Broadly-neutralizing Anti-HIV antibodies

Abstract

To provide a broad and potent antibody against human immunodeficiency virus (HIV).SOLUTION: This invention relates to a new category of broadly-neutralizing anti-HIV antibodies and provides an antibody carrying a specific amino acid sequence, comprising consensus heavy chain and light chain amino acid sequences of said antibody.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61 / 715,642, filed October 18, 2012, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT The invention disclosed herein was made, at least in part, with government support under Grant No. P01 AI081677 from the National Institutes of Health. Accordingly, the United States Government has certain rights in this invention.

[0003] The present invention relates to broadly potent antibodies against the human immunodeficiency virus ("HIV"). [Background technology]

[0004] HIV is a condition in humans characterized by clinical manifestations including a wasting syndrome, central nervous system degeneration, and profound immunosuppression that leads to acquired immune deficiency syndrome (AIDS), resulting in life-threatening opportunistic infections and malignancies. Since its discovery in 1981, HIV type 1 (HIV-1) has caused at least 25 million deaths worldwide. Even if HIV infections decline by 2.5% each year, it is projected that 20 to 60 million people will become infected over the next 20 years. There is a need for therapeutic agents and methods for treating or suppressing HIV infection.

[0005] Some HIV-infected individuals exhibit broadly neutralizing IgG antibodies in their serum. However, little is known about the specificity and activity of these antibodies, despite their potential importance in designing effective vaccines. In animal models, passive transfer of neutralizing antibodies can contribute to protection against viral challenge. Neutralizing antibody responses can also be developed in HIV-infected individuals, but the detailed composition of the serum response has not yet been fully characterized. Summary of the Invention

[0006] The present invention relates to a novel category of broadly neutralizing anti-HIV antibodies, the consensus heavy and light chain amino acid sequences of which are listed below and shown in Figures 3a and 3b: QVQLQESGPGLVKPSETLSLTCSSVSGX1SX2X3DX4YWSWIRQSPGKGLEWIGYVHDSGDTNYNPSLKSRVX5X6SLDTSKNQVSLKLX7X8VTAADSAX9YYCARAX 10 HGX 11 RIYGIVAFGEX 12 FTYFYMDVWGKGTTVTVSS (SEQ ID NO: 1) SX1VRPQPPSLSVAPGETARIX2CGEX3SLGSRAVQWYQQRPGQAPSLIIYNNQDRPSGIPERFSGSPDX4X5FGTTATLTITX6VEAGDEADYYCHIWDSRX7PTX8WVFGGGTTLTVL (SEQ ID NO: 2).

[0007] In the sequence of SEQ ID NO: 1 or 2, each "X" can be any amino acid residue or no amino acid. Preferably, each X can be the residue at the corresponding position in clonal variants 10-259, 10-303, 10-410, 10-847, 10-996, 10-1074, 10-1121, 10-1130, 10-1146, 10-1341, and 10-1369, as well as an artificially modified version of the 10-1074 antibody, 10-1074GM, as shown in Figures 3a and 3b.

[0008] 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 SEQ ID NOs: 33-38, with the proviso that the antibody is not antibody PGT-121, 122, or 123. SEQ ID NOs: 33-38 refer to the sequences of heavy chain CDRs (CDRH) 1-3 and light chain CDRs (CDRL) 1-3 according to the Kabat system as shown in Figures 3a and 3b. In one embodiment, the CDRs can contain sequences selected from the group consisting of SEQ ID NOs: 39-104, i.e., CDR sequences according to the KABAT system as shown in Table 1 below. Alternatively, the CDRs can contain sequences selected from the CDR sequences of their corresponding antibodies according to the IMGT system as shown in Table 1 below.

[0009] In one embodiment, the isolated anti-HIV antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, wherein the CDRH1, CDRH2, and CDRH3 comprise the sequences of SEQ ID NOs: 33 to 35, respectively. The CDRH1, CDRH2, and CDRH3 can also comprise the sequences of a set of CDRHs selected from the group consisting of SEQ ID NOs: 39 to 41, 45 to 47, 51 to 53, 57 to 59, 63 to 65, 69 to 71, 75 to 77, 81 to 83, 87 to 89, 93 to 95, 99 to 101, and 131 to 133. Alternatively, the CDRHs can comprise the sequences selected from the CDR sequences of their corresponding antibodies according to the IMGT system, as shown in Table 1 below.

[0010] In another embodiment, the isolated anti-HIV antibody, or antigen-binding portion thereof, comprises a light chain variable region including CDRL1, CDRL2, and CDRL3, wherein the CDRL1, CDRL2, and CDRL3 comprise the sequences of SEQ ID NOs: 36-38, respectively. For example, the CDRL1, CDRL2, and CDRL3 can comprise the sequences of a CDRL set selected from the group consisting of SEQ ID NOs: 42-44, 48-50, 54-56, 60-62, 66-68, 72-74, 78-80, 84-86, 90-92, 96-98, 102-104, and 134-136. Alternatively, the CDRLs can comprise sequences selected from the CDR sequences of their corresponding antibodies according to the IMGT system, as shown in Table 1 below.

[0011] In yet another embodiment, the isolated anti-HIV antibody, or antigen-binding portion thereof, described above, comprises (i) a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, and (ii) a light chain variable region comprising CDRL1, CDRL2, and CDRL3. The CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 can comprise respective sequences of CDR sets selected from the group consisting of SEQ ID NOs: 39-44, 45-50, 51-56, 57-62, 63-68, 69-74, 75-79, 81-86, 87-92, 93-98, 99-104, and 131-136. Alternatively, the CDRHs and CDRLs can comprise respective sequences selected from the CDR sequences of their corresponding antibodies according to the IMGT system, as shown in Table 1 below.

[0012] In further embodiments, the isolated anti-HIV antibody, or antigen-binding portion thereof, contains 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: 2. The heavy chain is 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 can contain 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 the light chain can each contain the sequence of SEQ ID NOs: 3 to 4, SEQ ID NOs: 5 to 6, SEQ ID NOs: 7 to 8, SEQ ID NOs: 9 to 10, SEQ ID NOs: 11 to 12, SEQ ID NOs: 13 to 14, SEQ ID NOs: 15 to 16, SEQ ID NOs: 17 to 18, SEQ ID NOs: 19 to 20, SEQ ID NOs: 21 to 22, SEQ ID NOs: 23 to 24, and 129 to 130.

[0013] In a preferred embodiment, the isolated anti-HIV antibody is one selected from the group consisting of 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 corresponding heavy chain variable regions, light chain variable regions, CDRH1-3, and CDRL1-3 are shown in Figures 3a and 3b. In a further preferred embodiment, the isolated anti-HIV antibody is a 10-1074-like antibody, i.e., one selected from the group consisting of 10-847, 10-996, 10-1074, 10-1074GM, 10-1146, and 10-1341. Antibodies in this group are more potent than PGT121 in neutralizing current viruses. The antibodies discussed above can be human, humanized, or chimeric.

[0014] 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 an anti-HIV antibody, or antigen-binding portion thereof, discussed above. Also featured are a vector containing the nucleic acid and a cultured cell containing the vector.

[0015] The nucleic acids, vectors, and cultured cells can be used in a method for producing anti-HIV antibodies or fragments thereof, which method includes, among other steps, obtaining the cultured cells described above, culturing the cells in a medium under conditions that allow 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 cells or the cell medium.

[0016] In a third aspect, the invention features a pharmaceutical composition including (i) at least one anti-HIV antibody, or antigen-binding portion thereof, described above, and (ii) a pharmaceutically acceptable carrier.

[0017] In a fourth aspect, the present invention provides a method for preventing or treating HIV infection or an HIV-related disease, comprising, among other steps, identifying a patient in need of such prevention or treatment; and administering to the patient a first therapeutic agent containing a therapeutically effective amount of at least one anti-HIV antibody, or antigen-binding portion thereof, as described above. The method can further comprise administering a second therapeutic agent, e.g., an antiviral agent.

[0018] In a fifth aspect, the present invention provides a kit comprising a pharmaceutically acceptable dosage unit of a pharmaceutically effective amount of at least one isolated anti-HIV antibody, or antigen-binding portion thereof, as described above, and a pharmaceutically acceptable dosage unit of a pharmaceutically effective amount of an anti-HIV drug. The two pharmaceutically acceptable dosage units can optionally be in the form of a single pharmaceutically acceptable dosage unit. Exemplary anti-HIV drugs can be selected from the group consisting of non-nucleoside reverse transcriptase inhibitors, protease inhibitors, entry or fusion inhibitors, and integrase inhibitors.

[0019] In a sixth aspect, the present invention provides a kit for the diagnosis, prognosis or treatment monitoring of HIV infection in a subject, the kit comprising: The kit may further comprise reagents for performing PCR or mass spectrometry.

[0020] The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. [Brief explanation of the drawings]

[0021] [Figure 1-1] Figure 1 shows the neutralizing activity of PGT121-like and 10-1074-like mutants. (A) Heatmap comparing the neutralizing potency of PGT121-like and 10-1074-like antibodies in the TZM-bl assay. Darker colors = stronger neutralization; white colors = no neutralization. (B) Correlation between the mean IC80 (y-axis) against 9 viruses and the apparent KD values for binding to gp120 and gp140 (x-axis). [Figure 1-2] (C) Graph comparing the neutralization breadth and potency of PGT121, 10-996, and 10-1074 antibodies in the TZM-bl 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 web graph (top left corner) shows the frequency distribution of neutralized viruses by HIV-1 clade. (D) Dot plot showing the molar neutralization rate (MNR; IC50 concentration ratio of Fab to IgG). The horizontal bar represents the average IC50 across all viruses. [Figure 1-3] Neutralizing activity of PGT121-like and 10-1074-like mutants. (E) Bar graph comparing the neutralizing potency of PGT121 (dark gray) and 10-1074 (light gray) against viruses isolated from past (Hist.) and current (Cont.) seroconverters. ns, not significant; **, p<0.005. Fold differences between median IC50s for neutralization of current viruses by PGT121 and 10-1074 are shown. [Figure 2]Figure 7. Binding and neutralization activity of PGT121GM and 10-1074GM mutant antibodies. (A) Bar graph comparing apparent KD values for binding of 10-1074, PGT121, PGT121GM, and 10-1074GM antibodies to gp120 and gp140. Error bars indicate SEM of KD values from three independent experiments. Fold differences between KD values of "wild-type" versus "glycomutant" antibodies are shown. (B) Bar graph comparing binding of glycans (Figure 7A) by PGT121 and 10-1074 with that by mutant antibodies (PGT121GM and 10-1074GM). Numerical binding scores are measured as fluorescence intensity (average of duplicate spots) for arrayed probes at 5 fmol per spot. (C) Coverage graph comparing the neutralization breadth and potency of PGT121, PGT121GM, 10-1074, and 10-1074GM antibodies in the TZM-bl assay against a panel of 40 viruses. [Figure 3-1] Figure 1 shows sequence alignment of PGT121 and 10-1074 clonal variants. (A) Amino acid alignment of the heavy chains (IgH) of PGT121-like and 10-1074-like antibodies and the predicted germline (GL) VH for all clonal variants. Amino acid numbering based on the crystal structure, framework (FWR) and complementarity-determining regions (CDRs) as defined by Kabat (J Exp Med 132(2):211-250) and IMGT (Nucleic Acids Res 37(Database issue):D1006-1012) is shown. Color shading indicates acidic (red), basic (blue), and tyrosine (green) amino acids. [Figure 3-2] Sequence alignment of PGT121 and 10-1074 clone variants. (B) Same as A, but for the light chain (IgL). [Figure 4]Figure 1 shows the binding affinity of PGT121 and 10-1074 clonal variants. (A) Binding affinity of the PGT121 IgG antibody variants interacting with the YU-2 gp140 and gp120 ligands as measured by surface plasmon resonance (SPR). M, mol / L; s, seconds; RU, response units; / , no detected binding. A chi-square value (X2) <10 indicates that the 1:1 binding model used to fit the curve adequately describes the experimental data. The equilibrium and rate constants shown are considered "apparent" constants to account for avidity effects resulting from IgG bivalent binding. (B) Dot plots showing the association (k) and dissociation (kd) rate constants for PGT121-like (blue shading) and 10-1074-like (green shading). (C) Linear regression graph comparing the k a and k d values (x-axis) for IgG antibodies binding to gp120 and gp140 against their neutralization potency (mean IC 80 values) (y-axis) against the nine viruses shown in Table 4. [Figure 5-1] Figure 1 shows binding of PGT121 mutants to gp120 "core" protein, gp120GD324-5AA mutant, and linear gp120V3 peptide. (A) ELISA-based binding analysis of PGT121-like and 10-1074-like antibodies to HXB2 gp120 core and 2CC-core proteins compared to intact YU-2 gp120. The x-axis indicates the antibody concentration (M) required to obtain the ELISA value (OD405nm) shown on the y-axis. The anti-CD4bs antibody VRC01 (Science 329(5993):856-861), anti-V3 loop antibody 10-188 (PLoS One 6(9):e24078), and non-HIV-reactive antibody mGO53 (Science 301(5638):1374-1377) were used as controls. All experiments were performed at least in duplicate. Representative data are shown. [Figure 5-2] Figure 1 shows binding of PGT121 mutants to gp120 "core" protein, gp120GD324-5AA mutant, and linear gp120V3 peptide. (B) Same as (A), but for binding to gp120GD324-5AA mutant protein. All experiments were performed at least twice. Representative data are shown. [Figure 5-3] Figure 1 shows binding of PGT121 variants to the gp120 "core" protein, the gp120GD324-5AA mutant, and the linear gp120V3 peptide. (c) Bar graph comparing the ELISA reactivity of PGT121-like and 10-1074-like antibodies and control antibodies (positive controls, 10-188, 1-79, 2-59, and 2-1261 (Nature 458(7238):636-640)) and negative control, mGO53) to the gp120V3-C3 overlapping peptide. The y-axis shows ELISA values (OD405nm) obtained by testing 2 μg / mL of IgG antibody. The amino acid sequences of individual peptides are shown in the lower right. All experiments were performed at least twice. Representative data are shown. [Figure 6-1] Figure 1 shows binding of PGT121 to gp120 glycosylation mutants and deglycosylated gp120. (A) ELISA-based binding analysis of PGT121 and 10-1074 antibody variants to gp120, gp120NNT301-303AAA, gp120N332A, and gp120N332A / NNT301-303AAA. The x-axis indicates the antibody concentration (M) required to obtain the ELISA value (OD405nm) shown on the y-axis. The dashed and continuous black lines indicate the average reactivity against the four antigens for the positive (10-188) and negative (mGO53) antibody control groups. All experiments were performed at least twice. [Figure 6-2] Figure 1 shows binding of PGT121 to gp120 glycosylation mutants and deglycosylated gp120. (B) Silver-stained SDS-PAGE gel comparing untreated gp120 (WT, wild-type), PNGaseF-digested gp120, and EndoH-digested gp120. L, protein ladder. All experiments were performed at least twice. [Figure 6-3] Figure 1 shows PGT121 binding to gp120 glycosylation mutants and deglycosylated gp120. (C) Same as (A), but comparing untreated gp120 with PNGase F-treated gp120. All experiments were performed at least twice. [Figure 6-4]Figure 1 shows PGT121 binding to gp120 glycosylation mutants and deglycosylated gp120. (D) Same as (A), but comparing untreated gp120 with EndoH-treated gp120. All experiments were performed at least twice. [Figure 7-1] Figure 1 shows the binding of PGT121 and 10-1074 clonal variants to glycans. (A) Monosaccharide sequences of a set of 15 N-glycan probes used in glycan microarray analysis to investigate direct binding to N-glycans for PGT121-like and 10-1074-like antibodies. DH indicates the lipid tag 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE), to which the N-glycan was attached by reductive amination. Important features to note are that (i) PGT121 group antibodies bound monoantennary N-glycan probe 10 (N2), which has a galactose-terminal antenna linked to the core mannose by a 1-3 linkage, but not the isomeric N-glycan probe 11 (designated N4), which has an antenna linked 1-6 to the core mannose; (ii) as in the case of biantennary probe 13 (NA2), the presence of this galactose-terminal 1-6 linked antenna allowed binding, as did the presence of α2-6 linked (rather than α2-3) linked sialic acid; and (iii) biantennary probe 12 (NGA2), which lacks galactose and terminates with N-acetylglucosamine, did not bind. [Figure 7-2] Figure 1 shows binding of PGT121 and 10-1074 clonal variants to glycans. (B) Bar graph comparing glycan binding by PGT121-like, 10-1074-like, and germline (GL) antibodies. 10-188, an anti-V3 loop antibody, was used as a negative control. Numerical binding scores were measured as fluorescence intensity (average of duplicate spots) for probes arrayed at 2 fmol (white) and 5 fmol (gray) per spot. [Figure 8-1]Antibody binding and neutralizing activity against high-mannose-only gp120 and virus. (A) Silver-stained SDS-PAGE gel comparing YU-2 gp120 (gp120kif) produced in cells treated with kifunensine with gp120 (WT, wild-type) produced in untreated cells. L, protein ladder. (B) ELISA comparison of binding of PGT121-like (blue label) and 10-1074-like (green label) antibodies to YU-2 gp120 (gp120WT) and gp120kif. The x-axis indicates the antibody concentration (M) required to obtain the ELISA value (OD405nm) shown on the y-axis. [Figure 8-2] Antibody binding and neutralization activity against high-mannose-only gp120 and virus. (C) Neutralization curve for PGT121 assessed against selected PGT121-susceptible / 10-1074-resistant pseudoviruses produced in the presence (virus kif) or absence (virus WT) of kifunensine. The dashed horizontal line indicates 50% neutralization, and IC50 values can be obtained from the antibody concentration on the x-axis. Experiments were performed in triplicate. Error bars indicate the SD of triplicate determinations. (D) Bar graph comparing the neutralization activity of selected antibodies against YU-2 and PVO.4 pseudoviruses produced in HEK293S GnTI- / - cells (virus GnT- / -) or wild-type cells (virus WT). The y-axis indicates the mean IC50 value (μg / mL) for neutralization of the virus indicated on the x-axis. Error bars indicate the SEM of IC50 values obtained from two independent experiments. [Figure 9-1] Figure 1 shows the neutralizing activity of PGT121, 10-996, and 10-1074. (A) Graph comparing the neutralizing potency (determined using the TZM-bl assay and a panel of 119 pseudoviruses) of PGT121, 10-996, and 10-74 against viruses of the indicated HIV-1 clades. 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 indicated on the x-axis. [Figure 9-2](A) Neutralizing activity of PGT121, 10-996, and 10-1074. (B) Graph comparing the neutralizing breadth and potency of PGT121, 10-996, and 10-1074 against an expanded panel of 119 viruses as determined by TZM-bl neutralization assay. The y-axis shows the cumulative frequency of IC80 values up to the concentration indicated on the x-axis. [Figure 9-3] Figure 1 shows the neutralizing activity of PGT121, 10-996, and 10-1074. (C) The graph shows the neutralization curves of selected viruses by PGT121 and 10-1074. The dashed horizontal line indicates 50% neutralization, and IC50 values can be obtained from the antibody concentration on the x-axis. Experiments were performed in triplicate. Error bars indicate the SD of triplicate determinations. [Figure 10] Figure 1. Historical vs. current neutralizing activity against clade B viruses. Dot plots comparing neutralizing potency for select bNAbs against clade B viruses isolated from historical (Hist.) and current (Cont.) seroconverters. Horizontal bars represent median IC50s across all viruses per patient. Differences between groups were assessed using the Mann-Whitney test. ns, not significant. [Figure 11] Table showing neutralization of two R5-tropic SHIVs with a panel of 11 broadly active anti-HIV-1 mAbs. Calculated IC50 values for neutralizing SHIVAD8EO (A) and SHIVDH12-V3AD8 (B). [Figure 12] 1 shows the relationship between plasma concentrations of passively administered neutralizing mAbs and viral acquisition after two different R5SHIV in macaques. Filled circles indicate protected (non-acquired) monkeys; open circles indicate infected animals. [Figure 13-1]1 is a table showing plasma concentrations of bNAbs. mAb concentrations were determined by measuring neutralizing activity in plasma samples. (A) ID50 values measured in the TZM.bl neutralization assay for 10-1074 and 3BNC117 against HIV-1 strains sensitive to one bNAb but not the other (i.e., HIV-1 strain X2088_9 (10-1074 sensitive); HIV-1 strain Q769_d22 (3BNC117 sensitive)). (B) Neutralizing activity of plasma before antibody administration (preP) but spiked with 0.01, 0.1, 1, 10, and 100 μg / mL of antibody 10-1074 (blue) or 3BNC117 (green). Based on the measured ID50 values in (A), neutralizing activity is reported as plasma ID50 titers (left column) and converted to antibody concentrations (right column). [Figure 13-2] (C) ID50 titers (left column) and concentrations (right column) of bNAbs measured in the indicated macaque plasma samples before (prebleed) and after (days) bNAb administration. DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention is based, at least in part, on the unexpected discovery of a novel category of broadly neutralizing antibodies (bNAbs) against HIV that can recognize carbohydrate-dependent epitopes, including complex N-glycans, on gp120.

[0023] Antibodies are essential for the success of most vaccines, and antibodies to HIV appear to be the only correlate of protection in the recent RV144 anti-HIV vaccine trial. Some HIV-1-infected patients develop broadly neutralizing serum activity against the gp160 viral spike 2–4 years after infection, but because of autologous viral escape through mutation, these antibodies generally do not protect infected humans. Nevertheless, broadly neutralizing activity exerts selective pressure on the virus, and passive transfer of broadly neutralizing antibodies (bNAbs) to macaques protects against SHIV infection. It has therefore been proposed that vaccines eliciting such antibodies may be protective against HIV infection in humans.

[0024] The development of single-cell antibody cloning methods has revealed that bNAbs target several different epitopes on the HIV-1 gp160 spike. Most potent HIV-1 bNAbs recognize the CD4 binding site (CD4bs) (Science 333(6049):1633-1637; Nature 477(7365):466-470; Science 334(6060):1289-1293), including the V1 / V2 (PG9 / PG16) (Science 326(5950):285-289) and V3 loop (PGT) (Nature 477(7365):466-470). They recognize carbohydrate-dependent epitopes involving variable loops (Nature 477(7365):466-470; Science 326(5950):285-289; Science 334(6059):1097-1103; Nature 480(7377):336-343). Because the antibodies studied to date are unique examples or members of small clonal families, little is known about carbohydrate-dependent epitopes.

[0025] To better understand the neutralizing antibody response to HIV-1 and the epitopes targeted by PGT antibodies, we isolated members of a large clonal family that dominated gp160-specific IgG memory responses from a clade A-infected patient who produced PGT121. As disclosed herein, PGT121 antibodies are divided into two groups, the PGT121-like group and the 10-1074-like group, according to sequence, binding affinity, neutralizing activity, and carbohydrate and V3 loop recognition. 10-1074 and related family members exhibit unusually potent neutralization, including broad reactivity against newly circulating viruses. Unlike previously characterized carbohydrate-dependent bNAbs, PGT121 binds to complex N-glycans rather than high-mannose N-glycans in glycan microassay experiments. The crystal structures of PGT121 and 10-1074, compared with those of their germline precursors and the structure of PGT121 bound to complex N-glycans, rationalize their unique properties.

[0026] In one example, assays were performed to isolate B cell clones encoding PGT121, which are unique among glycan-dependent bNabs in that they recognize complex N-glycans rather than high-mannose N-glycans. PGT121 clones are divided into PGT121-like and 10-1074-like groups, which are distinguished by sequence, binding affinity, carbohydrate recognition, and neutralizing activity. The 10-1074 group exhibits remarkable potency and breadth despite not detectably binding to protein-free glycans. Crystal structures of unliganded PGT121, 10-1074, and their germline precursors revealed that the carbohydrate recognition difference maps to the cleft between CDRH2 and CDRH3, which in the isolated PGT121 structure is occupied by complex N-glycans. Exchange of glycan contact residues between PGT121 and 10-1074 confirmed the importance of these residues for neutralizing activity. The HIV envelope displays a variety of high-mannose and complex N-glycans, and therefore these results, including the first structural characterization of complex N-glycan recognition by anti-HIV bNAbs, are important for understanding how antibodies and ultimately vaccines can achieve broadly neutralizing activity.

[0027] The term "antibody" (Ab), as used herein, includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific and polyreactive antibodies), and antibody fragments. Thus, the term "antibody," as used in any context herein, is intended to include, but is not limited to, any specific binding member, immunoglobulin class and / or isotype (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM); and biologically relevant fragments or specific binding members thereof, including, but not limited to, Fab, F(ab'), Fv, and scFv (single chain or related entities). Antibodies are understood in the art to be glycoproteins having at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. The heavy chain is composed of a heavy chain variable region (VH) and heavy chain constant regions (CH1, CH2, and CH3). The light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). Both the heavy and light chain variable regions contain framework regions (FWR) and complementarity determining regions (CDR). The four FWR regions are relatively conserved, while the CDR regions (CDR1, CDR2, and CDR3) correspond to hypervariable regions and are arranged from the NH2-terminus 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 an antigen, while the constant region(s), depending on the isotype, may mediate the binding of the immunoglobulin to host tissues or factors.

[0028] The definition of "antibody" as used herein also includes chimeric, humanized and recombinant antibodies, human antibodies produced from transgenic non-human animals, and antibodies selected from libraries using enrichment techniques available to those skilled in the art.

[0029] The term "variable" refers to the fact that certain segments of the variable (V) domains differ significantly in sequence among antibodies. V domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the 110-amino acid span of the variable regions. Instead, V regions consist of relatively invariant stretches of 15-30 amino acids called framework regions (FRs), separated by shorter regions of hypervariability called "hypervariable regions," each 9-12 amino acids long. Native heavy and light chain variable regions each contain four FRs, primarily adopting a β-sheet structure, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β-sheet structure. The hypervariable regions in each chain are held in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0030] The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. A hypervariable region generally comprises amino acid residues from the "complementarity determining regions" (CDRs).

[0031] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The term "polyclonal antibody" refers to a preparation containing different antibodies directed against different determinants ("epitopes").

[0032] The monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as the fragments exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). The described invention provides variable region antigen-binding sequences derived from human antibodies. Thus, chimeric antibodies of primary interest herein include antibodies having one or more human antigen-binding sequences (e.g., CDRs) and containing one or more sequences, e.g., FR or C-region sequences, derived from a non-human antibody. Additionally, chimeric antibodies included herein are those that contain human variable region antigen-binding sequences of one antibody class or subclass and other sequences, such as FR or C region sequences, derived from another antibody class or subclass.

[0033] A "humanized antibody" is generally considered to be a human antibody having one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which typically come from an "import" variable region. Humanization may be performed according to the method of Winter and coworkers by substituting the imported hypervariable sequences for the corresponding sequences of a human antibody (see, e.g., Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)). Thus, such "humanized" antibodies are chimeric antibodies (see, e.g., U.S. Pat. No. 4,816,567) in which substantially less of the variable region of an intact human antibody has been substituted by the corresponding sequence from a non-human species.

[0034] "Antibody fragments" comprise a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see, e.g., U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8(10):1057-1062

[1995] ); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0035] An "Fv" is the minimum antibody fragment containing a complete antigen-recognition and antigen-binding site. This fragment contains a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. Folding of these two domains yields six hypervariable loops (three loops each from the H and L chain), which contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. 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, albeit with lower affinity than the entire binding site.

[0036] A "single-chain Fv" ("sFv" or "scFv") is an antibody fragment comprising the VH and VL antibody domains connected into a single polypeptide chain. The sFv polypeptide can further comprise a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. For reviews of sFvs, see, e.g., Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra.

[0037] The term "diabody" refers to small antibody fragments prepared by constructing sFv fragments with a short linker (approximately 5-10 residues) between the VH and VL domains such that interchain, rather than intrachain, pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are more fully described, for example, in EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0038] Domain antibodies (dAbs), which can be produced in fully human form, are the smallest known antigen-binding fragments of antibodies, ranging from about 11 kDa to about 15 kDa. dAbs are the robust variable regions of the heavy and light chains (VH and VL, respectively) of immunoglobulins. They are widely used in the detection of microbial pathogens. dAbs are highly expressed in human cell culture, exhibit favorable biophysical properties, including (but not limited to) solubility and temperature stability, and are well suited to selection and affinity maturation by in vitro selection systems such as phage display. dAbs are biologically active as monomers, and due to their small size and inherent stability, can be formatted into larger molecules to create drugs with extended serum half-lives or other pharmacological activity. Examples of these techniques are described, for example, in WO 9425591 for antibodies derived from camelid heavy chain Ig; in addition, U.S. Patent Application Publication No. 20030130496 describes the isolation of single-domain fully human antibodies from phage libraries.

[0039] Fv and sFv are the only species with intact binding sites that lack any constant region. Therefore, they are suitable for reducing nonspecific binding during in vivo use. sFv fusion proteins can be constructed to fuse an effector protein to either the amino or carboxy terminus of an sFv. See, for example, Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment can also be, for example, a "linear antibody," as described, for example, in U.S. Pat. No. 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

[0040] In certain embodiments, the antibodies of the present invention are bispecific or multispecific. Bispecific antibodies are antibodies with binding specificities for at least two different epitopes. For example, bispecific antibodies can bind to two different epitopes of a single antigen. Other such antibodies can combine a first antigen-binding site with a binding site for a second antigen. Alternatively, anti-HIV arms can be combined with arms that bind to trigger molecules on leukocytes, such as T cell receptor molecules (e.g., CD3), or Fc receptors for IgG (Fc gamma R), such as Fc gamma RI (CD64), Fc gamma RII (CD32), and Fc gamma RIII (CD16), to focus and localize cellular defense mechanisms to infected cells. Bispecific antibodies can also be used to localize cytocidal agents to infected cells. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). For example, WO 96 / 16673 describes a bispecific anti-ErbB2 / anti-Fc gamma RIII antibody, and U.S. Pat. No. 5,837,234 discloses a bispecific anti-ErbB2 / anti-Fc gamma RI antibody. For example, a bispecific anti-ErbB2 / anti-Fc alpha antibody is reported in WO 98 / 02463; U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2 / anti-CD3 antibody. See, 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. U.S.A. 2012 See also Jan 17;109(3):875-80.

[0041] Methods for producing bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (see, e.g., Millstein et al., Nature, 305:537-539 (1983)). Similar procedures are disclosed, for example, in WO 93 / 08829; Traunecker et al., EMBO J., 10:3655-3659 (1991); Mouquet et al., Enhanced HIV-1 neutralization See also “by antibody heteroligation” Proc Natl Acad Sci U S A. 2012 Jan 17;109(3):875-80.

[0042] Alternatively, antibody variable regions with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion is with an Ig heavy chain constant region, including at least part of the hinge, CH2, and CH3 regions. According to some embodiments, the first heavy chain constant region (CH1), containing the site necessary for light chain binding, is present in at least one of the fusions. DNA encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and cotransfected into suitable host cells. This allows greater flexibility in adjusting the relative proportions of the three polypeptide fragments in embodiments where unequal ratios of the three polypeptide chains used in the construction provide optimal yields of the desired bispecific antibody. However, it is possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when expression of equal ratios of at least two polypeptide chains results in high yields or when the ratio does not significantly affect the yield of the desired chain combination.

[0043] Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. For example, Brennan et al., Science, 229:81 (1985) describes a procedure in which intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of a dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The generated Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The generated bispecific antibody can be used as an agent for the selective immobilization of enzymes.

[0044] Other modifications of antibodies are contemplated herein. For example, antibodies can be conjugated to one of a variety of non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. Antibodies can also be entrapped in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by coacervation or interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).

[0045] Typically, the antibodies of the described invention are recombinantly produced using vectors and methods available in the art. Human antibodies can also be produced by in vitro activated B cells (see, e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275). General methods of molecular genetics and genetic engineering useful in the present invention are described in detail in 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”i n Methods in Enzymology (MPDeutshcer, 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 These techniques are described in the current editions of Basic Technique, 2nd Ed. (RI 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, NJ). Reagents, cloning vectors, and kits for genetic manipulation are available from commercial vendors, such as BioRad, Stratagene, Invitrogen, ClonTech, and Sigma-Aldrich Co.

[0046] Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array into such germline mutant mice results in the production of human antibodies upon antigen challenge. See, for example, Jakobovits et al. 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); U.S. Patent Nos. 5,545,806, 5,569,825, and 5,591,669 (all GenPharm); U.S. Patent No. 5,545,807; and WO 97 / 17852. Such animals can be genetically engineered to produce human antibodies comprising the polypeptides of the described invention.

[0047] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were obtained by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and ScFv antibody fragments can all be expressed in and secreted from E. coli, allowing the facile production of large amounts of these fragments. Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (see, e.g., Carter et al., Bio / Technology 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragments with increased in vivo half-lives that contain salvage receptor binding epitope residues are described in U.S. Patent No. 5,869,046. Other methods for producing antibody fragments will be apparent to those skilled in the art.

[0048] Phage display, ribosome 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 10:1 Other techniques known in the art for selecting antibody fragments from libraries using enrichment methods, including, but not limited to, those disclosed in U.S. Pat. Nos. 3,033,303-1310, ... See US Patent Nos. 2,160, 6,521,404, 6,544,731, 6,555,313, 6,582,915, 6,593,081, and other members of the U.S. patent family, or technology from Cambridge Antibody Technology (CAT) as disclosed in the priority application based on British Patent No. 9206318, filed May 24, 1992; see also Vaughn, et al. 1996, Nature Biotechnology 14:309-314.) Single-chain antibodies may be designed and constructed using available recombinant DNA technology, such as DNA amplification methods (e.g., PCR), or possibly by using the respective hybridoma cDNA as a template.

[0049] Mutant antibodies are also within the scope of the present invention. Thus, variants of the sequences described in this application are also within the scope of the present invention. Further variants of antibody sequences with improved affinity can be obtained using methods known in the art, and these variants are within the scope of the present invention. For example, amino acid substitutions can be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of nucleotide sequences can be used to improve the translation efficiency in expression systems for antibody production.

[0050] Such variant antibody sequences will share 70% or greater (i.e., 80%, 85%, 90%, 95%, 97%, 98%, 99% or greater) sequence identity with the sequences described herein. Such sequence identity is calculated relative to the full length of the reference sequence (i.e., the sequence described herein). As referred to herein, percent identity is as determined using BLAST version 2.1.3 with the default parameters specified by NCBI (National Center for Biotechnology Information; www.ncbi.nlm.nih.gov / ) [Blosum 62 matrix; gap opening penalty = 11 and gap extension penalty = 1]. For example, peptide sequences comprising at least about 5, 10, 15, 20, 30, 40, 50, 75, 100, 150 or more contiguous peptides of one or more of the sequences disclosed herein, and all intermediate lengths therebetween, are provided by the present invention. As used herein, the term "intermediate length" is intended to describe any length between the cited values, for example, 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.

[0051] The present invention provides antibodies with broad neutralizing activity in serum, alone or in combination with other antibodies, such as, but not limited to, VRC01, anti-V3 loop, CD4bs and CD4i antibodies, and PG9 / PG16-like antibodies.

[0052] According to another embodiment, the present invention provides methods for preparing and administering HIV antibody compositions suitable for administration to human or non-human primate patients infected with or at risk of HIV infection in amounts and on schedules sufficient to induce a protective immune response against HIV or reduction of HIV virus in humans.

[0053] According to another embodiment, the present invention provides a vaccine comprising at least one antibody of the present invention and a pharmaceutically acceptable carrier. According to one embodiment, the vaccine is a vaccine comprising at least one antibody described herein and a pharmaceutically acceptable carrier. The vaccine may comprise any combination of antibodies having the properties described herein, and may further comprise an antibody that neutralizes HIV, as known in the art.

[0054] It should be understood that the composition may be a single antibody disclosed herein or a combination of the same or different antibodies disclosed herein to prophylactically or therapeutically treat the progression of HIV infection of various subtypes after vaccination. Such combinations can be selected according to the desired immunity. When the antibody is administered to an animal or human, it can be combined with one or more pharmaceutically acceptable carriers, excipients, or adjuvants as known to those of ordinary skill in the art. The composition may further comprise broadly neutralizing antibodies known in the art, including, but not limited to, VRC01, b12, anti-V3 loop, CD4bs, and CD4i antibodies, and PG9 / PG16-like antibodies.

[0055] Furthermore, particularly with regard to determining the efficacy level of HIV treatment in patients, suitable animal models are available and have been widely implemented to evaluate the in vivo efficacy of various gene therapy protocols against HIV (Sarver et al. (1993b), supra). These models include mice, monkeys, and cats. Even though these animals are not naturally susceptible to HIV disease, chimeric mouse models (e.g., SCID, bg / nu / xid, NOD / SCID, SCID-hu, immunocompetent SCID-hu, myeloablated BALB / c) reconstituted with human peripheral blood mononuclear cells (PBMCs), lymph nodes, fetal liver / thymus, or other tissues can be infected with lentiviral vectors or HIV and used as models of HIV pathogenesis. Similarly, simian immunodeficiency virus (SIV) / monkey models and feline immunodeficiency virus (FIV) / cat models can also be used. When used for the therapeutic treatment of AIDS, the pharmaceutical composition can contain other pharmaceutical agents in addition to the vectors of the present invention. These other pharmaceutical agents can be used in their traditional manner (i.e., as agents for treating HIV infection).

[0056] According to another embodiment, the present invention provides an antibody-based pharmaceutical composition comprising an effective amount of an isolated HIV antibody, or an affinity-matured version thereof, which provides a prophylactic or therapeutic treatment option for reducing infection with the HIV virus. The antibody-based pharmaceutical composition of the present invention may be formulated by any number of strategies known in the art (see, for example, McGoff and Scher, 2000, Solution Formulation of Proteins / Peptides: In McNally, EJ, ed. Protein Formulation and Delivery. New York, NY: Marcel Dekker; pp. 139-158; Akers and Defilippis, 2000, Peptides and Proteins as Parenteral Solutions. In: Pharmaceutical Formulation Development of Peptides and Proteins. Philadelphia, PA: Talyor and Francis; pp. 145-177; Akers, et al., 2002, Pharm. Biotechnol. 14:47-127). Pharmaceutically acceptable compositions suitable for administration to a patient will contain an effective amount of antibody in a formulation that retains biological activity and also promotes maximum stability during storage within an acceptable temperature range. The pharmaceutical composition may contain a pharmaceutically acceptable diluent, a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient, or a pharmaceutically acceptable carrier for animal or human administration, depending on the desired formulation. Vehicles commonly used in formulating pharmaceutical compositions may also be included. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, phosphate-buffered saline, Ringer's solution, dextrose solution, and Hank's solution. The amount of excipient useful in the pharmaceutical composition or formulation of the present invention is an amount that serves to uniformly distribute the antibody throughout the composition so that the composition can be uniformly dispersed when administered to a subject in need thereof. It may be useful to dilute the antibody to a concentration that provides the desired beneficial palliative or curative results while minimizing any adverse side effects that may result from excessively high concentrations. It may also have a preservative effect. Thus, for antibodies with high biological activity, more excipient will be used. Conversely, for any active ingredient(s) exhibiting lower biological activity, less excipient will be used.

[0057] The above antibodies, as well as antibody or vaccine compositions comprising at least one or a combination of the antibodies described herein, can be administered for the prophylactic and therapeutic treatment of HIV viral infection.

[0058] The present invention also relates to isolated polypeptides comprising the novel amino acid sequences of the light and heavy chains, as well as the consensus sequences for the heavy and light chains of SEQ ID NOs: 1 and 2, as listed in FIG.

[0059] In other related embodiments, the present invention provides polypeptide variants that encode the amino acid sequences of the HIV antibodies listed in Figure 3; the consensus sequences for the heavy and light chains of SEQ ID NOS: 1 and 2. These polypeptide variants have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the polypeptide sequences of the present invention as determined using the methods described herein (e.g., BLAST analysis using standard parameters). Those skilled in the art will recognize that these values can be appropriately adjusted to determine the corresponding identity of the encoded proteins, taking into account amino acid similarity and the like.

[0060] The term "polypeptide" is used in its conventional sense, i.e., as a sequence of amino acids. A polypeptide is not limited to a particular length of the product. Peptides, oligopeptides, and proteins are included in the definition of polypeptide, and such terms can be used interchangeably herein unless specifically indicated otherwise. The term also includes post-expression modifications of the polypeptide, both naturally occurring and non-naturally occurring, such as glycosylation, acetylation, phosphorylation, and other modifications known in the art. A polypeptide can be an entire protein or a subsequence thereof. A particular polypeptide of interest in the context of the present invention is an amino acid subsequence comprising a CDR, a VH, and a VL that is capable of binding to an antigen or an HIV-infected cell.

[0061] Polypeptide "variants," as that term is used herein, are polypeptides that typically differ from the polypeptides specifically disclosed herein by one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or may be synthetically produced, for example, by modifying one or more of the above polypeptide sequences of the invention and evaluating one or more biological activities of the polypeptides described herein, and / or by using any of a number of techniques well known in the art.

[0062] For example, certain amino acids can be substituted for other amino acids in a protein structure without appreciable loss of its ability to bind to other polypeptides (e.g., antigens) or cells. Because the binding ability and properties of a protein define its biological functional activity, certain amino acid sequence substitutions can be made in the protein sequence and, therefore, in the underlying DNA code. It is contemplated that various modifications can be made to the peptide sequences of the disclosed compositions, or the corresponding DNA sequences encoding said peptides, without significant loss of their biological utility or activity.

[0063] In many cases, a polypeptide variant will contain one or more conservative substitutions, where one amino acid is substituted for another amino acid with similar properties, such that one skilled in the art of peptide chemistry would predict that the secondary structure and hydropathic properties of the polypeptide will remain substantially unchanged.

[0064] Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. For example, substitutions that take into account the various aforementioned properties are well known to those of skill in the art and include arginine for lysine; glutamate for aspartate; serine for threonine; glutamine for asparagine; and valine for leucine and isoleucine.

[0065] "Homology" or "sequence identity" refers to the percentage of residues in a polynucleotide or polypeptide sequence variation that are identical to the non-variant sequence after aligning the sequences and, if necessary, inserting gaps to achieve maximum homology. In certain embodiments, 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% polynucleotide or polypeptide homology with the polynucleotides or polypeptides described herein.

[0066] Such variant polypeptide sequences will share 70% or more (i.e., 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) sequence identity with the sequences described herein. In further embodiments, the described invention provides polypeptide fragments comprising contiguous stretches of various lengths of the amino acid sequences disclosed herein. For example, peptide sequences comprising at least about 5, 10, 15, 20, 30, 40, 50, 75, 100, 150 or more contiguous peptides of one or more of the sequences disclosed herein, and all intermediate lengths therebetween, are provided by the invention.

[0067] The present invention also includes nucleic acid sequences encoding part or all of the light and heavy chains of the antibodies of the present invention, and fragments thereof. Due to redundancy in the genetic code, variants of these sequences will exist that encode the same amino acid sequences.

[0068] The present invention also includes isolated nucleic acid sequences encoding the heavy and light chain peptides of the HIV antibodies listed in FIG. 3, as well as the heavy and light chain consensus sequences of SEQ ID NOs: 1 and 2.

[0069] In other related embodiments, the described invention provides polynucleotide variants that encode the heavy and light chain polypeptide sequences of the HIV antibodies listed in Figure 3; the heavy and light chain consensus sequences of SEQ ID NOs: 1 and 2. These polynucleotide variants have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity compared to the polynucleotide sequences of the invention as determined using methods described herein (e.g., BLAST analysis using standard parameters). Those skilled in the art will understand that these values can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences, taking into account codon degeneracy, amino acid similarity, reading frame alignment, and the like.

[0070] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to single- or double-stranded RNA, DNA, or mixed polymers. Polynucleotides can include genomic sequences, additional genomic and plasmid sequences, and smaller engineered gene segments that express or can be adapted to express a polypeptide.

[0071] An "isolated nucleic acid" is a nucleic acid that is substantially separated from other genomic DNA, as well as from proteins or complexes, e.g., ribosomes and polymerases, that naturally accompany the native sequence. This term encompasses a nucleic acid sequence that has been removed from its naturally occurring environment, including recombinant or cloned DNA isolates, as well as chemically synthesized analogs, or analogs biosynthesized by heterologous systems. A substantially pure nucleic acid includes the nucleic acid in its isolated form. Thus, it refers to a nucleic acid as originally isolated and does not exclude genes or sequences later added to the isolated nucleic acid by the hand of man.

[0072] Polynucleotide "variant," as the term is used herein, refers to a polynucleotide that typically differs from the polynucleotides specifically disclosed herein by one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or may be synthetically produced, for example, by one or more modifications of a polynucleotide sequence of the invention and evaluating one or more biological activities of the encoded polypeptides described herein, and / or by using any of a number of techniques well known in the art.

[0073] Modifications can be made to the structure of the polynucleotides of the presently described invention to still obtain functional molecules that encode variant or derivative polypeptides with desired properties. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent or even improved variant or portion of a polypeptide of the invention, one skilled in the art will typically change one or more of the codons in the encoding DNA sequence.

[0074] Typically, polynucleotide variants contain one or more substitutions, additions, deletions and / or insertions such that the immunogenic binding properties of the polypeptides encoded by the variant polynucleotides are not substantially diminished compared to the polypeptides encoded by the polynucleotide sequences specifically set forth herein.

[0075] In further embodiments, the described invention provides polynucleotide fragments comprising contiguous stretches of various lengths of sequences identical to or complementary to one or more of the sequences disclosed herein, for example, polynucleotides comprising at least about 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, or 1000 or more contiguous nucleotides of one or more of the sequences disclosed herein, and all intermediate lengths therebetween, including any length between the recited values, e.g., 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 all integers between 200 and 500, 500 and 1000, are provided by the present invention.

[0076] In another embodiment of the present invention, polynucleotide compositions are provided that are capable of hybridizing to the polynucleotide sequences provided herein, or fragments thereof, or complementary sequences thereof, under moderate to high stringency conditions. Hybridization methods are well known in the art of molecular biology. By way of illustration, suitable moderately stringent conditions for testing hybridization of the polynucleotides of the present invention with other polynucleotides are: The conditions include a prewash in a solution of 5x SSC, 0.5% SDS, and 1.0 mM EDTA (pH 8.0); overnight hybridization at 50-60°C in 5x SSC; followed by two 20-minute washes at 65°C, each in 2x, 0.5x, and 0.2x SSC containing 0.1% SDS. Those 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 highly stringent hybridization conditions include those described above, except that the hybridization temperature is increased, for example, to 60-65°C or 65-70°C.

[0077] In some embodiments, the polypeptides encoded by the polynucleotide variants or fragments have the same binding specificity (i.e., specifically or preferentially bind to the same epitope or HIV strain) as the polypeptide encoded by the native polynucleotide. In some embodiments, the presently described polynucleotides, polynucleotide variants, fragments, and hybridization sequences encode polypeptides having a level of binding activity that is at least about 50%, at least about 70%, and at least about 90% of that for the polypeptide sequences specifically set forth herein.

[0078] Regardless of the length of the coding sequence itself, the total length of the polynucleotides of the described invention, or fragments thereof, can vary considerably because they can be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc. Nucleic acid fragments of almost any length can be used. For example, exemplary polynucleotide segments having lengths 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, etc. (including all intermediate lengths) are included in many embodiments of the invention.

[0079] Vectors, such as expression vectors, containing the nucleic acid sequences according to the invention are also included within the scope of the present invention. Cells transformed with such vectors are also included within the scope of the present invention.

[0080] The present invention also provides vectors and host cells containing the nucleic acids of the invention, as well as recombinant methods for producing the polypeptides of the invention. Vectors of the invention include those capable of replicating in any type of cell or organism, including, for example, plasmids, phages, cosmids, and minichromosomes. In some embodiments, a vector containing a polynucleotide of the described invention is a vector suitable for growth or replication of the polynucleotide, or a vector suitable for expression of a polypeptide of the described invention. Such vectors are known in the art and commercially available.

[0081] "Vector" includes shuttle vectors and expression vectors. Typically, a plasmid construct will also include an origin of replication (e.g., the ColE1 origin of replication) and a selectable marker (e.g., ampicillin or tetracycline resistance) for replication and selection, respectively, of bacterial plasmids. "Expression vector" refers to a vector containing control sequences or regulatory elements necessary for expression of antibodies, including antibody fragments, of the invention in bacterial or eukaryotic cells.

[0082] As used herein, the term "cell" can be any cell, including, but not limited to, of a eukaryotic, multicellular species such as, but not limited to, a mammalian cell or a human cell (as opposed to, for example, a unicellular yeast cell). A cell can exist as a single entity or can be part of a larger collection of cells. Such a "larger collection of cells" can include, for example, cell cultures (mixed or pure), tissues (e.g., endothelial, epithelial, mucosal, or other tissues), organs (e.g., lung, liver, muscle, and other organs), organ systems (e.g., circulatory system, respiratory system, etc.). , gastrointestinal system, urinary system, nervous system, integumentary system or other organ system), or organism (e.g., bird, mammal, etc.).

[0083] Polynucleotides of the invention may be synthesized in whole or in portions and assembled and inserted into vectors using routine molecular and cell biology techniques, including, for example, subcloning the polynucleotide into a linear vector using appropriate restriction sites and restriction enzymes. Polynucleotides of the described invention are amplified by the polymerase chain reaction using oligonucleotide primers complementary to each strand of the polynucleotide. These primers also contain restriction enzyme cleavage sites to facilitate subcloning into a vector. Replicable vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, and one or more marker or selectable genes.

[0084] To express a polypeptide of the present invention, a nucleotide sequence encoding the polypeptide, or a functional equivalent, may be inserted into an appropriate expression vector, i.e., a vector containing the necessary elements for the transcription and translation of the inserted coding sequence. Methods well known to those skilled in the art may be used to construct an expression vector containing a sequence encoding the polypeptide of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA methods, synthetic methods, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook, J., et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY, and Ausubel, F M et al. al.(1989)Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY.

[0085] The present invention also provides kits useful for performing diagnostic and prognostic assays using the antibodies, polypeptides, and nucleic acids of the present invention. The kits of the present invention include a suitable container containing the HIV antibodies, polypeptides, or nucleic acids of the present invention, either in labeled or unlabeled form. In addition, when the antibodies, polypeptides, or nucleic acids are provided in a labeled form suitable for an indirect binding assay, the kits further include reagents for performing the appropriate indirect assay. For example, the kits may include one or more suitable containers containing an enzyme substrate or derivatizing agent, depending on the nature of the label. Control samples and / or instructions may also be included. The present invention also provides kits for detecting the presence of HIV antibodies of the present invention or the nucleotide sequence of an HIV antibody in a biological sample by PCR or mass spectrometry.

[0086] As used herein, a "label" refers to a detectable compound or composition that is attached directly or indirectly to an antibody to produce a "labeled" antibody. Labels can also be attached to the polypeptides and / or nucleic acid sequences disclosed herein. The label can be detectable alone (e.g., radioisotope labels or fluorescent labels) or, in the case of enzymatic labels, can catalyze chemical alteration of a substrate compound or composition that is detectable. The antibodies and polypeptides of the described invention can also be modified to contain epitope tags or labels, for example, for use in purification or diagnostic applications. Suitable detection means include the use of labels, such as, but not limited to, radionucleotides, enzymes, coenzymes, fluorescers, chemiluminescers, chromogens, enzyme substrates or cofactors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes, etc.

[0087] According to another embodiment, the present invention provides a method of diagnosis, which generally involves contacting a biological sample taken from a patient, such as blood, serum, saliva, urine, sputum, a cell swab sample or a tissue biopsy, with HIV antibodies, and detecting the presence of the antibodies in the sample, a control sample or includes comparing the antibody to a predetermined cutoff value to determine whether it selectively binds, thereby indicating the presence of HIV virus.

[0088] According to another embodiment, the present invention provides a method for detecting the presence of an HIV antibody of the present invention in a biological sample from a patient. The detection method generally involves collecting a biological sample from the patient, such as blood, serum, saliva, urine, sputum, a cell swab sample, or a tissue biopsy, isolating an HIV antibody or a fragment thereof, or a nucleic acid encoding an HIV antibody, and testing the biological sample for the presence of the HIV antibody. The present invention also provides a method for detecting the nucleotide sequence of an HIV antibody in a cell. The nucleotide sequence of the HIV antibody may be detected using the primers disclosed herein. The presence of the HIV antibody in the biological sample from the patient may be determined by known recombinant methods and / or by using a mass spectrometer.

[0089] In another embodiment, the present invention provides a method for detecting an HIV antibody comprising a heavy chain containing a highly conserved consensus sequence and a light chain containing a highly conserved consensus sequence in a biological sample, the method comprising: obtaining an immunoglobulin-containing biological sample from a mammalian subject; isolating an HIV antibody from the sample; and identifying the highly conserved consensus sequences of the heavy and light chains. The biological sample may be blood, serum, saliva, urine, sputum, a cell swab sample, or a tissue biopsy. The amino acid sequence may be determined by methods known in the art, including, for example, PCR and mass spectrometry.

[0090] The term "assessing" includes any form of measurement and includes determining whether an element is present or not. The terms "determining," "measuring," "evaluating," "assessing," and "assaying" are used interchangeably and include quantitative and qualitative determinations. Assessment can be relative or absolute. "Assessing the presence of" includes determining the amount of something present and / or determining whether it is present or absent. As used herein, the terms "determining," "measuring," "assessing," and "analyzing" are used interchangeably and include both quantitative and qualitative determinations.

[0091] II. Methods of Reducing Viral Replication Further provided are methods for reducing an increase in HIV viral titer, viral replication, viral growth, or the amount of HIV viral proteins in a subject. According to another embodiment, a method includes administering to a subject an HIV antibody in an amount effective to reduce an increase in HIV titer, viral replication, or the amount of HIV proteins of one or more HIV strains or isolates in the subject.

[0092] According to another embodiment, the present invention provides a method for reducing viral replication or reducing the spread of HIV infection to additional host cells or tissues, the method comprising contacting a mammalian cell with an antibody or portion thereof that binds to an antigenic epitope on gp120.

[0093] III. Treatment method According to another embodiment, the present invention provides a method for treating a mammal infected with a viral infection, such as HIV, comprising administering to said mammal a pharmaceutical composition comprising an HIV antibody as disclosed herein. According to one embodiment, the method for treating a mammal infected with HIV comprises administering to said mammal a pharmaceutical composition comprising an antibody or fragment thereof of the present invention. The composition of the present invention can be administered to said mammal a pharmaceutical composition comprising an antibody or fragment thereof as disclosed herein. The composition may include more than one antibody (e.g., multiple antibodies or a pool of antibodies) with the same specificity. The composition may also include other HIV-neutralizing antibodies as known in the art, such as, but not limited to, VRC01, PG9, and b12.

[0094] Passive immunization has proven to be an effective and safe strategy for the prevention and treatment of viral diseases. (See, e.g., 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); and Igarashi et al., Nat. Med. 5:211-16 (1999)). Passive immunization using human monoclonal antibodies offers an emergency therapeutic strategy for the acute prevention and treatment of HIV.

[0095] A subject at risk for an HIV-related disease or disorder includes a patient who has been in contact with an infected person or who has been exposed to HIV in some other way. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of an HIV-related disease or disorder in order to prevent or slow the progression of the disease or disorder.

[0096] For in vivo treatment of human and non-human patients, pharmaceutical formulations comprising the HIV antibodies of the present invention are administered or provided to the patient. When used for in vivo therapy, the antibodies of the present invention are administered to the patient in a therapeutically effective amount (i.e., an amount that eliminates or reduces the patient's viral load). The antibodies are administered to human patients according to known methods, for example, intravenously, e.g., as a bolus or by continuous infusion over a period of time, intramuscularly, intraperitoneally, intracerebrospinal, subcutaneously, intra-articularly, intrasynovially, intrathecally, orally, topically, or by inhalation. The antibodies can be administered parenterally, at the target cell site if possible, or intravenously. In some embodiments, the antibodies are administered intravenously or subcutaneously. The therapeutic compositions of the present invention can be administered systemically, parenterally, or locally to the patient or subject. The above parameters for assessing successful treatment and disease improvement are easily measured by routine procedures well known to physicians.

[0097] For parenteral administration, the antibody may be formulated in a unit dosage form for injection (solution, suspension, emulsion) with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles include, but are not limited to, fixed oils and ethyl oleate. Liposomes can be used as carriers. The vehicle may also contain small amounts of additives, such as substances that improve isotonicity and chemical stability, such as buffers and preservatives. The antibody can be formulated in such a vehicle at a concentration of about 1 mg / mL to 10 mg / mL.

[0098] The dosage and administration regimen will depend on various factors readily determined by a physician, such as the nature of the infection, e.g., its therapeutic index, the patient, and the patient's medical history. Generally, a therapeutically effective amount of antibody is administered to a patient. In some embodiments, the amount of antibody administered ranges from about 0.1 mg / kg to about 50 mg / kg of patient body weight. Depending on the type and severity of the infection, about 0.1 mg / kg to about 50 mg / kg of body weight (e.g., about 0.1-15 mg / kg / dose) of antibody is an initial candidate dosage for administration to a patient, e.g., by one or more separate administrations or by continuous infusion. The progress of this treatment is easily monitored by conventional methods and assays and based on criteria known to physicians or others skilled in the art. The above parameters for assessing treatment success and disease improvement are readily measurable by routine procedures known to physicians.

[0099] Other therapies may be administered in combination with the HIV antibodies of the present invention. Combination administration includes simultaneous administration using separate formulations or a single pharmaceutical formulation, and sequential administration in any order, preferably with a period during which both (or all) active agents simultaneously exert their biological activities. Such combination therapy can produce a synergistic therapeutic effect. The above parameters for assessing treatment success and disease improvement can be easily measured by routine procedures well known to physicians.

[0100] The terms "treat" or "treatment" or "palliative" are used interchangeably and refer to both therapeutic treatment and prophylactic or preventative measures, where the goal is to prevent or slow (alleviate) the targeted pathological condition or disorder. Those in need of treatment include those who already have the disorder, as well as those who are susceptible to the disorder or those in whom the disorder must be prevented. A subject or mammal is successfully "treated" for an infection if, after receiving a therapeutic amount of an antibody according to the methods of the invention, the patient exhibits an observable and / or measurable reduction in one or more of the following, or the absence of one or more of the following: a reduction in the number of infected cells or the absence of infected cells; a reduction in the percentage of total cells that are infected; and / or some alleviation of one or more of the symptoms associated with the particular infection; reduced morbidity and mortality, and improved quality of life issues. The above parameters for assessing successful treatment and improvement of a disease are readily measurable by routine procedures known to physicians.

[0101] The term "therapeutically effective amount" refers to an amount of an antibody or drug effective to treat a disease or disorder in a subject or mammal.

[0102] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and sequential administration in any order.

[0103] As used herein, a "carrier" includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is nontoxic to the cells or mammals exposed at the dosages and concentrations employed. Often, the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants, including but not limited to ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as, but not limited to, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as, but not limited to, polyvinylpyrrolidone; amino acids, such as, but not limited to, glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates (including, but not limited to, glucose, mannose, or dextrin); chelating agents, such as, but not limited to, EDTA; sugar alcohols, such as, but not limited to, mannitol or sorbitol; salt-forming counterions, such as, but not limited to, sodium; and / or non-ionic surfactants, such as, but not limited to, TWEEN, polyethylene glycol (PEG), and PLURONICS.

[0104] Where a range of values is given, it is understood that each intervening value between the upper and lower limit of that range, to one-tenth of the unit of the lower limit, and any other stated or intervening value in the stated range, is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges, which may be independently included in these smaller ranges, are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both limits, ranges excluding either of both of those included limits are also included in the invention. [Example]

[0105] Example 1 This example describes the materials and methods used in Examples 2-5 below.

[0106] HIV antibodies were cloned and generated according to gp140-specific single B cell capture as previously described (Mouquet, H. et al. PLoS One 6, e24078 (2011); Tiller, T. et al. J Immunol Methods 329, 112-24 (2008); and Scheid, JF et al. Nature 458, 636-40 (2009)). GM and 10-1074 GM "Glycomutant" antibodies were generated by substituting residues 10-1074 at HC positions 32, 53, 54, 58, 97, 1001 with PGT121 and vice versa. The binding properties of anti-gp140 antibodies to HIV Env proteins have been previously described (Scheid, JF et al. Science 333, 1633-7 (2011); Walker, L M et al. Nature 477, 466-70 (2011); and Mouquet, H. et al. PLoS Neutralization was assessed using ELISA, SPR, and glycan microarray assays as previously described (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 primary HIV-1 mutants as previously described (Li, M. et al. J Virol 79, 10108-25 (2005); Euler, Z. et al. Journal of Virology 85, 7236-45 (2011); and Bunnik, E. M. et al. Nature Medicine 16, 995-7 (2010)). The structures of PGT121 (unliganded and liganded), 10-1074, and GL Fab fragments were solved by molecular replacement to 2.8 Å, 2.3 Å, 1.8 Å, and 2.4 Å resolution, respectively.

[0107] Single B cell RT-PCR and Ig gene analysis gp140 from PBMCs of patient 10 (pt10; referred to as patient 17 in Nature 477(7365):466-470) + CD19 + IgG + Single-cell sorting of B cells, cDNA synthesis, and nested PCR amplification of Ig genes were performed as previously described (PLoS One 6(9):e24078). The Igλ genes expressed by the PGT121 clonal variants were amplified using a forward primer (L-Vλ3-21) further upstream within the leader region to avoid regions potentially susceptible to mutation (31). * The sequence was PCR amplified using the nucleotide sequence 02:5'CTGGACCGTTCTCCTCCTCG 3'. All PCR products were sequenced and analyzed for Ig gene usage, CDR3 analysis, and VH / Vκ somatic hypermutation counts (IgBLAST; http: / / www.ncbi.nlm.nih.gov / igblast and IMGT®; http: / / www.imgt.org). Multiple sequence alignments were performed using the MacVector program (v. 12.5.0) with the ClustalW analysis function (default parameters) and used to generate phylogenetic trees by the neighbor-joining method (best-tree mode and outgroup-rooted method). Alternatively, phylogenetic trees were generated using the UPGMA method (best-tree mode).

[0108] Germline (GL) precursor gene segments 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 / ). H 4-59 * 01, J H 6 * 03. V L 3-21 * 02 and J L 3 *02. (These gene segments are the most frequently used in the human antibody repertoire (PLoS One 6(8):e22365; Immunogenetics 64(5):337-350). To construct a representative GL ancestral sequence, we The IgH and IgL sequences of 10-996 (an antibody containing minimal somatic hypermutation) were aligned to the GL sequence using IgBLAST (http: / / www.ncbi.nlm.nih.gov / igblast). The GL IgH sequence was derived from the mature V H and J. H The gene segments are replaced with their GL counterparts, and the N region nucleotides and D H The GL IgL sequence was constructed by using the 10-996 sequence for the CDRH3 region containing the gene segment. L 3-21 * 02 and J L 3 * It was constructed from 02 gene segment sequences.

[0109] Antibody cloning and production The purified and digested PCR products were cloned into human Igγ1 or Igλ expression vectors (J Immunol Methods 329(1-2):112-124). The vectors containing the IgH and Igλ genes were then sequenced and compared with the original PCR product sequences. PGT121 and 10-303 share the same Igλ gene and have one amino acid difference at position 2 of the IgH gene (Figure 4). Therefore, to produce PGT121 IgG, we used the 10-303Igλ and PGT121 IgH genes, which were generated by introducing a single substitution (V2M) into the 10-303IgH gene by site-directed mutagenesis (QuikChange Site-Directed Mutagenesis Kit; Stratagene). To generate His-tagged Fab, we used the IgG1 C HPGT121 and 10-1074 V were synthesized by modifying our standard Igγ1 vector (Science 301(5638):1374-1377) to encode the 1 domain followed by a 6x-His tag. H The gene was subcloned into the 6xHis-IgCγ1 expression vector. GM (S32Y, K53D, S54R, N58T, H97R, T100lY) and 10-1074 GM IgH DNA fragments encoding the (Y32S, D53K, R54S, T58N, R97H, Y1001T) mutant antibodies were obtained as synthetic minigenes (IDT) and subcloned into an Igγ1 expression vector.

[0110] 10-1074 GM The heavy chain sequences for 10-1074 are listed below with mutations underlined. GM The light chain sequence of this antibody is the same as that of 10-1074. QVQLQESGPGLVKPSETLSVTCSVSGDSMNN S YWTWIRQSPGKGLEWIGYIS KS ESA N YNPSLNSRVVISRDTSKNQLSLKLNSVTPADTAVYYCATAR H GQRIYGVVSFGEFF T YYSMDVWGKGTTVTVSS.

[0111] Antibodies and Fab fragments were purified using polyethyleneimine (PEI)-precipitation (PLoS One IgH and IgL antibodies were produced by transient transfection of exponentially growing HEK293T cells (ATCC, CRL-11268) with IgH and IgL expression plasmids using the ELISA kit (Protein G, 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 HisPur™ Cobalt resin (Thermo Scientific) as described below.

[0112] HIV-1 Env protein Alanine mutations were introduced into positions 301 to 303 (Asn-Asn-Thr), 324 to 325 (Gly-Asp), and 332 (Asn) (HXBc2 amino acid numbering) of the pYU-2 gp120 vector (a gift from J. Sodroski, Harvard Medical School) using the QuikChange Site-Directed Mutagenesis kit (Stratagene) according to the manufacturer's instructions. The same procedure was used to introduce alanine mutations into the pYU-2 gp120 vector (a gift from J. Sodroski, Harvard Medical School) at positions 301 to 303 (Asn-Asn-Thr), 324 to 325 (Gly-Asp), and 332 (Asn) (HXBc2 amino acid numbering). N332A Asn262 in the vector gp120 and Asn406 gp120 For each PNGS located between A "double glycan" mutant was generated by introducing a single alanine mutation. Site-directed mutations were confirmed by DNA sequencing.

[0113] YU-2 gp140 (Journal of virology 74(12):5716-5725), YU-2 gp120, HXB2c gp120 コア HEK 293T cells were transfected with expression vectors encoding the high-mannose-only YU-2 gp120 protein (Nature 393(6686):648-659), HXB2c 2CC core protein (PLoS Pathog 5(5):e1000445), and the YU-2 gp120 mutant protein. kif To produce α-glucanase (α-glucanase), 25 μM kifunesine (Enzo Life Sciences) was added at the time of transfection. Culture supernatants were collected and concentrated using a centrifugation-based filtration device (Vivacell 100, Sartorius Stedim Biotech GmbH) that allowed for buffer exchange of the sample into 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.

[0114] For the deglycosylation reaction, 50 μg of HEK 293T cell-produced YU-2 gp120 in PBS was incubated overnight at 37°C with 200 U of PNGase F (New England Biolabs) or 10,000 U of Endo H in the respective reaction buffer without denaturant. f After buffer exchange with PBS using a centrifugal filter (Amicon® Ultra, Millipore), glycosidase-treated gp120 (200 ng) was examined by SDS-PAGE using 4–12% NuPAGE gels (Invitrogen) followed by silver staining (Pierce Silver Stain Kit, Thermo Scientific).

[0115] ELISAs High-binding 96-well ELISA plates (Costar) were coated overnight with 100 ng / well of purified gp120 in PBS. After washing, the plates were blocked for 2 hours with 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 for the ELISA using the YU-2 gp120 double glycan mutant), diluted 1:4 seven times serially in PBS. After washing, the plates were developed by a 1-hour incubation with goat HRP-conjugated anti-human IgG antibody (Jackson ImmunoResearch) at 0.8 μg / mL in blocking buffer and the addition of HRP chromogenic substrate (ABTS solution, Invitrogen) (PLoS One 6(9):e24078). Selected gp120 V3 Antibody binding to the overlapping peptides was tested using the peptide-ELISA method described previously.

[0116] For competitive ELISAs, gp120-coated plates were blocked with blocking buffer for 2 hours and then incubated with biotinylated antibodies (PGT121 at 26.6 nM, 10-1074 at 0.21 nM, 10-996 at 0.43 nM, and 10-1369 at 1.67 nM) in 1:2 serial dilutions of antibody competitors in PBS (IgG concentration range of 5.2 to 667 nM). Plates were developed as described above using HRP-conjugated streptavidin (Jackson ImmunoResearch) at 0.8 μg / mL in blocking buffer. All experiments were performed at least in duplicate.

[0117] Glycan microarray analysis Microarrays were generated by robotically printing lipid-conjugated glycan probes (neoglycolipids) at two levels (2 and 5 fmol / spot) in replicates onto nitrocellulose-coated glass slides (Methods Mol Biol 808:117-136). Binding assays were performed using microarrays containing 15 neoglycolipids derived from high-mannose and complex-type N-glycans. The probe sequences are shown in Figure 7A. Briefly, antibodies were tested at 50 μg / mL, and binding was detected with biotinylated anti-human IgG (Vector) followed by AlexaFluor 647-labeled streptavidin (Molecular Probes).

[0118] Surface plasmon resonance Experiments were performed using a Biacore T100 (Biacore, Inc) (Nature 467(7315):591-595). Briefly, YU-2 gp140 and gp120 proteins were primary amine coupled onto a CM5 chip (Biacore, Inc) at a coupling density of 300 RU. Anti-gp120 IgG and germline precursor (GL) were injected onto the flow cell at 1 μM and 10 μM, respectively, at a flow rate of 35 μL / min with a 3-minute association phase and a 5-minute dissociation phase. The sensor surface was regenerated by a 30-second injection of 10 mM glycine-HCl pH 2.5 at a flow rate of 50 μL / min. Dissociation (k d (seconds -1 )), meeting (k a (M -1 seconds -1 ) and the binding constant (K D (M) or K A (M -1 ) was calculated from kinetic analysis after background subtraction using a 1:1 binding model without bulk reflection coefficient (RI) correction (Biacore T100 Evaluation software). The binding constant for bivalent IgG calculated using the 1:1 binding model was calculated as K D To emphasize that the values include potential avidity effects, they are referred to in this document as "apparent" affinities.

[0119] Neutralization assay Virus neutralization was assessed using a luciferase-based assay in TZM.bl cells (J Virol 79(16):10108-10125). The HIV-1 pseudoviruses tested primarily contained tier-2 and tier-3 viruses (Journal of Virology 84(3):1439-1452) (Tables 4 and 5). High-mannose-only pseudoviruses were neutralized in wild-type cells (Enzo Life Sciences) treated with 25 μM kifunesine (Figure 8C) or HEK293S GnTI. - / - Nonlinear regression analysis was used to determine the concentration at which half-maximal inhibition was observed (IC 50We calculated the seroconversion values (S values) using a previously characterized PBMC-based assay (Journal of Virology 85(14):7236-7245; Nat Med 2014) using infections with primary HIV-1 variants (n = 95) isolated from clade B-infected donors with known seroconversion dates between 1985 and 1989 ("past seroconverters," n = 14) or clade B-infected donors with known seroconversion dates between 2003 and 2006 ("current seroconverters," n = 21). The neutralizing activity of each antibody was evaluated using IC values ranging from 0.001 to 50 μg / mL, which were calculated as the percentage of virus neutralized. 50 The relative area under the curve (RAUC) was calculated by normalizing all AUC values by the highest value (obtained at 10-1074).

[0120] statistical analysis Statistical analysis was performed with GraphPad Prism software (v5.0b). The apparent binding affinity of antibodies to gp120 and gp140 relative to their neutralization potency in the TZM-bl assay against a panel of nine selected virus strains was analyzed using the Spearman correlation test. The Mann-Whitney test was used to compare (i) the affinity of antibodies belonging to the PGT121 or 10-1074 groups to gp120 / gp140, and (ii) the past and future associations. The neutralizing activity against viruses isolated from current and current seroconverters was compared.

[0121] Crystallization and structure determination 6x-His-tagged PGT121, 10-1074, and 10-996GL Fabs were expressed for crystallization. The Fabs were extracted from the supernatant of transiently transfected HEK293-6E cells by sequential Ni addition. 2+Crystals of the ligand-free PGT121 Fab were purified by β-NTA affinity chromatography (Qiagen) and Superdex200 10 / 300 (GE Healthcare) size-exclusion chromatography. For the preparation of the ligand-free PGT121 Fab, PGT121 IgG was isolated from the supernatant of transiently transfected HEK293-6E cells by Protein A affinity chromatography (Pierce), and the Fab fragment was obtained by papain digestion of the IgG and further purified by Superdex200 10 / 300 (GE Healthcare) size-exclusion chromatography.

[0122] Purified Fab was concentrated to 8-20 mg / mL in PBS buffer ("unliganded" PGT121, 8 mg / mL; 10-1074 and GL, 20 mg / mL). "Liganded" PGT121 Fab crystals were prepared from protein samples (final concentration: 15 mg / mL) mixed with a 3-fold molar excess of NA2 glycan and incubated at 20 °C for 2 h. Crystallization conditions were screened using a Mosquito® crystallization robot (TTP labs) in 400 nL droplets at 20 °C using a 1:1 protein-to-reservoir ratio. Crystals of "unliganded" PGT121 Fab (P212121; a = 56.8, b = 74.7, c = 114.9 Å) were obtained in 24% PEG 4,000, 0.1 M Tris-HCl pH 8.5, 10 mM CuCl2, and crystals of "liganded" PGT121 Fab (P212121; a = 67.8, b = 67.8, c = 94.1 Å) were obtained in 17% PEG 10,000, 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 Å; β = 95.39°) were obtained in 25% PEG 3,350, 0.1 M Bis-Tris pH 5.5, 0.2 M NaCl, and crystals of GL Fab (P21; a = 54.9, b = 344.7, c = 55.2 Å; β = 91.95°) were obtained in 20% PEG 3,350, 0.24 M sodium malonate pH 7.0, 10 mM MnCl. Crystals were cryoprotected by immersion in mother liquor containing 20% glycerol (unliganded and liganded PGT121 Fab) or 20% ethylene glycol (10-1074 Fab and GL Fab), followed by flash cooling with liquid nitrogen.

[0123] Diffraction data were collected at beamline 12-2 (wavelength = 1.029 Å) using a Pilatus 6M pixel detector (Dectris) at the Stanford Synchrotron Radiation Lightsource (SSRL). Data were indexed, integrated, and scaled using XDS. Using data from the "ligand-free" PGT121 Fab crystal, we used Phenix to generate two search models after removing residues in the CDRH3 and CDRL3 loops: the C of PGT128 Fab, and the C of PGT128 Fab. H -C L domain (PDB code 3PV3) and V of 2F5 H -V L We used the domain (PDB code 3IDJ) to find molecular replacement solutions for one Fab per asymmetric unit (chain H and L for heavy and light chains, respectively). We then used the "unliganded" PGT121 structure as a search model to find molecular replacement solutions for the "liganded" PGT121 Fabs (one Fab per asymmetric unit), 10-1074 Fabs (one Fab per asymmetric unit), and GL (four Fabs per asymmetric unit).

[0124] Iterative refinement (including non-crystallographic symmetry constraints for GL) was performed using Phenix. The models were manually fitted to the electron density map using Coot. Atomic models were fitted to 3.0 Å resolution (R work =21.6%, R free =26.4%), and for 10-1074 Fab, the resolution was 1.9 Å (R work =18.7%, R free =22.3%) and 2.4 Å resolution (R work =19.4%, R free =23.7%) and for the "liganded" PGT121 Fab at 2.4 Å resolution (R work =20.1%, R freeThe PGT121 Fab atomic model contained 95.2%, 4.9%, and 0.0% of residues within the favored, allowed, and disallowed regions of the Ramachandran plot, respectively (10-1074 Fab: 98.8%, 0.9%, 0.2%; GL Fab: 96.0%, 3.8%, 0.23%; PGT121 Fab with ligand: 96.7%, 3.1%, 0.2%). PyMOL was used for molecular visualization and to generate diagrams of the Fab structure. Buried surface area calculations were performed with Areamol (CCP4 suite) using a 1.4 Å probe.

[0125] Fab structures were aligned using the Super script in PyMOL. Pairwise Cα alignments were performed using PDBeFold.

[0126] Example 2 Predominance and Diversity of PGT121 Clonotypes We isolated gp140-specific IgG memory B cells from clade A-infected African donors using YU-2 gp140 trimers as bait. We identified 87 matched immunoglobulin heavy (IgH) and light (IgL) chain genes corresponding to 23 unique clonal families. One clonal family dominated the IgH anti-gp140 repertoire, representing approximately 28% of all expanded B cell clones. This B cell family corresponds 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 their IgH nucleotide sequences, the PGT121 family divided into two groups: a PGT121-like group containing PGT121-123 and nine closely related variants, and a second group, 10-1074-like, containing 20 members. Although our previous primers (J Immunol Methods 329(1-2):112-124; Science 301(5638):1374-1377) did not amplify IgL genes expressed by the PGT121B cell clone due to a nucleotide deletion in the region encoding framework region 1, we used new Igλ-specific primers designed to amplify highly somatically mutated genes to obtain 24 of the 38 Igλ genes (Table 3). Consistent with the high level of hypermutation in IgH genes (average of 18.2% of VH genes), the amplified Igλ genes were highly mutated (average of 18.2% of Vλ genes) and contained nucleotide deletions (12 to 21 nucleotides) in framework region 1 (FWR1) and 9-nucleotide insertions in framework region 3 (FWR3) (Figure 3B and Table 3).

[0127] Sequence alignments of three PGT antibodies (PGT-121, -122, and -123), eleven PGT121 and 10-1074 clonal variants (10-259, 10-303, 10-410, 10-847, 10-996, 10-1074, 10-1121, 10-1130, 10-1146, 10-1341, 10-1369, and 10-1074GM), putative germline (GL), and consensus sequences are shown in Figures 3(a) and 3(b). Sequences for the corresponding heavy chain variable regions, light chain variable regions, heavy chain CDRs, and light chain CDRs according to both the IMGT and KABT systems are listed in Table 1 below. Sequence identification numbers assigned to the sequences according to the KABT system are listed in Table 2 below.

[0128] [Table 1-1]

[0129] [Table 1-2]

[0130] [Table 1-3]

[0131] [Table 1-4]

[0132] [Table 2]

[0133] Eleven novel, unique mutants were expressed (Table 3) and demonstrated binding to YU-2 gp120 and gp140 by ELISA and surface plasmon resonance (SPR). Unless otherwise noted, gp120 and gp140 proteins for these and other experiments were expressed in mammalian cells capable of binding either complex or high-mannose N-glycans to PNGS. The level of reactivity with gp120 differed between the PGT121 and 10-1074 group antibodies, with the latter exhibiting higher apparent affinity (Figure 3A), primarily due to slower dissociation from gp120 / gp140 for the 10-1074-related antibody (Figure 4B).

[0134] Example 3 PGT121 and 10-1074 epitopes Asn332 near the V3 loop stem gp120 It has been reported that V3 is crucial for binding and virus neutralization by PGT121 (Nature 477(7365):466-470). Therefore, we investigated the role of V3 in antigen recognition by PGT121-like and 10-1074-like antibodies. コア ) or the HXB2 gp120 "core" protein, which retains a portion of V3 (2CC-core), and the YU-2 gp120 mutant protein with a double alanine substitution in the V3 stem (gp120 GD324-5AA ) was used to perform ELISA. The antibodies tested were those that inhibited the V3 loop-deleted mutant and gp120 compared to intact YU-2 gp120. GD324-5AA Both antibodies showed decreased reactivity to V3, with binding of the 10-1074 group antibodies being the most affected (Figure 5A and B). These results suggest that recognition by both antibody groups requires protein determinants near the V3 loop. None of the antibodies bound to overlapping peptides spanning V3, suggesting that the target epitope is discontinuous and / or requires a specific conformation not achieved by the isolated peptide (Figure 5C).

[0135] Asn332 gp120(Previous numbering (J Proteome Res 7(4):1660-1674) Asn337 gp120 ) has the sequence Asn-X-Ser / T Asn332 is the N-terminal residue of a potential N-glycosylation site (PNGS) defined as hr. gp120 To determine whether the N-linked glycans and / or their N-linked glycans are required for the gp120 reactivity of the novel PGT121 and 10-1074 group antibodies, we analyzed the YU-2 gp120 antibody by ELISA. N332A The N332A substitution reduced binding of PGT121 and all novel antibody variants, but not of a mutant gp120 lacking the adjacent glycosylation site (gp120 NNT301-3AAA Their reactivity to the Asn332 mutant was unchanged. gp120 To determine whether PNGS, in addition to PNGS, affects recognition by novel antibodies, we compared the N332A mutation in YU-2 gp120 with Asn262. gp120 and Asn406 gp120 A series of 11 double glycan mutants were constructed, combining mutations in PNGS located between them. All PGT121-like and 10-1074-like antibodies were found to be ubiquitous on gp120. N332A The glycans bound to each of the double glycan mutants with comparable affinity to each other.

[0136] To compare total glycan recognition by PGT121-like and 10-1074-like antibodies, we used PNGase, which cleaves both complex-type and high-mannose N-glycans. We investigated their binding to YU-2 gp120 treated with PNGase F. Because gp120 cannot be enzymatically deglycosylated completely unless it is denatured, PNGase F is a promising candidate for this purpose. PNGase F treatment resulted in partial deglycosylation of natively folded gp120 (Fig. 6). Nevertheless, the reactivity of the two groups of antibodies differed in that partial deglycosylation of gp120 with PNGase F reduced the binding activity of all PGT121-like antibodies but not any of the 10-1074-like antibodies (Fig. 6C). Similar experiments performed with YU-2 gp120 treated with Endo H, which cleaves high-mannose N-glycans but not complex-type N-glycans, affected the binding of 10-1074-like antibodies more than PGT121-like antibodies (Fig. 6D).

[0137] N-glycan microarrays revealed that six of the seven tested PGT121-like antibodies exhibited detectable binding to complex mono- or biantennary N-glycans terminated with galactose or α2-6-linked sialic acid, but not to high-mannose glycans. This confirms and extends previous reports of the lack of binding of PGT121-123 to high-mannose N-glycans and the lack of competition by the Man4 and Man9 dendrons for gp120 binding (Figure 7). In contrast, there was no detectable binding to protein-free glycans by the 10-1074-like antibody (Figure 7). PGT121-like antibodies bound to protein-free complex N-glycans but not to high-mannose N-glycans, whereas PGT121-like antibodies bound to YU-2 gp120 (gp120) produced in cells treated with kifunesin, a mannosidase inhibitor that results in exclusive binding of high-mannose glycans to PNGS. kif ) (Fig. 8B). Most of the PGT121-like antibodies retained binding to gp120 kif On the other hand, the 10-1074-like antibody exhibited a small but reproducible decrease in binding to gp120. kif These results are consistent with the hypothesis that complex-type N-glycans, as well as high-mannose N-glycans, may contribute to the epitope of PGT121-like antibodies.

[0138] Epitope mapping experiments were performed by competitive ELISA using two representative members of each group (PGT121 and 10-1369 for the PGT121-like group; 10-1074 and 10-996 for the 10-1074-like group). All four antibodies showed cross-competition, but PGT121 inhibited the binding of 10-996 and 10-1074 to gp120 more slowly than the reverse. To further map the target epitope, we investigated the crown of the V3 loop (Figure 5), CD4bs, the coreceptor binding site (CD4-induced; CD4i), and a group of high-mannose N-glycans (2G12) (Journal of Virology 76(14):7293-7305; Proc Natl Acad Sci USA, 2014). i USA 102(38):13372-13377) or an anti-gp120 antibody that recognizes the V3 loop and N-linked glycans at positions 301 and 332 (PGT128) was used. An anti-V3 crown antibody inhibited the binding of PGT121 and 10-1369 but did not interfere with the binding of 10-996 and 10-1074. PGT128, and to a lesser extent 2G12, reduced the binding of all four antibodies to gp120, but CD4bs and CD4i antibodies did not.

[0139] Taken together, these data suggest that the PGT121 clone members contain the V3 loop and Asn332. gp120 This suggests that they recognize sites containing protein determinants in the vicinity of related glycans. However, the clones fall into two families, the PGT121-like and the 10-1074-like, which differ in their affinity for gp120 and the role of the glycans in epitope formation.

[0140] Example 4 Broad and potent HIV neutralization To evaluate the neutralizing activity of novel PGT121 mutants, we measured their ability to inhibit HIV infection of TZM-bl cells using 10 virus strains, including R1166.c1, which lacks PNGS at gp120 position 332. All PGT121 mutants, including the 10-1074-like antibody, neutralized nine pseudoviruses, and none neutralized the R1166.c1 control (Figure 1A and Table 4). Neutralizing activity correlated with affinity for the HIV spike, with the 10-1074 group showing slightly greater potency than the PGT121 group (Figure 1B and Figure 4C). A representative germline version (GL) of the PGT121 / 10-1074 antibody clonotype was unable to bind gp120 / gp140 or neutralize any of the viruses in the panel, implying that somatic mutations are required for binding and neutralization. Pairing of the GL light chain with the mutant 10-1074- or 10-996-group heavy chains failed to rescue binding or neutralization, suggesting that both mutant chains contribute to the correct construction of the antibody paratope.

[0141] The following assays were performed to compare the neutralizing activity of PGT121 with that of 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) (Tables 4 and 5). 10-996 and 10-1074 demonstrated neutralization potency and breadth similar to PGT121 (Figure 1C, Figure 9, and Tables 5 and 6). As expected, most viruses with amino acid changes at gp120 positions 332 and / or 334 (spanning Asn332-X-Ser334 / Thr334) were resistant to neutralization (83.8% resistant to PGT121 and 100% resistant to 10-1074 and 10-996). The majority of viruses resistant to neutralization (68.5% for 10-996, 72.5% for 10-1074, and 60.8% for PGT121) were due to mutations in this PNGS (Table 7). Comparable neutralizing activity was observed for the IgG and Fab forms of PGT121 and 10-1074, suggesting that bivalency is not important for their activity (Figure 1D).

[0142] To assess the potential role of complex N-glycans on the HIV envelope in neutralization by PGT121 and 10-1074, we produced high-mannose-only virions by two different methods: by assembly of pseudoviruses in cells treated with kifunesin, resulting in Man9GlcNAc2 N-linked glycans, or by HEK293S GnTI - / - This method results in Man5GlcNAc2 N-linked glycans assembled in cells. We found that PGT121 neutralized two of the three kifunesine-derived PGT121-sensitive / 10-1074-resistant strains equivalently to their counterparts produced in wild-type cells (Fig. 8C). - / - In cells Two PGT121-sensitive / 10-1074-resistant virus strains produced in GnTI cells were equally sensitive to PGT121 and 10-1074 as their counterparts produced in wild-type cells. Consistent with previous reports that complex N-glycans protect the CD4 binding site to some extent from antibody binding, GnTI - / - Virus produced in cells was detected by CD4 binding site antibody (NIH45-46 G54W and 3BNC60) (Fig. 8D).

[0143] Example 5: Newly circulating HIV-1 Next, we investigated the activity of PGT121 and 10-1074 against the transmitted founder virus by assessing neutralization in a peripheral blood mononuclear cell (PBMC)-based assay using 95 clade B viruses isolated from a cohort of individuals who seroconverted between 1985 and 1989 ("past seroconverters," n = 14) or between 2003 and 2006 ("current seroconverters," n = 25) (51, 52). We compared PGT121 and 10-1074 with anti-CD4bs bNAbs and other bNAbs, including VRC01, PG9 / PG16, b12, 2G12, 4E10, and 2F5. Clustering analysis of neutralizing activity showed separation into two groups; the PGT121 / 10-1074 group contained the most active HIV neutralizers, including anti-CD4bs and PG9 antibodies (Table 8). Surprisingly, 10-1074 demonstrated exceptional neutralization potency against this panel of clade B viruses, exhibiting the greatest breadth (67% of the 95 clade B viruses) at 0.1 μg / mL for all bNAbs tested (Table 8). While 10-1074 demonstrated higher potency (~20-fold difference) than PGT121 against current clade B viruses, both antibodies were more effective against historical viruses than current viruses (Figure 1E and Figure 10).

[0144] Example 6 Crystal structures of PGT121, 10-1074 and GL To investigate the structural determinants of the differences between PGT121- and 1074-like antibodies, we solved the crystal structures of the Fab fragments of PGT121, 10-1074, and a representative germline precursor (GL) at 3.0 Å, 1.9 Å, and 2.4 Å resolution, respectively (Table 9). The heavy and light chain variable domains (V) in the three Fabs were significantly different from each other. H and V L ) overlay revealed that differences relative to GL were limited to small displacements in the CDRH3 and CDRL3 loops of the affinity-matured Fab, with conservation of main-chain structure (Table 10).

[0145] An unusual feature shared by antibodies is their long (25 residue) CDRH3 loop, which is located at the V H A two-stranded antiparallel β-sheet extends domains F and G. In each Fab, the tip of the extended CDRH3 loop contains primarily non-polar residues. Similar structural features were observed for the CDRH3 of PGT145, a carbohydrate-sensitive antibody whose epitope contains the gp120 V1V2 loop. However, the extended two-stranded β-sheet of CDRH3 of PGT145 contains primarily negatively charged residues, including two sulfated tyrosines at the tip. The V1V2 loops of PGT121 and PGT145 H -V L The alignment (Table 10) is PGT145 Previously, CDRH3 PGT121 and its tip and V H The domains are aligned, but CDRH3 of PGT121, 10-1074, and GL are V L V L CDRH3 for those PGT121 / CDRH3 10-1074 / CDRH3 GL The slope of opens a cleft between CDRH2 and CDRH3, a feature not shared by related antibodies.

[0146] PGT121 and 10-1074 differ significantly from GL and from each other (132 residues in PGT121 VH is 10-1074VH and G.L. VH and 10-1074, which differ by 36 and 45 residues, respectively. VH and GL VH (29 differences). Most of these PGT121 / 10-1074 differences are due to the CDR VH Interestingly, six substitutions in CDRH3 (residues 100d, 100f, 100h, 1 00j, 100l, 100n) alternate so that every two residues are replaced, resulting in V L The cleft between CDRH2 and CDRH3 resurfaces due to the tilt of CDRH3 toward the nucleus. This region likely contributes to the different fine specificities of PGT121 and 10-1074. HC Five other solvent-exposed substitutions in GL (residues 64, 78, 80-82; chains D and E) are potential antigen contact sites, given that the framework regions in HIV antibodies can contact gp120. Other differences that may contribute to subtle specificity differences include Asp56, which is absent in 10-1074 or GL. HC The negative patch on PGT121 (10-1074 and Ser56 in GL) HC ), as well as positive patches on CDRL1 and CDRL3 that are not found on the analogous surface of GL.

[0147] Somatic mutations common to PGT121 and 10-1074 may contribute to the shared characteristics of their epitopes. The heavy chains of PGT121 and 10-1074 share only three common mutations (out of 36 PGT121-GL differences and 29 10-1074-GL differences). In contrast, PGT121 and 10-1074 share 18 common light chain mutations (out of 37 PGT121-GL differences and 36 10-1074-GL differences), including an insertion in light chain FWR3 that causes a bulge in the loop connecting chains D and E, and a CDRL2 mutation that results in a low-negative surface. GL Asp50 in LC -Asp51 LCAsn50 in both PGT121 and 10-1074 LC -Asn51 LC Contains substitutions for LC PGT121 and L.C. 10-1074 The majority of common substitutions (approximately 50% of the LC substitutions) introduced into CDRL1, CDRL2, and FWR2 are potential contact regions for the epitope shared by PGT121 and 10-1074. LC points out.

[0148] Next, Asn332 gp120 Bond and Asn301 gp120 Comparison was made with the structure of PGT128, which recognizes the linked glycan and V3 and was solved in complex with the ectodomain / mini-V3 loop gp120 expressed in cells unable to produce complex N-glycan modified proteins. Unlike the CDRH3 loop of PGT121 and 10-1074, PGT128 CDRH3 PGT128 VL It is not leaning towards CDRH3 PGT128 does not contain a two-stranded β-sheet. PGT128 (18 residues) is shorter than the CDRH3 of PGT121 and 10-1074 (24 residues), but CDRH2 PGT128 contains a six-residue insertion that is not found in PGT121 or 10-1074. Because of these differences, CDRH2 is the most prominent feature in PGT128, whereas CDRH3 is the most prominent feature in PGT121 and 10-1074. PGT128 and CDRL3 PGT128 together, Asn332 gp120 Man bound to 8 / 9 Recognizes CDRH3 PGT128 makes contact with the base of the V3 loop. This mode of gp120 recognition is not possible for PGT121 and 10-1074 because the structural features of their CDRH2 and CDRH3 loops differ significantly from those of PGT128, consistent with the ability of PGT128, but not PGT121 and 10-1074, to recognize protein-free high-mannose glycans (Figure 7).

[0149] Example 7 Crystal structure of PGT121-glycan complex A 2.4 Å resolution structure of PGT121 with a complex sialylated biantennary glycan was solved (Table 9) using crystals obtained under conditions containing NA2, a complex asialyl biantennary glycan (Figure 7). Surprisingly, the glycan bound to PGT121 in our crystal structure is not NA2, but rather a complex N-glycan from the adjacent PGT121 Fab in the crystal lattice, specifically Asn105. HC The identity of this glycan was an N-glycan attached to Asn105. HC This is clear because there is electron density for the glycosidic bond to the Manα1-3Man antenna and for the terminal sialic acid on the Manα1-3Man antenna (M The galactose and sialic acid moieties of the anα1-6Man antenna were not elucidated. The composition of the linked glycans corresponded to the portion of the α2-6 sialylated A2(2-6) glycans attached by PGT121 in the microarray experiments (Fig. 7) and corresponded to the predicted sialyl linkages in the complex-type N-glycans attached to PNGS on proteins expressed in HEK293T cells. The V of this structure ("liganded" PGT121) H -V L The domain is V in the PGT121 structure without the attached N-glycan ("unliganded" PGT121). H -V L The domains overlapped without significant difference (Fig. 10), but the elbow bend angle (V H -V L and C H 1-C L The angle between the pseudo-dyads differs between the structures, likely reflecting the flexibility of Fab to adopt elbow bend angles that can vary depending on the crystal lattice forces.

[0150] Given that binding of complex N-glycans was observed in one crystal structure (the "liganded" PGT121 structure) but not in another (the "unliganded" PGT121 structure), we speculate that the affinity of PGT121 for complex N-glycans not bound to gp120 is within the range of the PGT121 concentration in the crystal (~10 mM). The K for binding isolated glycans D 1.6 mM K obtained for PG9 binding to Man5GlcNAc2-Asn D The K for PGT121 binding of isolated glycans is assumed to be in the range of 1–10 mM, comparable to D represents only a small contribution to the affinity of PGT121 for gp120, which is in the nM range (Fig. 4A).

[0151] The glycans in the "liganded" PGT121 structure are V H It interacts exclusively with the PGT121 domain and makes extensive contacts with residues in all three CDRs ( HC Top buried surface area = 600 Å 2 The contacts involve 10 direct hydrogen bonds with nine amino acids and 18 water-mediated hydrogen bonds (Figure 11), anchoring the glycan between the N-acetylglucosamine moiety attached to the mannose at the branch site and the terminal sialic acid on the 1-3 antenna. Asp31 HC In addition to the water-mediated hydrogen bond with PGT121 residue Asp31 HC and His97 HC This sialic acid makes several contacts with PGT121, including three direct hydrogen bonds with . Sialic acids also contribute to the water-mediated intraglycan hydrogen bond network. Direct contacts with sialic acids may explain the stronger binding of PGT121 to sialylated A2(2-6) glycans than to nonsialylated NA2 glycans in our glycan microarray analysis (Figure 7). Extensive water-mediated protein contacts established by the N-acetylglucosamine and galactose moieties of the 1-3 antennae could explain the observed binding of nonsialylated mono- and biantennary glycans to PGT121 (Figure 7).

[0152] Six of the residues (Ser32) contribute to direct or likely amino acid side chain contacts to the glycan. HC-CDRH1 , Lys53 HC-CDRH2 , Ser54 HC-CDRH2 , Asn58 HC-CDRH2 , His97 HC-CDRH3 , Thr100l HC-CDRH3 ) is 10-1074 (Tyr32 HC-CDRH1 , Asp53 HC-CDRH2 , Arg54 HC-CDRH2 , Thr58 HC-CDRH2 , Arg97 HC-CDRH3 , Tyr100l HC-CDRH3 ), which is highly conserved among PGT121-like antibodies but not among 10-1074-like antibodies. The 10-1074 residue lacks a corresponding functional group to make the observed glycan contacts or to have a bulky side chain that would cause a steric clash. Four of these residues are similar to those in GL (Tyr32 HC-CDRH1 , Tyr53 HC-CDRH2 , Gln97 HC-CDRH3 , Tyr100l HC-CDRH3 ), which suggests that the lack of binding of 10-1074-like antibodies and GL to protein-free complex glycans in our glycan microarrays may be due to hydrogen bonding and / or steric clashes (e.g., Arg97 10-1074 In contrast, His97 PGT121 ;Tyr100l 10-1074 In contrast, Thr100l PGT121 ) Like most of the sequence differences between PGT121 and 10-1074 groups in the CDRH loops, particularly for the surface of the cleft between CDRH2 and CDRH3 where we observed bound complex-type N-glycans, differential recognition of complex-type glycans on gp120 may account for some or all of the observed differences in their fine specificity.

[0153] Example 8 Substitution of Glycan-Containing Antibody Residues Affects Neutralization To assess the contribution of complex-type N-glycan-containing residues identified from the "liganded" PGT121 structure, we generated two mutant antibodies designed to exchange complex-type glycan-containing residues between PGT121 and 10-1074: 10-1074 IgG with six substitutions at PGT121 residues (IgH Y32S, D53K, R54S, T58N, R97H, Y1001T), and PGT121 IgG with the reciprocal substitutions. GM and PGT121 GM ) displayed near-wild-type apparent affinity for YU-2 gp120 / gp140 as measured by SPR (Fig. 2A), demonstrating that the substitutions did not disrupt binding to envelope spikes from virus strains neutralized by both PGT121 and 10-1074 (Fig. 1A). The ability to accommodate PGT121 complex-type N-glycan-containing residues within the 10-1074 background without disrupting binding to gp120 / gp140 bound by both wild-type antibodies implies overall similar antigen binding despite subtle specificity differences.

[0154] Unlike wild-type PGT121, PGT121 GM showed no glycan binding in microarray experiments. This confirms that residues at substitution position 10-1074 are not compatible with protein-free glycan binding (Figure 2B) and supports the suggestion that residues in contact with glycans in the "liganded" PGT121 structure are involved in the recognition of complex glycans in the microarray. GM also showed no binding to protein-free glycans (Fig. 2B), indicating that residues other than those substituted are involved in generating binding sites for protein-free complex N-glycans.

[0155] Next, we compared the neutralization of wild-type and "glycomutant" antibodies using a TZM-bl-based assay. We tested 40 virus strains, including strains with different resistance to PGT121 or 10-1074, as well as strains sensitive to both wild-type antibodies (Figure 2C and Table 12). PGT121 binding to purified YU-2 envelope protein GM and 10-1074 GM Consistent with binding, both mutants neutralized YU-2 virus, but 64% of the PGT121-susceptible strains were neutralized by PGT121. GM (Figure 2C and Table 12). This suggests that the glycan-containing residues identified in the "liganded" PGT121 structure are involved in the neutralizing activity of PGT121. Conversely, 10-1074 GM exhibited higher average potency against 10-1074-susceptible strains than wild-type 10-1074, including a greater than 3-fold increase in potency against four 10-1074-susceptible strains (WITO4160.33, ZM214M.PL15, Ce1172_H1, and 3817.v2.c59) (Figure 2C and Table 12). In general, PGT121 substitution into 10-1074 significantly increased the potency of 10-1074 in PGT121-susceptible / 10-1074-resistant strains. GM Although two of these strains (CNE19 and 62357_14_D3_4589) do not confer susceptibility to 10-1074 GM (IC 50 = 0.19 μg / mL and 40.8 μg / mL). Interestingly, these were gp120 This is the only PGT121-susceptible / 10-1074-resistant strain that contains the combined PNGS. The other PGT121-susceptible / 10-1074-resistant strains have Asn332 gp120 Lacking the linking glycan and PGT121 GM and 10-1074 GM These antibodies were resistant to wild-type PGT121, implying that their sensitivity to wild-type PGT121 requires compensation by adjacent N-glycans and / or the protein portion of the epitope. A dramatic gain of function was observed in 10-1074 strains (CNE19).GM This result was observed only for the 10-1074 strain against the 10-1074 susceptible strain. GM Together with the general improvement observed for 10-1074 (Figure 2C), this is consistent with the interpretation that crystallographically identified glycan-containing residues may transfer PGT121-like recognition properties to 10-1074 in some situations and / or affect its potency in other situations. GM The loss of neutralizing activity of PGT121 provides evidence that the neutralizing activity of PGT121 requires residues identified in the "liganded" PGT121 structure as contacting complex N-glycans.

[0156] result PGT121 is a glycan-dependent bNAb originally identified in the serum of a clade A-infected donor in a functional screen that yields only two clonally related members. Using gp140 trimers as "bait" for single-cell sorting, 29 new clonal variants were isolated. The PGT121 clonal family contains distinct groups of closely related antibodies: the PGT121 group and the 10-1074 group. Results show that the epitopes of both groups are located at Asn332. gp120 The PGT121-like and 10-1074-like antibody groups differ in amino acid sequence, gp120 / gp140 binding affinity, and neutralizing activity. The 10-1074-like antibody exhibits neutralization at Asn332. gp120 Although PGT121-like antibodies are completely dependent on intact PNGS in gp120 It was able to neutralize some virus strains lacking PNGS.

[0157] A notable difference between the two antibody groups is that PGT121-like antibodies bound complex N-glycans in the carbohydrate array, whereas 10-1074-like antibodies showed no detectable binding to any of the protein-free N-glycans tested (Figure 7). Protein-free glycan binding by anti-HIV antibodies is not always detected; for example, PG9 recognizes gp120-associated high-mannose glycans, but binding to any protein-free glycans was not detected in the microarray. Thus, a positive result in the glycan microarray implies engagement of a specific glycan with the antibody epitope, whereas a negative result does not exclude glycan recognition. For example, high-mannose glycans can engage the PGT121 epitope, even if not detectable in the glycan microarray experiment, which is consistent with binding and neutralization of the high-mannose-only form of gp120 protein and virions (Figure 8).

[0158] The molecular basis for the differences between PGT121, 10-1074, and their GL precursors was partially revealed by their crystal structures. The finding that the majority of light chain somatic mutations are shared between PGT121 and 10-1074, while the heavy chain mutations are distinct, suggests that the light chains contact a shared portion of the gp120 epitope, while the heavy chains recognize distinct features. All three antibodies display an extended CDRH3 with a nonpolar tip that may allow access to cryptic epitopes. Differences in the antigen-binding sites of the two mature Fabs were primarily confined to the cleft between CDRH2 and the extended CDRH3. Interestingly, the putative antigen-binding cleft between CDRH2 and CDRH3 was also found in representative germline precursors of PGT121 and 10-1074.

[0159] V HStructural information regarding glycan recognition by PGT121-like antibodies was obtained from the crystal structure, in which complex sialylated N-glycans attached to domain residues interact with the binding site of the adjacent PGT121Fab. Several features of the "liganded" PGT121 structure suggest that it is suitable for understanding the recognition of complex N-glycans on gp120 by PGT121-like antibodies. First, the glycan in the structure corresponding to the α2-6 sialylated glycan A2(2-6)PGT121 binds in the microarray (Figure 7). Second, The glycan interacts with PGT121 using the cleft between CDRH3 and CDRH2, which structural analysis suggests is involved in epitope recognition, as seen in the V in the PGT121 and 10-1074 structures. L This potentially explains the unusual tilt of CDRH3 toward V. Third, V was identified as interacting with glycans. H The majority of residues differ between PGT121 and 10-1074, which rationalizes the different binding profiles in glycan microarrays and potentially explains the different fine specificities revealed in protein binding experiments. Fourth, the exchange of crystallographically identified glycan contact residues between PGT121 and 10-1074 partially transferred their properties: PGT121 GM Like 10-1074, it did not bind to protein-free glycans, but PGT121 GM and 10-1074 GM Both PGT121 and PGT122 conserved near-wild-type binding to purified YU-2 gp120 / gp140. GM PGT121 retained the ability to neutralize some virus strains neutralized by wild-type PGT121 and 10-1074, but failed to neutralize strains that were PGT121-sensitive / 10-1074-resistant. This provides evidence that the glycan-binding motif is essential for PGT121's neutralizing activity against 10-1074-resistant strains. Consistent with the transfer of crystallographically identified glycan motifs for reciprocal exchange and the hypothesis that the epitopes of PGT121- and 10-1074-like antibodies are related, 10-1074 GMThe neutralizing potency of 10-1074 was increased or unaffected relative to 10-1074, and in some cases, GM potently neutralized PGT121-susceptible / 10-1074-resistant strains. In analyses of gp120 sequences from strains for which PGT121 neutralization data were available, apart from interactions with PNGS at Asn332gp120 for viruses susceptible to PGT121-like and 10-1074-like antibodies, no clear patterns of PNGS utilization emerged for different categories of virus strains (PGT121-susceptible / 10-1074-susceptible, PGT121-susceptible / 10-1074-resistant, PGT121-resistant / 10-1074-susceptible), except that 10-1074-resistant strains generally lacked Asn332gp120-associated PNGS.

[0160] Example 9 Passive transfer of anti-HIV-1 neutralizing mAb in vivo Five isolated, potent, and broadly active anti-HIV neutralizing monoclonal antibodies were administered to rhesus macaques, who were then challenged intrarectally with one of two different SHIVs 24 hours later. Combined results from 60 challenged animals demonstrated protective neutralizing titers in plasma of approximately 1:100, preventing viral acquisition in 50% of exposed monkeys.

[0161] Animal experiments The macaques used in this study were of MHC class I Mamu-A * The test was negative for the 01 allele.

[0162] Construction of R5-tropic SHIVDH12-V3AD8 These V3 sequences were introduced into the genetic background of the pSHIVDH12_CL7 molecular clone (J. of Virology 78, 5513-5519 (2004)) using PCR mutagenesis with primers corresponding to the 5' and 3' halves of the gp120 V3 coding region of SHIVAD8EO (PNAS 109, 19769-19774 (2012)) (forward primer: AGAGCATTTTATACAACAGGAGACATAATAGGAGATATAAGACAAGCACATTGCAACATTAGTAAAGTAAAATGGC, and reverse primer: TCCTGGTCCTATATGTATACTTTTCCTTGTATTGTTGTTGGGTCTTGTACAATTAATTTCTACAGTTTCATTC) using Platinum PFX DNA polymerase (Invitrogen). After gel purification, the PCR product was treated with T4 polynucleotide kinase (GibcoBRL) and blunt-end ligated to generate pSHIVDH12_V3AD8, which was used to transform competent cells.

[0163] virus First, viral stocks were prepared by transfecting 293T cells with the SHIVAD8EO or SHIVDH12-V3AD8 molecular clone using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Culture supernatants were harvested 48 hours later, and aliquots were stored at -80°C until use. Concanavalin A-stimulated rhesus monkey PBMCs (2 × 10 in 500 μL) were used. 6 Cells) were infected with transfected cell supernatant by spinoculation for 1 hour (J. of Virology 74, 10074-10080 (2000)), mixed with an equal number / volume of activated PBMCs, and cultures were maintained for at least 12 days with daily replacement of culture medium. Samples of supernatant medium were pooled around the time of peak RT production to prepare individual virus stocks.

[0164] antibody Eleven monoclonal antibodies (VRC01, NIH45-46, 45-46G54W, 45-46m2, 3BNC117, 12A12, 1NC9, and 8ANC195, 10-1074, PGT121, and PGT126) were isolated and produced. DEN3, a dengue virus NS1-specific human IgG1 monoclonal antibody (PNAS 109, 18921-18925 (2012)), or a control human IgG (NIH Nonhuman Primate Reagent Resource, http: / / www.nhpreagents.org) was used as a negative control antibody in this study. Selected monoclonal antibodies were administered intravenously for preexposure passive transfer 24 hours before virus challenge infection.

[0165] Quantification of plasma viral RNA levels Viral RNA levels in plasma were determined by real-time reverse transcription-PCR (ABI Prism 7900HT Sequence Detection System; Applied Biosystems).

[0166] Plasma antibody concentration The concentrations of administered monoclonal antibodies in monkey plasma were measured by enzyme-linked immunosorbent assay (ELISA) using recombinant HIV-1 JRFL gp120 (Progenics Pharmaceuticals) or HIV IIIB (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. The plates were washed with PBS / 0.05% Tween-20 and blocked with 1% (vol / vol) BSA. After blocking, serial dilutions of antibody or plasma samples were added to the plates and incubated for 1 hour at room temperature. Binding was detected with alkaline phosphatase-conjugated goat anti-human IgG F(ab)2 fragment (Pierce) and visualized with SIGMAFAST OPD (Sigma-Aldrich). The decay half-life of neutralizing monoclonal antibodies was calculated using a monoexponential decay equation based on plasma concentrations starting on the 5th or 7th day after antibody administration (J. of Virology 84, 1302-1313 (2010)).

[0167] Neutralization assay The in vitro potency and neutralizing activity of each mAb present in plasma samples collected from rhesus macaques was assessed by two types of neutralization assays: 1) the TZM-bl entry assay with pseudotyped challenge virus (AIDS Res Hum Retroviruses 26, 89-98 (2010)) or 2) a 14-day PBMC replication assay with replication-competent virus (J. of Virology 76, 2123-2130 (2002)). For the TZM-bl assay, pseudotyped viruses expressing the env gene from SHIVAD8EO or SHIVDH12_V3AD8, each containing the respective envelope tag, were used. Serially diluted mAb or plasma samples were incubated with pseudotyped virus prepared by cotransfecting 293T cells with pNLenv1 and pCMV vectors expressing the protein (J. of Virology 84, 4769-4781 (2010)). The 50% neutralizing inhibitory dose (IC50) titer was calculated as the dilution producing a 50% decrease in relative luminescence units (RLU) compared to the level in the virus control wells after subtraction of the cell control RLU (J. of Virology 84, 1439-1452 (2010)). The neutralization phenotype (titer level) of the SHIVDH12_V3AD8 molecular clone was determined by TZM-bl cell assay using plasma samples from a cohort study that exhibits broad neutralizing activity against subtype B HIV-1 isolates (J. of General Virology 91, 2794-2803 (2010)).

[0168] Determination of animal protective titers and statistical analysis Calculations of plasma neutralizing titers for each R5 SHIV that would result in prevention of virus acquisition in 50 or 80% of challenged animals were performed using the method of Reed and Muench (Am J Hyg 27, 493-497 (1938)). One significant outlier (DEW7) was omitted from the calculations. Probit regression was used to model the relationship between the plasma titers required to confer sterilizing immunity in vivo using all 60 passively immunized monkeys (Cambridge University Press, Cambridge, England, ed. 3rd, 2007), including p-values from this model based on likelihood ratio tests. Plasma titers required for various in vivo protection levels (33%, 50%, 80%, 90%, and 95%) were determined from the probit model estimates, and 90% confidence intervals were constructed using bootstrap methods.

[0169] result: SHIVDH12_V3AD8, like SHIVAD8EO, had Tier 2 anti-HIV-1 neutralization sensitivity characteristics (Table 13). Rhesus macaques inoculated intravenously or intrarectally with SHIVDH12_V3AD8 exhibited peak viremia ranging from 10 to 10 viral RNA copies / mL in plasma at 2 to 3 weeks postinfection (PI). In most SHIVDH12_V3AD8-infected animals, plasma viral loads declined to background levels between 8 and 20 weeks PI.

[0170] The neutralization sensitivity of SHIVAD8EO to 11 recently reported broadly reactive anti-HIV-1 mAbs was initially determined in the TZM-bl assay system (Figures 11A and B). Eight of these antibodies, VRC01, NIH45-46(23), 45-46G54W, 45-46m2, 3BNC117, 12A12, 1NC9, and 8ANC195, targeted the gp120 CD4 bs (Science 333, 1633-1637 (2011)), and three, 10-1074, PGT121, and PGT126 (Nature 477, 466-470 (2011)), were dependent on the presence of the HIV-1 gp120 N332 glycan. When tested against SHIVAD8EO, all three glycan-dependent mAbs exhibited greater potency than the CD4 bs mAb (Figure 11A). The IC50 values for the three mAbs targeting the gp120 N332 glycan ranged from 0.09 to 0.15 μg / mL. The CD4 bs mAbs exhibited a much broader range of IC50 neutralization activity (0.14 to 6.34 μg / mL), with 3BNC117 being the most potent. A similar hierarchy of neutralizing mAb potency (glycan-dependent > CD4 bs-dependent) was observed for SHIVDH12-V3AD8, although its neutralization activity was distributed over a much broader (>100-fold) range compared to the IC50 values observed for SHIVAD8EO (Figure 11B). SHIVDH12-V3AD8 was somewhat more sensitive to glycan-targeting mAbs and more resistant to CD4 bs-neutralizing mAbs than SHIVAD8EO.

[0171] Based on the results shown in Figure 11, five neutralizing mAbs were selected for pre-exposure passive transfer studies: VRC01 (because it was the first CD4bs NAb of the newly isolated broadly acting NAbs to be characterized); CD4bs mAbs 45-46m2 and 3BNC117 (both of which exhibited strong neutralizing activity against SHIVAD8EO and SHIVDH12-V3AD8); and the gp120 N332 glycan-dependent mAbs, PGT121 and 10-1074.

[0172] The protocol for passive transfer experiments involved intravenous administration of decreasing doses of neutralizing mAbs and intrarectal challenge of the animals 24 hours later. The goal was to prevent viral acquisition, and we selected an SHIV challenge dose large enough to establish in vivo infection after a single inoculation, knowing that repeated administration of humanized anti-HIV mAbs to individual macaques may reduce their efficacy and / or possibly induce anaphylactic reactions. In this regard, we previously performed intrarectal titration of SHIV AD8 in rhesus macaques and reported that an inoculation of 1 x 10 TCID50, as determined by endpoint dilution in rhesus macaque PBMCs, was equivalent to administering approximately 3 animal infectious doses (AID50) (J. of Virology 86, 8516-8526 (2012)). Indeed, a single intrarectal inoculation of 3 AID50 resulted in successful establishment of infection with SHIVAD8EO and SHIVDH12-V3AD8 in 10 of 10 rhesus macaques.

[0173] As a control for the first passive transfer experiment, animals were intravenously administered an anti-dengue virus NS1 IgG1 mAb and challenged with SHIVAD8EO 24 hours later. Both monkeys (ML1 and MAA) became rapidly infected, with peak levels of plasma viremia occurring 2 weeks PI. VRC01, the first anti-HIV-1 neutralizing mAb tested for protection against viral acquisition, was administered to two macaques at a dose of 50 mg / kg. One of the two inoculated macaques (DEGF) was completely protected from SHIVAD8EO challenge, with no evidence of plasma viremia or cell-associated viral DNA over the 45-week observation period. The other 50 mg / kg VRC01 recipient (DEH3) became infected, but peak plasma viremia was delayed until 5 weeks PI. Two additional macaques receiving a lower dose of VRC01 (20 mg / kg) were not protected from SHIVAD8EO challenge. These results are summarized in Table 13.

[0174] The protective properties of PGT121 against SHIVAD8EO challenge infection were next investigated. PGT121 was one of the most potent glycan-targeting neutralizing mAbs measured in the TZM-bl assay (Figure 11). Based on the results obtained with VRC01, we chose to start with in vivo PGT121 mAb titration at 20 mg / kg. Two monkeys (KNX and MK4) that received challenge infection survived the SHIVAD8EO challenge infection. When lower doses of PGT121 were administered (i.e., 5 mg / kg, 1 mg / kg, or 0.2 mg / kg), 1 of 2 animals, 2 of 2 animals, and 0 of 2 animals were protected, respectively (Table 13).

[0175] The ability of VRC01 and PGT121 mAbs to prevent SHIVDH12-V3AD8 acquisition was similarly evaluated (Table 13). Results obtained with VRC01 were comparable to those observed with SHIVAD8EO challenge infection: one of two recipients at 30 mg / kg was protected from establishing SHIVDH12-V3AD8 infection. PGT121 mAb was significantly more effective than VRC01 in preventing SHIVDH12-V3AD8 acquisition: two of two recipients at 0.2 mg / kg PGT121 survived infection. PGT121 also appeared to be somewhat effective in preventing SHIVDH12-V3AD8 against SHIVAD8EO in vivo infection (Table 13). This result is consistent with the in vivo This is consistent with the 8-fold difference in IC50 values of PGT121 for neutralization of the two SHIVs in the vitro assay (Figure 11).

[0176] The results of passive transfer of 10-1074, 3BNC117, or 45-46m2 neutralizing mAbs to rhesus macaques, followed by challenge with either SHIV AD8EO or SHIV DH12-V3AD8, are summarized in Table 13. 10-1074 mAb potently blocked in vivo acquisition of both SHIVs. CD4bs 3BNC117 and 45-46m2 mAbs were selected for passive transfer to macaques based on their IC50 values against both SHIVs in the in vitro neutralization experiments shown in Figure 11. 3BNC117 successfully blocked SHIV AD8EO infection in two of two monkeys at 5 mg / kg but not in the other two animals given a dose of 1 mg / kg (Table 13). This was similar to the results observed when the same amount of 3BNC117 was administered to macaques challenged with SHIVDH12-V3AD8 (one of two infected at 5 mg / kg and one of two infected at 1 mg / kg).

[0177] Plasma samples collected at various time points from passively transmitted macaques were analyzed to determine the HIV-1 gp120 Neutralizing mAb concentrations were determined by ELISA. In general, the plasma concentration of each mAb at the time of challenge infection (24 hours after antibody administration) correlated with the dose of antibody administered (Table 13).

[0178] The relationship between plasma mAb concentration and in vivo protection is shown in Figure 12. Of the five neutralizing mAbs evaluated, PGT121 was clearly the most effective against both viruses, with SHIV DH12-V3AD8 exhibiting somewhat greater sensitivity to this mAb (two of two monkeys were protected at plasma concentrations of 0.2 μg / mL). In contrast, plasma concentrations of VRC01 of approximately 400 μg / mL were required to protect one of two animals from the same SHIV DH12-V3AD8 challenge virus (Table 13). The most potent CD4 bs mAb administered to macaques in this study, 3BNC117, was also approximately 6- to 10-fold more effective than VRC01 in preventing acquisition of either SHIV (Figure 12, Table 13).

[0179] The circulating half-lives of PGT121, 10-1074, 3BNC117, and VRC01 mAbs were quite similar: 3.5 days, 3.5 days, 3.3 days, and 3.1 days, respectively. In contrast, the half-life of 45-46m2 was extremely short and could not be determined. Based on plasma mAb concentrations in several macaques 24 hours after administration of 20 mg / kg of the humanized neutralizing mAb (i.e., approximately 250 μg / mL [Table 13]), the two monkeys that received 20 mg / kg of 45-46m2 had plasma mAb concentrations of only 15.0 and 17.6 μg / mL, which was a greater than 95% decline compared to the other neutralizing mAbs at 24 hours.

[0180] When macaques were challenged with SHIVAD8EO and SHIVDH12-V3AD8, neutralization titers were measured using plasma samples collected 24 hours after mAb administration. As shown in Table 13, a good correlation was observed between antiviral plasma neutralization titers and protection from SHIV infection. Administration of two glycan-dependent mAbs (PGT121 and 10-1074) clearly resulted in the highest titers of anti-HIV-1 neutralization activity at the time of viral challenge. Titers measured in recipients of 45-46m2 mAb were at the limit of detection or were undetectable due to its extremely short in vivo half-life.

[0181] The neutralizing titers, measured in plasma, required to prevent virus acquisition in 50% of challenged monkeys were calculated using the method described by Reed and Muench (Am J Hyg 27, 493-497 (1938)). 28 monkeys challenged with SHIVAD8EO or SHIVDH12-V3AD These protective titers were derived separately for the 32 macaques challenged with SHIVAD8EO or SHIVDH12-V3AD8 (Tables 15 and 16). The plasma neutralization titers required to protect 50% of animals challenged with SHIVAD8EO or SHIVDH12-V3AD8 were calculated to be 1:115 and 1:96, respectively. Because these similar titers were obtained 1) after SHIV challenge infection by the same route and inoculum size and 2) after administration of the same neutralizing mAb panel, the neutralization data from all 60 animals were combined and subjected to probit regression to examine the relationship between plasma neutralization titers and in vivo protection. As further confirmation, when a term for the SHIV virus was included 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 the entire group of 60 macaques, the probit regression estimated that a plasma neutralization titer of 1:104 would prevent virus acquisition in 50% of the animals. Providing analysis of the data also estimated that a 50% plasma neutralization titer of 1:57 or 1:329 would protect 33% or 80% of exposed animals, respectively.

[0182] Example 10. Administration of Neutralizing mABs to an HIV In Vivo Model of Chronic Infection Summary of Methods: The neutralizing activity of the broadly active 3BNC11724 and 10-107423 neutralizing mAbs against SHIVAD8EO was initially determined in the TZM-bl cell system against SHIVAD8EO. Their ability to prevent viral acquisition or control plasma viremia in chronically infected animals challenged with R5-tropic SHIVAD8EO was assessed by monitoring plasma viral load and cell-associated viral nucleic acid; levels of CD4+ T cell subsets were measured by flow cytometry. SGA analysis of circulating viral variants and determination of plasma antibody levels were performed. Plasma concentrations of NAbs were determined by measuring neutralizing activity against HIV-1 pseudovirus preparations that were only sensitive to either 10-1074 or 3BNC117.

[0183] result: Two groups of chronically infected macaques were evaluated. The first group consisted of two clinically asymptomatic animals (DBZ3 and DC99A) that had been infected for 159 weeks and continued to show similar and significant declines in circulating CD4+ T cells (Table 17). The treatment for ongoing SHIV infection was the combined administration of 101074 and 3BNC117 at a 10 mg / kg dose. At the time of mAb administration, plasma viral loads in macaques DBZ3 and DC99A were 1.08 × 10 and 7.6 × 10 RNA copies / mL, respectively. Both monkeys responded to combination anti-HIV-1 mAb treatment, with plasma viremia immediately and rapidly decreasing to undetectable levels within 7 to 10 days. Suppression of measurable SHIVAD8EO in the plasma of macaques DBZ3 and DC99A after a single administration of the two mAbs lasted for 27 and 41 days, respectively. In each case, plasma viremia returned to pretreatment levels.

[0184] A second group of three animals (DBX3, DCF1, and DCM8), each of which had also been infected with SHIVAD8EO for more than three years and had clinical symptoms of intermittent diarrhea and / or decreased appetite, were treated with the two neutralizing antibodies (Table 17). At the time of mAb administration, the level of circulating CD4+ T cells in macaque DCM8 was only 43 cells / μL and was somewhat higher in animals DCF1 (10 cells / μL) and DBXE (158 cells / μL). Plasma viral loads exceeded 10 RNA copies / mL in animals DBXE and DCF1 and were significantly lower in monkey DCM8 (1.59 × 10 RNA copies / mL). Administration of the two mAbs to monkey DBXE resulted in a biphasic decline from 2.0 × 10 RNA copies on day 0 to undetectable plasma levels on day 20. This was followed within a few days by a return of high circulating viral levels in DBXE. Macaque DCM8, which had a lower plasma viral load and very low circulating CD4+ T cell counts, experienced a rapid decline in viremia to undetectable levels between days 6 and 20 after initiation of mAb treatment Finally, DCF1, an animal previously reported to produce broadly reactive anti-HIV-1 NAbs, exhibited a transient and relatively modest 27-fold decrease in plasma viremia by day 6 in response to combination mAb therapy, after which viral load returned to high pretreatment levels.

[0185] PBMC-associated viral RNA and DNA levels were also determined before and after antibody administration (Table 18). For each animal, mAb treatment resulted in a decrease in cell-associated viral RNA levels that correlated well with plasma viral load measurements. No consistent pattern was observed for cell-associated viral DNA levels as a result of antibody treatment. Administration of neutralizing mAbs to chronically SHIVAD8EO-infected monkeys also had a beneficial effect on circulating CD4+ T cell levels, particularly in animals with very high viral loads. CD4+ T cell counts in macaques DBXE and DCF1 increased two- to three-fold during the period of mAb-mediated viral suppression but gradually declined to pretreatment levels as viremia became detectable again.

[0186] The plasma concentration of each mAb was determined by measuring its plasma neutralizing activity against selected HIV-1 pseudovirus strains sensitive to one or the other antibody but not both (Figure 13A). Suppression of SHIVAD8EO viremia was maintained in all treated animals until a threshold plasma mAb concentration of approximately 1 to 3 μg / mL was reached (Figures 13B and 13C). This was also true for macaque DCF1, in which a modest, transient decrease in plasma viral RNA levels was observed. Interestingly, mAbs administered to clinically symptomatic macaques DCM8 and DCF1 had shortened or undetectable half-lives. As previously mentioned, macaque DCM8 had extremely low CD4+ T cell levels (43 cells / μL plasma), and macaque DCF1 had to be euthanized 56 days after the initiation of treatment due to its deteriorating clinical condition. Necropsy of DCF1 revealed severe enteropathy characterized by disseminated gastrointestinal cryptosporidiosis, pancreatitis, and cholangitis.

[0187] SGA analysis was used to determine whether amino acid substitutions occurred in the gp120 region previously shown to affect susceptibility to the 10-1074 or 3BNC117 mAb. In each case, rebound virus present in plasma after immunotherapy remained unchanged. To further test the susceptibility of re-emerging virus, two clinically asymptomatic monkeys (DBZ3 and DC99A) were again administered a combination of 10-1074 and 3BNC117 (10 mg / kg each). The viral load in each animal again rapidly declined and became undetectable by day 7 of the second immunotherapy cycle. Viremia was suppressed for 7 days in the DBZ3 macaque and for more than 21 days in the DC99A monkey. Taken together, these results suggest that the re-emergence of virus after the first treatment cycle in these two animals represented insufficient mAb levels in vivo rather than antibody-selected viral resistance.

[0188] [Table 3]

[0189]

Table 4

[0190]

Table 5

[0191]

Table 6

[0192]

Table 7

[0193]

Table 8

[0194]

Table 9

[0195]

Table 10

[0196]

Table 11

[0197]

Table 12

[0198]

Table 13

[0199]

Table 14

[0200] [Table 15]

[0201] [Table 16]

[0202] [Table 17]

[0203] [Table 18]

[0204] The above examples and descriptions of preferred embodiments should be considered illustrative, rather than limiting, of the invention defined by the claims. As will be readily appreciated, numerous variations and combinations of the features described above can be used without departing from the invention as set forth in the claims. Such variations will not be considered a departure from the scope of the invention, and all such variations are to be construed as falling within the scope of the following claims. All references cited herein are incorporated by reference in their entirety.

Claims

1. (i) (1) a complementarity determining region (CDR) H1 comprising the amino acid sequence of SEQ ID NO: 69, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 70, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 71, a CDRL1 comprising the amino acid sequence of SEQ ID NO: 72, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 73, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 74; (2) CDRH1 comprising the amino acid sequence of SEQ ID NO: 39, CDRH2 comprising the amino acid sequence of SEQ ID NO: 40, CDRH3 comprising the amino acid sequence of SEQ ID NO: 41, CDRL1 comprising the amino acid sequence of SEQ ID NO: 42, CDRL2 comprising the amino acid sequence of SEQ ID NO: 43, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 44; (3) CDRH1 comprising the amino acid sequence of SEQ ID NO: 51, CDRH2 comprising the amino acid sequence of SEQ ID NO: 52, CDRH3 comprising the amino acid sequence of SEQ ID NO: 53, CDRL1 comprising the amino acid sequence of SEQ ID NO: 54, CDRL2 comprising the amino acid sequence of SEQ ID NO: 55, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 56; (4) CDRH1 comprising the amino acid sequence of SEQ ID NO: 57, CDRH2 comprising the amino acid sequence of SEQ ID NO: 58, CDRH3 comprising the amino acid sequence of SEQ ID NO: 59, CDRL1 comprising the amino acid sequence of SEQ ID NO: 60, CDRL2 comprising the amino acid sequence of SEQ ID NO: 61, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 62; (5) CDRH1 comprising the amino acid sequence of SEQ ID NO: 63, CDRH2 comprising the amino acid sequence of SEQ ID NO: 64, CDRH3 comprising the amino acid sequence of SEQ ID NO: 65, CDRL1 comprising the amino acid sequence of SEQ ID NO: 66, CDRL2 comprising the amino acid sequence of SEQ ID NO: 67, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 68; (6) CDRH1 comprising the amino acid sequence of SEQ ID NO: 75, CDRH2 comprising the amino acid sequence of SEQ ID NO: 76, CDRH3 comprising the amino acid sequence of SEQ ID NO: 77, CDRL1 comprising the amino acid sequence of SEQ ID NO: 78, CDRL2 comprising the amino acid sequence of SEQ ID NO: 79, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 80; (7) CDRH1 comprising the amino acid sequence of SEQ ID NO: 81, CDRH2 comprising the amino acid sequence of SEQ ID NO: 82, CDRH3 comprising the amino acid sequence of SEQ ID NO: 83, and CDRH4 comprising the amino acid sequence of SEQ ID NO:

84. CDRL1 comprising the amino acid sequence of SEQ ID NO:85, CDRL2 comprising the amino acid sequence of SEQ ID NO:86; (8) CDRH1 comprising the amino acid sequence of SEQ ID NO: 87, CDRH2 comprising the amino acid sequence of SEQ ID NO: 88, CDRH3 comprising the amino acid sequence of SEQ ID NO: 89, CDRL1 comprising the amino acid sequence of SEQ ID NO: 90, CDRL2 comprising the amino acid sequence of SEQ ID NO: 91, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 92; (9) CDRH1 comprising the amino acid sequence of SEQ ID NO: 93, CDRH2 comprising the amino acid sequence of SEQ ID NO: 94, CDRH3 comprising the amino acid sequence of SEQ ID NO: 95, CDRL1 comprising the amino acid sequence of SEQ ID NO: 96, CDRL2 comprising the amino acid sequence of SEQ ID NO: 97, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 98; (10) CDRH1 comprising the amino acid sequence of SEQ ID NO: 99, CDRH2 comprising the amino acid sequence of SEQ ID NO: 100, CDRH3 comprising the amino acid sequence of SEQ ID NO: 101, CDRL1 comprising the amino acid sequence of SEQ ID NO: 102, CDRL2 comprising the amino acid sequence of SEQ ID NO: 103, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 104; or (11) CDRH1 comprising the amino acid sequence of SEQ ID NO: 131, CDRH2 comprising the amino acid sequence of SEQ ID NO: 132, CDRH3 comprising the amino acid sequence of SEQ ID NO: 133, CDRL1 comprising the amino acid sequence of SEQ ID NO: 134, CDRL2 comprising the amino acid sequence of SEQ ID NO: 135, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 136; and (ii) a pharmaceutically acceptable carrier.

2. The pharmaceutical composition of claim 1 , wherein the anti-HIV antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 3, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 129.

3. The pharmaceutical composition of claim 1 , wherein the anti-HIV antibody or antigen-binding portion thereof comprises a light chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 4, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 130.

4. The pharmaceutical composition of claim 1, wherein the anti-HIV antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region comprising each of the amino acid sequences of SEQ ID NOs: 13-14, 3-4, 7-8, 9-10, 11-12, 15-16, 17-18, 19-20, 21-22, 23-24, and 129-130.

Images