodies tested showed detectable binding to one- or two-antennary complex-type N-glycans ending in galactose or α2-6-linked sialic acid, but did not show detectable binding to high-type glycans. mannose content, confirming and extending previous reports of the lack of binding of PGT121-123 to high-mannose N-glycans and the lack of competition with Man4 and Man9 dendrons for binding to gp120 (Fig. 7). In contrast, there is no detectable binding of 10-1074-like antibodies to protein-free glycans (Fig. 7). Although PGT121-like antibodies bound to protein-free complex-type N-glycans but did not bind to high-mannose N-glycans, PGT121-like antibodies retained the ability to bind to gp120 YU-2 produced in cells treated with kifunensin (gp120kif) , a mannosidase inhibitor that results in the exclusive binding of high-mannose glycans to PNGS (Figure 8B). For most PGT121-like antibodies, a small but reproducible decrease in binding to gp120kif was observed. In contrast, 101074-like antibodies completely retained binding to gp120kif (Fig. 8B). The results obtained are consistent with the hypothesis that high-mannose N-glycans, as well as complex-type N-glycans, may be involved in the formation of the epitope for PGT121-like antibodies.

Epitope mapping experiments were performed using two representative representatives of each group (PGT121 and 10-1369 for the PCT121-like group; 10-1074 and 10-996 for the 10-1074-like group) in a competitive ELISA. All four antibodies exhibited cross-competition, but PGT121 inhibited the binding of 10-996 and 10-1074 to gp120 more weakly than vice versa. To further map target epitopes, we used anti-gp120 antibodies that recognize the V3 loop crown (Figure 5), CD4bs, the coreceptor binding site (CD4-inducible; CD4i), the high-mannose N-glycan array (2G12) ( Journal of virology 76(14): 7293-7305; Proc. Natl. Acad. Sci. USA 102(38): 13372-13377)) or the V3-linked glycans at positions 301 and 332 (PGT128). Antibodies against corona V3 inhibited the binding of PGT121 and 10-1369 but did not interfere with the binding of 10-996 and 10-1074. Antibodies PGT128 and to a lesser extent 2G12, but not antibodies CD4bs and CD4i, reduced the binding of all four antibodies to gp120.

Taken together, the data obtained indicate that representatives of the PGT121 clone recognize a region that involves a protein determinant near the V3 loop and the glycan associated with Asn332gp120. However, the clone is divided into two families, the PGT121-like and 10-1074-like groups, which differ in their affinities for gp120 and the role of glycans in epitope formation.

Example 4: Broad and effective neutralization of HIV.

To evaluate the neutralizing activity of the new PGT121 variants, we measured their ability to inhibit HIV infection of TZM-b1 cells using 10 viral strains, including R1166.cl, which lacks the PNGS at position 332 of gp120. All PGT121 variants, including 10-1074-like antibodies, neutralized 9 pseudoviruses and none neutralized the R116 6.cl control (Fig. 1A and Table 4). Neutralizing activity correlated with affinity for HIV spike, with slightly higher efficiencies observed for the 10-1074 group than for the PGT121 group (Fig. 1B and Fig. 4C). The typical germline (GL) variant antibody clonotype PGT121/10-1074 failed to bind gp120/gp140 or neutralize any viruses included in the panel, indicating that a somatic mutation is required for binding and neutralization. Pairing of GL light chains with 10-1074- or 10-996-mutant heavy chains could not rescue binding or neutralization, suggesting that both mutant chains contribute to the correct assembly of the antibody paratope.

The following assays were performed to compare the neutralizing activities of PGT121 and two 10-1074-like antibodies (10-996 and 10-1074) against an expanded panel of 119 difficult-to-neutralize pseudoviruses (classified as tier-2 and tier-3) (Table 4 and 5). 10-996 and 10-1074 had neutralization efficiencies and breadths similar to PGT121 (Figure 1C, Figure 9, and Tables 5 and 6). As expected, the majority of viruses carrying amino acid changes in gp120 at positions 332 and/or 334 (spanning PNGS Asn332-X-Ser334/Thr334) were resistant to neutralization (83.8% were reactive).

- 23 046696 resistant to PGT121, 100% were resistant to 10-1074 and 10-996). Mutation in this PNGS was responsible for the resistance of most viruses to neutralization (68.5% in the case of 10-996, 72.5% in the case of 10-1074 and 60.8% in the case of PGT121) (Table 7). Comparable neutralization activities were observed for the IgG and Fab forms of PGT121 and 10-1074, suggesting that bivalency is not critical for their activity (Fig. 1D).

To evaluate the possible role of complex-type N-glycans on the HIV envelope in neutralization by antibodies PGT121 and 10-1074, we produced virions only with high mannose content in two different ways: the assembly of pseudoviruses in cells treated with kifunensine, which leads to the formation of N-linked glycans Man9GlcNAc2 , or assembly in HEK 293S GnTI −/− cells, which leads to the formation of N-linked glycans Man5GlcNAc2. We found that PGT121 neutralized 2 of 3 kifunensin-derived PGT121-sensitive/10-1074-resistant strains equivalent to their counterparts produced in wild-type cells (Fig. 8C). Two PGT121-sensitive/10-1074-sensitive viral strains obtained in GnTI −/− cells were equally sensitive to PGT121 and 10-1074 as their counterparts obtained in wild-type cells. Consistent with previously reported findings that complex-type N-glycans partially protect the CD4 binding site from antibody binding, viruses produced in GnTI −/− cells were more sensitive to CD4 binding site antibodies (NIH45-46G54W and 3BNC60) (Fig. 8D).

Example 5: Newly transmitted HIV-1.

We then examined the activity of PGT121 and 10-1074 against transmitted founder viruses by assessing neutralization in a peripheral blood mononuclear cell (PBMC)-based assay using 95 clade B viruses isolated from a cohort of people who seroconverted from 1985 to 1989 (historical seroconverters , n=14) or from 2003 to 2006 (modern seroconverters, n=25) (51, 52). The authors compared PGT121 and 10-1074 with antiCD4bs-bN-AT and other bN-Abs, including VRC01, PG9/PG16, b12, 2G12, 4E10 and 2F5. Cluster analysis of neutralizing activity showed segregation into two groups; The PGT121/10-1074 group contained the most active HIV neutralizers, including anti-CD4bs and PG9 antibodies (Table 8). It is noteworthy that in the case of 10-1074, exceptional neutralization efficiency was observed on this panel of clade B viruses, with the greatest breadth observed at 0.1 μg/ml (67% of 95 clade B viruses) of all bN-Abs tested (Table 8) . Although 10-1074 was more potent against modern clade B viruses than PGT121 (∼20-fold difference), both antibodies were more effective against historical than modern viruses ( Fig. 1E and Fig. 10 ).

Example 6: Crystal structures of PGT121, 10-1074 and GL.

To investigate the structural determinants of the differences between PGT121-like and 1074-like antibodies, we determined the crystal structures of Fab fragments of PGT121, 101074, and a typical germline (GL) precursor at 3.0 Å, 1.9 Å, and 2.4 Å resolution, respectively ( table 9). Superposition of the heavy and light chain variable domains (VH and VL) for the three Fabs revealed a conserved backbone structure, with differences limited to minor rearrangements of the CDRH 3 and CDRL 3 loops in affinity matured Fabs compared to GL (Table 10).

An unusual feature of the antibodies is their long (25 residues) CDRH loop 3, which forms a double-stranded antiparallel β-sheet spanning the F and G strands of the VH domain. In each Fab, the tip of the extended CDRH loop 3 mainly contains nonpolar residues. A similar structural feature was observed in the case of CDRH 3 PGT145, a carbohydrate-sensitive antibody whose epitope includes the V1V2 loop of gp120. However, the extended double-stranded β-sheet of CDRH 3 PGT145 mainly contains negatively charged residues, including two sulfated tyrosines at the tip. The VH-VL alignment of PGT121 and PGT145 (Table 10) shows that CDRH 3PGT145 extends beyond CDRH 3PGT121, and that its tip and VH domains align, whereas CDRH 3 of PGT121, 101074, and GL tilts toward VL. The tilt of CDRH 3PGT121/CDRH 310-1074/CDRH 3GL toward VL opens a gap between CDRH2 and CDRH 3, a feature not present in related antibodies.

PGT121 and 10-1074 are highly divergent with respect to GL and each other (of 132 residues, PGT121VH differs from 10-1074VH and GLVH by 36 and 45 residues, respectively, and 10-1074VH and GLVH differ by 29 residues). Most of the differences in PGT121/10-1074 are located in the loops of CDRVH and CDRL 3. Interestingly, the six substitutions in CDRH 3 (residues 100d, 100f, 100h, 100j, 1001, 100n) alternate so that every other residue is replaced, causing a change in surface with the formation of a gap between CDRH2 and CDRH 3, which results from the tilt of CDRH 3 towards VL. Such a region likely contributes to the different subtle specificities of PGT121 and 10-1074. Five other solvent-exposed substitutions in heavy chain framework region 3 (FWR3HC) (residues 64, 78, 80-82; strands D and E) are possible antigen contact sites, given that framework regions in HIV antibodies can contact gp120 . Other differences that may contribute to subtle differences in specificity include a negative patch on PGT121 near Asp56HC that is absent in 10-1074 or GL (Ser56HC in 10-1074 and GL) and positive patches on the surface of CDRL 1 and CDRL 3 that are not found on a similar surface GL.

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Somatic mutations shared by PGT121 and 10-1074 may be involved in the shared features of their epitopes. Heavy chains PGT121 and 10-1074 share only three mutations (out of 36 differences in PGT121GL and 29 differences in 10-1074-GL). In contrast, PGT121 and 10-1074 share 18 light chain mutations (out of 37 PGT121-GL differences and 36 10-1074-GL differences), including an insertion in FWR3 light chain that causes the formation of a loop connecting strands D and E and a substitution Asp50LC-Asp51LC in CDRL 2GL to Asn50LC-Asn51LC in both antibodies PGT121 and 10-1074, resulting in a less negatively charged surface. The large number of common substitutions introduced in LCPGT121 and LC10-1074 (approximately 50% of LC substitutions) indicates that CDRL 1, CDRL 2, and FWR2LC are potential contact regions for epitopes shared by PGT121 and 10-1074.

Comparisons were then made with the structure of PGT128, which recognizes Asn332gp120- and Asn301gp120-linked glycans and V3 and was determined to be complexed with gp120 with the V3 outer domain/miniloop expressed in cells that cannot produce proteins modified by complex-type N-glycans. Unlike the CDRH 3 loops of PGT121 and 10-1074, PGT128CDRH 3 does not tilt toward PGT128VL, and CDRH 3PGT128 does not have a double-stranded β-sheet. In addition, CDRH 3PGT128 (18 residues) is shorter than CDRH 3 PGT121 and 10-1074 (24 residues), whereas CDRH2PGT128 contains a six-residue insertion not found in PGT121 or 10-1074. Because of these differences, CDRH2 is the most expressed feature in PGT128, whereas CDRH 3 is the most expressed in PGT121 and 10-1074. CDRH2PGT128 and CDRL 3PGT128 together recognize Man8/9 bound to Asn332gp120, and CDRH 3PGT128 contacts the base of the V3 loop. This method of recognition of gp120 is not possible in the case of PGT121 and 10-1074, since the structural characteristics of their CDRH2 and CDRH 3 loops differ significantly from those of PGT128, which is consistent with the ability of PGT128, but not PGT121 and 10-1074 (Fig. 7), to recognize free from proteins glycans with high mannose content.

Example 7. Crystal structure of the PGT121-glycan complex.

A 2.4 A resolution structure of the PGT121 antibody associated with a sialylated biantennary complex-type glycan (Table 9) was obtained using crystals obtained under conditions including the presence of NA2, a non-sialylated biantennary complex-type glycan (FIG. 7). Surprisingly, the glycan associated with PGT121 in the crystal structure obtained by the authors was not NA2, but was a complex-type N-glycan from the neighboring PGT121 Fab in the crystal lattice; in particular, the N-glycan associated with Asn105HC. Glycan identification was obvious as electron density was observed for the glycosidic linkage to Asn105HC and for the terminal sialic acid on the Mana1-3Man antenna (the galactose and sialic acid residues of the Mana1-6Man antenna were not resolved in the structure). The composition of the bound glycan corresponds to the portion of the α2-6-sialylated A2(2-6) glycan that was bound by the PGT121 antibody in microarray experiments (Fig. 7) and the expected sialyl linkage in complex-type N-glycans associated with PNGS on proteins expressed in HEK293T cells. Although the VH-VL domains of this structure (bound to the PGT121 ligand) overlap without significant differences with the VH-VL domains of the PGT121 structure without the bound N-glycan (not bound to the PGT121 ligand) (Table 10), the crank angle (the angle between the VHVL pseudopairs and CH1-CL) are different in structures. This difference likely reflects the flexibility that allows Fabs to accept variable crank angles depending on lattice stresses.

Given that the authors observed complex-type N-glycan binding in one crystal structure (the ligand-bound PGT121 structure) but not in another structure (the non-ligand-bound PGT121 structure), we estimated that the affinity of PGT121 for the complex-type N-glycan , not associated with gp120, is in the concentration range of PGT121 in crystals (~10 mM). Assuming that the KD for isolated glycan binding is in the range of 1-10 mM, comparable to the 1.6 mM KD obtained for PG9 binding to Man5GlcNAc2-Asn, then the KD for PGT121 antibody binding of the isolated glycan makes only a small contribution to the affinity of PGT121 for relative to gp120, which is in the nanomolar range (Fig. 4A).

The glycan in the structure of ligand-bound PGT121 interacts exclusively with the VH domain and makes extensive contacts with residues in all three CDRs (buried surface area on PGT121HC=600 A2). The contacts include 10 direct and 18 water-mediated hydrogen bonds (Table 11) with 9 amino acids anchoring the glycan between the N-acetylglucosamine residue linked to the branch point mannose and the terminal sialic acid on the 1-3 antenna. Several contacts with PGT121 are made through this sialic acid, including three direct hydrogen bonds with PGT121 residues Asp31HC and His97HC in addition to water-mediated hydrogen bonds with Asp31HC. Sialic acid also contributes to the water-mediated hydrogen bonding network within the glycan. Direct contacts with sialic acid may explain the stronger binding of PGT121 to the sialylated A2(2-6) glycan than to the non-sialylated NA2 glycan in our glycan microarray assay (FIG. 7).

Extensive water-mediated contacts with proteins created by N residues

- 25 046696 acetylglucosamine and galactose 1-3 antennas may explain the binding observed for non-sialylated one- and two-antennary glycans with PGT121 (Fig. 7).

Six residues promoting direct or likely contacts through amino acid side chains with glycans (Ser32HC-CDRH 1, Lys53HC-CDRH2, Ser54HC-CDRH2, Asn58HCCDRH2, His97HC-CDRH 3, Thr1001HC-CDRH 3) differ from those in 10-1074 (Tyr32HC -CDRH 1, Asp53HC-CDRH2, Arg54HC-CDRH2, Thr58HC-CDRH2, Arg97HC-CDRH 3, Tyrl001HC-CDRH 3) and are highly conserved among PGT121-like, but not among 10-1074-like antibodies. Residues 10-1074 do not have the appropriate functional groups to make observed glycan contacts or have bulky side chains that may cause steric conflicts. Four of these residues also differ from those in GL (Tyr32HC-CDRH 1, Tyr53HC-CDRH2, Gln97HC-CDRH 3, Tyr1001HC-CDRH 3), suggesting that the lack of binding of 10-1074-like antibodies and GL to protein-free glycans of the complex type on glycan microarrays results from a lack of hydrogen bonding and/or steric conflicts (e.g., His97PGT121 versus Arg9710-1074; Thr1001PGT121 versus Tyr100110-1074). Since most of the sequence differences between PGT121 and 10-1074 are clustered in the loops of CDRH, particularly towards the cleft surface between CDRH2 and CDRH 3, where we observed a bound complex-type N-glycan, differential recognition of complex-type glycans on gp120 may account for some or all differences in their observed subtle specificity.

Example 8. Replacement of glycan-contacting antibody residues affects neutralization.

To evaluate the contributions of complex-type N-glycan-contacting residues determined from the ligand-bound structure of PGT121, we generated two mutant antibodies to exchange complex-type glycan-contacting residues between PGT121 and 10-1074: IgG 10-1074 with residues of PGT121 (six substitutions in IgH Y32S, D53K, R54S, T58N, R97H, Y1001T) and IgG PGT121 with reciprocal substitutions. The glycomutant antibodies (10-1074GM and PGT121GM) had near-wild-type apparent affinity for YU-2 gp120/gp140 as measured using SPR (Fig. 2A), indicating that the substitutions did not disrupt binding to the shell spike , derived from a virus strain neutralized by both PGT121 and 10-1074 (Fig. 1A). The fact that PGT121 residues contacting complex-type N-glycans can be accommodated in the 10-1074 backbone without interfering with gp120/gp140 bound by both wild-type antibodies suggests an overall similarity in antigen binding despite subtle differences in specificity .

In contrast to wild-type PGT121, PGT121GM did not bind glycans in microarray experiments, confirming that residues 10-1074 at substitution positions are not compatible with protein-free glycan binding (Fig. 2B) and confirming that glycan-contacting residues in a ligand-bound structure, PGT121 are involved in the recognition of complex-type glycans on microarrays. 10-1074GM also did not bind to protein-free glycans (Fig. 2B), suggesting that some residues in addition to the replaced residues are involved in creating the complex-type protein-free N-glycan binding site.

A TZM-bl-based assay was then used to compare neutralization by wild-type and glycomutant antibodies. We tested 40 viral strains, including strains differentially resistant to PGT121 or 10-1074 and strains sensitive to both wild-type antibodies (Fig. 2C and Table 12). Consistent with the binding of PGT121GM and 10-1074GM to purified YU-2 envelope proteins, both mutants neutralized YU-2 virus; however, 64% of PGT121-sensitive strains were resistant to PGT121GM (Fig. 2C and Table 12), suggesting that the glycan-contacting residues identified in the ligand-bound structure of PGT121 are relevant to the neutralizing activity of PGT121. Conversely, 10-1074GM exhibited higher average potency than wild-type 10-1074 against 10-1074-susceptible strains (Fig. 2C and Table 12), including a more than 3-fold increase in potency against four 10-1074-susceptible strains (WITO4160.33, ZM214M.PL15, Ce1172_H1 and 3817.v2.c59). In general, PCT121 substitutions in 10-1074 did not confer sensitivity to 10-1074GM PGT121-sensitive/101074-resistant strains, but two such strains (CNE19 and 62357_14_D3_4589) became susceptible to 10-1074GM (IC50 = 0.19 μg/ml and 40.8 μg/ml, respectively). Interestingly, there were only PGT121-sensitive/10-1074-resistant strains that contained an intact PNGS associated with Asn332gp120. Other PGT121-sensitive/10-1074-resistant strains lack the Asn332gp120-linked glycan and are resistant to PGT121GM and 10-1074GM, suggesting that their sensitivity to wild-type PGT121 involves an adjacent N-glycan and/or compensation by protein moieties epitope. Although significant gain of function was observed only for 101074GM against one strain (CNE19), this result, together with the overall improvement observed for 10-1074GM against 10-1074-sensitive strains (Fig. 2C), is consistent with this explanation that the crystallographically identified glycan-contacting residues may transfer the recognition properties of the PGT121-like antibody to 10-1074 in some contexts and/or affect its effectiveness in other ways. In addition, the loss of neutralizing activity in the case of

- 26 046696

PGT121GM against PGT121-4yBCTBHTe.ibHbix strains suggests that the neutralizing activity of PGT121 involves residues identified as contacting a complex-type N-glycan in the ligand-bound structure of PGT121.

Results.

PGT121 is a glycan-dependent bN Ab that was initially identified in the serum of a clade A virus-infected donor, yielding only two clonally related members in a functional screen. gp140 trimers were used as bait for single-cell sorting, yielding 2 9 new clonal variants. The PGT121 family of clones includes distinct groups of closely related antibodies: PGT121 and 10-1074 groups. The results indicate that the epitopes of both groups involve PNGS at position Asn332gp120 and the base of the V3 loop. The PGT121-like and 10-1074-like antibody groups differ in amino acid sequences, gp120/gp140 binding affinities, and neutralizing activities, with 101074-like antibodies being completely dependent on an intact PNGS at position Asn332gp120 for neutralization, whereas PGT121-like antibodies are capable of neutralize some viral strains that lack PNGS in Asn332gp120.

A significant difference between the two groups of antibodies is that the PGT121-like antibodies bind complex-type N-glycans on the carbohydrate matrix, whereas the 10-1074-like antibodies show no detectable binding to any protein-free N-glycans tested (Fig. .7). Binding of protein-free glycans by anti-HIV antibodies cannot always be detected; for example, although PG9 recognizes the gp120-associated high-mannose glycan, microarrays do not show binding to protein-free glycans. Thus, although a positive result obtained on a glycan microarray implies the involvement of a particular glycan in the antibody epitope, a negative result does not exclude glycan recognition. For example, although high-mannose glycans are not detected in glycan microarray experiments, they may be involved in the PGT121 epitope, consistent with data that only high-mannose forms of gp120 protein and virions bind and neutralize (Fig. 8).

The molecular basis for the differences between PGT121, 10-1074 and their precursor GL was revealed, in part, using their crystal structures. The finding that most somatic light chain mutations are common to PGT121 and 10-1074, whereas heavy chain mutations differ, suggests that the light chain contacts common parts of the gp120 epitope and the heavy chain recognizes distinct features.

All three antibodies have an extended CDRH 3 with a nonpolar tip that may provide access to cryptic epitopes. Differences in the antigen binding region of the two mature Fabs are mainly located in the cleft between CDRH2 and extended CDRH 3. Interestingly, the putative antigen binding cleft between CDRH2 and CDRH 3 is also found in the typical germline precursor for PGT121 and 10-1074.

Structural information regarding the recognition of PGT glycans by 121-like antibodies was obtained from a crystal structure in which a complex-type sialylated N-glycan bound to a VH domain residue interacted with the binding site of an adjacent PGT121 Fab. Several features of the ligand-bound structure of PGT121 suggest that it is suitable for understanding the recognition of complex-type N-glycans on gp120 by PGT121-like antibodies. First, the glycan structure corresponds to the α2-6-sialylated A2(2-6) glycan that antibody PGT121 binds on microarrays (Figure 7). Second, the glycan interacts with PGT121 using the gap between CDRH 3 and CDRH2, which was confirmed by structural analyzes to be involved in epitope recognition, possibly explaining the unusual tilt of CDRH 3 towards VL in the structures of PGT121 and 10-1074. Third, most of the VH residues identified as glycan-interacting residues are different in PGT121 and 10-1074, which explains the different binding profiles on glycan microarrays and possibly explains the different subtle specificities revealed in protein binding experiments. Fourth, the exchange of crystallographically identified glycan contact residues between PGT121 and 10-1074, in part, leads to a transfer of their properties: PGT121GM, like 10-1074, did not bind to protein-free glycans, but both antibodies PGT121GM and 10-1074GM retained Near-wild-type binding to purified YU-2 gp120/gp140. Although PGT121GM retained the ability to neutralize some viral strains that were neutralized by wild-type PGT121 and 10-1074, it was unable to neutralize strains that were PGT121-sensitive/10-1074-resistant, suggesting that the glycan binding motif is important for neutralizing activity of PGT121 against 101074-resistant strains. In the case of reciprocal exchange, the neutralization efficiency of antibody 10-1074GM was increased or unaffected compared to 10-1074, and in one case, 101074GM potentially neutralized a PGT121-sensitive/10-1074-resistant strain, consistent with the transfer of a crystallographically identified glycan motif and the hypothesis that the epitopes of PGT121- and 10-1074-like antibodies are related. In the analyzes the follower

- 27 046696 gp120 features from strains for which there is other data on the neutralization of PGT121, other than the correlation with PNGS in Asn332gp120 in the case of viruses sensitive to PGT121-like and 10-1074-like antibodies, there is no clear picture of the use of PNGS for different categories of viral strains (PGT121 -sensitive/10-1074-sensitive, PGT121 -sensitive/10-1074-resistant, PGT121-resistant/10-1074-sensitive), with the exception of 10-1074-resistant strains, which typically lack Asn332gp120-associated PNGS.

Example 9. Passive transfer of neutralizing anti-HIV-1 mAbs in-vivo.

Five isolated potent and broad-spectrum monoclonal neutralizing anti-HIV antibodies were administered to rhesus monkeys and challenged intrarectally with either of two different SHIVs 24 hours later. Combining the results obtained from 60 infected animals, the titer for protective neutralization in plasma that prevents the emergence of viruses in exposed macaques by 50% is approximately 1:100.

Experiments on animals.

The macaques used in this study were negative for the Mamu-A*01 MHC class I allele.

Construction of R5-tropic SHIVDH12-V3AD8.

PCR mutagenesis was used using primers corresponding to the 5' and 3' halves of the V3 gp120 SHIVAD8EO coding region (PNAS 109, 19769-19774 (2012)) (forward primer:

AGAGCATTTTATACAACAGGAGACATAATAGGAGATATAAGACAAGCACATTGCAACATTAGT

AAAGTAAAATGGC and reverse primer:

TCCTGGTCCTATATGTATACTTTTCCTTGTATTGTTGTTGGGTCTTGTACAATTAATTTCTAC

AGTTTCATTC) to introduce such V3 sequences into the genetic background of the molecular clone pSHIVDH12_CL7 (J. of Virology 78, 5513-5519 (2004)) using Platinum PFX DNA polymerase (Invitrogen). After gel purification, the PCR product was treated with T4 polynucleotide kinase (GibcoBRL) and blunt-end ligated, creating pSHIVDH12.V3AD8, which was used to transform competent cells.

Viruses.

Virus stocks were prepared by first transfecting 293T cells with molecular clones SHIVAD8EO or SHIVDH12-V3AD8 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Culture supernatants were collected after 48 hours and aliquots were stored at -80°C until use. Concanavalin A-stimulated rhesus macaque PBMCs (2x106 cells in 500 μl) were infected with supernatants of transfected cells by centrifugation inoculation (J. of Virology 74, 10074-10080 (2000)) for 1 hour, mixed with the same number/volume of activated PBMCs and cultures were maintained for at least 12 days with daily replacement of culture medium. Supernatant samples were pooled near peak production time points to obtain individual viral stocks.

Antibodies.

Eleven monoclonal antibodies were isolated and obtained (VRC01, NIH45-46, 45-46G54W, 4546m2, 3BNC117, 12A12, 1NC9 and 8ANC195, 10-1074, PGT121 and PGT126). DEN3, human monoclonal IgG1 antibody specific for dengue virus NS1 (PNAS 109, 18921-18925 (2012)) or control human IgG (NIH Nonhuman Primate Reagent Resource http://www.nhpreagents.org) were used as negative control antibodies in this study. research. Monoclonal antibodies selected for passive transfer prior to exposure were administered intravenously 24 hours before viral challenge.

Quantification of viral RNA levels in plasma.

Plasma viral RNA levels were determined using real-time reverse transcription-PCR (ABI Prism 7900HT Sequence Detection System; Applied Biosystems).

Plasma antibody concentrations.

Concentrations of administered monoclonal antibodies in macaque plasma were determined by enzyme-linked immunosorbent assay (ELISA) using recombinant gp120 HIV-IJRFL (Progenies Pharmaceuticals) or HIV III B (Advanced Biotechnology inc.) (J. of Virology 75, 8340-8347 (2001)) . Briefly, microtiter plates were coated with HIV-1 gp120 (2 μg/ml) and incubated overnight at 4°C. Plates were washed with PBS/0.05% Tween 20 and blocked using 1% (v/v) BSA. After blocking, serial dilutions of antibodies or plasma samples were added to the plate and incubated for 1 hour at room temperature. Binding was detected using goat anti-human IgG F(ab)2 fragments coupled to alkaline phosphatase (Pierce) and visualized using SIGMAFAST OPD (Sigma-Aldrich). The half-life of neutralizing monoclonal antibodies was calculated using the exponential decay formula based on plasma concentrations starting at

- 28 046696 day or 7 days after administration of the antibody (J. of Virology 84, 1302-1313 (2010)).

Neutralization assay.

The in vitro potency of each mAb and the neutralizing activity present in plasma samples collected from rhesus monkeys were assessed in two types of neutralization assays: 1) a TZM-bl penetration assay using a pseudotyped challenge virus (AIDS Res. Hum. Retroviruses 26 , 89-98 (2010)) or 2) a 14-day replication assay in PBMC using a replication competent virus (J. of virology 76, 2123-2130 (2002)). For TZM-bl assays, serially diluted mAbs or plasma samples were incubated with pseudotyped viruses expressing the env gene derived from SHIVAD8EO or SHIVDH12.V3AD8 and obtained by cotransfection of 293T cells with pNLenvl and pCMV vectors expressing the corresponding envelope proteins (J. of Virology 84, 4769-4781 (2010)). The titer for the inhibitory dose producing 50% neutralization (IC50) was calculated as the dilution causing a 50% reduction in relative luminescence units (RLU) compared to levels in virus control wells after subtracting the RLU of cell control (J. of Virology 84, 1439 -1452 (2010)). The neutralization phenotype (tier levels) of the SHIVDH12_V3AD8 molecular clone was determined in a TZM-bl cell assay using plasma samples from a cohort study that exhibited a wide range of neutralizing activity against DBX-1 subtype B isolates (J. of General Virology 91, 2794- 2803 (2010)).

Determination of protective titers in animals and statistical analyses.

Calculation of the neutralization titer in plasma against each SHIV R5 resulting in a 50 or 80% prevention of virus infection in animals challenged with the virus was carried out using the method of Reed and Muench (Am. J. Hyg. 27, 493-497 (1938)) . One animal with significantly outlier values (DEW7) was excluded from the calculation. Probit regression was used to model the relationship between plasma titers required to confer sterilizing immunity in vivo using all 60 passively immunized monkeys (Cambridge University Press, Cambridge, England, ed. 3rd, 2007), with p-values in such The models are based on the likelihood ratio test. Plasma titers required for different levels of in vivo protection (33%, 50%, 80%, 90% and 95%) were determined based on probit model estimates and bootstrapping to generate 90% confidence intervals.

Results.

SHIVDH12-V3AD8, similar to SHIVAD8EO, has properties that are sensitive to neutralization by anti-HIV-1 tier 2 antibodies (Table 13). Rhesus monkeys inoculated intravenously or intrarectally with SHIVDH12-V3AD8 exhibited peak viremia ranging from 105 to 107 viral RNA copies/ml plasma at 2 to 3 weeks postinfection (PI). In the majority of SHIVDH12-V3AD8-infected animals, plasma viral loads decreased to background levels from 8 to 20 weeks postinfection.

The sensitivity of SHIVAD8EO to neutralization by 11 recently described broadly reactive anti-HIV-1 mAbs was first determined in the TZM-bl assay system (Fig. 11A and B). Eight of these antibodies, VRC01, NIH45-46 (23), 45-46G54W, 45-46m2, 3BNC117, 12A12, 1NC9 and 8ANC195, were targeted by CD4bs gp120 (Science 333, 1633-1637 (2011)) and three antibodies, 10 -1074, PGT121 and PGT126 (Nature 477, 466-470 (2011)), were dependent on the presence of the HIV-1 gp120 I332 glycan. When tested against SHIVAD8EO, all three glycan-dependent mAbs were more potent than CD4bs-MAT (Fig. 11 A). IC50 values for the three mAbs targeting the I332-glycan gp120 ranged from 0.09 to 0.15 μg/ml. CD4bs-MATs exhibited a much wider range (0.14 to 6.34 μg/mL) of IC50 neutralizing activity, with 3BNC117 being the most effective. A similar hierarchy (glycan-dependent > CD4bs-dependent) mAb neutralizing potency was also observed using SHIVDH12-V3AD8, but the neutralizing activity was distributed over a much wider range (>100-fold) compared to the IC50 values observed for SHIVAD8EO (Figure 11B ). SHIVDH12-V3AD8 were slightly more sensitive to glycan-targeting mAbs and more resistant to CD4bs-neutralizing mAbs than SHIVAD8EO.

Based on the results shown in FIG. 11, five neutralizing mAbs were selected for the preexposure passive transfer study: VRC01 because it was the first characterized CD4bs-N-Ab among the newly isolated broad-range N-Abs; CD4bs-MAT 45-46m2 and 3BNC117, which both exhibited strong neutralizing activity against SHIVAD8EO and SHIVDH12-V3AD8; and I332-glycan-dependent mAbs gp120, PGT121 and 10-1074.

The protocol for passive transfer experiments consisted of intravenous administration of decreasing amounts of neutralizing mAbs and intrarectal challenge of animals 24 hours later. The goal was to block the emergence of the virus in the body, and due to evidence that repeated administration of humanized anti-HIV mAbs to individual macaques may reduce their effectiveness and/or possibly induce anaphylactic responses, select a SHIV challenge dose of a sufficient size to ensure infection in vivo after one inoculation. To this end, the authors previously performed intrarectal titration of SHIVAD8 in rhesus monkeys and reported that inoculation of 1x103 TCID50, as determined by the final dilution of rhesus monkey PBMC, was equivalent to inoculation of approximately 3

- 29 046696 doses infecting animals by 50% (AID50) (J. of virology 86, 8516-8526 (2012)). Indeed, single intrarectal inoculations of 3 AID50 resulted in successful infection of 10 out of 10 rhesus monkeys with SHIVAD8EO or SHIVDH12-V3AD8 viruses.

As a control, in the first passive transfer experiment, animals were intravenously injected with IgG1 mAb against dengue virus NS1 and challenged using SHIVAD8EO 24 h later. Both monkeys (ML1 and MAA) rapidly became infected, producing peak levels of plasma viremia at 2 weeks postinfection. VRC01 was the first neutralizing anti-HIV-1 mAb tested for protection against viral emergence and was administered to two macaques at a dose of 50 mg/kg. One (DEGF) of two inoculated macaques was completely protected from SHIVAD8EO challenge with no evidence of plasma viremia or cell-associated viral DNA over the 45-day follow-up period. Another recipient inoculated with 50 mg/kg VRC01 (DEH3) became infected, but peak plasma viremia was delayed until 5 weeks postinfection. Two other macaques administered smaller amounts (20 mg/kg) of VRC01 were not protected from SHIVAD8EO challenge. The results obtained are summarized in table. 13.

The protective properties of PGT121 against SHIVAD8EO infection were then examined. PGT121 was one of the most effective glycan-targeting neutralizing mAbs as measured in the TZM-bl assay (Fig. 11). Based on the results obtained using VRC01, a dose of 20 mg/kg was selected to initiate in vivo titration of mAb PGT121. Two infected monkeys (KNX and MK4) were resistant to SHIVAD8EO challenge. When animals were administered lower amounts (i.e., 5 mg/kg, 1 mg/kg, or 0.2 mg/kg) of PGT121, 1 of 2, 2 of 2, and 0 of 2 animals, respectively, were protected (Table . 13).

The ability of mAbs VRC01 and PGT121 to block the appearance of SHIVDH12-V3AD8 in the body was assessed in a similar way (Table 13). The results obtained with VRC01 were comparable to those observed with SHIVAD8EO infection: 1 of 2 recipients receiving 30 mg/kg were protected from developing SHIVDH12-V3AD8 infection. The PGT121 mAb was significantly more effective than VRC01 in preventing SHIVDH12-V3AD8 infection: 2 of 2 recipients receiving 0.2 mg/kg PGT121 were resistant to infection. PGT121 was also slightly more effective in preventing in vivo infection by HIVDH12-V3AD8 compared to SHTVAD8EO (Table 13). This result is consistent with the 8-fold difference in IC50 values for PGT121 in neutralizing the two SHIVs in in vitro assays (Fig. 11).

The results of passive transfer of neutralizing mAbs 10-1074, 3BNC117 or 45-46m2 rhesus macaques followed by infection with either SHIVAD8EO or SHIVDH12-V3AD8 are summarized in Table. 13. mAb 10-1074 effectively blocked in vivo infection by both SHIVs. CD4bs mA 3BNC117 and 45-46m2 were selected for passive transfer to macaques based on their IC50 values against both SHIVs in the in vitro neutralization experiments shown in FIG. 11. 3BNC117 successfully blocked SHIVAD8EO infection in 2 of 2 monkeys at 5 mg/kg, but not in the other 2 animals that received 1 mg/kg (Table 13). The findings were similar to those observed when equal amounts of 3BNC117 were administered to macaques challenged with SHIVDH12-V3AD8: 1 in 2 became infected at the 5 mg/kg dose; 1 in 2 became infected at the 1 mg/kg dose.

Plasma samples collected at various time points from passive transfer macaques were analyzed in an HIV-1 gp120 ELISA to determine neutralizing mAb concentrations. In general, plasma concentrations of each mAb at the time of infection (24 hours after antibody administration) correlated with the dose of antibody administered (Table 13).

The relationships of plasma mAb concentrations with in vivo protection are shown in FIG. 12. Of the 5 neutralizing mAbs evaluated, PGT121 was clearly the most effective against both viruses, with SHIVDH12-V3AD8 showing slightly higher sensitivity to this mAb (2 of 2 monkeys were protected at a plasma concentration of 0.2 μg/ml). In contrast, a plasma concentration of VRC01 of almost 400 μg/ml was required to protect 1 of 2 animals from the same SHIVDH12-V3AD8 virus used for infection (Table 13). The most effective CD4bs-MAT administered to macaques in this study, 3BNC117, was approximately 6-10 times more effective than VRC01 in preventing infection with any SHIV (Fig. 12, Table 13).

The calculated half-lives of mAbs PGT121, 10-1074, 3BNC117, and VRC01 were very similar: 3.5 days, 3.5 days, 3.3 days, and 3.1 days, respectively. In contrast, the half-life of 45-4bt2 was extremely short and could not be determined. Based on the plasma mAb concentrations in several macaques 24 hours after administration of 20 mg/kg humanized neutralizing mAbs (i.e., approximately 250 μg/mL [Table 13]), and two monkeys receiving 20 mg/mL kg 45-46m2 had plasma mAb concentrations of only 15.0 and 17.6 μg/ml, decay was more than 95% relative to other neutralizing mAbs at the 24 h time point.

Neutralization titers were measured in plasma samples collected 24 hours after mAb administration when macaques were infected with SHIVAD8EO or SHIVDH12-V3AD8 viruses. As shown in table. 13, observed a good correlation between plasma antiviral neutralizing antibody titers and protection

- 30 046696 from SHIV infection. Administration of two glycan-dependent mAbs (PGT121 and 10-1074) clearly resulted in the highest titers of neutralizing anti-HIV-1 activity during viral infection. Titers measured in mAb 45-46m2 recipients were within the reporting limits or were not reported due to the extremely short half-life in vivo.

The method described by Reed and Muench (Am. J. Hyg. 27, 493-497 (1938)) was used to calculate the neutralization titers, measured in plasma, required to prevent the acquisition of viruses in 50% of infected monkeys. Such protective titers for the 28 SHIVAD8EO-challenged monkeys or the 32 SHIVDH12-V3AD8-challenged monkeys were calculated separately (Tables 15 and 16). Plasma neutralization titers required to protect 50% of animals challenged with SHIVAD8EO or SHIVDH12-V3AD8 were calculated as 1:115 and 1:96, respectively. Because these similar titers were obtained following: 1) SHIV infection via identical routes and using an identical inoculum size and 2) administration of the same set of neutralizing mAbs, neutralization data for all 60 animals were pooled and subjected to probit regression analysis to examine the relationship between neutralization titers in plasma and protection in vivo. When tested next, when SHIV was included as a term in the probit regression model for all 60 macaques, there was no evidence of a difference between the two SHIV viruses (p=0.16). When applied to an entire group of 60 macaques, probitregression estimated that plasma neutralization titers of 1:104 could prevent viral acquisition in 50% of animals. Probit analysis estimates that plasma 50% neutralization titers of 1:57 or 1:329 may protect 33% or 80%, respectively, of exposed animals.

Example 10 Administration of neutralizing mAbs in in vivo models chronically infected with HIV.

Summary of Methods: The neutralization activities of the broad-spectrum anti-SHIVAD8EO neutralizing mAbs 3BNC11724 and 10107423 were first determined in the anti-SHIVAD8EO TZM-bl cell system. Their abilities to block viral acquisition or control plasma viremia in chronically infected animals challenged with R5-tropic SHIVAD8EO were assessed by monitoring plasma viral load and cell-associated viral nucleic acids; CD4+ T cell subset levels were measured by flow cytometry. SGA analyzes of circulating viral variants and determination of antibody levels in plasma were performed. Plasma concentrations of N Abs were determined by measuring neutralizing activity against HIV-1 pseudovirus drugs sensitive to either 10-1074 alone or 3BNC117 alone.

Results.

Two groups of chronically infected macaques were evaluated. The first group consisted of two asymptomatic animals (DBZ3 and DC99A) that were infected for 159 weeks and had similar and significant reductions in the number of circulating CD4+ T cells (Table 17). The treatment regimen for current SHIV infection consisted of coadministration of 101074 and 3BNC117 at a dose of 10 mg/kg. During mAb administration, plasma viral loads in DBZ3 and DC99A macaques were 1.08 x 104 and 7.6 x 103 RNA copies/ml, respectively. Both macaques responded to combination anti-HIV-1 mAb treatment with an immediate and rapid reduction in plasma viremia to undetectable levels within 7–10 days. Suppression of measured SHIVAD8EO in plasma of DBZ3 and DC99A macaques after a single administration of the two mAbs lasted 27 and 41 days, respectively. In each case, plasma viremia returned to pretreatment levels.

A second group of three animals (DBX3, DCF1, and DCM8), each of which had also been infected with SHIVAD8EO for more than 3 years, and which had clinical signs with intermittent diarrhea and/or anorexia, were treated with two neutralizing antibodies (Table 17 ). During mAb administration, the level of circulating CD4+ T cells in DCM8 macaques was only 43 cells/μl, and was slightly higher in DCF1 (105 cells/μl) and DBXE (158 cells/μl) animals. Plasma viral loads exceeded 105 RNA copies/ml in DBXE and DCF1 animals and were significantly lower (1.59λ103 RNA copies/ml) in monkey DCM8. Administration of two DBXE mAbs to a monkey resulted in a biphasic decrease in viremia from 2.0 x 105 RNA copies on day 0 to undetectable plasma levels on day 20. This was followed by a return of high levels of circulating virus in DBXE over several days. In DCM8 macaques, with a lower plasma viral load and very few circulating CD4+ T cells, a rapid decline in viremia to undetectable levels was observed from days 6 to 20 after initiation of mAb treatment. Finally, animal DCF1, which was previously reported to produce broad-spectrum anti-HIV-1 N-Abs, observed a transient and relatively small 27-fold reduction in plasma viremia by day 6 in response to mAb combination therapy, followed by viral load returned to high levels that were before treatment.

Levels of PBMC-associated RNA and DNA were also determined before and after antibody administration (Table 18). In each animal, mAb treatment resulted in reduced levels of cell-associated viral RNA, which correlated well with plasma viral load measurements. No pattern was observed in cell-associated viral DNA levels resulting from antibody treatment. Administration of neutralizing mAbs to chronically SHIVAD8EO-infected monkeys also had beneficial effects on circulating CD4+ T cell levels, particularly in animals with

- 31 046696 very high viral load. The number of CD4+ T cells in DBXE and DCF1 macaques increased 2- to 3-fold during the period of monoclonal antibody-mediated viral suppression, but gradually decreased to levels observed before treatment as viremia could again be detected.

Plasma concentrations of each mAb were determined by measuring neutralizing activity in plasma against selected strains of HIV-1 pseudoviruses sensitive to one or the other, but not both, antibodies (Fig. 13A). In each treated animal, suppression of SHIVAD8EO viremia was maintained until a threshold plasma mAb concentration of approximately 1 to 3 μg/ml was reached (FIGS. 13B and 13C). There was even a case in DCF1 macaque where a small and transient decrease in plasma viral RNA levels was observed. Interestingly, mAbs administered to symptomatic DCM8 and DCF1 macaques had a shortened half-life or were undetectable. As stated previously, macaque DCM8 had extremely low levels of CD4+ T cells (43 cells/μl plasma) and macaque DCF1 was euthanized on day 56 after initiation of treatment due to its deteriorating clinical condition. DCF1 necropsy revealed severe enteropathy characterized by disseminated gastrointestinal cryptosporidiosis, pancreatitis, and cholangitis.

SGA analysis was used to determine whether amino acid substitutions occurred in regions of gp120 that were previously shown to affect sensitivity to mAb 10-1074 or 3BNC117. In each case, the recurrent virus present in the plasma after immunotherapy was unchanged. To further test the sensitivity of emerging viruses, combination therapy of 10-1074 plus 3BNC117 (10 mg/kg each) was repeated in two asymptomatic monkeys (DBZ3 and DC99A). The viral load in each animal dropped rapidly again, becoming undetectable on day 7 of the second immunotherapy cycle. Viremia was suppressed within 7 days in macaque DBZ3 and over 21 days in monkey DC99A. Taken together, these results suggest that viral reappearance after the first treatment cycle in these two animals reflects insufficient mAb levels in vivo rather than viral resistance resulting from selection in the presence of antibodies.

Table 3

Repertoire of clonal variants PGT121 and 10-1074

pH 0 mAb# VH D.H. JH CDR3 ¹ VHmut Length ¹ (-) (+) Y Lc νλ ΰλ CDR3 ¹ VKmut FRW1_del FRW3. Jns Length ¹ (-) (+) Y 10-160 4-59 3-3/16 6 ARRGQRIYGWSFGEFFYYYSMDV 49 24 2 3 4 / / / / / // / / 10-186 4-59 3-3/16 6 ARRGQRIYGWSFGEFFYYYSMDV 52 24 2 3 4 λ 3-21 3 HMWDSRSGFSWS 47 12 3 12 1 2 0 10-248 4-59 3-3/16 6 ARRGQRIYGWSSGEFFYYYSMDV 46 24 2 3 4 / / / / / // / / 10-259* 4-59 / 6 TKHGRRIYGIVAFNEWFTYFYMDV 63 24 2 4 3 λ 3-21 3 HIYDARGGTNWV 58 21 3 12 1 2 1 10-266 4-59 3-3/16 6 ARRGQRIYGWSFGEFFYYYSMDV 49 24 2 3 4 λ 3-21 3 HMWDSRSGFSWS 46 12 3 12 1 2 0 10-267 4-59 / 6 AQQGKRIYGIVSFGELFYYYYMDA 58 24 2 2 5 / / / / / // / / 10-303* 4-59 3-3/9 6 TLHGRRIYGIVAFNEWFTYFYMDV 54 24 2 3 3 λ 3-21 3 HIWDSRVPTKWV 50 21 3 12 1 3 0 10-354 4-59 3-3/16 6 ARRGQRIYGWSFGEFFYYYSMDV 48 24 2 3 4 / / / / / // / / 10-410’ 4-59 3-10/3 6 ALHGKRIYGIVALGELFTYFYMDV 63 24 2 3 3 λ 3-21 3 HIWDSRRPTNWV 44 21 3 12 1 3 0 10-416 4-59 3-3/16 6 ARRGQRIYGWSFGEFFYYYSMDV 47 24 2 3 4 λ 3-21 3 HMWDSRSGFSWS 45 12 3 12 1 2 0 10-468 4-59 3-3/16 6 ARRGQRIYGWSFGEFFYYYSMDV 47 24 2 3 4 λ 3-21 3 HMWDSRSGFSWS 46 12 3 12 1 2 0 10-543 4-59 3-10/3 6 ALHGKRIYGIVALGELFTYFYMDV 60 24 2 3 3 / / / / / // / / 10-570 4-59 3-3/16 6 ARRGQRIYGWSFGEFFYYYSMDV 42 24 2 3 4 / / / / / // / / 10-621 4-59 3-3/9 6 TLHGRRIYGIVAFNEWFTYFYMDV 54 24 2 3 3 / / / / / // / / 10-664 4-59 3-3/16 6 ARRGQRIYGWSFGEFFYYYSMDV 47 24 2 3 4 / / / / / // / / 10-720 4-59 3-3/16 6 ARRGQRIYGWSFGEFFYYYSMDV 49 24 2 3 4 / / / / / // / / 10-730 4-59 3-3/16 6 ARRGQRIYGWSFGEFFYYYSMDV 48 24 2 3 4 / / / / / // / / 10-814 4-59 3-10 6 TQQGKRIYGIVSFGEFFHYYYMDA 43 24 2 3 4 λ 3-21 3 HKWDSRSPLSWV 52 15 3 12 1 3 0 10-847* 4-59 3-3/16 6 ARRGQRIYGWSFGEFFYYYSMDV 47 24 2 3 4 λ 3-21 3 HMWDSRSGFSWS 46 12 3 12 1 2 0 10-948 4-59 3-3/9 6 TLHGRRIYGIVAFNEWFTYFYMDV 55 24 2 3 3 λ 3-21 3 HIWDSRVPTKWV 46 21 3 12 1 3 0 10-996 4-59 3-3/10 6 TQQGKRIYGWSFGEFFHYYYMDA 41 24 2 3 4 λ 3-21 3 HKWDSRSPLSWV 50 15 3 12 1 3 0 10-1022 4-59 3-3 6 ARRGQRIYGWSFGEFFYYYSMDV 51 24 2 3 4 / / / / / // / / 10-1059 4-59 3-3/16 6 TKHGRRIYGWAFGEWFTYFYMDV 59 24 2 4 3 λ 3-21 3 HIYDARRPTNWV 46 21 3 12 1 3 1 10-1074* 4-59 3-3/16 6 ARRGQRIYGWSFGEFFYYYSMDV 49 24 2 3 4 λ 3-21 3 HMWDSRSGFSWS 45 12 3 12 1 2 0 10-1074* 4-59 3-3/16 6 ARRGQRIYGWSFGEFFYYYSMDV 49 24 2 3 4 λ 3-21 3 HMWDSRSGFSWS 45 12 3 12 1 2 0 10-1121* 4-59 3-10/3-3 6 ALHGKRIYGIVALGELFTYFYMDV 63 24 2 3 3 λ 3-21 3 HIWDSRRPTNWV 44 21 3 12 1 3 0 10-1130* 4-59 3-10/3-3 6 ALHGKRIYGIVALGELFTYFYMDV 60 24 2 3 3 λ 3-21 3 HIWDSRRPTNWV 42 21 3 12 1 3 0 10-1141* 4-59 3-10/3-3 6 ALHGKRIYGIVALGELFTYFYMDV 63 24 2 3 3 λ 3-21 3 HIWDSRRPTNWV 45 21 3 12 1 3 0 10-1146’ 4-59 3-10 6 AQQGKRIYGIVSFGEFFYYYYMDA 58 24 2 2 5 λ 3-21 3 HYWDRSRSPISWV 61 15 3 12 1 2 1 W-1151 4-59 3-3 6 ARRGQRIYGWSFGEFFYYYSMDV 48 24 2 3 4 λ 3-21 3 HMWDSRSGFSWS 46 12 3 12 1 2 0 10-1167 4-59 3-3/16 6 ARRGQRIYGWSFGEFFYYYSMDV 49 24 2 3 4 / / 3 / I / I / / 10-1223 4-59 3-3/16 6 ARRGQRIYGWSFGEFFYYYSMDV 47 24 2 3 4 / / / I / // I / 10-1232 4-59 3-10/3-3 6 ALHGKRIYGIVALGELFTYFYMDV 58 24 2 3 3 / 3-21 / / / // / / 10-1263 4-59 3-3/16 6 ARRGQRIYGWSFGEFFYYYSMDV 49 24 2 3 4 λ 3-21 3 HMWDSRSGFSWS 46 12 3 12 1 2 0 10-1294 4-59 3-3/16 6 ARRGQRIYGWSFGEFFYYYSMDV 49 24 2 3 4 λ 3-21 3 HMWDSRSGFSWS 45 12 3 12 1 2 0 10-1341* 4-59 3-3/16 6 ARRGQRIYGWSFGEFFYYYSMDV 49 24 2 3 4 λ 3-21 3 HMWDSRSGFSWS 45 12 3 12 1 2 0 10-1342 4-59 3-3/16 6 ARRGQRIYGWSFGEFFYYYSMDV 48 24 2 3 4 λ 3-21 3 HMWDSRSGFSWS 46 12 3 12 1 2 0 10-1369* 4-59 3-3/16 6 TKHGRRIYGWAFGEWFTYFYMDV 57 24 2 4 3 λ 3-21 3 HIYDARRPTNWV 43 21 3 12 1 3 1 10-1476 4-59 3-3/16 6 ARRGQRIYGWSFGEFFYYYSMDV 47 24 2 3 4 λ 3-21 3 HMWDSRSGFSWS 46 12 3 12 1 2 0

VHmut and VXxmut indicate the total number of mutations in the VH and VL Ig genes. (-) and (+) indicate the amounts of negatively and positively charged amino acids in the complementarity determining region (CDR3) of Ig, respectively. Y indicates the number of tyrosine residues in CDR5 IgH/L (green). 1Based on Kabat nomenclature (IgBLAST, http://www.ncbl.nim.nih.gov/igblast). Acidic and basic amino acids are indicated in red and blue, respectively). FRW1_del, number of nucleotides deleted in framework region 1 (FRW1) of IgL. FRW3_ins, number of integrated nucleotides in framework region 3 (FRW3) of IgL. Clonal members with identical IgH sequences are color coded, and among these, the identity of the IgL sequences that defines the clones is indicated in bold. * Typical antibody variants that were obtained and analyzed are indicated. IgL 10-266 was not cloned, and IgG 10-1141 was not obtained.

Table 4

In vitro neutralization assay using TZM-bl in the main panel

IC50 10-1369 10-259 PGT121 10-303 10-410 10-1130 10-1121 10-1146 10-996 10-1341 10-847 10-1074 BaL.26 0.069 0.021 0.021 0 043 0.016 0 013 0.04G 0.064 0 045 3,032 0.022 0 033 SS1196.1 0.033 0.012 0.008 001b 0.008 0 008 0.029 0.02/ 0 00/ 0.011 0 006 0 010 6535.3 0 023 0 005 0 007 0014 0 003 0 003 0 008 0 022 0 018 3,009 0011 0 007 QH0692.42 E.boz 0.155 1.08b 3.122 2.630 1 8/1 4.18/ 0.b9E 0.39b 0.33b 0.259 0.259 TRJO4551.58 0.560 0.189 3.896 14.401 '8.511 36.880 15.360 0.543 0.516 0.333 0.210 0.170 SC422661.8 0.195 0.096 0.263 0.333 3.132 0 0/0 0.1 /3 0.19b 0.2bb 3.189 0.137 0.145 PVO.4 0.225 0.1/5 0.'4/ 0 6/0 3.494 0 385 0.5/0 0.313 0.211 3 236 0 1/2 0 1/8 CAAN5342.A2 0.070 0 020 0 013 0 020 3012 0 009 0 033 0.032 0 007 3,009 0 006 0 007 YU-2 0.210 0.135 0.098 0.190 0.089 0 078 0.152 0.275 0.25G 0.234 0.161 0.143 R1166.C1 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 MuLV >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40

IC80

BaL.26 0.268 0.101 0.081 0.156 3.066 0 062 0.154 0.203 0.228 3.159 0 112 0 124 SS1196.1 0 033 0 037 0 030 0 055 3,030 0 037 0 098 0.073 0 040 3,040 0 026 0 027 6535.3 0.060 0.022 0.011 0 053 3.021 0013 0.033 0.078 0 085 3,038 0 044 0 044 QH0692.42 1.714 0.551 14.976 18 122 12,071 >40 21,943 1.993 1 404 1 100 0 908 0 861 TRJO4551.58 3.818 0.96b 26.93C : 10 >40 >40 : 23 2.601 4.265 1.226 0./G8 0.G93 SC422661.8 PVO.4 CAAN5342.A2 YU-2 0.910 0./8/ 0.186 3./38 0.333 0./16 0 063 0.382 0.711 1.09/ 0 056 0.3b6 1.156 2.199 0 092 0.502 0.449 1.b/2 3,055 3.243 0 2G1 1/83 0 045 0313 0.741 2.-1Gb 0 095 0.340 0.GG3 1.319 0 088 0./50 0.815 1..4b 0 060 0.891 0.501 0./b4 0 054 0./66 0.386 0.//4 0 035 0b3/ 0.392 O./GG 0 044 0.398 R1166.C1 >40 >40 >40 >40 >40 >40 >23 >40 >40 >40 >40 >40 MuLV >40 >40 >40 >40 >40 >40 >23 >40 >40 >40 >40 >40

The numbers indicate the concentrations of IgG antibodies in μg/mL to achieve IC50 (top) and IC80 (bottom) in the TZM-bl neutralization assay. IC50/80 values are color coded and increasing sensitivity to neutralization is indicated from dark green to dark red. > indicates that the IC50 for a given virus was not achieved at the concentration tested. Murine leukemia virus (MuLV) and R1166.C1 (clade AE) are negative controls.

- 33 046696

In vitro neutralization assay using TZM-bl on ___________expanded IC ₅₀ value panel ___________

Table 5

Virus ID Treasure 0-996 10-1074 PGT121 Virus ID Treasure 10-996 10-1074 PGT121 6535.3 IN MillMjl 11II111 MIIM CNE58 Sun 0.570 0.267 >50 QH0692.42 IN 0 396 0 191 1.041 MS208.A1 A >50 >50 >50 SC422661.8 IN Ιίίβ iliill 0 101 Q23.17 A |Mii BIB lillillll PVO.4 V 0 186 iiiis 0 131 Q461.e2 A >60 >60 >60 TRO. eleven V iiiiiii » Q769.d22 A. >ou >50 AS10.029 V 0С67 Q259.d2.17 ft >60 8,990 RHPA4259.7 V 0 C34 eleven··· Q842.d12 ft. THRO4156.18 V >50 >50 3415.v1.c1 A Iiiiiii 1 >60 >50 REJO4541.67 V >60 >50 3.607 3365.v2.c2 A iil® I "iiiiii TRJO4551.58 V iii 0 170 3,728 0260.v5.c36 A ilili and® 0 054 WITO4160.33 V 0 538 0 185 0.459 191955_A11 A(T/F) >50 >50 >50 CAAN5342.A2 V AND·· 191084 B7-19 A(T/F) 0 057 Iiiiii YU-2 V 0 256 0 143 Itiisifi 9004SS_A3_4 A (T/F) IDDI WEAU„d15_410__787 V (T/F) 0 147 0 104 iiiiii T257-31 CRF02_AG 1006 11 03 1601 V (T/F) andiimi ^: · 0 603 928-28 CRF02_AG 1054 07 TC4 1499 B (77F) 0 260 0 129 0.115 263-8 CRF02_AG 1056_10_TA11__1826 V (T/F) 0 117 Mib Iiiiii T250-4 CRFO2__AG I 1012 11 TC21 3257 V (T/F) SSJ ocae 0 Oilfl T251-18 CRFO2_AG iiii 6240 08 TA5 4622 BCT7F) 0С95 0.068 ifiiBU T278-50 CRF02_AG iiiiii 6244_.13__B5_.4576 in CF/F) 0 353 11th| ΐΐβίβΐ T255-34 CRFO2„AG jiMii 62357_14_D3_4569 In CF/F Hi® i >5P 1,036 211-9 CRF02_AG iiiiii SC05 8C11 2344 BCF/F 0С69 diva 0 093 235-47 CRF02_AG Biili DI1| eiilil Du156.12 WITH 1Ι1·ΒΙ! ιιιΐί·· OOP? 620345.cO1 CRF01_AE >50 >50 >50 DU172.17 With 0.173 0.121 0.115 CNE8 CRF01__AE >50 >50 >50 Du422.1 With 0 C56 0 029 C1080.c03 CRF01__AE >50 >50 >50 ZM197M.PB7 With >50 >50 >50 R2184.C04 CRF01.....AE >50 >50 >50 ZM214M.PL15 With 0413 0 174 ββΐί R1166.C01 CRF01_AE >50 >50 >50 ZM233M.PB6 With 0 856 yashsh 1.451 C2101 c01 CRF01_AE >50 >50 >50 ZM249M.PL1 With >50 >50 >50 C3347.C11 CRF01_AE >50 >50 >50 ZM53M.PB12 With -"JOQIJ C4118.C09 CRF01__AE >50 >50 >50 ZM109F.PB4 With >50 >50 7,894 CNE5 CRF01__AE >50 >50 >50 ZM135M.PL10a With 0С99 BJOX009000.02.4 CRF01_AE >50 >50 3.626 CAP45.2.00.G3 With >50 BJOX015000.11.5 CRF01_AE (T/F) >50 >50 >50 CAP210.2.00.E8 With iiji 1 >50 BJOX010000.06.2 CRF01_AE (T/F) >50 >50 >50 HIV-001428-2.42 With 0С40 Ш1И11 BJ 0X025000.01.1 CRF01_AE (T/F) >50 >50 >50 HIV-0013095-2.11 With IV >50 BJOX028000.10.3 CRF01_AE (T/F) >50 >50 >50 HIV-16055-2.3 With >50 >50 X1193__c1 G iait ii· 0 045 HIV-16845-2.22 With 1.325 1.16( P0402 c2 11 G Miijill 0 0-2 I Ce1086_B2 With CT7F) >50 >50 X1254__c3 G 0 056 CeO393_C3 C(T/F) >50 >50 >50 X2088_c9 G "— IMII Ce1176__A3 C (T/F) 0 0-13 iiiiii X2131 C1 B5 G ill Ce2010_F5 C(T/F) >50 >50 >50 P1981_C5_3 G M AI V&I CeO682_E4 C (T/F) >50 >50 >50 X1632 S2 B10 G >50 >50 >50 Ce1172_H1 C (T/F) 0.05ο 1111111 1I1MI11 3016.v5.c45 D >60 Ce2060_G9 C(T/F) >50 A07412M1.vrc12 D ΙΜΐΙΙ MY1 08703010054-2A2 C(T/F) >50 >50 >50 231965.C01 IN >50 >50 >50 BF1266.431a C (T/F) >50 >50 >50 231966.C02 D >50 >50 >50 246FC1G C (T/F) 0.092 D1I1 0.083 191821_E6_1 V (T/F) >50 >50 >50 249M B10 C(T/F) >50 >50 >50 3817.v2.c59 CD JAD® ®3i1A§i >50 ZM247v1 (Rev-) C(T/F) 0 C55 6480.v4.c25 CD 1Msh1 Oh yeah I·· 7030102001E5(Rev-) C (T/F) iiiiii ιι···ι 6952.v1.c20 CD ii—g iiii 0 085 1394C9G1(Rev-) C(T/F) 0 086 0 050 0 486 6811.v7.c18 CD Ι··| iiiiiii : 0 004 Ce704809221_1B3 C (T/F) ιβββ 0 139 89-F1 2 25 CD >50 >50 >60 CNE19 Sun is 3301.v1.c24 A.C. IMilli 11II1I CNE20 Sun MB· iMyi 6041.v3.c23 A.C. >50 >50 >50 CNE21 Sun 0 086 iiiiii II···· 6540.v4.c1 A.C. >50 >50 >50 CNE17 Sun 1II ϋ·1 6545.v4.c1 A.C. >50 >50 >50 CNE30 Sun sftil ilMlSI 0 101 0815.v3.c3 ACD IMI MiMMI V1III111 CNE52 Sun 4.525 1 226 3,741 3103.v3.c10 ACD IMI! II||| 0 1142 CNE53 Sun 0 057 liiji|| 0 055

The numbers indicate the concentrations of IgG antibodies in μg/ml to achieve IC ₅₀ in the TZM-bl neutralization assay. IC ₅₀ values are color coded and increasing sensitivity to neutralization is indicated from dark green to dark red. > indicates that the IC ₅₀ for a given virus was not achieved at the concentration tested.

- 34 046696

In Vitro Neutralization Assay Using TZM-bl Extended Panel - IC80 Values

Table 6

Virus ID Treasure 10-996 10-1074 PGT121 Virus ID Treasure 10-996 10-1074 PGT121 6535.3 V 0.046 0-1 CNE58 Sun 2.220 0.968 >50 QH0692.42 V 1.854 0.929 8.545 MS208.A1 A >50 >50 >50 SC422661.8 V .- ; 0.418 0L60 Q23.17 A PV0.4 V 0.952 0.360 0.945 Ο461.Θ2 A >50 >50 >50 TRO.11 V 0.081 Ι·Ι1Ι iiHiiii Q769.d22 A >50 >50 >50 ASYU.0.29 V g.--.g. G '10 0.169 Q259.d2.17 A >50 >50 >50 RHPA4259.7 V |Ym1|| YI· Q842.d12 A >50 >50 1 - ·'' THRO4156.18 V >50 >50 3415.v1.c1 A >50 >50 >50 REJO4541.67 V >50 >50 >50 3365.v2.c2 A 1.380 0.450 7.353 TRJO4551.58 V 0.634 3 Z ^l 4 0260.v5.c36 A 0.436 0.160 0.152 WITO4160.33 V G 2.112 6.007 191955A11 A (T/F) >50 >50 >50 CAAN 5342. A2 V 0.079 ιΐιΐ·ιιΙι 191084 B7-19 A (T/F) (> 1 - 0.128 0.128' YU-2 V AND· I 9004SSA34 A (T/F) 0.050 0.030 0.026 WEAU_d15_410_787 V (T/F) L. 0.375 0.295 T257-31 CRF02AG >50 >50 >50 1006—11_C3_1601 in (T/F) O.()'1'>: : 0.013. 0.023. 928-28 CRF02AG 7.15' 4.696 >50 105407TS41499 B(T/F) 0.901 0.563 0.696 263-8 CRF02_AG >50 6.527 2 -..-:^6 1056—10-TA11-1826 V (T/F) '.. · 0.272 0.303 T250-4 CRF02AG 0 '0'.· ( 0.005 101211 TS21 3257 V (T/F) 0.111 0.059 T251-18 CRF02 AG 7.399 IlMlll . .eleven 624008TA54622 V (T/F) 0.348 0.306 0.584 T278-50 CRF02_AG >50 18.276 -l|| 62441ZV54576 V (T/F) 1.296 0.922 1.878 T255-34 CRF02 AG >50 >50 >50 62357 14 D3 4589 B (T/F) >50 :·ο 45.559 211-9 CRF02_AG 3.848 0 '25 8.840 SC05_8C11_2344 V (T/F) fgllll A· 0.275 235-47 CRF02_AG 0.38' 0.163 jiil Du156.12 WITH O. ·ο· ΠΓ.7Γ. 0.033 620345.c01 CRF01AE >50 >50 >50 DU172.17 WITH 0.430 0.890 CNE8 CRF01_AE >50 >50 >50 Du422.1 With 0.166 C1080.c03 CRF01AE >50 >50 >50 ZM197M.PB7 With >50 -jU >50 R2184.C04 CRF01_AE >50 >50 >50 ZM214M.PL15 With 3.251 2.367 3.150 R1166.C01 CRF01_AE >50 >50 >50 ZM233M.PB6 With 4.524 0.349 = 97 C2101.C01 CRF01AE >50 >50 >50 ZM249M.PL1 With >50 - -.π >50 C3347.C11 CRF01_AE >50 >50 >50 ZM53M.PB12 With >50 π C4118.c09 CRF01AE >50 >50 >50 ZM109F.PB4 With >50 - LI >50 CNE5 CRF01_AE >50 >50 >50 ZM135M.PL10a With 0.553 0.367 5.885 BJGX009000.02.4 CRF01 AE >50 >50 CAP45.2.00.G3 With >50 -.0 BJOX015000.11.5 CRF01 AE (T/F) >50 >50 >50 CAP210.2.00.E8 With >50 >50 >50 BJOX010000.06.2 CRF01AE (T/F) >50 >50 >50 HIV-001428-2.42 With 0.204 0.261 0.156 BJOX025000.01.1 CRF01 AE (T/F) >50 >50 >50 HIV-0013095-2.11 With >50 •-ill >50 BJOX028000.10.3 CRF01AE (T/F) >50 >50 >50 HIV-16055-2.3 With >50 >50 4.290 X1193_c1 G 0.482 II-' . 0.202 HIV-16845-2.22 With 5.835 >50 P0402_c2_11 G ()<>;>> 0.039 0.D56, Ce1086_B2 s (T/F) >50 >50 lillillll X1254_c3 G 0.420 II -!lj 0.199 SeOZEZ C3 s (T/F) >50 - .eleven >50 X2088-C9 G 1shm 0.014 0.029 Ce1176_A3 s (T/F) 0.151 X2131C1B5 G 0.085 0.064 0.058 Ce2010 F5 C (T/F) >50 >50 >50 P1981 C5 3 G 0.018 iiiiii·! O.D15 CeO682_E4 C (T/F) >50 >50 >50 X1632S2B10 G >50 >50 thirty Ce1172_H1 C (T/F) w—r 0.166 3016.v5.c45 D >50 >50 >50 Ce2060 G9 C (T/F) >50 >50 >50 A07412M1.vrc12 D 0.070 0.048 0.406 Ce703010054_2A2 C (T/F) >50 >50 >50 231965.C01 D >50 >50 -li BF1266.431a C (T/F) >50 >50 >50 231966.C02 D >50 >50 - .0 246F C1G C (T/F) 0.270 iliili. 0.287 191821E61 D (T/F) >50 >50 >50 249M B10 C (T/F) >50 >50 >50 3817.v2.c59 CD ΜΙΜΙΙΙ 14.880 >50 ZM247v1 (Rev-) C (T/F) 0.252 0.186 11I|II1 6480.v4.c25 CD 0.049 0.041 0.079 7030102001 E5(Rev-) C (T/F) 0.044 d.021 them·· 6952.v1.c20 CD 0.188 0.138 0.605 1394C9G1 (Rev-) C (T/F) c.v.- 0.191 3.372 6811.v7.c18 CD llii'l iiiiii 0.017 Ce704809221_1B3 C (T/F) 1.208 0.696 0.492 89-F1 2 25 CD >50 >50 ->11 CNE19 Sun >50 >50 0.189 3301.v1.c24 A.C. 0.054 II 0.043 CNE20 Sun OOO 0.005 0.008 ^; 6041.v3.c23 A.C. >50 >50 >50 CNE21 Sun liiiii 0.181 il 6540.v4.c1 A.C. >50 >50 >50 CNE17 Sun 24.701 13.297 >50 6545.v4.c1 A.C. >50 >50 >50 CNE30 Sun 1.200 0.559 0815.v3.c3 ACD 0.25' 0.138 0.105 CNE52 Sun 4;;.-:- 13.147 32.935 3103.v3.c10 ACD 0.150 0.101 0.110 CNE53 Sun ..ίί;. 0.141 0.200

The numbers indicate the concentrations of IgG antibodies in μg/ml to achieve IC80 in the TZM-bl neutralization assay. IC80 values are color coded and increasing sensitivity to neutralization is indicated from dark green to dark red. > indicates that the IC80 for a given virus was not reached at the concentration tested.

- 35 046696

Table 7

Neutralization sensitivity according to N332-PNGS

Virus ID Treasure N332 5334 10-996 10-1074 PGT121 Virus ID Treasure N332 S334 10-996 10-1074 PGT121 6535.3 IN 88¾¾ CNE58 Sun CD QH0692.42 V MS208.A1 A SC422661.8 V Q23.17 A CD PVO.4 V Q461.e2 A V N TRO.11 V Q769.d22 A N ASYU.0.29 V Q259.d2.17 A T N RHPA4259.7 V Q842.d12 & N THRO4156.18 V T N 3415.v1.c1 A REJO4541.67 V T N 3365.v2.c2 A TRJO4551.58 V 0260.V5.C36 A WITO4160.33 V 191955 A11 A(T/F) To N CAAN5342.A2 V 191084 B7-19 A(T/F) YU-2 V 9004SS A3_4 A (T/F) WEAU d15 410 787 in (T/F) T257-31 CRF02 AG To N 1006 11 SZ 1601 V (T/F) Mag 928-28 CRF02AG 1054J)7_TC4_1499 V (T/F) 263-8 CRF02AG 1056 10 TA11 1826 V (T/F) T250-4 CRF02AG 1012 11 TS21 3257 V (T/F) T251-18 CRF02AG 624008TA54622 V (T/F) T278-50 CRF02AG 6244 13 В5 4576 V (T/F) T255-34 CRF02AG 6235714D34589 V (T/F) 211-9 CRF02AG SC05 8С11 2344 V (T/F) 235-47 CRF02AG Du156.12 WITH 620345.c01 CRF01 AE E N Du172.17 WITH CNE8 CRF01AE E N DU422.1 With C1080.C03 CRF01AE E N ZM197M.PB7 With D R2184.C04 CRF01 AE V N ZM214M.PL15 With R1166.C01 CRF01 AE E N ZM233M.PB6 With C2101.C01 CRF01 AE E N ZM249M.PL1 With C3347.c11 CRF01 AE E N ZM53M.PB12 With I N C4118.O09 CRF01 AE E D ZM109F.PB4 With TO N CNE5 CRF01AE E N ZM135M.PL10a With BJQX009000.02.4 CRF01_AE E N CAP45.2.00.G3 With N BJOX015000.11.5 CRF01AE (T/F) E N CAP210.2.00.E8 With BJOX010000.06.2 CRF01AE (T/F) TO N HIV-001428-2.42 With BJQX025000.01.1 CRF01_AE (T/F) E N HIV-0013095-2.11 With BJOX028000.10.3 CRF01AE (T/F) HIV-16055-2.3 With TO X1193_c1 G HIV-16845-2.22 With P0402 c2 11 G Ce1086_B2 C (T/F) N AND X1254_c3 S SeOSEZSZ s (T/F) D X2088_c9 G (T) Ce1176_A3 C (T/F) X2131 C1 B5 G Ce2010 F5 C (T/F) T N P1981 C5 3 G Ce0682 E4 C (T/F) X1632 S2 B10 G N Cs1172 H1 C (T/F) And· 3016.v5.c45 D T D Ce2060 G9 C (T/F) (T) A07412M1.vrc12 D Ce703010054_2A2 C (T/F) D N 231965.C01 D BF1266.431a C (T/F) T N 231966.C02 D 246F C1G C (T/F) 191821 E6 1 D(T/F) 249M B10 C (T/F) 3817.v2.c59 CD ZM247v1(Rev-) s (T/F) (T) 6480.V4.C25 CD 7030102001 E5(Rev-} C (T/F) 6952.v1.c20 CD 1394C9G1 (Rev-) C (T/F) 6811.v7.c18 CD Ce704809221_1B3 s (T/F) 89-F1 2 25 CD T CNE19 Sun 3301.v1.c24 A.C. (T) CNE20 Sun G, 6041.v3.c23 A.C. D N CNE21 Sun 6540.v4.c1 A.C. T N CNE17 Sun 6545.v4.c1 A.C. T N CNE30 Sun 0815.v3.c3 ACD CNE52 Sun 3103.v3.c10 ACD CNE53 Sun

The dots indicate the presence of an asparagine residue and a serine residue at positions 332 and 334, respectively. Mutations at positions 332 and 334 (HXB2N sequence numbering) are indicated by amino acid substitution. IC50 values are color coded and increasing sensitivity to neutralization in the TZM-bl assay.

- 36 046696

PBMC-based in vitro neutralization assay

Table 8

Virus ID 3BNC55 3BNC60 3BNC117 8ANC134 1NC9 45-46 8ANC195 12A12 4E10 N2 2G12 2F5 PG 9 PG16 VRC01 45-46 54W PGT121 10-1074 P035.6.E4 0£'34- 0 45 P G-I7 1,043 1,865 3 430 .=. '..1. 1' r. ' P035.6.N11 0?39· SHYAYSH 0 053 ··. 2.94' 0.756 >1 P035.6.D10 >50 >50 MMM 1D||| <QC<|32_ <0.0.1’32 0 79? • ·; · 1 900 >1 3 390 ' 1020.1 3.174. 0 1'3 R151.37.S7 P.1 26 l λ vft 0 1 33 *• ^eleven 2.94 3 35P Ministry of Foreign Affairs P151.37.F1 0 733 0 136 -8.043 0 869 >50 >50 P151.37.F10 1IIIMII! 8,863 9.314 1'- <0 39 MSB <3.016 1.-6С- 0.172 >50 >53 P1 53.1 0.2.A9 eMeiSiiiiiiiliiiBili 46.735 0 D39 . 1 .' I·.'» 0 91 8 0.431 >5 34.912 42.623 1.466 P153.10.2.DB 23.1 24 0 030- . ! . 0 86 24.193 .·.·. . ·, . P153.102E10 IM|MI|»Yu11M^^I >·. 0 PM 0 79? iii • .· 1MI YIM >5 >50 >50 >50 P1BS. 1 2.1.010 0 1 35 them • .·. i 2.492 >1 >5 Pl SS.1 2.2.F4 1 ·.. . •' ; । ·. Ί. l. ·, ·. -.ι DC P1B6.12.2.G2 1.890 ' fe0 ^, .0&-32 ¹ <0XXJ32' 0.046 igi 21.988 >1 >5 6.385 · 3.ϋίβ 0.108. R195.31.Ab Μ·®™ 0.399' 2.934 I·'·. 3.884 ' ,' 1 ι , 1 , 1 , 1 R195.31.AYU -1.. M·· 1.243 1 '.·" . >1 4.800 >50 >50 >50 P1 95.31.F11 2.3J6 • ' 'ι··. । . . P019.1 D2 ' ·1 - '. ^eleven >50 P019.1.D6 . 14 · 1 ·. · M^m P019.1.G7 ..-..1 -.-/-. 1·.. . ·>!)(· . 1 6,932 ··. 20 t'9? 6./64 go >50. P1 75.10.D7 '..I . ··.. ··... . 42.6E5 35.805 1 P175.10.D12 11¾¾¾¾¾ 1 ' .'Ί Ό.643 0 9:»3 P175.10.G10 ·. 1.. . ·..·. 1 · ' 33.008 8 364 ΙΙΙΜΜΙΜΙΙ 1.14/ P013.18.A9 Р154.44.С8 P154.44.G8 0/25 0.102 - 0Ώ79.. >50 0.683 -.042 <JL' 16 4d0 25 3'6 -' 1_·_. 1-1·. -- Z.f oo - J.'eJ ·_<·11 -.'J 0.600 >5 >50 >50 >50 P1B3.50.2.H3 12.049 1 '-:" .,|... And I'·· . -4 · 'Ъ >1 1.03' 0.19υ 2.UB 0.185 P1 B3.3.2.B9 , · .. 0 142 ··. 1·. P0D1.35.F5 0 133 Yaymmshv <0 39 ιιιιιΐ >5. Р001.35.Н4 .· · » And- pOD2.39.CB 2.154 0 546 >50 1.955 . -·. p002.39.FB . . . " 1' .·. 1 1 3 090 •·. 1Ί ·. >50 0.864 MmiM! <3016 1 610 3625 22 125 0349: f P0D2.39.H1D 1472 0.785 0.726 iiiiiiii 0.147 0 L r - 1 ·.' 1 - eleven X P034.6.D6 iiiiiei· 0 735 1 164 .eleven ||j|| S 8. £Ώ P034.6.G10 22 4.8: I 423 ·. > press® 8 598 . 1>1 5 P034.6.N5 - . i 1. ·. 20 /30 slurry 0 /45 . - · Eevee t) T/1 E31G- 3 17/ 0 03/ ί·7Η8 ' P1D1.20.1.F1 0 634 -.III ABOUT ABOUT P1D1 20.1.Н8 P1 27.46.A6 0 090 ·eleven about yijz .1·· 1 /1U > 1 1 470 3 86 1 ·<0Ό[)32 ¹ <0 J032; P127.46.D1 1242 ;0E24-·: 0.043 ' 5 403 2,558 SHI t 09? IN 0150 - 0 230 0 203 >50 >50 P127.46.D2 1.125 0 1/3 0.221 YaZHI··- ··. 0 494 ' <(}ΐΜ8 <00/8 0023 ^s >50 >50 P174.28.E11 liMiiiiiiiiiiiiie *ί 0.894 2 104 lOOWgi -. 1' .:.. | -I <0 01 3 913 ‘ 863 '0 085 0.0&3 P177.25.1.G9 0.980 0 191 0.189 iiliiiiiB Р177.25.2.В4 1 179 0 080 ' 0 041 ί 23 6 39 ' ::1 eleven - 1 . ' 1 3 ⁷ 29 - %H 1 241 > >l 0 453 3 530 '13029' p 023 · P177.25.2.D1 >50 1 U4U .359 46.82b ·eleven '.Ί 1.149 R180.14. Av 1 389 0 098 ! OOSR' .· Mmm ·. - >· P180.14.G6 2.449 Mig i··! 4.009 7,988 <Ί Air P1S0.14.G7 Ί Ί Ί I" Μ··Ι ·. —Λ, 0 799 15 75Q . - .-, '|-·-. .-.-, . Ί G twd. 1 243 - 0-2 3 928 0 03.6 I Р197.25.1.D2 >50 1.480 And -And — • ^eleven . 1 < 'ί ί. P197.25.1.D7 >50 0 789 ' Gy G· 19 ' 0 05? 7 Γ 5β yiiiiii amides •ι· 0 943 . ι.1 · g <0 01 Fr <0 078 0 05? ^s ?0 337 0 057 Р197.25.1,Н1 >50 > ί. . -ί . <0 DD32 ² shi 0 754 Mdiy P405.18.D3 MIIIIII· 0 09υ 0.048 i 0 031 '. 1 0.4 <Ό 39 "by them >1 >1 >5 3.254 . ,0.0'- , 0.013 ^: P405.18.F10 p405.18.H5 P405.19.A8 P405.19.B12 5 646 1.100 immy 1 19.220 • 0 278 iiiill 1111111 ΙιΙΙΙΙΙί IM^ 0 469 43 684 Ό 037 1 350 1.323 2012 C- 718 0.49/ Iiilii 0.555 <0 39 .-. .- ·. 1 0 968 . Ί 0.815 0 878 1.029 HI- 3.141 >1 AND III. ·. ·. . ... |||I......>...... >50 VI11^SH 0.044! 0473 ·. 3.031 > 0917 <0-0032; >5. 1iMMvY|IImI p405.19.F11 0 689 .0.018. 0.109 ΐίΐβίιι iii" .-.. iiiiiiii 0 413 <0 39 3 630 |Μ|ί ' ' .4 HI Nl Nl 1140.6F5 lllliill 5.9 5 liiiiit ilMill . -G*· . >25 Bill MiiYVV^^WIIIIIIIIIIIIMI1 1140.6G9 0 748 0 1-6 , 0 114 4 222 3,096 0.147 ·.. 0 220 6713 3 270 1 520 Them . , .·., , . ..·, ., ·. . .> 1 R116.2 1 1 ' 1 . 40.937 -ι· 0.042' .-.. 1 g .. 1/.3/0 4.1/3 ·.' ' > ...... ·->.; I ·.'· 1.· βΜ»πΜΜΙΜΜ«ΜΜ··Ι P116.3.F6 22.297 0.235 0 255 4 Ι·. 0 728 0.158 15 52 iiiili 1 ' · AI mi P11R.3.G9 'eleven. having 1.385 0 540 fiiiiil iiiiiiii 0 650 • 1 chic R116.4. II IMI iliiiilsi 11111 whether · Jiilil' ΙΜΜ 3.-20 >25 tut ive 1^mI|^D8IIIIV^Inshmi|ii 1234.ZA9 2 623 0 226 · 0 032 1 Ί1 research institute 4.- ·4 iiiiiiii 5 >?5 >25 111|1i 1234.3D9 4/34 0 υ39 0.08/ iiiiiiii dim . ' >25 ! d - 6 658.8L6 |"M1ViiMV|iiiiMMi" >50 2,057 0.379 lillMlilB 314-6 >25 >12.5 >25 658.8D2 Ivzhv^^^^· 40 6-7 1 651 0.347 ii·· 3 291 7 300 AI 658.8F6 vYI^I······ .<>W 0 994 AND·· >50 ni)ia' >25 . >25 >25 O.fa YmM1^shYvNYNYiM№yan 526.17-2С11 IIIIiMMYMIv^P eMim iiiiiiiii iiiiiiiii -. . III ND ND ND ND Nvya 526.17-2G1 :W 5,002 iiiiiiii ND ND ND ND iii <30.-a· 0840 .00/4 '<0.3032' ^: 0ΰ18 1 526.17-2G3 2.825 0.170' 0 120 ^: 4.692 veins iiiiiiii ιΐβϋ ND ND ND ND 1I1| 5.3.0: S 0 354 1 <3.00327 0 087 <0 60321 ¹ 424.9F4 0 539 2,534 iiiili -.. iiiili ND ND ND ND lllliill ,<:3θ· 0310 --.:9.021 - 0032. ' b ₍ 919 :, 424. EN 1 1/.1U1 1.385 1114 19.990 I i reveal .-.. 1 ί ND ND ND ND 1IN1 >& I1MIVI1I1y!1В1вв 139.19.A6 >50 fYIINv· liiliu .. . 0.090 .-.. 0 520 ND ND ND ND Ii 139.19.С10 >60 5.Μυ - . and|||1 ·.. I··· ND ND ND ND 139 19.F2 >50 0.755 iBiliil AI >50 0 525 ND ND ND ND Ishi mN" 20В.9.Св |D||1IV 1 510 -'50 ND ND ND ND o o|vz 0 248 0 303 0.100 - 0 125 0 137- 208.9.F12 5.263 0.265 0.314 MMiti 4 460 0 4-4 BIT >50 Ml. ND ND HI' >1 ·Μ4·Μ1^Μ1^··Μ|Ι··ΙΒ S 208.9.G10 6 842 3151 0 35' ‘ 2.896 - ΐΒιΙϋι And··· >50 Nl. ND ND HI- 0 882 >1 Me O 1031.12.6C4 lllllMjiU 97-7 0 057- >53 >50 IMI 1 130 >25 6 230 >1 t 1031.1 2.7D5 0023' 19 210 0 23- ^59 '50 ίί··! 0 980 ’ - 8 530 U "1 1031.12.9D9 <ι·.ο:.'32 0Q30. >53 >50 itMlI 3 540 >25 10,770 .>1. >1 b 1.7.1A7 >50 iiOll 0 176 1I1I1I11 0 743 >25 >25 >25 Mtllll <0.316 3,640 ND ND ND S 1.7.1D2 1 7.1G10 232.7.1В2 232.7.1C3 232.7.1С11 458.5.12V1 458.5.12E1 D1M||aM||8||MShV >50 JMMMMMSH >50 >50 IgBiieiiliJIHiMJI -50 0 241 0 226 |1|DM11I|MI|MIM 5 263 0 265 0 314 >50 iiii >50 >50 iilii |||M|I| 1I1I111 -.. iiiilil f-0 o№ >50 go 1 5-0 4 460 0 669 0 745 0 158- >50 MM 0 537 04-4 dmI AND··· AND·· 10 2'3 iiiiiijj Ή 057 0 525 >50 ·. >25 IVI lllliil 13 370 0.880 iiiili Iiiilii >12.5 1111(1 18 480 lj 111 >25 >25 iilii Mill jiffU >25 22 620 >25 AI · ..... 5 460 3 820 1 660 1¾¾¾ Iiya1 m mi <0.316 3.700 3.303 0.345, ; E|046 ζ <s:is <0 078 3 282 - p .11 ?!obc'33 0 370 3090 '; -3.024 ί β 323 ebzv ί oz-- -.370 I 3.035- I (:>:;>: 1з]018 ^: ' 458.5.12G9 6.842 0.151 0.35' llliflll! iii 0 632 >50 '44 4 9-0 >25 2 350 172.7С6 Mil· 9 /- ' 0 05? -oo Ml. ND ND HI' Iiv .<0.318 3.431 « 0.01E ’ ij.325. e|e27. 172 7F11 19 210 di·· >53 >50 HI ND ND HI. Shi 172.7G5 YYYYBIYYYIN -. kob^j mMI 0 030, >59 >5 U Nl. ND ND HI- And·· 11619G11 >50 i!i·· Μ··Ι 0 /43 Nl ND ND HI- >1 3 3/8 3 02* . ', DoZ'.G ir20 " 1161.ES 1 iiiiiiiiiiiiiiiiiii >50 --.. 0 669 10 2-3 mi.· ND ND HI' - >1 >5 3.618 ¢.365: ojo-jl ' 537.8.A11 >50 iililie 0 745 wiiMiii* ItliiiM HI ND ND HI. >1 3459 3273 ' 0.131 Ж eb· ί 537.8.E6 >50 M·····! >50 0.001’ 0 158 HI ND ND HI. >1 3.331 , 3 065' 0 386 e]o27. 537.8.E10 >50 0.9/3 1.7“ "50 - ' .'1 . · ·.: HI. ND ND Ill '1 2.0/1 3,-'4 ¹ 0.325 obi·

The numbers indicate the concentrations of IgG antibodies in µg/ml to achieve the IC50 in the PBMC-based neutralization assay. IC50 values are color coded and increasing sensitivity to neutralization is indicated from dark green to dark red. > indicates that the IC50 for a given virus was not achieved at the concentration tested. ND - not determined

Table 9

Data collection and refinement statistics (molecular substitution)

PGT121 Fab "unliganded" 10-1074 Fab GLFab PGT121 Fab "ligand bound" Data collection Spatial Ρ2ι Ρ2ι P2 ₁ 2 ₁ 2 ₁ Dimensions „, unit cell a, b, c (A) 56.75, 74.67, 114.917 61.38, 40.26, 84.46 54.93, 344.74, 55.23 67.79, 67.79, 94.11 α, β, Y (°) 90.00, 90.00, 90.00 90.00, 95.39, 90.00 90.00, 91.95, 90.00 90.00, 90.00, 90.00 Resolution (A) 2.78-35.5 (2.78-2.93) 1.80-36.31 (1.80-1.91) 2.42-38.60 (2.42-2.55) 2.33-38.66 (2.33-2.47) E>''merge 0.099 (0.293) 0.075 (0.558) 0.072 (0.482) 0.161 (0.603) // σ, 8.8 (3.1) 8.7(1.8) 11.0(1.9) 8.7 (2.9) Completeness(%) 96.7 (84.8) 93.49 (98.0) 95.5 (80.1) 92.2 (98.9) Redundancy 3.2 (2.7) 2.7 (2.8) 3.1 (2.6) 5.3 (5.8) Clarification Resolution (A) 3.0 1.9 2.42 2.4 Reflection amount 10,076 31,363 74.237 16,831 ^work ^free 0.216/0.264 0.187/0.223 0.194/0.237 0.201/0.249 Number of atoms Protein 3,276 3,346 12,881 3.127 Ligand/ion 0 0 0 129 Water 0 300 527 203 B-factor Protein 32.78 29.17 44.67 31.48 Ligand/ion - - - 45.1 Water - 37.37 40.27 36.78 Standard deviations Bond lengths (A) 0.005 0.007 0.005 0.006 Bond angles C) 0.971 1.234 0.951 0.949

* Data for each structure were obtained from one crystal * Values in parentheses are for the highest resolution shell

Table 10

RMSD values for Ca Fab alignments

Fab1/Fab2 RMSD _VH (A) gg RMSD _VL (A) ^K O°C ^L D ^V ° RMSDVHWL (A) ^k S ^C q ^T _b ^B0

PGT121/PGT128 PGT121/PGT145 1.159 2.93 116/130 124/130 1.63 1.91 95/100 94/105 1.462 1.75 207/235 206/235 PGT121/10-1074 0.74 128/130 1.2 102/105 1.26 226/235 PGT121/GL 1.33 129/130 1.37 94/105 1.6 225/235 10-1074/GL 1.38 130/130 1.35 92/105 1.39 220/235 PGT121 /PGT12 _{connected sligazzo} „ 0.79 125/128 0.5 100/100 0.78 225/228

- 38 046696

Contacts between Fab PGT121 and bound glycan

Table 11

glycan atom Protein atom Water Distance(A) glycan atom Atom squirrel Water Distance(A) GlcNAc ⁶ -O3 Asn ⁵⁸ -N62 2.91 Sia ¹⁰ -O8 Asp ³¹ -0 2.72 GlcNAc ⁶ -O7 Asn ⁵⁸ -O61 2.94 Sia ¹ °-O10 His ⁹⁷ -N 3.18 GlcNAc ⁶ -O6 _H2O471 ^​ 3.15 Sia ¹ °-O10 His ⁹⁷ -O 3.19 GlcNAc ⁶ -O4 _H2O477 ^​ 3.05 Sia ¹⁰ -O9 _H2O489 ^​ 3.19 GlcNAc ⁶ -O3 _H2O481 ^​ 2.94 Sia ¹⁰ -O8 Ser ³² -Oy 3.70* Man ¹ -O4 _H2O410 ^​ 3.02 Man ³ -O3 Asn ⁵⁸ -O61 2.58 Man ¹ -O4 _H2O420 ^​ 2.66 Man ³ -O6 _H2O477 ^​ 3.35 Man ¹ -O3 _H2O410 ^​ 3.35 GlcNAc4 ^- O5 Thr ⁵⁷ -O 3.33 Man ¹ -O2 _H2O477 ^​ 3.14 GIcNAc ⁴ -N2 _H2O479 ^​ 3.2 Man ¹ -O5 _H2O477 ^​ 2.62 Fuc ⁹ -O2 _H2O471 ^​ 2.57 Man ² -O6 Thr ¹⁰⁰ -Oy1 3.34 Asp ³¹ -0 _H2O435 ^​ 3.09 Man ² -O2 _H2O410 ^​ 3.41 Asp ³¹ -O61 H ₂ O ⁴⁸⁰ 3.32 Man ² -O5 _H2O446 ^​ 2.95 Tyr ⁵⁰ -OH _H2O481 ^​ 2.8 Man ² -O6 _H2O446 ^​ 3.26 His ⁵² -Ne2 _H2O435 ^​ 3.16 GlcNAc ⁷ -N2 Tyr ³³ -OH 2.72 Ser ⁵⁴ -Oy _H2O410 ^​ 3.2 GIcNAc ⁷ -O5 _H2O410 ^​ 3.38 Ser ⁵⁴ -O _H2O420 ^​ 3.16 GIcNAc ⁷ -O7 _H2O411 ^​ 3.00 Gly ⁵⁵ -O _H2O479 ^​ 2.85 GIcNAc ⁷ -O3 His ⁹⁷ -N62 3.60* Asp ⁵⁸ -O61 _H2O481 ^​ 3.49 GIcNAc ⁷ -O7 His ⁹⁷ -No. 2 3.70* Asp ⁵⁶ -O62 _H2O448 ^​ 3.07 Gal ⁸ -O3 Lys ⁵³ -N< 2.97 Asn ⁸⁸ -N52 _H2O481 ^​ 3.15 Gal ⁸ -O4 H ₂ O ⁴⁸⁰ 3.47 Arg-No. _H2O411 ^​ 2.58 Gal ⁸ -O4 _H2O438 ^​ 2.76 Thr ^100l -Oy1 _H2O411 ^​ 2.94 Gal ⁸ -O5 _H2O435 ^​ 3.17

hydrogen bonding criteria: bond distance < 3.5 A, O-H-O/N-H-O angle > 90° * Contacts are close to the distance cutoff limit for hydrogen bonding and are included as possible interactions

- 39 046696

Table 12 _________Neutralizing activity of PGT121GM u10-1074 _gm in vitro_________

Virus ID Clade PGT121 PGT121 _gm 10-1074 10-1074 _GM

Q842.d12 A 0.074 >50 >50 >50 3365.v2.c2 A 7.353 >50 0.450 0.467 0260.v5.c36 A . 0.152 >50 0.160 0.618 YU.2 IN 0.356 1.355 0.398 0.262 TRO.11 IN 0.051 0.258 ('.057 0.049 TRJO4551.58 IN 35.291 >50 (/634 0.721 QH0692.42 IN 8.545 >50 0.929 0.3/n PVO.4 IN 0.945 4 (.564 (.'.360 0.138 RHPA4259.7 IN 0.054 20.801 0.118 0.087 WITO4160.33 IN 6.007 >50 2.112 0.406 1054_07_TS4_1499 in (T/F) 0.696 >50 0.563 0.193 6244_13_В5_4576 V (T/F) 1.878 46.680 0.922 0.394 6235714D34589 V (T/F) 45.559 >50 >50 40 782 CNE19 Sun 0/89 48 092 0.379 CNE17 Sun >50 >50 13.297 4.8'6 CNE58 Sun >50 >50 0.968 1.158 CNE30 Sun 0.559 8.401 1.200 1.045 CNE52 Sun 32.935 >50 13.147 6.664 ZM233M.PB6 With 8.977 >50 0.349 0.232 ZM53M.PB12 With 0.002 >50 >50 >50 CAP45.2.00.G3 With 6.54'1 >50 >50 >50 HIV-16055-2.3 With 4.290 >50 >50 >50 HIV-16845-2.22 With >50 >50 5.835 2.678 ZM214M.PL15 With 3/50 >50 2.367 0.200 ZM135M.PL10a With 5.885 >50 0.367 0.184 Ce1086_B2 s (T/F) 0.006 >50 >50 >50 Ce1172_H1 s (T/F) 0.088 0.180 0.166 0.054 1394C9G1(Rev-) C (T/F) 3.372 2.120 0.191 0.075 3817.v2.c59 QD >50 >50 14.880 3.423 6952.v1.c20 CD 0 605 >50 0.138 0.134 BJOX009000.02.4 CRF01_AE 37.289 >50 >50 >50 211-9 CRF02 AG 8.840 >50 0.425 0.976 928-28 CRF02_AG >50 >50 4.696 3.121 T251-18 CRF02_AG >50 >50 7.395 3.459 T278-50 CRF02_AG >50 >50 18.276 12.017 263-8 CRF02AG 24.576 >50 6.527 7.779 235-47 CRF02_AG 1.676 >50 0.163 0.069 A07412M1.vrc12 D 0.406 16 94 ⁷ (.'.048 0.044 X1193_c1 G 0.202 11.859 0.475 0.195 X1254_c3 G 0/99 0.222 0.297 0.112

The numbers indicate the concentrations of IgG antibodies in μg/ml to achieve the IC50 in the TZM-bl neutralization assay. IC50 values are color coded and increasing sensitivity to neutralization is indicated from dark green to dark red. > indicates that the IC50 for a given virus was not achieved at the concentration tested.

- 40 046696

Table 13

Animal identification number At SHIVAD8EO At concentration (µg/ml) on day 0 titer (TZMbl) on day 0 Animal identification number At SHIVDH12-V3AD8 At concentration (µg/ml) on day 0 Title (TZM-bl) on day 0 Dose protected dose protected RHDEGF VRC01 50 mg/kg Yes 586.9 1:162 RHDEJ3 VRC01 thirty mg/kg Yes 395.8 1:52 RHDEH3 No 711.0 1:176 RHKZ1 No 306, 0 1:70 RHDE1L 20 mg/kg No 206.5 1:65 RHKZA PGT121 20 mg/kg Yes 215.1 1:13120 RHJBN No 188.1 1:68 RHDECT Yes 200.7 1:13805 RHKNX PGT121 20 mg/kg Yes 267.9 1:2495 RHKTL Yes 282.7 1:12669 RHMK4 Yes 253.6 1:2773 RHPZ9 Yes 133, 1 1:12055 RHDE9J 5 mg/kg Yes 55.7 1:563 RHK2Z 1 mg/kg Yes 15.1 1:422 RHPNR No 47.2 1:618 RHMT8 No 29.3 1:539 RHDCGI 1 mg/kg Yes 24.0 1:116 RHDEEB 0.2 mg/kg Yes 3, 1 1:159 RHKNE Yes 19, 7 1:55 RHDEP2 Yes 16 1:101 RHK4 4 0.2 No 1.8 < 1:20 RHMFD 0.05 No 10 <1:20 mg/kg mg/kg RHK4 9 No 1.8 1:17 RHKIA No 1.3 <1:20 RHDEEM 10-1074 20 mg/kg Yes 289, 8 1:2004 RHKIM 10- 1074 20 mg/kg Yes 290.3 1:1972 RHKIL Yes 257.7 1:2075 RHKWM Yes 173.3 1:2282 RHME1 5 mg/kg Yes 112.9 1:633 RHMJW 5 mg/kg Yes 96, 6 1:420 RHPNV Yes 117.5 1:384 RHMJT Yes 95.3 1:376 RHPID 1 mg/kg No 19, 9 1:56 RHDENI 1 mg/kg Yes 28.4 1:106 RHDCHX No 24.8 1:53 RHJHZ No 18.6 1:136 RHPZE 3BNC117 5 mg/kg Yes 105.4 1:272 RHHE8 0.2 mg/kg No 19, 4 1:39 RHPM5 Yes 76, 1 1:372 RHKCZ No 19.7 1:35 RHKMH 1 mg/kg No 39, 6 1:55 RHMFBA 3BNC11 7 20 mg/kg Yes 294.9 1:143 RHMJ5 No 15.1 1:75 RHMER Yes 272.7 1:142 RHPLD 45-46t2 20 mg/kg No 15.0 1:27 RHKIV 5 mg/kg Yes 114.6 1:80 RHMA9 No 17.6 < 1:20 RHKPI No 133, 1 1:90 RHMC6 5 mg/kg No 2.3 < 1:20 RHDE9D 1 mg/kg No 23.3 1:20 RHDE0CA No 2.2 < 1:20 RHDEW7 Yes 29.6 1:18 RHML1 DEN3 20 mg/kg No ND < 1:20 RHMEV 0.2 mg/kg No 3.9 < 1:20 RHMAA No ND < 1:20 RHMF9 No 5.7 < 1:20 RHKZMA 45- 4 6t2 5 mg/kg No 2.1 ND RHKNP No 4.0 ND RHJII hu-IgG 100 mg/kg No ND ND RHJK1 No ND ND

- 41 046696

IC50 in TZM-b11 cells

Table 14

Sample ID number S321 S500 B520 G435 T520b M263 MbOOs HIVIG (µg/ml) phenotype Tier R5 SHIVDH12-V3AD8 321 289 77 172 168 429 134 132 2 R5 SHIVAD8-EO 48 36 39 31 41 44 48 1768 2 X4 SHIVDH12-CL7 110 94 50 65 109 115 65 530 2 HIV-1CAAN5342.A2 84 <20 27 <20 <20 77 185 638 2 HIV-1MN.3 13944 9152 822 8432 3968 43722 1709 1.81 1

Values represent the serum dilution at which relative fluorescence units (RLU) were reduced by 50% compared to control virus wells (without test sample)

Table 15 SHIVAD8EO

Final neutralization - Number of animals Accumulated value Protected plasma titer Protected Infected Protected ^a Infected ^b Attitude % 2773 1 0 12 0 12/12 100% 2495 1 0 eleven 0 11/11 100% 2075 1 0 10 0 10/10 100% 2004 1 0 9 0 9/9 100% 633 1 0 8 0 8/8 100% 618 0 1 7 1 7/8 88% 563 1 0 7 1 7/8 88% 384 1 0 6 1 6/7 86% 372 1 0 5 1 5/6 83% 272 1 0 4 1 4/5 80% 176 0 1 3 2 3/5 60% 162 1 0 3 2 3/5 60% 115 1 0 2 2 2/4 50%c 75 0 1 1 3 1/4 25% 68 0 1 1 4 1/5 20% 65 0 1 1 5 1/6 17% 56 0 1 1 6 1/7 14% 55 1 0 1 6 1/7 14% 55 0 1 0 7 0/7 0% 53 0 1 0 8 0/8 0% 27 0 1 0 9 0/9 0% 20 0 1 0 10 0/10 0% 20 0 1 0 eleven 0/11 0% 20 0 1 0 12 0/12 0% 20 0 1 0 13 0/13 0% 17 0 1 0 14 0/14 0%

and the sum is below.

b sum above.

c the calculated final protection titer (50% protective titer) was 1:115

Table 16

SHIVDH12-V3AD8

Final Neutralization Number of Animals__________Cumulative Value______________________________Protected___________________________

Plasma titer Protected Infected Protected" Infected Attitude _____% 13805 1 0 16 0 16/16 100% 13120 1 0 15 0 15/15 1SSR/o 12669 1 0 14 0 14/14 100% 12055 1 0 13 0 13/13 100% 2282 1 0 12 0 12/12 100% 1972 1 0 eleven 0 11/11 1SSR/0 539 0 1 10 1 10/11 91% 422 1 □ 10 1 10/11 91% 420 1 O 9 1 9/10 90% 376 1 0 8 1 8/9 89% 159 1 0 7 1 7/8 88% 143 1 0 6 1 6/7 86% 142 1 0 5 1 5/6 83% 136 0 1 4 2 4/6 67% 106 1 0 4 2 4/6 67% 101 1 0 3 2 3/5 60% ^from 90 0 1 2 3 2/5 40%c 80 1 0 2 3 2/5 40% 70 0 1 1 4 1/5 20% 52 1 0 1 4 1/5 20% 39 0 1 0 5 0/5 0% 35 0 1 0 6 0/6 0% 20 0 1 0 7 0/7 0% 20 0 1 0 8 0/8 0% 20 0 1 0 9 0/9 0% 20 0 1 0 10 0/10 0% 20 0 1 0 eleven 0/11 0% 20 0 1 0 12 0/12 0% 20 0 1 0 13 0/13 0%

sum below

- 42 046696 ^b sum on top ^with the calculated final protection titer (50% protective titer) was 1:95.5 Table 17

Before infection Before mAb treatment

Animal e Weeks T cells after CD4+ infection cells/µl T cells CD4+ cells/µl viral load RNA copies/ml clinical status DBZ3 159 650 118 1.08E+04 no symptoms DC99A 159 623 165 7.60E+03 no symptoms DBXE 163 1585 158 1.96E+05 Intermittent diarrhea DCF1 157 1203 105 1.44E+05 Intermittent diarrhea DCM8 163 608 43 1.59E+03 Periodic

diarrhea Table 18

Animal Time Number of RNA copies Number of copies of treatment Gag SIV per 108 DNA Gag SIV per 108 (days) cells cells

DBZ3 0 9000 6700 DBZ3 10 360 7500 DBZ3 20 2400 14000 DC99A 0 31000 1400 DC99A 14 18000 5600 DC99A 20 8100 2700 DBXE 0 470000 71000 DBXE 14 17000 33000 DBXE 17 11000 22000 DCM8 0 110000 8600 DCM8 14 1700 1600 DCM8 20 22000 6600 DCF1 0 240000 15000 DCF1 14 190000 11000 DCF1 20 1100000 14000

The above examples and description of preferred embodiments are to be taken as illustrative and not limiting of the present invention as defined by the claims. As may be readily understood, numerous variations and combinations of the features mentioned above may be used without departing from the scope of the present invention as set forth in the claims. Such changes should not be construed as departing from the scope of the invention, and all such changes are intended to be included within the scope of the following claims. All publications cited in this description are included in the description in their entirety.

Claims (22)

1. An isolated bispecific anti-HIV antibody, or antigen-binding portion thereof, comprising a first antigen-binding arm and a second antigen-binding arm, wherein the first antigen-binding arm and the second antigen-binding arm specifically bind to various epitopes or molecules, wherein the first antigen-binding arm binds to an antigen on HIV and contains CDRH 1, CDRH 2, CDRH 3, CDRL 1, CDRL 2 and CDRL 3, - 43 046696 where CDRH 1, CDRH 2, CDRH 3, CDRL 1, CDRL 2 and CDRL 3 contain the corresponding sequences of a set of CDRs selected from the group consisting of SEQ ID NO: 69-74, SEQ ID NO: 57-62 and SEQ ID NO: 93-98. 2. An isolated bispecific anti-HIV antibody, or an antigen-binding portion thereof, according to claim 1, wherein the first antigen-binding arm comprises a heavy chain variable region and a light chain variable region with the sequences SEQ ID NO: 13-14, SEQ ID NO: 9-10 or SEQ ID NO: 21-22 respectively. 3. An isolated bispecific anti-HIV antibody, or an antigen-binding portion thereof, claim 2, wherein the heavy chain variable region and the light chain variable region comprise the corresponding sequences of SEQ ID NO: 13-14. 4. The isolated bispecific anti-HIV antibody, or antigen-binding portion thereof, according to claim 1, wherein CDRH 1, CDRH 2, CDRH 3, CDRL 1, CDRL 2 and CDRL 3 contain the sequences of SEQ ID NO: 69-74. 5. An isolated bispecific anti-HIV antibody, or an antigen-binding portion thereof, according to any one of claims 1 to 4, wherein the second antigen-binding arm binds to CD3. 6. An isolated bispecific anti-HIV antibody, or an antigen-binding portion thereof, according to any one of claims 1 to 5, wherein the antibody is a human antibody, a humanized antibody or a chimeric antibody. 7. An isolated bispecific anti-HIV antibody, or an antigen binding portion thereof, according to any one of claims 1 to 6, wherein the first antigen binding arm is selected from Fab, F(ab')2, Fv and scFv. 8. An isolated nucleic acid molecule containing at least one sequence encoding a bispecific anti-HIV antibody according to any one of claims 1 to 7 or an antigen-binding portion thereof. 9. A vector for the expression of a bispecific anti-HIV antibody or an antigen-binding part thereof according to any one of claims 1 to 7, containing a nucleic acid according to claim 8. 10. A cultured cell for expressing a bispecific anti-HIV antibody or an antigen-binding part thereof according to any one of claims 1 to 7, containing a nucleic acid according to claim 8 or a vector according to claim 9. 11. A pharmaceutical composition containing (i) at least one anti-HIV antibody, or an antigen-binding part thereof, according to any one of claims 1 to 7, or a nucleic acid according to claim 8, or a vector according to claim 9; and (ii) a pharmaceutically acceptable carrier. 12. The pharmaceutical composition according to claim 11, also containing a second therapeutic agent. 13. The pharmaceutical composition according to claim 12, wherein the second therapeutic agent comprises an antiviral agent, or a second anti-HIV antibody, or an antigen-binding portion thereof. 14. A method of preventing or treating HIV infection or an HIV-related disease in a patient, comprising administering to the patient a therapeutic agent containing a therapeutically effective amount of an anti-HIV antibody according to any one of claims 1 to 7 or an antigen-binding portion thereof. 15. The method of claim 14, further comprising administering a second therapeutic agent. 16. The method according to claim 15, also including the administration of an antiviral agent. 17. Use of at least one bispecific anti-HIV antibody or antigen-binding portion thereof according to any one of claims 1 to 7 for the prevention or treatment of HIV infection. 18. Use of a nucleic acid according to claim 8 for the prevention or treatment of HIV infection. 19. Use of the vector according to claim 9 for the prevention or treatment of HIV infection. 20. A method for producing a bispecific anti-HIV antibody or an antigen-binding part thereof, comprising obtaining a cultured cell according to claim 10; culturing the cell in a medium under conditions allowing expression of the polypeptide encoded by the vector and purification of the antibody or antigen-binding portion thereof from the cultured cell or cell medium. 21. A kit for the prevention or treatment of HIV infection, containing: (i) a pharmaceutically acceptable dosage form of a pharmaceutically effective amount of at least an isolated bispecific anti-HIV antibody or antigen-binding portion thereof according to any one of claims 1 to 7, or a nucleic acid according to claim 8, or a vector according to claim 9, and (ii) a container containing a pharmaceutically acceptable dosage dose of a pharmaceutically effective amount of at least one isolated bispecific anti-HIV antibody, or antigen binding portion, or nucleic acid, or vector. 22. The kit of claim 21, further comprising a pharmaceutically acceptable dosage form of a pharmaceutically effective amount of any anti-HIV agent, wherein the two pharmaceutically acceptable dosage forms may optionally be in the form of a common pharmaceutically acceptable dosage form.