Broad-spectrum neutralizing anti-HIV antibody

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 of related applications This application claims priority to U.S. Provisional Patent Application No. 61 / 715,642, filed October 18, 2012, pursuant to Section 119(e) of the U.S. Patent Act, which is incorporated herein by reference in its entirety.

[0002] Statements relating to federally funded research or development The inventions disclosed herein were, at least in part, made with government support under grant number P01 AI081677 from the National Institutes of Health. Therefore, the Government of the United States has certain rights to these inventions.

[0003] This invention relates to a broad-spectrum and potent antibody against human immunodeficiency virus ("HIV"). [Background technology]

[0004] HIV is a human condition characterized by a clinical picture that includes wasting syndrome, central nervous system degeneration, and severe immunosuppression, leading to acquired immunodeficiency syndrome (AIDS), life-threatening opportunistic infections, and malignant diseases. Since its discovery in 1981, at least 25 million people worldwide have died from HIV-1. Even if HIV infection decreases by 2.5% annually, it is estimated that 20 to 60 million people will be infected over the next 20 years. There is a need for therapeutic drugs and methods to treat or control HIV infection.

[0005] Some HIV-infected individuals exhibit broad-spectrum 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 may contribute to defense against viral attack. While a neutralizing antibody response can also be expressed in HIV-infected individuals, the detailed composition of the serological response is still not fully understood. [Overview of the project]

[0006] This invention relates to a novel category of broad-spectrum neutralizing anti-HIV antibodies. The consensus weight and light chain amino acid sequence of the antibodies are listed below and shown in Figures 3a and 3b: QVQLQESGPGLVKPSETLSLTCSSVSGX1SX2X3DX4YWSWIRQSPGKGLEWIGYVHDSGDTNYNPSLKSRVX5X6SLDTSKNQVSLKLX7X8VTAADSAX9YYCARAX 10 HGX 11 RIYGIVAFGEX 12 FTYFYMDVWGKGTTVTVSS(Sequence ID 1) SX1VRPQPPSLSVAPGETARIX2CGEX3SLGSRAVQWYQQRPGQAPSLIIYNNQDRPSGIPERFSGSPDX4X5FGTTATLTITX6VEAGDEADYYCHIWDSRX7PTX8WVFGGGTTLTVL(Sequence ID 2).

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

[0008] Accordingly, one aspect of the present invention features an isolated anti-HIV antibody, or its antigen-binding moiety, having at least one complementarity-determining region (CDR) having a sequence selected from the group consisting of SEQ ID NOs. 33-38, provided that the antibody is not antibody PGT-121, 122, or 123. SEQ ID NOs. 33-38 refer to 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 CDR may contain a sequence selected from the group consisting of SEQ ID NOs. 39-104, i.e., a CDR sequence according to the Kabat system, as shown in Table 1 below. Alternatively, the CDR may contain a sequence 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 its antigen-binding moiety, contains a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, where CDRH1, CDRH2, and CDRH3 each contain the sequences of SEQ ID NOs. 33-35. CDRH1, CDRH2, and CDRH3 may also contain sequences from a set of CDRHs selected from the group consisting of SEQ ID NOs. 39-41, 45-47, 51-53, 57-59, 63-65, 69-71, 75-77, 81-83, 87-89, 93-95, 99-101, and 131-133. Alternatively, the CDRHs may contain sequences selected from the CDR sequences of their corresponding antibodies using the IMGT system as shown in Table 1 below.

[0010] In another embodiment, the isolated anti-HIV antibody, or its antigen-binding moiety, contains a light chain variable region comprising CDRL1, CDRL2, and CDRL3, where CDRL1, CDRL2, and CDRL3 each contain the sequences of SEQ ID NOs. 36-38. For example, CDRL1, CDRL2, and CDRL3 may contain sequences from a set of CDRLs 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 may contain sequences selected from the CDR sequences of their corresponding antibodies using the IMGT system as shown in Table 1 below.

[0011] In yet another embodiment, the isolated anti-HIV antibody described above, or its antigen-binding moiety, comprises (i) a heavy chain variable region including CDRH1, CDRH2, and CDRH3, and (ii) a light chain variable region including CDRL1, CDRL2, and CDRL3. The CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 may include sequences from a set of CDRs 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 CDRH and CDRL may contain sequences selected from the CDR sequences of their corresponding antibodies using an IMGT system as shown in Table 1 below.

[0012] In a further embodiment, the isolated anti-HIV antibody, or its antigen-binding moiety, contains either 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 derived from SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 and 129. The light chain may contain sequences 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 may contain the sequences SEQ ID NOs: 3-4, 5-6, 7-8, 9-10, 11-12, 13-14, 15-16, 17-18, 19-20, 21-22, 23-24, and 129-130, respectively.

[0013] In a preferred embodiment, the isolated anti-HIV antibody is 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., 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 the current virus. The antibodies discussed above can be human antibodies, humanized antibodies, or chimeric antibodies.

[0014] In a second embodiment, the present invention provides isolated nucleic acids having sequences encoding a CDR, heavy chain variable region, or light chain variable region of the anti-HIV antibody or its antigen-binding moiety discussed above. The invention also features vectors having the nucleic acids and cultured cells having the vectors.

[0015] The nucleic acids, vectors, and cultured cells can be used in a method for producing anti-HIV antibodies or fragments thereof. This method includes, among many other steps, obtaining the cultured cells described above, culturing the cells in a culture medium under conditions that enable the expression of polypeptides encoded by the vector and the construction of antibodies or fragments thereof, and purifying the antibodies or fragments from the cultured cells or the culture medium of the cells.

[0016] In a third aspect, the present invention is characterized by a pharmaceutical composition comprising (i) at least one anti-HIV antibody or its antigen-binding moiety as described above, and (ii) a pharmaceutically acceptable carrier.

[0017] In a fourth embodiment, the present invention provides a method for preventing or treating HIV infection or HIV-related disease. The method includes, among many, 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 of the aforementioned anti-HIV antibodies or its antigen-binding moiety. The method may further include administering a second therapeutic agent, for example, an antiviral agent.

[0018] In a fifth embodiment, the present invention provides a kit comprising a pharmaceutically acceptable dose unit of at least one isolated anti-HIV antibody or its antigen-binding moiety described above, and a pharmaceutically acceptable dose unit of an anti-HIV drug. The two pharmaceutically acceptable dose units may optionally take the form of a single pharmaceutically acceptable dose unit. Exemplary anti-HIV drugs may 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 contains anti-HIV components in a biological sample from the subject. The kit contains one or more detection reagents that specifically bind to the antibody. The kit may further contain reagents for performing PCR or mass spectrometry.

[0020] Details of one or more embodiments of the present invention are described below. Other features, purposes, and advantages of the present invention will become clear from the description and claims. [Brief explanation of the drawing]

[0021] [Figure 1-1] This figure shows the neutralizing activity of PGT121-like and 10-1074-like variants. (A) Heatmap comparing the neutralizing efficacy of PGT121-like and 10-1074-like antibodies in the TZM-bl assay. Darker colors = stronger neutralization; white = no neutralization. (B) Correlation between mean IC80 against 9 viruses (y axis) and apparent KD values ​​for binding to gp120 and gp140 (x axis). [Figure 1-2] This figure shows the neutralizing activity of PGT121-like and 10-1074-like variants. (C) Graph comparing the neutralizing width and potency of PGT121, 10-996, and 10-1074 antibodies in a TZM-bl assay against an expanded panel of HIV-119 virus. The y-axis shows the cumulative frequency of IC50 values ​​up to the concentration shown on the x-axis. The spiderweb graph (upper left corner) shows the frequency distribution of neutralized viruses by the HIV-1 clade. (D) Dot plot showing molar neutralization rate (MNR; ratio of IC50 concentrations of Fab to IgG). Horizontal bars represent the average IC50 of all viruses. [Figure 1-3] This figure shows the neutralizing activity of PGT121-like and 10-1074-like variants. (E) Bar graph comparing the neutralizing efficacy of PGT121 (dark gray) and 10-1074 (light gray) against viruses isolated from past (Hist.) and present (Cont.) seroconverts. ns, not significant;**, p<0.005. Shows the fold difference in multiples between median IC50 for neutralization of present viruses by PGT121 and 10-1074. [Figure 2]This figure shows the binding and neutralizing activity of PGT121GM and 10-1074GM mutant antibodies. (A) Bar graph comparing the apparent KD values ​​for binding of 10-1074, PGT121, PGT121GM, and 10-1074GM antibodies to gp120 and gp140. Error bars show SEM of KD values ​​obtained from three independent experiments. The difference in multiples between the KD values ​​of "wild-type" and "glycomutant" antibodies is shown. (B) Bar graph comparing the binding of glycan (Figure 7A) by PGT121 and 10-1074 with that of mutant antibodies (PGT121GM and 10-1074GM). The numerical score of binding is measured as the fluorescence intensity (average of two replicate spots) for probes aligned at 5 fmol per spot. (C) Coverage graph comparing the neutralization width and potency of PGT121, PGT121GM, 10-1074, and 10-1074GM antibodies in a TZM-bl assay against a panel of 40 viruses. [Figure 3-1] This figure shows the sequence alignment of PGT121 and 10-1074 clonal variants. (A) Amino acid alignment of the heavy chain (IgH) of PGT121-like and 10-1074-like antibodies, as well as putative germline (GL)VH for all clonal variants. Amino acid numbering is shown based on crystal structure, framework (FWR), and complementarity-determining region (CDR) as defined by Kabat (J Exp Med 132(2):211-250) and IMGT (Nucleic Acids Res 37 (Database issue):D1006-1012). Color shading indicates acidic (red), basic (blue), and tyrosine (green) amino acids. [Figure 3-2] This figure shows the sequence alignment of PGT121 and 10-1074 clone mutants. (B) Same as A, but for the light chain (IgL). [Figure 4]This figure shows the binding affinity of PGT121 and 10-1074 clonal mutants. (A) Binding affinity of the interaction between PGT121 IgG antibody mutant and YU-2 gp140 and gp120 ligands as measured by surface plasmon resonance (SPR). M, mol / L; s, seconds; RU, response units; / , no binding detected. A chi-2 value (X²) < 10 indicates that the 1:1 binding model used to fit the curve adequately explains the experimental data. The equilibrium and rate constants shown are considered "apparent" constants to explain the binding activity effect resulting from the divalent binding of IgG. (B) Dot plots showing the association (ka) and dissociation (kd) rate constants for PGT121-like (blue shaded) and 10-1074-like (green shaded) mutants. (C) A linear regression graph comparing the Ka and KD values ​​(x axis) of IgG antibodies for their binding to gp120 and gp140 with their neutralizing efficacy (mean IC80 value) (y axis) against the nine viruses shown in Table 4. [Figure 5-1] This figure shows the binding of PGT121 variants to the 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 shows the antibody concentration (M) required to obtain the ELISA value (OD405nm) shown on the y-axis. 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 in at least two replicates. Representative data are shown. [Figure 5-2] This figure shows the binding of PGT121 mutants to the gp120 "core" protein, the gp120GD324-5AA mutant, and the linear gp120V3 peptide. (B) is the same as (A), but shows the binding to the gp120GD324-5AA mutant protein. All experiments were performed at least twice. Representative data are shown. [Figure 5-3] This figure shows the binding of PGT121 mutants 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 the 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] This figure shows the binding of PGT121 to glycosylated and deglycosylated gp120 mutants. (A) ELISA-based binding analysis of PGT121 and 10-1074 antibody variants to gp120, gp120NNT301-303AAA, gp120N332A, and gp120N332A / NNT301-303AAA. The x-axis shows the antibody concentration (M) required to obtain the ELISA values ​​(OD405nm) shown on the y-axis. The black discontinuous and continuous lines show the mean reactivity to the four antigens in the positive (10-188) and negative (mGO53) antibody control groups. All experiments were performed at least twice. [Figure 6-2] This figure shows the binding of PGT121 to glycosylated and deglycosylated gp120 mutants. (B) Silver-stained SDS-PAGE gels 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] This figure shows the binding of PGT121 to glycosylated and deglycosylated gp120 mutants. (C) is the same as (A), but compares untreated gp120 with PNGase F-treated gp120. All experiments were performed at least twice. [Figure 6-4]This figure shows the binding of PGT121 to glycosylated and deglycosylated gp120 mutants. (D) is the same as (A), but compares untreated gp120 with EndoH-treated gp120. All experiments were performed at least twice. [Figure 7-1] This figure 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 bound by reductive amination. The notable and important features are: (i) the PGT121 group antibody bound to monoantenna N-glycan probe 10 (N2), which has a galactose-terminal antenna linked to coremannose by 1-3 linkages, but not to isomer N-glycan probe 11 (designated N4), which has an antenna linked to coremannose by 1-6 linkages; (ii) as with biantenna probe 13 (NA2), the presence of this galactose-terminal 1-6 linkage antenna allowed binding, and also allowed the presence of α2-6 linked sialic acid (not α2-3 linkage); and (iii) biantenna probe 12 (NGA2), which lacks galactose and ends with N-acetylglucosamine, did not bind. [Figure 7-2] This figure shows the binding of PGT121 and 10-1074 clone variants to glycans. (B) Bar graph comparing glycan binding by PGT121-like, 10-1074-like, and germline type (GL) antibodies. 10-188, i.e., anti-V3 loop antibody, was used as a negative control. Numerical binding scores were measured as fluorescence intensity (average of two spots) for probes arranged at 2 fmol (white) and 5 fmol (gray) per spot. [Figure 8-1]This figure shows the antibody binding and neutralizing activity against high-mannose-only gp120 and the virus. (A) Silver-stained SDS-PAGE gel comparing YU-2 gp120 (gp120kif) produced in kifunensin-treated cells 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 shows the antibody concentration (M) required to obtain the ELISA value (OD405nm) shown on the y-axis. [Figure 8-2] This figure shows the antibody binding and neutralizing activity of high-mannose-only gp120 against viruses. (C) Neutralization curves for PGT121 evaluated against selective PGT121-sensitive / 10-1074-resistant pseudoviruses produced in the presence (virus kif) or absence (virus WT). Horizontal discontinuities indicate 50% neutralization, and the IC50 value can be obtained from the antibody concentration on the x axis. The experiment was performed three times. Error bars show the SD of the three measurements. (D) Bar graph comparing the neutralizing activity of selective antibodies against YU-2 and PVO.4 pseudoviruses produced in HEK293S GnTI- / - cells (virus GnT- / -) or wild-type cells (virus WT). The y axis shows the mean IC50 value (μg / mL) for virus neutralization shown on the x axis. Error bars show SEM of IC50 values ​​obtained from two independent experiments. [Figure 9-1] This figure shows the neutralizing activity of PGT121, 10-996, and 10-1074. (A) A graph comparing the neutralizing efficacy of PGT121, 10-996, and 10-74 against the virus of the HIV-1 clade shown (determined using TZM-bl assay and a panel of 119 pseudoviruses). The x-axis shows the antibody concentration (μg / mL) required to achieve 50% neutralization (IC50). The y-axis shows the cumulative frequency of IC50 values ​​up to the concentration shown on the x-axis. [Figure 9-2](B) A graph comparing the neutralizing activity of PGT121, 10⁻⁹⁶⁹⁶⁹⁷⁴ and 10⁻⁹⁷⁴ against an expanded panel of 119 virus as determined by the TZM-bl neutralization assay. The y-axis shows the cumulative frequency of IC80 values ​​up to the concentration shown on the x-axis. [Figure 9-3] This figure shows the neutralizing activity of PGT121, 10⁻⁹⁶, and 10⁻¹⁰⁷⁴. (C) The graph shows the neutralization curves of selected viruses by PGT121 and 10⁻¹⁰⁷⁴. The horizontal discontinuous line indicates 50% neutralization, and the IC50 value can be obtained from the antibody concentration on the x axis. The experiment was performed three times. Error bars indicate the standard deviation of the three measurements. [Figure 10] This figure shows the neutralizing activity against clade B virus in the past versus present. The dot plot compares the neutralizing efficacy of selected bNAb against clade B virus isolated from seroconverted antibody patients in the past (Hist.) and present (Cont.). Horizontal bars represent the median IC50 for total virus per patient. Group differences were assessed using the Mann-Whitney U test. ns indicates not significant. [Figure 11] This table shows the neutralization of two R5-directed SHIVs in a panel of 11 broad-acting anti-HIV-1 mAbs. Calculated IC50 values ​​for neutralizing SHIVAD8EO(A) and SHIVDH12-V3AD8(B). [Figure 12] This figure shows the relationship between plasma concentrations of passively administered neutralizing mAbs and viral acquisition in macaques after two different R5SHIV infections. Black circles indicate protected (unacquired) monkeys; white circles indicate infected animals. [Figure 13-1]This table shows the 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 that are sensitive to one bNAb but not to another (i.e., HIV-1 strain X2088_9 (sensitive to 10-1074); HIV-1 strain Q769_d22 (sensitive to 3BNC117)). (B) Neutralizing activity of plasma spiked with 10-1074 (blue) or 3BNC117 (green) antibodies at 0.01, 0.1, 1, 10, and 100 μg / mL, but before antibody administration (preP). Neutralizing activity is reported as plasma ID50 titer (left column) and converted to antibody concentration (right column) based on the measured ID50 values ​​in (A). [Figure 13-2] This table shows the plasma concentrations of bNAb. The concentration of mAb was determined by measuring the neutralizing activity in plasma samples. (C) ID50 titer (left column) and bNAb concentration (right column) measured before (pre-blood collection) and after (days) bNAb administration in the macaque plasma samples shown. [Modes for carrying out the invention]

[0022] This invention is at least in part based on the unexpected discovery of a novel category of broad-spectrum 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 against HIV appear to be the only ones correlated with protection in recent RV144 anti-HIV vaccine trials. Some HIV-1 infected individuals develop broad-spectrum neutralizing serum activity against the gp160 viral spike 2-4 years after infection, but due to autoviral escape through mutation, these antibodies generally do not protect infected individuals. Nevertheless, broad-spectrum neutralizing activity exerts selective pressure on the virus, and passive transfer of broad-spectrum neutralizing antibodies (bNAbs) to macaques provides protection against SHIV infection. Therefore, it has been proposed that vaccines that induce 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) and include V1 / V2 (PG9 / PG16) (Science 326(5950):285-289) and V3 loop (PGT) (Nature 477(7365):466-470). It recognizes carbohydrate-dependent epitopes with 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 epitopes targeted by PGT antibodies, we isolated members of a large clonal family that governs the gp160-specific IgG memory response from clade A-infected patients producing PGT121. As disclosed herein, PGT121 antibodies are divided into two groups, namely the PGT121-like group and the 10-1074-like group, according to their sequence, binding affinity, neutralizing activity, and recognition of carbohydrates and the V3 loop. 10-1074 and related family members exhibit unusually potent neutralization, including broad reactivity to novel transmissible viruses. Unlike previously characterized carbohydrate-dependent bNAbs, PGT121 binds to complex N-glycans rather than high-mannose ones in glycan microassay experiments. The crystalline structures of PGT121 and 10-1074, compared to the structures of their germline precursors and the structure of PGT121 bound to complex N-glycans, reasonably explain their unique properties.

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

[0027] As used herein, the term “antibody” (Ab) includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies and multireactive antibodies), and antibody fragments. Therefore, as used herein, the term “antibody” 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 their biologically related fragments or specific binding members (Fab, F(ab')2, Fv, and scFv (single-chain or related entities)). In the art, an antibody is understood to be a glycoprotein having at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or its antigen-binding portion. The heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH1, CH2, and CH3). The light chain consists of a variable light chain region (VL) and a constant light chain region (CL). Both the heavy and light chain variable regions include a framework region (FWR) and a complementarity-determining region (CDR). While the four FWR regions are relatively conserved, the CDR regions (CDR1, CDR2, and CDR3) represent hypervariable regions and are arranged from the NH2 terminus to the COOH terminus as follows: FWR1, CDR1 FWR2, CDR2, FWR3, CDR3, and FWR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens, while, depending on their isotype, the constant regions (one or more) may mediate the binding of immunoglobulins to host tissues or factors.

[0028] As used herein, the definition of “antibody” includes chimeric antibodies, humanized antibodies and recombinant antibodies, human antibodies produced from transgenic non-human animals, and antibodies selected from a library 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) domain differ significantly in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody to that particular antigen. However, its variability is not uniformly distributed across the entire 110-amino acid span of the variable region. Rather, the V region consists of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids, separated by shorter hypervariable regions called "hypervariable regions," each 9-12 amino acids long. The variable regions of the natural heavy and light chains each contain four FRs, primarily taking the form of β-sheet structures, connected by three hypervariable regions, which form loops connecting the β-sheet structures and, in some cases, forming parts of them. The hypervariable regions within each chain are held in extremely close proximity to one another by FR (Functional Retention), and together with hypervariable regions from other chains, they contribute to the formation of the antibody's antigen-binding site (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0030] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are involved in antigen binding. The hypervariable region generally includes amino acid residues from the "complementarity-determining region" (CDR).

[0031] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting that population are identical except for possible spontaneous mutations that may be present in small amounts. The term "polyclonal antibody" refers to a preparation containing different antibodies oriented to different determinants ("epitopes").

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

[0033] A “humanized antibody” is generally considered to be a human antibody that has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often called “implant” residues, and these implant residues are typically obtained from “implant” variable regions. Humanization may be carried out by substituting the corresponding sequence of the human antibody with the implant hypervariable sequence, according to the method of Winter and collaborators (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 a “humanized” antibody is a chimeric antibody in which substantially fewer variable regions than the intact human variable region are replaced by corresponding sequences from a non-human species (see, e.g., U.S. Patent No. 4,816,567).

[0034] An "antibody fragment" is a portion of an intact antibody, for example, containing the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see, for example, U.S. Patent 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] "Fv" is the minimal antibody fragment containing a complete antigen recognition and antigen-binding site. This fragment contains a dimer of tightly linked, non-covalently associated single-chain and single-light-chain variable region domains. The folding of these two domains gives rise to six hypervariable loops (three from the H and L chains), which donate amino acid residues to antigen binding, conferring antigen-binding specificity to the antibody. However, even a single variable region (or half of Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to the antigen, albeit with lower affinity than the full binding site.

[0036] A "single-stranded Fv" ("sFv" or "scFv") is an antibody fragment containing VH and VL antibody domains linked to a single polypeptide chain. The sFv polypeptide may further contain a polypeptide linker between the VH and VL domains, which allows the sFv to form a structure desirable for antigen binding. For a review of sFv, see, for example, 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 a small antibody fragment prepared by constructing an sFv fragment having a short linker (approximately 5-10 residues) between the VH and VL domains, such that interchain pairing rather than intrachain pairing of the V domain is achieved, resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites. A bispecific diabody is a heterodimer of two "cross-linked" sFv fragments in which the VH and VL domains of two antibodies reside on different polypeptide chains. Diabodies are described more adequately, for example, in European Patent No. 404,097; International Publication No. 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 approximately 11 kDa to approximately 15 kDa. dAbs are robust variable regions of the heavy and light chains (VH and VL, respectively) of immunoglobulins. They are microorganisms. They are highly expressed in physical cell cultures, exhibit desirable biophysical properties including (but not limited to) solubility and temperature stability, and are well-suited for selection and affinity maturation by in vitro selection systems such as phage displays. dAbs are bioactive as monomers and, thanks to their small size and inherent stability, can be formatted into larger molecules to produce drugs with long serum half-lives or other pharmacological activities. Examples of these techniques are described, for example, in International Publication No. 9425591 for antibodies derived from camel heavy chain Ig, and in addition, U.S. Patent Application Publication No. 20030130496 describes the isolation of single-domain fully human antibodies from a phage library.

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

[0040] In some embodiments, the antibodies of the present invention are bispecific or multispecific. A bispecific antibody is an antibody that has binding specificity to at least two different epitopes. For example, a bispecific antibody can bind to two different epitopes of a single antigen. Other such antibodies can combine a binding site for a first antigen with a binding site for a second antigen. Alternatively, an anti-HIV arm can be combined with an arm that binds to a trigger molecule on leukocytes, e.g., a T cell receptor molecule (e.g., CD3), or to Fc receptors (Fc gamma R) of IgG, e.g., Fc gamma RI (CD64), Fc gamma RII (CD32), and Fc gamma RIII (CD16), to concentrate and localize cellular defense mechanisms to infected cells. Bispecific antibodies can also be used to localize cytotoxic 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, International Publication No. 96 / 16673 describes a bispecific anti-ErbB2 / anti-Fc gamma RIII antibody, and U.S. Patent 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 International Publication No. 98 / 02463; and a bispecific anti-ErbB2 / anti-CD3 antibody is taught in U.S. Patent No. 5,821,337. 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 US A. 2012. See also Jan 17;109(3):875-80.

[0041] Methods for producing bispecific antibodies are known in the art. Conventional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, in which case the two chains have different specificities (see, for example, Millstein et al., Nature, 305:537-539 (1983)). Similar procedures are disclosed, for example, in International Publication No. 93 / 08829, Traunecker et al., EMBO J., 10:3655-3659 (1991), and Mouquet et al., Enhanced HIV-1 neutralization. See also “by antibody heteroligation” Proc Natl Acad Sci US A.2012 Jan 17;109(3):875-80.

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

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

[0044] Other modifications of antibodies are intended herein. For example, antibodies can be conjugated to one of various non-proteinoid polymers, such as polyethylene glycol, polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol. Antibodies can also be captured in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared 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 present 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, for example, U.S. Patents No. 5,567,610 and No. 5,229,275). General molecular genetics and genetic engineering methods useful in the present invention are found in Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press) and 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 This is 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. EJ 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 can produce the entire repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, homozygous deletion of antibody heavy chain linkage region (JH) genes in chimeric and germline mutant mice has been shown to result in complete inhibition of endogenous antibody production. Transplantation of human germline immunoglobulin gene arrays into such germline mutant mice results in the production of human antibodies upon antigen challenge. For example, Jakobovits et al. See also 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 International Publication No. 97 / 17852. Such animals can be genetically engineered to produce human antibodies containing the polypeptide of the invention described herein.

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

[0048] Phage displays, ribosome displays (Hanes and Pluckthun, 1997, Proc. Nat. Acad. Sci. 94:4937-4942), bacterial displays (Georgiou, et al., 1997, Nature Biotechnology 15:29-34) and / or yeast displays (Kieke, et al., 1997, Protein Engineering 10: 1 Single-chain antibodies may be selected by using other techniques known in the art for selecting antibody fragments from a library using enrichment methods (including, but not limited to, 303-1310) as an alternative to the techniques discussed previously. Single-chain antibodies are selected from a library of single-chain antibodies produced directly using filamentous phage techniques. Phage display techniques are known in the art (e.g., U.S. Patents No. 5,565,332, 5,733,743, 5,871,907, 5,872,215, 5,885,793, 5,962,255, 6,140,471, 6,225,447, 6,291,650, 6,49 See the specifications of U.S. Patent Nos. 2,160, 6,521,404, 6,544,731, 6,555,313, 6,582,915, and 6,593,081, as well as other members of the U.S. Patent Family, or the technology from Cambridge Antibody Technology (CAT) disclosed in the application for priority under U.S. Patent No. 9206318 filed on 24 May 1992 (see also Vaughn, et al. 1996, Nature Biotechnology 14:309-314). Single-stranded antibodies may be designed and constructed using available recombinant DNA techniques, such as DNA amplification methods (e.g., PCR), or possibly by using each hybridoma cDNA as a template.

[0049] Mutant antibodies are also included in the scope of the present invention. Therefore, variants of the sequence described in this application are also included in 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, antibodies with further improved affinity can be obtained by amino acid substitution. Alternatively, translation efficiency in an expression system for antibody production can be improved by codon optimization of the nucleotide sequence.

[0050] Such mutant antibody sequences will share 70% or more (i.e., 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more) sequence identity with the sequences described herein. Such sequence identity is calculated relative to the full length of the reference sequence (i.e., the sequences described herein). The identity rates referred to herein are those determined using BLAST version 2.1.3 with default parameters specified by NCBI (National Center for Biotechnology Information; www.ncbi.nlm.nih.gov / ) [Blosum 62 matrix; gap start penalty = 11 and gap extension penalty = 1]. For example, the present invention provides peptide sequences containing at least about 5, 10, 15, 20, 30, 40, 50, 75, 100, 150 or more adjacent peptides and all intermediate-length peptides between them for one or more of the sequences disclosed herein. As used herein, the term “intermediate length” is intended to describe any length between reference 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 having broad-spectrum neutralizing activity in serum, either alone or in combination with other antibodies, such as VRC01, anti-V3 loop, CD4bs and CD4i antibodies, and PG9 / PG16-like antibodies (but not limited to these).

[0052] According to another embodiment, the present invention provides a method for preparing and administering an HIV antibody composition suitable for administration in a quantity and schedule sufficient to induce a protective immune response against HIV or a reduction of the HIV virus in humans to human or non-human primate patients who are infected with HIV or at risk of HIV infection.

[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 a plurality of antibodies having the properties described herein, and may further comprise an HIV-neutralizing antibody 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, for the purpose of prophylactically or therapeutically treating the progression of various subtypes of HIV infection after vaccination. Such combinations may be selected according to the desired immunization. When the antibody is administered to an animal or a human, it may be used in combination with one or more pharmaceutically acceptable carriers, excipients, or adjuvants, as are generally known to those skilled in the art. The composition may further include, but is not limited to, broad-spectrum neutralizing antibodies known in the art, including (but not limited to) VRC01, b12, anti-V3 loop, CD4bs, and CD4i antibodies, as well as PG9 / PG16-like antibodies.

[0055] Furthermore, regarding the determination of the level of efficacy in patients for HIV treatment, appropriate animal models are particularly available and have been widely used to evaluate the in vivo efficacy of various gene therapy protocols against HIV (Sarver et al. (1993b), cited above). These models include mice, monkeys, and cats. Even if 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, bone marrow excision BALB / c) reconstituted using 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 HIV pathogenesis models. Similarly, simian immunodeficiency virus (SIV) / monkey models and feline immunodeficiency virus (FIV) / cat models can be used. When used in the therapeutic treatment of AIDS, the pharmaceutical composition may contain other pharmaceuticals together with the vector according to the present invention. These other pharmaceuticals can be used in their conventional forms (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 isolated HIV antibody, or an affinity-matured version, which provides an option for prophylactic or therapeutic treatment to reduce HIV virus infection. 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: Taylor and Francis; pp. 145-177; Akers, et al., 2002, Pharm. Biotechnol. 14: 47-127). A pharmaceutically acceptable composition suitable for administration to a patient will contain an effective amount of antibody in a formulation that maintains biological activity during storage within an acceptable temperature range and also promotes maximum stability. The pharmaceutical composition may, depending on the desired formulation, contain a pharmaceutically acceptable diluent, a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient, or for administration to animals or humans. This may also include vehicles commonly used in the formulation of pharmaceutical compositions. 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 Hanks' solution. The amount of excipient useful in the pharmaceutical composition or formulation of the present invention is the amount that helps to uniformly distribute the antibody throughout the composition so that the composition can be uniformly dispersed when it is to be administered to a subject who needs it. It may help to dilute the antibody to a concentration that produces the desired beneficial relief or healing result while minimizing any adverse side effects that may occur from concentrations that are too high. It may also have a preservative effect. Therefore, more excipients will be used for antibodies with high biological activity. On the other hand, fewer excipients will be used for any active ingredient(s) that exhibit lower biological activity.

[0057] Antibody compositions or vaccine compositions comprising the above-mentioned antibodies, and at least one or a combination of the antibodies described herein, can be administered for prophylactic and therapeutic treatment of HIV virus infection.

[0058] The present invention also relates to isolated polypeptides comprising novel amino acid sequences for light and heavy chains, as well as consensus sequences for the heavy and light chains of SEQ ID NOs. 1 and 2, as shown in Figure 3.

[0059] In other related embodiments, the present invention provides polypeptide variants encoding the amino acid sequence of the HIV antibody shown in Figure 3; the consensus sequences for the heavy and light chains of SEQ ID NOs: 1 and 2. These polypeptide variants exhibit at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity compared to the polypeptide sequence of the present invention when determined using the methods described herein (e.g., BLAST analysis using standard parameters). It will be understood by those skilled in the art that these values ​​can be appropriately adjusted, taking into account amino acid similarity, etc., to determine the corresponding identity of the encoded protein.

[0060] The term "polypeptide" is used in its conventional sense, i.e., as a sequence of amino acids. Polypeptides are not limited to products of a specific length. Peptides, oligopeptides, and proteins are included in the definition of polypeptide, and such terms may be used interchangeably herein unless otherwise indicated. The term also includes post-expression modifications of polypeptides, both naturally occurring and non-natural, such as glycosylation, acetylation, phosphorylation, and other modifications known in the art. Polypeptides may be entire proteins or partial sequences thereof. The specific polypeptides relevant to the present invention are amino acid partial sequences comprising CDR, VH, and VL that can bind to antigens or HIV-infected cells.

[0061] When this term is used herein, a polypeptide "mutant" is a polypeptide that is typically different from the polypeptides specifically disclosed herein by one or more substitutions, deletions, additions, and / or insertions. Such mutants may be of natural origin or may be synthetically produced, for example, by one or more modifications of the polypeptide sequences described herein, and by the evaluation of one or more biological activities of the polypeptides described herein, and / or by the use of any of the many techniques known in the art.

[0062] For example, one amino acid can be substituted for another amino acid in the protein structure without significant loss of the ability to bind to other polypeptides (e.g., antigens) or cells. Since a protein's binding ability and properties determine its biological functional activity, a certain amino acid sequence substitution can affect the protein sequence and, therefore, its underlying DNA coding. It is possible to create a DNA sequence, thereby obtaining a protein with similar properties. Therefore, it is conceivable that various modifications can be made to the peptide sequence of the disclosed composition, or the corresponding DNA sequence encoding the peptide, without significantly losing their biological utility or activity.

[0063] In many cases, polypeptide variants will contain one or more conservative substitutions. A “conservative substitution” is the substitution of one amino acid with another amino acid having similar properties, such that those skilled in peptide chemistry would expect the polypeptide’s secondary structure and hydropathicity to remain substantially unchanged.

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

[0065] "Homologousity" or "sequence identity" refers to the percentage of residues in a polynucleotide or polypeptide sequence variant that is identical to a non-mutant 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 polynucleotide or polypeptide 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 present invention provides polypeptide fragments comprising continuous stretches of various lengths of the amino acid sequences disclosed herein. For example, the present invention provides peptide sequences comprising continuous stretches of at least about 5, 10, 15, 20, 30, 40, 50, 75, 100, 150, or more peptides and all intermediate-length peptides between them of one or more of the sequences disclosed herein.

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

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

[0069] In other related embodiments, the present invention provides polynucleotide variants encoding the heavy and light chain polypeptide sequences of the HIV antibody shown 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 present invention when determined using the methods described herein (e.g., BLAST analysis using standard parameters). It will be understood by those skilled in the art that these values ​​can be appropriately adjusted by taking into account codon degeneracy, amino acid similarity, leading frame arrangement, etc., to determine the corresponding identity of proteins encoded by the two nucleotide sequences.

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

[0071] "Isolated nucleic acids" are nucleic acids that are substantially isolated not only from other genomic DNA but also from proteins or complexes, such as ribosomes and polymerases, that naturally have their own sequences. This term encompasses nucleic acid sequences removed from their naturally occurring environment and includes recombinant or cloned DNA isolates, and chemosynthetic analogs or analogs biosynthesized by heterologous systems. Substantially pure nucleic acids include the isolated forms of said nucleic acids. Therefore, this refers to nucleic acids that are isolated from the outset and does not exclude genes or sequences later added to nucleic acids isolated by human hands.

[0072] When the term "polynucleotide variant" is used herein, it refers to a polynucleotide that is typically different in one or more substitutions, deletions, additions, and / or insertions from the polynucleotides specifically disclosed herein. Such variants may be of natural origin or may be synthetically produced, for example, by one or more modifications of the polynucleotide sequences of the present invention, and by the evaluation of one or more biological activities of the encoded polypeptides described herein, and / or by the use of any of the many techniques known in the art.

[0073] The structure of the polynucleotide of the present invention can be modified to create functional molecules encoding mutant or derivative polypeptides having desired properties. When it is desired to modify the amino acid sequence of the polypeptide to produce an equivalent or even improved mutant or portion of the polypeptide of the present invention, those skilled in the art will typically modify one or more codons in the encoding DNA sequence.

[0074] Typically, a polynucleotide variant contains one or more substitutions, additions, deletions, and / or insertions such that the immunogenicity of the polypeptide encoded by the variant polynucleotide is not substantially reduced compared to the polypeptide encoded by the polynucleotide sequence specifically shown herein.

[0075] In further embodiments, the present invention provides polynucleotide fragments comprising continuous stretches of various lengths of sequences identical to or complementary to one or more sequences disclosed herein. For example, the present invention provides polynucleotides comprising continuous stretches of at least about 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more nucleotides and all intermediate lengths between them, encompassing any length between the cited values, for example, 16, 17, 18, 19, etc., 21, 22, 23, etc., 30, 31, 32, etc., 50, 51, 52, 53, etc., 100, 101, 102, 103, etc., 150, 151, 152, 153, etc., and all integers between 200 and 500, 500 and 1000.

[0076] In another embodiment of the present invention, a polynucleotide composition is provided that can hybridize to a polynucleotide sequence, or a fragment thereof, or a complementary sequence thereof, under medium to high stringency conditions. Hybridization methods are well known in the field of molecular biology. For illustrative purposes, a suitable moderately stringent condition is provided for testing the hybridization of the polynucleotide of the present invention with other polynucleotides. The procedure includes pre-washing in a solution of 5x SSC, 0.5% SDS, and 1.0 mM EDTA (pH 8.0); hybridization overnight in 5x SSC at 50-60°C; followed by two washes at 65°C for 20 minutes each in 2x, 0.5x, and 0.2x SSC containing 0.1% SDS, respectively. It will be understood by those skilled in the art 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 to, for example, 60-65°C or 65-70°C.

[0077] In some embodiments, the polypeptide encoded by the polynucleotide variant or fragment has the same binding specificity as the polypeptide encoded by the native polynucleotide (i.e., it binds specifically or preferentially to the same epitope or HIV strain). In some embodiments, the polynucleotides, polynucleotide variants, fragments and hybridization sequences described herein encode polypeptides having binding activity levels of at least about 50%, at least about 70%, and at least about 90% of that of the polypeptide sequences specifically shown herein.

[0078] The polynucleotides or fragments thereof of the present invention can be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc., regardless of the length of their own coding sequences, so their total length can vary considerably. Nucleic acid fragments of almost any length can be used. For example, exemplary polynucleotide segments having total lengths (including all intermediate lengths) of about 10,000, about 5,000, about 3,000, about 2,000, about 1,000, about 500, about 200, about 100, and about 50 base pairs are included in many embodiments of the present invention.

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

[0080] The present invention also provides vectors and host cells containing the nucleic acids of the present invention, as well as recombinant methods for the production of the polypeptides of the present invention. The vectors of the present invention include those that can replicate in any type of cell or organism, including, for example, plasmids, phages, cosmids, and minichromosomes. In some embodiments, the polynucleotide-containing vectors of the present invention are vectors suitable for the growth or replication of the polynucleotides, or vectors suitable for the expression of the polypeptides of the present invention. Such vectors are known and commercially available in the art.

[0081] "Vectors" include shuttle vectors and expression vectors. Typically, plasmid constructions will also include a replication origin (e.g., ColE1 replication origin) and a selection marker (e.g., ampicillin or tetracycline resistance) for replication and selection of bacterial plasmids, respectively. "Expression vector" refers to a vector containing regulatory sequences or modulogen necessary for the expression of antibodies containing the antibody fragment of the present invention in bacterial or eukaryotic cells.

[0082] As used herein, the term “cell” can be any cell, including but not limited to eukaryotic, multicellular species such as mammalian cells or human cells (as opposed to, for example, unicellular yeast cells). A cell may exist as a single entity or as part of a larger collection of cells. Such a “larger collection of cells” could be, for example, a cell culture (mixed or pure), a tissue (e.g., endothelium, epithelium, mucosa or other tissue), an organ (e.g., lungs, liver, muscles and other organs), or an organ system (e.g., the circulatory system, respiratory system). This may include the gastrointestinal system, urinary system, nervous system, endothelial system or other organ systems, or living organisms (e.g., birds, mammals, etc.).

[0083] The polynucleotides of the present invention may be synthesized as a whole, or synthesized in small amounts and combined, and inserted into a vector using routine molecular and cell biological methods, for example, including subcloning of the polynucleotides into a linear vector using appropriate restriction sites and restriction enzymes. The polynucleotides of the present invention are amplified by polymerase chain reaction using oligonucleotide primers complementary to each chain of the polynucleotide. These primers also include restriction enzyme cleavage sites to facilitate subcloning into a vector. The 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 the polypeptide of the present invention, the nucleotide sequence encoding the polypeptide, or a functional equivalent, may be inserted into a suitable expression vector, i.e., a vector containing the elements necessary 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 the target polypeptide and the sequences encoding appropriate transcription and translation regulatory 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, FMet. al.(1989)Current Protocols in Molecular It is listed in 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 antibody, polypeptide, or nucleic acid of the present invention in either a labeled or unlabeled form. In addition, when the antibody, polypeptide, or nucleic acid is supplied in a labeled form suitable for indirect binding assays, the kit further includes reagents for performing the appropriate indirect assay. For example, the kit may include one or more suitable containers containing an enzyme substrate or a derivatizer, depending on the nature of the labeling. A control sample and / or instructions may also be included. The present invention also provides kits for detecting the presence of the HIV antibody of the present invention or the nucleotide sequence of the HIV antibody in a biological sample by PCR or mass spectrometry.

[0086] As used herein, “label” refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody in order to produce a “labeled” antibody. The label may also be conjugated to polypeptides and / or nucleic acid sequences disclosed herein. The label may be detectable on its own (e.g., radioisotope labeling or fluorescent labeling), or, in the case of enzymatic labeling, may catalyze the chemical denaturation of a detectable substrate compound or composition. The antibodies and polypeptides of the present invention may also be modified to include epitope tags or labels for use, for example, in purification or diagnostic applications. Suitable detection means include, but are not limited to, the use of labels such as radionucleotides, enzymes, coenzymes, fluorescent agents, chemiluminescent agents, chromogens, enzyme substrates or cofactors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, and dyes.

[0087] According to another embodiment, the present invention provides a diagnostic method. The diagnostic method generally involves contacting a biological sample taken from a patient, such as blood, serum, saliva, urine, sputum, cell swab sample, or tissue biopsy material, with an HIV antibody, and the antibody reacting with the sample with a control sample or This includes determining whether a sample selectively binds to a predetermined cutoff value, thereby indicating the presence of the HIV virus.

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

[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 collecting 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, cell swab sample, or tissue biopsy material. 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 determining whether or not an element exists. The terms “determining,” “measuring,” “evaluating,” “assessing,” and “assaying” are used alternately and include quantitative and qualitative determinations. Assessments may be relative or absolute. “Assessing the existence” includes determining the quantity of something that exists and / or determining whether or not it exists. As used herein, the terms “determining,” “measuring,” and “assessing” and “assaying” are used alternately and include both quantitative and qualitative determinations.

[0091] II. Methods to reduce virus replication The present invention further provides methods for reducing an increase in HIV viral titer, viral replication, viral growth, or the amount of HIV viral protein in a subject. In another embodiment, one method comprises administering to a subject an amount of HIV antibody effective in reducing an increase in HIV titer, viral replication, or the amount of HIV protein 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 further host cells or tissues, the method comprising contacting mammalian cells with an antibody or a portion thereof that binds to an antigen 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, the method comprising administering to the mammal a pharmaceutical composition comprising an HIV antibody disclosed herein. According to one embodiment, the method for treating an HIV-infected mammal comprises administering to the mammal a pharmaceutical composition comprising an antibody or fragment thereof of the present invention. The composition of the present invention is disclosed The composition may include one or more antibodies (e.g., multiple antibodies or a pool of antibodies) having the same properties. The composition may also include other HIV-neutralizing antibodies known in the art, such as, but not limited to, VRC01, PG9, and b12.

[0094] Passive immunization has been proven to be an effective and safe strategy for the prevention and treatment of viral diseases. (See, for example, Keller et al., Clin. Microbiol. Rev. 13:602-14 (2000); Casadevall, Nat. Biotechnol. 20:114 (2002); Shibata et al., Nat. Med. 5:204-10 (1999); and Igarashi et al., Nat. Med. 5:211-16 (1999)). Passive immunization using human monoclonal antibodies provides an emergency treatment strategy for the emergency prevention and treatment of HIV.

[0095] Subjects at risk of HIV-related disease or disability include patients who have been in contact with an infected person or who have been exposed to HIV in any other way. Prophylactic medication may be administered before the onset of symptoms characteristic of HIV-related disease or disability in order to prevent or slow the progression of the disease or disability.

[0096] For the in vivo treatment of human and non-human patients, a pharmaceutical formulation comprising the HIV antibody of the present invention is administered to or provided to said patients. When used in vivo therapy, the antibody of the present invention is administered to the patient in a therapeutically effective dose (i.e., a dose that eliminates or reduces the patient's viral load). The antibody is administered to human patients according to known methods, for example, by intravenous administration, for example, as a bolus or by continuous infusion over a period of time, via intramuscular, intraperitoneal, intraracerobrospinal, subcutaneous, intra-articular, synovial bursa, subarachnoid, oral, topical or inhalation routes. The antibody can be administered parenterally, and if possible, it can be administered to target cell sites or intravenously. In some embodiments, the antibody is administered intravenously or subcutaneously. The therapeutic composition of the present invention may be administered systemically, parenterally, or topically to a patient or subject. The above parameters for evaluating therapeutic success and improvement of disease are readily measured by routine procedures well known to physicians.

[0097] For parenteral administration, the antibody may be formulated in an injectable unit dosage form (solution, suspension, emulsion) accompanied by 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. Examples of non-aqueous vehicles include, but are not limited to, non-volatile 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 drug administration plan depend on various factors readily determined by the physician, such as the nature of the infection, its therapeutic index, the patient, and the patient's medical history. Generally, a therapeutically effective dose of antibody is administered to the patient. In some embodiments, the amount of antibody administered ranges from about 0.1 mg / kg to about 50 mg / kg of the patient's body weight. Depending on the type and severity of the infection, an antibody dose of about 0.1 mg / kg to about 50 mg / kg (e.g., about 0.1–15 mg / kg / dose) is an initial candidate dose for administration to the patient, for example, by one or more separate doses or by continuous infusion. The course of this treatment is readily monitored by conventional methods and analyses, and based on criteria known to physicians or others skilled in the art. The above parameters for evaluating treatment success and disease improvement are readily measurable by routine procedures known to physicians.

[0099] Other therapies may be used in combination with the administration of the HIV antibody of the present invention. Combination therapy 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 exert their biological activity simultaneously. Such combination therapy can produce a synergistic therapeutic effect. The above parameters for evaluating treatment success and disease improvement are readily measurable by routine procedures well known to physicians.

[0100] The terms “to treat,” “to cure,” and “to alleviate” are used interchangeably and refer to both therapeutic and preventive or protective measures, the purpose of which is to prevent or delay (mitigate) the targeted pathological condition or disability. Those requiring treatment include those who already have a disability, as well as those who are susceptible to the disability or who need to prevent the disability. An infection is “successfully treated” if, after a subject or mammal receives a therapeutic dose of antibodies according to the method of the present invention, the patient shows an observable and / or measurable reduction or 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 all infected cells; and / or some degree of relief of one or more symptoms associated with a particular infection; a reduction in morbidity and mortality, and an improvement in quality of life. The above parameters for evaluating the success of treatment of a disease and improvement of a disease are readily measurable by routine procedures known to physicians.

[0101] The term "therapeutic dose" refers to the amount of antibody or drug that is effective in treating a disease or disorder in a subject or mammal.

[0102] Administration “in combination” with one or more additional therapeutic agents includes simultaneous administration and sequential administration in any order.

[0103] As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cells or mammals being exposed at the doses and concentrations used. In many cases, physiologically acceptable carriers are pH-buffered aqueous solutions. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants, including but not limited to ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, e.g., serum albumin, gelatin, or immunoglobulins, but not limited to these; hydrophilic polymers, e.g., polyvinylpyrrolidone, but not limited to these; amino acids, e.g., glycine, glutamine, asparagine, arginine, or lysine, but not limited to these; monosaccharides, disaccharides, and other carbohydrates (including but not limited to glucose, mannose, or dextrin); chelating agents, e.g., EDTA, but not limited to these; sugar alcohols, e.g., mannitol or sorbitol, but not limited to these; salt-forming counterions, e.g., sodium, but not limited to these; and / or nonionic surfactants, e.g., TWEEN, polyethylene glycol (PEG), and PLURONICS, but not limited to these.

[0104] If a range is given, unless otherwise explicitly indicated by the context, each intermediate value between the upper and lower limits of that range, up to one-tenth of the lower limit unit, and any other display or intermediate value within the range are understood to be included in the present invention. The upper and lower limits of these smaller ranges, which may be independently included within these smaller ranges, are also included in the present invention, depending on any specially excluded limits within the range. If the range includes one or both limits, the range excluding either of these included limits is also included in the present invention. [Examples]

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

[0106] HIV antibodies were cloned and generated according to previously described gp140-specific single B cell capture methods (Mouquet, H. et al. PLoS One 6, e24078 (2011); Tiller, T. et al. J Immunol Methods 329, 112-24 (2008); and Scheid, J. et al. Nature 458, 636-40 (2009)). PGT121 GM and 10-1074 GM The "glycomutant" antibody was produced by substituting 10-1074 residues at HC positions 32, 53, 54, 58, 97, and 100 with PGT121, and vice versa. The binding properties of the anti-gp140 antibody to the HIV Env protein have been previously described (Scheid, J.F. et al. Science 333, 1633-7 (2011); Walker, L. et al. Nature 477, 466-70 (2011); and Mouquet, H. et al. PLoS Neutralization was analyzed by ELISA, SPR, and glycan microarray assays, as in One 6, e24078 (2011). Neutralization was assessed using (i) luciferase-based analysis in TZM.bl cells, and (ii) PBMC-based analysis using infection with primary HIV-1 variants, 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. et al. Nature medicine 16, 995-7 (2010)). The structures of PGT121 ("ligand-less" and "ligand-possessed"), 10-1074, and GL Fab fragments were elucidated by molecular substitution to 2.8 Å, 2.3 Å, 1.8 Å, and 2.4 Å resolutions, respectively.

[0107] Single B-cell RT-PCR and Ig gene analysis gp140 from patient 10 (pt10; called patient 17 in Nature 477(7365):466-470) PBMC + CD19 + IgG + Single-cell sorting of B cells, cDNA synthesis, and nested PCR amplification of Ig genes were performed as described in a previous study (PLoS One 6(9):e24078). The Igλ gene expressed by the PGT121 clonal variant was PCR amplified using a more upstream forward primer (L-Vλ3-21 * 02: 5’CTGGACCGTTCTCCTCCTCG 3’) in the leader region to avoid regions (31) that might be mutated. All PCR products were sequenced and analyzed for Ig gene usage, CDR3 analysis, and VH / Vκ somatic hypermutation numbers (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 phylogenetic trees were generated from them by the neighbor-joining method (in best tree mode and outgroup rooting method). Alternatively, phylogenetic trees were generated using the UPGMA method (in best tree mode).

[0108] The germline (GL) precursor gene segments of PGT121-like and 10-1074-like antibodies were identified for V H 4-59 * 01, J H 6 * 03, V L 3-21 * 02 and J L 3 *Identified as 02. (These gene segments are among the most frequently used in the repertoire of human antibodies (PLoS One 6(8):e22365;Immunogenetics 64(5):337-350). To construct a representative GL ancestral sequence, we IgBLAST (http: / / www.ncbi.nlm.nih.gov / igblast) was used to align the IgH and IgL sequences of 10-996 (antibodies containing minimal somatic hypermutations) to the GL sequence. The GL IgH sequence was mature V H and J H The gene segments are replaced with their GL-corresponding regions, and the N region nucleotides and D H It was constructed by using 10-996 sequences for the CDRH3 region containing the gene segment. The GL IgL sequence is V L 3-21 * 02 and J L 3 * It was constructed from the O2 gene segment sequence.

[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 to the original PCR product sequences. PGT121 and 10-303 shared the same Igλ gene and had a single 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 generated by introducing a single substitution (V2M) into the 10-303IgH gene using site-directed mutation (QuikChange Site-Directed Mutagenesis Kit; Stratagene). To generate His-tagged Fab, IgG1 C HBy modifying our standard Igγ1 vector (Science 301(5638):1374-1377) to encode one domain followed by a 6x-His tag, we obtained PGT121 and 10-1074 V H The gene was subcloned into a 6xHis-IgCγ1 expression vector. PGT121 GM (S32Y, K53D, S54R, N58T, H97R, T100lY) and 10-1074 GM IgH DNA fragments encoding mutant antibodies (Y32S, D53K, R54S, T58N, R97H, Y100lT) were obtained as synthetic minigenes (IDTs) and subcloned into an Igγ1 expression vector.

[0110] 10-1074 GM The heavy chain sequences for each are listed below, with mutations underlined. 10-1074 GM The light chain sequence 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 obtained by polyethyleneimine (PEI) precipitation (PLoS One) IgH and IgL expression plasmids were produced by transient transfection of exponentially grown HEK293T cells (ATCC, CRL-11268) using 6(9):e24078). IgG antibodies were purified with affinity using Protein G Sepharose beads (GE Healthcare) according to the manufacturer's instructions. Fab fragments were purified with affinity using HisPur® cobalt resin (Thermo Scientific) as described below.

[0112] HIV-1 Env protein Using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's instructions, alanine mutations were introduced into the pYU-2 gp120 vector (donated by J. Sodroski, Harvard Medical School) at positions 301-303 (Asn-Asn-Thr), 324-325 (Gly-Asp), and 332 (Asn) (HXBc2 amino acid numbering). The same procedure was used to introduce pYU-2 gp120 N332A Asn262 in vectors gp120 and Asn406 gp120 Each PNGS located between them We generated "double glycan" mutants 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 using an expression vector encoding the HXB2c 2CC core (PLoS Pathog 5(5):e1000445) protein and the YU-2 gp120 mutant protein (Nature 393(6686):648-659). kif To produce ), 25 μM kifunesin (Enzo Life Sciences) was added during transfeeding. The culture supernatant was collected and concentrated using a centrifuge-based filtration system (Vivacell 100, Sartorius Stedim Biotech GmbH) that allowed for buffer exchange of the sample to 10 mM imidazole, 50 mM sodium phosphate, and 300 mM sodium chloride; pH 7.4. The protein was 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 production YU-2 gp120 in PBS is incubated overnight at 37°C, with 200 U of PNGase F (New England Biolabs) or 10,000 U of Endo H in their respective reaction buffers, which do not contain denaturants. f The sample was digested at New England Biolabs. After replacing the buffer with PBS using a centrifugal filter (Amicon® Ultra, Millipore), glycosidase-treated gp120 (200 ng) was examined by SDS-PAGE using a 4-12% NuPAGE gel (Invitrogen), followed by silver staining (Pierce Silver Stain Kit, Thermo Scientific).

[0115] ELISAs A high-binding 96-well ELISA plate (Costar) was coated overnight with purified gp120 at 100 ng / well in PBS. After washing, the plate was blocked for 2 hours with 2% BSA, 1 μM EDTA, and 0.05% Tween-PBS (blocking buffer), then incubated for 2 hours with IgG at a concentration of 26.7 nM (or 427.2 nM for ELISAs using the YU-2 gp120 double glycan mutant), and diluted 1:4 seven times sequentially with PBS. After washing, the plate was advanced by incubation for 1 hour with goat HRP-conjugated anti-human IgG antibody (Jackson ImmunoResearch) (0.8 μg / mL in blocking buffer) and by adding HRP dye protozoan (ABTS solution, Invitrogen) (PLoS One 6(9):e24078). Selected gp120 V3 Antibody binding to overlapping peptides was tested using the peptide-ELISA method described earlier.

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

[0117] Glycan microarray analysis Microarrays were generated by robotically printing lipid-bound glycan probes (neoglycolipids) repeatedly at two levels (2 and 5 fmol / spot) 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 N-glycans. The probe sequences are shown in Figure 7A. In short, antibody was 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 The experiment was performed using Biacore T100 (Biacore, Inc) (Nature 467(7315):591-595). In short, YU-2 gp140 and gp120 proteins were coupled onto a CM5 chip (Biacore, Inc) at a coupling density of 300 RU via primary amine coupling. Anti-gp120 IgG and germline precursor (GL) were injected into the flow cell at 1 μM and 10 μM concentrations, respectively, with a flow rate of 35 μL / min, a 3-minute association phase, and a 5-minute dissociation phase. The sensor surface was regenerated by injecting 10 mM glycine-HCl pH 2.5 at a flow rate of 50 μL / min for 30 seconds. Dissociation (k d (seconds -1 )), meeting (k a (M -1 seconds -1 ) and coupling constant (K D (M) or K A (M -1 The binding constant for divalent IgG calculated using a 1:1 binding model without bulk reflectance (RI) correction was determined from kinetic analysis after background subtraction (Biacore T100 Evaluation software). The binding constant for divalent IgG calculated using the 1:1 binding model was determined as K D To emphasize that the value includes potential avidity effects, this document refers to it as "apparent" affinity.

[0119] Neutralization assay Viral neutralization was evaluated using a luciferase-based assay in TZM.bl cells (J Virol 79(16):10108-10125). The HIV-1 pseudoviruses tested mainly 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 treated with 25 μM kyfunesin (Enzo Life Sciences) (Figure 8C) or HEK293S GnTI. - / - It was produced in cells (Figure 8D). Using nonlinear regression analysis, the concentration at which maximum half of the inhibition was observed (IC) was determined. 50The value was calculated. Infection with primary HIV-1 variants (n=95) isolated from clade B-infected donors with known seroconversion dates between 1985 and 1989 ("past seroconversions", n=14) or clade B-infected donors with known seroconversion dates between 2003 and 2006 ("current seroconversions", n=21) was performed using assays based on previously characterized PBMCs (Journal of virology 85(14):7236-7245; Nat Med The neutralizing activity was also evaluated in 16(9):995-997). The neutralizing activity of each antibody was evaluated by the percentage of neutralized virus in an IC50 test ranging from 0.001 to 50 μg / mL. 50 The area under the best-fit curve was calculated using GraphPad Prism software (v5.0b) to fit the values. The relative area under the curve (RAUC) value was derived by normalizing all AUC values ​​by the highest value (obtained at 10⁻¹⁷⁴).

[0120] statistical analysis Statistical analysis was performed using GraphPad Prism software (v5.0b). The neutralizing efficacy of antibodies against gp120 and gp140 in the TZM-bl assay against a selected panel of 9 viral strains, relative to their apparent binding affinity, was analyzed using the Spearman correlation test. The Mann-Whitney test was used to analyze (i) the affinity of antibodies belonging to PGT121 or group 10-1074 to gp120 / gp140, and (ii) past... We compared the neutralizing activity against viruses isolated from current seroconverted antibody patients.

[0121] Crystallization and structure determination For crystallization, 6x-His-tagged PGT121, 10-1074, and 10-996GL Fab were expressed. Fab was sequentially extracted from the supernatant of transiently transfected HEK293-6E cells. 2+-Purification was performed by NTA affinity (Qiagen) and Superdex200 10 / 300 (GE Healthcare) size exclusion chromatography. For ligand-less PGT121 Fab crystals, 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 cleavage of the IgG, which was further purified by Superdex200 10 / 300 (GE Healthcare) size exclusion chromatography.

[0122] Purified Fab was concentrated in PBS buffer to 8–20 mg / mL ("ligand-free" PGT121, 8 mg / mL; 10–1074 and GL, 20 mg / mL). "Ligand-containing" PGT121 Fab crystals were prepared from protein samples (final concentration: 15 mg / mL) mixed with 3-fold molar excess NA2 glycan and incubated at 20°C for 2 hours. Crystallization status was screened using a Mosquito® crystallization robot (TTP labs) in 400 nL droplets at 20°C with a 1:1 protein-to-reservoir ratio. Crystals of PGT121 Fab without ligand (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, and 10 mM CuCl2, while crystals of PGT121 Fab with ligand (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, and 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, and 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, and 10 mM MnCl2. The crystals were cryoprotected by immersion in a mother liquor containing 20% ​​glycerol ("ligand-free" and "ligand-containing" 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). The data were indexed, integrated, and scaled using XDS. Using data obtained from "ligand-free" PGT121 Fab crystals, we used Phenix to perform two search models after removing residues within the CDRH3 and CDRL3 loops, i.e., C of PGT128 Fab. H -C L Domain (PDB code 3PV3) and V of 2F5 H -V L Using the domain (PDB code 3IDJ), we found molecular substitution solutions for one Fab per asymmetric unit (heavy and light chain H and L, respectively). Subsequently, using the "ligand-less" PGT121 structure as a search model, we found molecular substitution solutions for "ligand-containing" PGT121 Fab (one Fab per asymmetric unit), 10-1074 Fab (one Fab per asymmetric unit), and GL (four Fabs per asymmetric unit).

[0124] Using Phenix, we perform iterative improvements (including non-crystallographic symmetry constraints on GL), The model was manually fitted to the electron density map using Coot. The atomic model was set to a resolution of 3.0 Å (R) for PGT121 Fab. work =21.6%, R free =26.4%), for 10-1074 Fab, the resolution is 1.9 Å (R work =18.7%, R free =22.3%), and for the four GL Fab molecules, 2.4 Å resolution (R work =19.4%, R free =23.7%) and for PGT121 Fab "having ligand", 2.4 Å resolution (R work =20.1%, R freeThe ratio was improved to 24.9%. The atomic model of PGT121 Fab contains 95.2%, 4.9%, and 0.0% of residues within the preferred, acceptable, and unacceptable 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 structures. The buried surface area was calculated using Areaimol (CCP4 suite) with a 1.4 Å probe.

[0125] The Fab structures were aligned using PyMOL's SuperScript. Pairwise Cα alignment was performed using PDBeFold.

[0126] Example 2: Dominance and diversity of PGT121 clones Using the YU-2 gp140 trimer as a "bait," gp140-specific IgG memory B cells were isolated from clade A-infected African donors. 87 matching immunoglobulin heavy (IgH) and light (IgL) chain genes corresponding to 23 unique clonal families were identified. One clonal family was dominant in the IgH anti-gp140 repertoire, accounting for approximately 28% of all proliferating B cell clones. This B cell family corresponds to the same clone as PGT121-123 (Nature 477(7365):466-470) and contains 38 members, 29 of which were unique at the nucleotide level (Table 3). Based on their IgH nucleotide sequences, the PGT121 family was divided into two groups: a PGT121-like group containing PGT121-123 and nine closely related mutants, and a second group containing 20 members, 10-1074-like. Our conventional primers (J Immunol Methods 329(1-2):112-124;Science 301(5638):1374-1377) did not amplify the IgL gene expressed by the PGT121B cell clone due to a nucleotide deletion in the region encoding framework region 1. However, using novel Igλ-specific primers designed to amplify highly somatically mutated genes, we obtained 24 of 38 Igλ genes (Table 3). Consistent with the high hypermutation levels of the IgH gene (average 18.2% of the VH gene), the amplified Igλ genes were highly mutated (average 18.2% of the Vλ gene), with nucleotide deletions (12 to 21 nucleotides) in framework region 1 (FWR1) and a 9-nucleotide insertion in framework region 3 (FWR3) (Figure 3B and Table 3).

[0127] The sequence alignments of three PGT antibodies (PGT-121, -122, and -123), 11 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). The sequences for the corresponding heavy chain variable region, light chain variable region, heavy chain CDR, and light chain CDR according to both the IMGT and KABT systems are listed in Table 1 below. The 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 their binding to YU-2 gp120 and gp140 was demonstrated by ELISA and surface plasmon resonance (SPR). Unless otherwise noted, the gp120 and gp140 proteins for these and other experiments were expressed in mammalian cells capable of binding either complex N-glycan or high-mannose N-glycan to PNGS. Reactivity levels with gp120 differed between PGT121 and antibodies belonging to the 10-1074 group, with the latter exhibiting higher apparent affinity, primarily due to slower dissociation from gp120 / gp140 for 10-1074 analog antibodies (Figure 4B) (Figure 3A).

[0134] Example 3: PGT121 and 10-1074 epitopes Asn332 near the V3 loop stem gp120 V3 has been reported to be extremely important for binding and virus neutralization by PGT121 (Nature 477(7365):466-470), and therefore we investigated the role of V3 in antigen recognition by PGT121-like and 10-1074-like antibodies. V1-V3 loop lacks (gp120 コア ) or uses the (2CC-core) HXB2 gp120 "core" protein which retains a portion of V3, and the YU-2 gp120 mutant protein (gp120) which has a double alanine substitution in the V3 stem. GD324-5AA ELISA was performed using ( ). The antibodies tested were a variant lacking the V3 loop and gp120 compared to intact YU-2 gp120. GD324-5AA The antibodies showed reduced reactivity to V3, with the binding of antibody groups 10-1074 being the most affected (Figures 5A and B). These results suggest that recognition by both antibody groups requires protein determinants in the vicinity of the V3 loop. No antibodies bound to the overlapping peptide extending to V3, suggesting that the target epitope is discontinuous and / or requires a specific higher-order structure not achieved by isolated peptides (Figure 5C).

[0135] Asn332 gp120(Previously numbered as Asn337 in J Proteome Res 7(4):1660-1674) gp120 ) is the sequence Asn-X-Ser / T Asn332 is the N-terminal residue of a promising N-glycosylation site (PNGS) defined as hr. gp120 To determine whether and / or its N-linked glycan is required for the gp120 reactivity of novel PGT121 and 10-1074 group antibodies, we performed ELISA on YU-2gp120 N332A The binding of these to was tested. The N332A substitution reduced the binding of PGT121 and all novel antibody variants, but the mutant gp120 (gp120) lacking the adjacent glycosylation site NNT301-3AAA Their responsiveness to mutants remained unchanged. (Asn332) gp120 In addition to PNGS, to determine whether PNGS affects recognition by novel antibodies, we used the N332A mutation in YU-2 gp120 as Asn262. gp120 and Asn406 gp120 A series of 11 double glycan mutants were constructed, combined with intermediate PNGS mutations. All of the PGT121-like and 10-1074-like antibodies were gp120 N332A It bound to each of the double glycan mutants with comparable affinity to the target.

[0136] To compare whole-glycan recognition by PGT121-like and 10-1074-like antibodies, we used PNGases that cleave both complex N-glycans and high-mannose N-glycans. We investigated their binding to YU-2 gp120 treated with 1F. Since gp120 cannot be enzymatically deglycosylated more completely unless it is denatured, PNGase Treatment with PNGase F resulted in partial deglycosylation of naturally folded gp120 (Figure 6). Nevertheless, the reactivity of the two antibody groups differed in that partial deglycosylation of gp120 by PNGase F reduced the binding activity of all PGT121-like antibodies, but not of any 10-1074-like antibodies (Figure 6C). Similar experiments using YU-2 gp120 treated with Endo H, which cleaves high-mannose N-glycans but not complex N-glycans, showed that binding of 10-1074-like antibodies was more affected than that of PGT121-like antibodies (Figure 6D).

[0137] N-glycan microarray analysis revealed that six of the seven PGT121-like antibodies tested showed detectable binding to complex mono- or biantennae N-glycans with galactose or α2-6 linked sialic acid at the terminus, but not to high-mannose glycans. This confirms and expands on previous reports of no binding of PGT121-123 to high-mannose N-glycans, and no competition for gp120 binding by Man4 and Man9 dendrons (Figure 7). In contrast, no detectable binding of 10-1074-like antibodies to protein-free glycans was observed (Figure 7). PGT121-like antibodies bound to proteinless complex N-glycans but not to high-mannose N-glycans. However, PGT121-like antibodies were produced in cells treated with kyfunesin, a mannosidase inhibitor that leads to exclusive binding of high-mannose N-glycans to PNGS, resulting in YU-2 gp120 (gp120) kif The antibodies retained binding to gp120 (Figure 8B). Most PGT121-like antibodies retain binding to gp120 kif It showed a small but reproducible decrease in binding to gp120. On the other hand, the 10-1074-like antibody showed a small but reproducible decrease in binding to gp120. kif Complete binding to was maintained (Figure 8B). These results are consistent with the hypothesis that not only high-mannose N-glycans but also complex N-glycans may be involved in the epitopes of PGT121-like antibodies.

[0138] Epitope mapping experiments were performed by competitive ELISA using two representative members from 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 mildly than the reverse. To further map the target epitopes, we mapped the V3 loop crown (Figure 5), CD4bs, coreceptor binding sites (CD4-inducible; CD4i), and a group of high-mannose N-glycans (2G12) (Journal of virology 76(14):7293-7305; Proc Natl Acad Sc We used anti-gp120 antibodies that recognize the V3 loop and N-linked glycan (PGT128) at positions 301 and 332 (i USA 102(38):13372-13377). Anti-V3 crown antibodies inhibited binding of PGT121 and 10-1369 but did not interfere with 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] Considering these factors, these data suggest that the PGT121 clone member is a V3 loop and Asn332 gp120 This suggests that the related glycans recognize sites containing protein determinants in their vicinity. However, the clones are divided into two families, the PGT121-like group and the 10-1074-like group, which differ in their affinity for gp120 and the role of the glycans in epitope formation.

[0140] Example 4: Broad-spectrum and potent HIV neutralization To evaluate the neutralizing activity of novel PGT121 variants, we measured their ability to inhibit HIV infection in TZM-bl cells using 10 viral strains, including R1166.c1 lacking PNGS at gp120 position 332. All PGT121 variants, 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). Representative germline versions (GLs) of the PGT121 / 10-1074 antibody clones were unable to bind gp120 / gp140 and were unable to neutralize any of the viruses in their panel. This implies 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. This suggests 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 against an expanded panel of 119 refractory pseudoviruses (classified as tier-2 and tier-3) with the neutralizing activity of two 10-1074-like antibodies (10-996 and 10-1074) (Tables 4 and 5). 10-996 and 10-1074 showed similar neutralizing efficacy and breadth 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 (extending to Asn332-X-Ser334 / Thr334PNGS) were resistant to neutralization (83.8% were resistant to PGT121, and 100% were 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 attributed to mutations in this PNGS (Table 7). Comparable neutralizing activity was observed for IgG and Fab forms of PGT121 and 10-1074. This suggests that divalent status is not important for their activity (Figure 1D).

[0142] To evaluate the potential role of complex N-glycans on the HIV envelope in neutralization by PGT121 and 10-1074, we produced high-mannose-only virions in two different ways: by constructing pseudoviruses in cells treated with kyfunesin, resulting in Man9GlcNAc2N-binding glycans, or by HEK293S GnTI - / - A method for obtaining Man5GlcNAc2N-binding glycans through cell-based construction. We found that PGT121 neutralizes two of three kyfunesin-derived PGT121-sensitive / 10-1074-resistant strains to an equivalent degree compared to their counterparts produced in wild-type cells (Figure 8C). GnTI - / - cells The two PGT121-sensitive / 10-1074-resistant virus strains produced were susceptible to PGT121 and 10-1074 to the same extent as their counterparts produced in wild-type cells. Consistent with previous reports that the complex N-glycan provides some protection to prevent antibody binding to the CD4 binding site, GnTI - / - The virus produced in the cell is CD4-binding site antibody (NIH45-46 G54W They were more sensitive to 3BNC60 (Figure 8D).

[0143] Example 5: Novel Infectious HIV-1 Next, we investigated the activity of PGT121 and 10-1074 against transmitted founder viruses by evaluating neutralization in peripheral blood mononuclear cell (PBMC)-based assays using 95 clade B viruses isolated from cohorts of individuals who became seroconverted between 1985 and 1989 ("past seroconverts," n=14) or individuals who became seroconverted between 2003 and 2006 ("current seroconverts," 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 revealed segregation into two groups; the PGT121 / 10-1074 group contained the most active HIV antidotes, including anti-CD4bs and PG9 antibodies (Table 8). Surprisingly, 10-1074 showed excellent neutralizing efficacy against this clade B virus panel, exhibiting a maximum range of 0.1 μg / mL for all bNAb tested (67% of 95 clade B viruses) (Table 8). 10-1074 showed higher efficacy (~20-fold difference) against current clade B viruses than PGT121, although both antibodies were effective against older 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 the PGT121-like 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). Superposition of the heavy and light chain variable domains (V H and V L ) among the three Fabs revealed that, along with the conservation of the main chain structure, the differences with respect to GL were limited to small displacements of the CDRH3 and CDRL3 loops of the affinity-matured Fabs (Table 10).

[0145] A distinctive feature shared by the antibodies is their long (25-residue) CDRH3 loops, which form a two-stranded antiparallel β-sheet that extends the F and G strands of the V H domain. In each Fab, the tip of the extended CDRH3 loop contains predominantly nonpolar residues. Similar structural features were observed for the CDRH3 of PGT145, a carbohydrate-sensitive antibody whose epitope includes the gp120 V1V2 loop. However, the extended two-stranded β-sheet of PGT145's CDRH3 contains predominantly negatively charged residues, including two sulfated tyrosines at the tip. Alignment of the V H -V L of PGT121 and PGT145 (Table 10) shows that CDRH3 PGT145 extends the previous CDRH3 PGT121 , and that its tip aligns with the V H domain, but that the CDRH3s of PGT121, 10-1074, and GL are tilted towards V L . The tilt of CDRH3 L towards V PGT121 / CDRH3 10-1074 / CDRH3 GL opens the creft between CDRH2 and CDRH3, which is a feature not shared by related antibodies.

[0146] PGT121 and 10-1074 are significantly different from each other and from GL (out of 132 residues, PGT121 VH is, 10-1074VH and GL VH These differ by 36 and 45 residues respectively, resulting in 10-1074 VH and GL VH (There are 29 differences). The majority of these differences in PGT121 / 10-1074 are due to CDR VH It is located in the loop and CDRL3. Interestingly, there are six substitutions in CDRH3 (residues 100d, 100f, 100h, 1 00j, 100l, 100n) are alternated so that they are substituted every two residues, and as a result, V L The tilt of CDRH3 causes the cleft between CDRH2 and CDRH3 to reappear on the surface. This region is likely to be one of the causes of the different subtle specificities of PGT121 and 10-1074. Heavy chain framework region 3 (FWR3) HC Five other solvent exposure substitutions in ) (residues 64, 78, 80-82; chains D and E) are potential antigen contact sites, given that the framework region in the HIV antibody can come into contact with gp120. Another difference that could contribute to subtle differences in specificity is Asp56, which is not present in 10-1074 or GL. HC Negative patch on PGT121 in the vicinity of (10-1074 and Ser56 in GL) HC ), as well as positive patches on CDRL1 and CDRL3 that are not found on similar surfaces of GL.

[0147] Somatic mutations common to PGT121 and 10-1074 may be responsible for their shared epitope features. 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 insertions into light chain FWR3, which cause bulging of the loop connecting chains D and E, and CDRL2, which results in a low negative charge surface. GL Asp50 inside LC -Asp51 LCAsn50 in both PGT121 and 10-1074 LC -Asn51 LC This includes substitution with LC. PGT121 and LC 10-1074 Many common substitutions introduced (approximately 50% of LC substitutions) are CDRL1, CDRL2, and FWR2, which are potential contact regions of epitopes shared by PGT121 and 10-1074. LC This is pointed out.

[0148] Next, Asn332 gp120 Joining and Asn301 gp120 We compared the structure of PGT128, which recognizes bound glycans and V3, with that of PGT128, which has been elucidated as a complex with an external domain / mini V3 loop gp120 expressed in cells unable to produce complex N-glycan-denatured proteins. Unlike the CDRH3 loops of PGT121 and 10-1074, PGT128 CDRH3 PGT128 VL It is not tilted in that direction, CDRH3 PGT128 It does not contain a double-stranded β-sheet. In addition, CDRH3 PGT128 (18 residues) is shorter than CDRH3 of PGT121 and 10-1074 (24 residues), but CDRH2 PGT128 It contains a 6-residue insertion not found in PGT121 or 10-1074. Due to these differences, CDRH2 is the most prominent feature in PGT128, while CDRH3 is the most prominent feature in PGT121 and 10-1074. CDRH2 PGT128 and CDRL3 PGT128 Together, Asn332 gp120 Man is connected 8 / 9 Recognizing CDRH3 PGT128 This contacts the V3 loop base. This gp120 recognition mode is not possible for PGT121 and 10-1074 because the structural features of their CDRH2 and CDRH3 loops are significantly different from those of PGT128, which is consistent with the ability of PGT128 to recognize protein-free high-mannose glycans (Figure 7), rather than PGT121 and 10-1074.

[0149] Example 7: Crystal structure of the PGT121-glycan complex The 2.4 Å resolution structure of PGT121 with complex sialylated bianthenticoglycans was elucidated using crystals obtained under conditions containing NA2 and complex asialyl bianthenticoglycans (Figure 7) (Table 9). Surprisingly, the glycan bound to PGT121 in our crystal structure was not NA2, but rather a complex N-glycan from the adjacent PGT121 Fab within the crystal lattice, specifically Asn105. HC It was an N-glycan bound to it. This glycan identity was Asn105 HC This is evident because there is electron density for the glycosidic bond to and for the terminal sialic acid on the Manα1-3Man antenna (M The galactose and sialic acid portions of the anα1-6Man antenna were previously unknown. The composition of the bound glycan corresponded to the α2-6 sialyzed A2(2-6) glycan portion bound by PGT121 in microarray experiments (Figure 7), and also to the sialyl bond predicted in the complex N-glycan bound to PNGS on the protein expressed in HEK293T cells. This structure ("ligand-bearing" PGT121) H -V L The domain is V of the PGT121 structure without bound N-glycan ("ligand-less" PGT121). H -V L The domains overlap without significant difference (Figure 10), but the elbow bend angle (V H -V L and C H 1-C L The angle between pseudo-dimolia (pseudo-dyads) differs between structures. This difference likely reflects the flexibility that allows Fab to adopt elbow bending angles that can vary depending on crystal lattice forces.

[0150] Given that binding of complex N-glycans was observed in one crystal structure ("ligand-containing" PGT121 structure) and not in another structure ("ligand-uncontaining" PGT121 structure), we hypothesize that the affinity of PGT121 for complex N-glycans not bound to gp120 is within the range of PGT121 concentration in the crystal (~10 mM). K for binding of isolated glycans D However, the 1.6 mM K result obtained for PG9 binding to Man5GlcNAc2-Asn D Assuming it is in the range of 1-10 mM, which is comparable to, the K for PGT121 binding in isolated glycans D This represents a very small contribution of PGT121 to gp120 affinity, in the nM range (Figure 4A).

[0151] Glycans within the PGT121 structure that "have ligands" are V H It interacts exclusively with the domain and makes extensive contact with residues within all three CDRs (PGT121). HC The buried surface area = 600 Å 2 The contact involves 10 direct hydrogen bonds and 18 water-mediated hydrogen bonds at 9 amino acids (Figure 11), fixing the glycan between the N-acetylglucosamine moiety bound to mannose at the branching site and the terminal sialic acid on the 1-3 antennas. Asp31 HC In addition to water-mediated hydrogen bonding, PGT121 residue Asp31 HC and His97 HC Several contacts with PGT121, including three direct hydrogen bonds, are made by this sialic acid. Sialic acid also contributes to the water-mediated hydrogen bonding network within the glycan. Direct contact with sialic acid may explain the stronger binding of PGT121 to sialyzed A2(2-6) glycans than to non-sialyzed 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 antennas may explain the observed binding to PGT121 for non-sialyzed mono and biantennae glycans (Figure 7).

[0152] Six of the residues that contribute to direct or possibly amino acid side chain contact with the glycan (Ser32 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 Unlike ), it is highly conserved among PGT121-like antibodies but not among 10-1074-like antibodies. The 10-1074 residues lack corresponding functional groups for making the observed glycan contact or for having bulky side chains that cause steric collisions. Four of these residues are the same as those in GL (Tyr32 HC-CDRH1 Tyr53 HC-CDRH2 , Gln97 HC-CDRH3 Tyr100l HC-CDRH3 Unlike other methods, this is due to a lack of binding of 10-1074-like antibodies and GL to protein-free complex glycans in our glycan microarray, resulting in hydrogen bonding and / or steric collisions (e.g., Arg97). 10-1074 In contrast, His97 PGT121 Tyr100l 10-1074 In contrast, Th100l PGT121 ) This suggests that the deficiency is the cause. In the CDRH loop, as is the case with the majority of the sequence differences between PGT121 and group 10-1074, particularly on the surface of the cleft between CDRH2 and CDRH3 where we observed the bound complex N-glycan, different recognition of complex glycans on gp120 may be the main cause of some or all of the observed subtle specificity differences.

[0153] Example 8 Substitution of glycan-containing antibody residues affects neutralization. To evaluate the contribution of complex N-glycan-containing residues identified from the "ligand-containing" PGT121 structure, we produced two mutant antibodies designed to exchange complex glycan-containing residues between PGT121 and 10-1074: 10-1074 IgG with PGT121 residues (6 substitutions at IgH Y32S, D53K, R54S, T58N, R97H, Y1001T) and PGT121 IgG with mutual substitution. GM and PGT121 GM The PGT121 complex N-glycan-containing residue exhibited near-wild-type apparent affinity for YU-2 gp120 / gp140 when measured by SPR (Figure 2A). This provides evidence that the substitution did not disrupt binding to envelope spikes derived from virus strains neutralized by both PGT121 and 10-1074 (Figure 1A). The ability to adapt the PGT121 complex N-glycan-containing residue within a 10-1074 background without disrupting binding to gp120 / gp140 bound by both wild-type antibodies implies that antigen binding is overall similar despite subtle differences in specificity.

[0154] Unlike wild-type PGT121, PGT121 GM The compound did not exhibit glycan binding in microarray experiments. This confirms that the substituted residues 10-1074 are incompatible with protein-free glycan binding (Figure 2B) and supports the suggestion that residues in contact with glycans within the "ligand-containing" PGT121 structure are involved in the recognition of complex glycans in the microarray. 10-1074 GM It also did not show binding to protein-free glycans (Figure 2B). This indicates that residues other than the substituted ones are involved in the formation of binding sites for protein-free complex N-glycans.

[0155] Next, we compared the neutralization of wild-type antibodies and "glycomutant" antibodies using a TZM-bl-based assay. We tested 40 viral strains, including strains with different resistances to PGT121 or 10-1074, as well as strains susceptible to both wild-type antibodies (Figure 2C and Table 12). PGT121 to purified YU-2 envelope protein GM and 10-1074 GM Consistent with the binding, both mutants neutralized the YU-2 virus, but 64% of PGT121-sensitive strains did not neutralize PGT121. GM It was resistant to (Figure 2C and Table 12). This suggests that the glycan-containing residues identified in the "ligand-containing" PGT121 structure are related to the neutralizing activity of PGT121. Conversely, 10-1074 GM It exhibited higher average potency than wild-type 10-1074 against 10-1074-sensitive strains, including a potency increase of more than three times against four 10-1074-sensitive strains (WITO4160.33, ZM214M.PL15, Ce1172_H1, and 3817.v2.c59) (Figure 2C and Table 12). In general, PGT121 substitution to 10-1074 resulted in 10-1074 resistance in PGT121-sensitive / 10-1074-resistant strains. GM Although they do not confer sensitivity to 10-1074, two of these strains (CNE19 and 62357_14_D3_4589) exhibit 10-1074 GM They became sensitive to (each, IC) 50 (=0.19 μg / mL and 40.8 μg / mL). Interestingly, these are intact Asn332 gp120 It is the only PGT121-sensitive / 10-1074-resistant strain that contains conjugated PNGS. Another PGT121-sensitive / 10-1074-resistant strain is Asn332. gp120 Lacking binding glycans, and PGT121 GM and 10-1074 GM They are resistant to this, which implies that their sensitivity to wild-type PGT121 requires compensation by adjacent N-glycans and / or protein portions of the epitope. A dramatic increase in functionality resulted in 10-1074 units of one stock (CNE19).GM Although this was observed only for 10-1074 susceptible strains, this result was observed for 10-1074 GM The general improvements observed (Figure 2C) are consistent with the interpretation that, in the presence of crystallographically identified glycan-containing residues, the PGT121-like recognition properties may be transferred to 10-1074, and / or its potency may be affected in other situations. In addition, PGT121 against PGT121-sensitive strains GM The loss of neutralizing activity provides evidence that the neutralizing activity of PGT121 requires the residue identified as being in contact with the complex N-glycan in the "ligand-containing" PGT121 structure.

[0156] result PGT121 is a glycan-dependent bNAb originally identified in the serum of clade A-infected donors during functional screening, giving rise to only two clonally related members. Using the gp140 trimer as a "bait" for single-cell sorting, 29 novel clonal variants were isolated. The PGT121 clonal family includes two distinct groups of closely related antibodies: the PGT121 group and the 10-1074 group. The results showed that the epitope in both groups was Asn332. gp120 This suggests the presence of PNGS at the base of the V3 loop. The PGT121-like and 10-1074-like antibody groups differ in amino acid sequence, gp120 / gp140 binding affinity, and neutralizing activity, with the 10-1074-like antibody neutralizing Asn332. gp120 While it is entirely dependent on intact PNGS in the case of Asn332, PGT121-like antibodies are used in Asn332 gp120 We were able to neutralize some virus strains that lacked PNGS.

[0157] A notable difference between the two antibody groups is that the PGT121-like antibody bound to complex N-glycans in the carbohydrate array, while the 10-1074-like antibody 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 detectable; for example, PG9 recognizes gp120-bound high-mannose glycans, but no binding to any protein-free glycans was detected in the microarray. Therefore, a positive result in a glycan microarray implies the involvement of a specific glycan in the antibody epitope, while a negative result does not rule out glycan recognition. For example, high-mannose glycans may be involved in the PGT121 epitope even if undetectable in glycan microarray experiments, which is consistent with the binding and neutralization of gp120 protein and virions in high-mannose-only forms (Figure 8).

[0158] The molecular basis for the differences between PGT121, 10-1074, and their GL precursors was partially elucidated by their crystal structures. The finding that the majority of light chain somatic mutants are shared between PGT121 and 10-1074, but that heavy chain mutations are different, suggests that the light chain contacts a common region of the gp120 epitope, while the heavy chain recognizes distinctly different feature regions. All three antibodies present an elongated CDRH3 with a nonpolar tip that may allow access to the latent epitope. Differences in the antigen-binding sites of the two mature Fabs were primarily localized to the cleft between CDRH2 and elongated 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 a PGT121-like antibody was obtained from a crystal structure in which a complex sialylated N-glycan bound to a domain residue interacts with the binding site of the adjacent PGT121Fab. Several features of the "ligand-bearing" PGT121 structure suggest that it is suitable for understanding the recognition of complex N-glycans on gp120 by a PGT121-like antibody. Firstly, the glycan in the structure corresponding to the α2-6 sialylated glycan A2(2-6)PGT121 binds in the microarray (Figure 7). Secondly, Furthermore, the glycan interacts with PGT121 using a cleft between CDRH3 and CDRH2, which structural analysis has suggested is involved in epitope recognition, and this is related to the V structure of PGT121 and 10-1074. L This potentially explains the anomalous slope of CDRH3 toward that direction. Thirdly, V, which has been identified to interact with glycans. H The majority of residues differ between PGT121 and 10-1074, which reasonably explains the different binding profiles in glycan microarrays and potentially the different subtle specificities revealed in protein binding experiments. Fourthly, 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 preserved near-wild-type binding to purified YU-2 gp120 / gp140. PGT121 GM It retained the ability to neutralize some viral strains neutralized by wild-type PGT121 and 10-1074, but was unable to neutralize strains that were PGT121-sensitive / 10-1074-resistant. This provides evidence that the glycan-binding motif is essential for the neutralizing activity of PGT121 against 10-1074-resistant strains. Due to reciprocal exchange, consistent with the crystallographically identified transfer of the glycan motif, and consistent with the hypothesis that the epitopes of PGT121-like and 10-1074-like antibodies are related, 10-1074 GMThe neutralizing effect of 10-1074 is increased or unaffected, and in some cases, 10-1074 GM This strongly neutralized PGT121-sensitive / 10-1074-resistant strains. Aside from the interaction of Asn332gp120 with PNGS for viruses sensitive to PGT121-like and 10-1074-like antibodies, analysis of gp120 sequences from strains for which PGT121 neutralization data is available did not reveal a clear pattern in PNGS utilization for different categories of viral strains (PGT121-sensitive / 10-1074-sensitive, PGT121-sensitive / 10-1074-resistant, PGT121-resistant / 10-1074-sensitive), except that 10-1074-resistant strains generally lack Asn332gp120-related PNGS.

[0160] Example 9 Passive in vivo transfer of anti-HIV-1 neutralizing mAbs Five isolated, potent, and broad-acting anti-HIV neutralizing monoclonal antibodies were administered to rhesus monkeys, and 24 hours later, these monkeys were challenged rectally with one of two different SHIVs. Combining the results from 60 challenged animals, the plasma protective neutralizing titer that prevented viral acquisition in 50% of the exposed monkeys was approximately 1:100.

[0161] Animal experiments The macaques used in this study were of MHC class IMamu-A * The result for the O1 allele was negative.

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

[0163] virus First, viral stocks were prepared by transfecting 293T cells with SHIVAD8EO or SHIVDH12-V3AD8 molecular clones using lipofectamine 2000 (Invitrogen, Carlsbad, California). After 48 hours, the culture supernatant was collected and stored at -80°C until aliquots were used. Concanavalin A-stimulated rhesus monkey PBMCs (2 × 10⁶ in 500 μL) were used. 6 Cells were infected with transmitted cell supernatant by 1 hour of spinocuration (J. of Virology 74, 10074-10080 (2000)), mixed with an equal number / volume of activated PBMCs, and the culture was maintained for at least 12 days with daily replacement of the culture medium. Samples of the 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, dengue virus NS1-specific human IgG1 monoclonal antibody (PNAS 109, 18921-18925 (2012)), or control human IgG (NIH Nonhuman Primate Reagent Resource http: / / www.nhpreagents.org) were used as negative control antibodies in this study. The selected monoclonal antibody for pre-exposure passive transfer was administered intravenously 24 hours prior to viral challenge infection.

[0165] Quantification of plasma viral RNA levels The levels of viral RNA in plasma were determined by real-time reverse transcription-PCR (ABI Prism 7900HT sequence detection system; Applied Biosystems).

[0166] Antibody concentration in plasma The concentration of administered monoclonal antibodies in monkey plasma was measured by enzyme-linked immunosorbent assay (ELISA) using recombinant HIV-1 JRFL gp120 (Progenics Pharmaceuticals) or HIVIIIB (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% (volume / volume) BSA. After blocking, serial dilutions of antibody or plasma samples were added to the plates and incubated at room temperature for 1 hour. Binding was detected using goat anti-human IgG F(ab)2 fragment (Pierce) bound to alkaline phosphatase and visualized with SIGMAFAST OPD (Sigma-Aldrich). The decay half-life of the neutralizing monoclonal antibody was calculated using a single-exponential decay formula based on plasma concentrations that began on day 5 or day 7 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 monkeys were evaluated using two types of neutralization assays: 1) TZM-bl entry assay with pseudotyped challenge-infecting viruses (AIDS Res Hum Retroviruses 26,89-98 (2010)) or 2) 14-day PBMC replication assay with replicable viruses (J. of virology 76,2123-2130 (2002)). For the TZM-bl assay, pseudotyped viruses expressing the env gene derived from SHIVAD8EO or SHIVDH12_V3AD8, with their respective envelopes Pseudotyped viruses (J. of Virology 84, 4769-4781 (2010)) prepared by co-transfecting 293T cells with pNLenv1 and pCMV vectors expressing the protein were incubated with serially diluted mAbs or plasma samples. The 50% neutralizing inhibitory dose (IC50) titer was calculated as the dilution that produced a 50% decrease in relative luminescence units (RLU) compared to the level in the virus control well after subtracting the cell control RLU (J. of Virology 84, 1439-1452 (2010)). 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)), the neutralizing phenotype (titer level) of the SHIVDH12_V3AD8 molecular clone was determined by TZM-bl cell assay.

[0168] Determination and statistical analysis of animal defense titers Plasma neutralizing titers for each R5 SHIV were calculated using the Reid and Münch method (Am J Hyg 27, 493-497 (1938)) to result in preventing 50% or 80% of viral acquisition in animals infected with the viral challenge. One animal with a significant outlier (DEW7) was excluded from the calculation. Probit regression was used to model the relationship between the plasma titers required to confer sterilizing immunity in vivo (Cambridge University Press, Cambridge, England, ed. 3rd, 2007) using all 60 passively immunized monkeys, 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 the bootstrap method.

[0169] result: SHIVDH12_V3AD8, like SHIVAD8EO, exhibited Tier 2 anti-HIV-1 neutralizing susceptibility characteristics (Table 13). Rhesus monkeys inoculated intravenously or rectally with SHIVDH12_V3AD8 presented peak viremia at 2 to 3 weeks post-infection (PI), with plasma levels ranging from 10⁵ to 10⁷ viral RNA copies / mL. In most SHIVDH12_V3AD8-infected animals, plasma viral loading decreased 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 first determined using the TZM-bl assay system (Figure 11A and B). Of these antibodies, eight, VRC01, NIH45-46(23), 45-46G54W, 45-46m2, 3BNC117, 12A12, 1NC9, and 8ANC195, targeted gp120 CD4 bs (Science 333, 1633-1637 (2011)), while three, 10-1074, PGT121, and PGT126 (Nature 477, 466-470 (2011)), were dependent on the presence of 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, with the most potent being 3BNC117, exhibited a much broader IC50 neutralizing activity (0.14 to 6.34 μg / mL). A similar hierarchy of neutralizing mAb potency (glycan-dependent > CD4 bs-dependent) was observed in SHIVDH12-V3AD8, but its neutralizing activity was distributed over a much wider range (>100 times) compared to the IC50 values ​​observed for SHIVAD8EO (Figure 11B). SHIVDH12-V3AD8 was slightly 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 the pre-exposure passive transmission study: VRC01 (as it was the first CD4bs NAb of newly isolated broad-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 the passive transmission experiment involved intravenous administration of a tapering dose of neutralizing mAb and rectal challenge infection of animals 24 hours later. The goal was to inhibit viral acquisition, and given the knowledge that repeated administration of humanized anti-HIV mAbs to individual macaques may reduce their potency and / or possibly induce anaphylactic reactions, a SHIV challenge infection dose of sufficient size to establish in vivo infection after a single dose was selected. In this regard, we previously performed rectal titer measurement of SHIVAD8 in rhesus monkeys and reported that inoculation of 1 × 10³ TCID50, determined by endpoint dilution in rhesus monkey PBMCs, was equivalent to administering approximately 3 animal infection doses of 50 (AID50) (J. of virology 86, 8516-8526 (2012)). In fact, a single rectal inoculation of 3 AID50 successfully established infection with SHIVAD8EO and SHIVDH12-V3AD8 in all 10 out of 10 rhesus monkeys.

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

[0174] The protective properties of PGT121 against SHIVAD8EO challenge infection were then investigated. PGT121 was one of the most potent glycan-targeted neutralizing mAbs measured in the TZM-bl assay (Figure 11). Based on the results obtained in VRC01, we chose to begin with in vivo PGT121 mAb titer measurement at 20 mg / kg. Two monkeys (KNX and MK4) that had undergone challenge infection tolerated SHIVAD8EO challenge infection. When PGT121 was administered at lower doses (i.e., 5 mg / kg, 1 mg / kg, or 0.2 mg / kg), one out of two animals, two out of two animals, and zero out of two animals were protected, respectively (Table 13).

[0175] The ability of VRC01 and PGT121 mAb to prevent SHIVDH12-V3AD8 acquisition was similarly evaluated (Table 13). The results obtained with VRC01 were comparable to those observed in SHIVAD8EO challenge infection: one of two recipients at 30 mg / kg was protected from the establishment of 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 tolerated the infection. PGT121 also appeared to be somewhat effective in preventing SHIVDH12-V3AD8 in SHIVAD8EO in vivo infection (Table 13). This result suggests that in vivo This is consistent with the 8-fold difference in PGT121 IC50 values ​​for neutralizing two SHIVs in the Toro assay (Figure 11).

[0176] Table 13 summarizes the results of passive transmission of 10-1074, 3BNC117, or 45-46m2 neutralizing mAbs to rhesus macaques, followed by challenge infection with either SHIVAD8EO or SHIVDH12-V3AD8. 10-1074 mAb potently inhibited in vivo acquisition of both SHIVs. CD4bs 3BNC117 and 45-46m2 mAbs were selected for passive transmission to macaques based on their IC50 values ​​against both SHIVs in in vitro neutralization experiments shown in Figure 11. 3BNC117 successfully inhibited SHIVAD8EO infection in two out of two monkeys at a dose of 5 mg / kg, but failed to inhibit infection 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 infected with the SHIVDH12-V3AD8 challenge (one of two macaques became infected at 5 mg / kg, and the other two became infected at 1 mg / kg).

[0177] Plasma samples collected at various time points from macaques that had received passive transmission were tested for HIV-1 gp120. The neutralizing mAb concentration was 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. Among the five neutralizing mAbs evaluated, PGT121 was clearly the most effective against both viruses, and SHIVDH12-V3AD8 showed somewhat greater sensitivity to this mAb (2 of 2 monkeys were protected at a plasma concentration of 0.2 μg / mL). In contrast, a plasma concentration of nearly 400 μg / mL of VRC01 was required to protect 1 of 2 animals from the same SHIVDH12-V3AD8 challenge virus (Table 13). The most potent CD4 bs mAb administered to macaques in this study, 3BNC117, was approximately 6 to 10 times more effective than VRC01 in preventing acquisition of either SHIV (Figure 12, Table 13).

[0179] The circulating half-lives of the 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 the plasma mAb concentration in several macaques 24 hours after administration of 20 mg / kg of a humanized neutralizing mAb (i.e., approximately 250 μg / mL [Table 13]), 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% decay compared to the other neutralizing mAbs at 24 hours.

[0180] When macaques were challenged with SHIVAD8EO and SHIVDH12-V3AD8, neutralizing 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 neutralizing titers and protection from SHIV infection. As a result of administration of two glycan-dependent mAbs (PGT121 and 10-1074), the highest titers of anti-HIV-1 neutralizing activity were clearly obtained at the time of viral challenge infection. The titers measured in recipients of the 45-46m2 mAb were at or below the detection limit because of its extremely short in vivo half-life.

[0181] The neutralizing titers, measured in plasma, required to prevent virus acquisition in 50% of the challenged monkeys were calculated using the method described by Reed and Muench (Am J Hyg 27, 493-497 (1938)). For 28 monkeys challenged with SHIVAD8EO or 32 monkeys challenged with SHIVDH12-V3AD 8, these protective titers were derived separately (Tables 15 and 16). The plasma neutralizing titers required to protect 50% of the animals challenged with SHIVAD8EO or SHIVDH12-V3AD8 were calculated to be 1:115 and 1:96, respectively. Since these similar titers were obtained 1) following SHIV challenge infection by the same route and inoculum size and 2) after administration of the same population of neutralizing mAbs, neutralization data from all 60 animals were combined and subjected to probit regression to investigate the relationship between plasma neutralizing titers and in vivo protection. As a further confirmation, when the terms for the SHIV viruses were 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, probit regression estimated that a plasma neutralizing titer of 1:104 would prevent virus acquisition in 50% of the animals. Probit analysis of the data also estimated that 50% plasma neutralizing titers of 1:57 or 1:329 would protect 33% or 80%, respectively, of the exposed animals.

[0182] Example 10: Administration of neutralizing mAB to an in vivo model of chronically infected HIV. Summary of Methods: The neutralizing activity of broad-acting 3BNC11724 and 10-107423 neutralizing mAbs against SHIVAD8EO was first determined in a TZM-bl cell system against SHIVAD8EO. Their ability to inhibit viral acquisition or control plasmaviremia in chronically infected animals challenged with R5-targeted SHIVAD8EO was assessed by monitoring plasma viral loading and cell-associated viral nucleic acids; levels of CD4+ T cell subsets were measured by flow cytometry; SGA analysis of circulating viral variants and determination of antibody levels in plasma were performed. Plasma concentrations of NAbs were determined by measuring their neutralizing activity against HIV-1 pseudovirus preparations 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 exhibit similar and significant decreases in circulating CD4+ T cells (Table 17). The treatment for ongoing SHIV infection was combination administration of 10 mg / kg doses of 101074 and 3BNC117. At the time of mAb administration, plasma viral loadings in macaque DBZ3 and DC99A were 1.08 × 10⁴ and 7.6 × 10³ RNA copies / mL, respectively. Both monkeys responded to combination anti-HIV-1 mAb treatment, with plasmaviremia immediately and rapidly reduced to undetectable levels within 7 to 10 days. Suppression of measurable SHIVAD8EO in the plasma of macaque DBZ3 and DC99A after a single dose of the two mAbs lasted for 27 and 41 days, respectively. In each case, plasmaviremia reverted to pre-treatment levels.

[0184] A second group of three animals (DBX3, DCF1, and DCM8) that had been infected with SHIVAD8EO for more than three years, and also presented with clinical symptoms of intermittent diarrhea and / or loss of appetite, were treated with two neutralizing antibodies (Table 17). At mAb administration, the circulating CD4+ T cell level in macaque DCM8 was only 43 cells / μL, while it was slightly higher in animal DCF1 (10⁵ cells / μL) and DBXE (158 cells / μL). Plasma viral loading exceeded 10⁵ RNA copies / mL in animal DBXE and DCF1, and was significantly lower in monkey DCM8 (1.59 × 10³ RNA copies / mL). Administration of the two mAbs to monkey DBXE resulted in a biphasic decrease from 2.0 × 10⁵ RNA copies on day 0 to undetectable plasma levels on day 20. Subsequently, high circulating viral levels reappeared in DBXE within a few days. Macaque DCM8s with lower plasma viral loads and very low circulating CD4+ T cell counts experienced a rapid decrease in viremia to undetectable levels between day 6 and day 20 after the initiation of mAb treatment. Finally, animal DCF1, which has been previously reported to produce a broadly reactive anti-HIV-1 NAb, showed a transient and relatively modest 27-fold decrease in plasmaviremia by day 6 in response to concomitant mAb therapy, after which the viral load returned to high pre-treatment levels.

[0185] PBMC-associated viral RNA and DNA levels were also determined before and after antibody administration (Table 18). In each animal, mAb treatment resulted in a decrease in cellular-associated viral RNA levels, which correlated well with plasma viral loading measurements. No consistent pattern was observed for cellular-associated viral DNA levels as a result of antibody treatment. Administration of neutralizing mAbs to chronic SHIVAD8EO-infected monkeys also had a beneficial effect on circulating CD4+ T cell levels, particularly in animals with very high viral loadings. CD4+ T cell counts in macaque DBXE and DCF1 increased 2 to 3 times during the mAb-mediated viral suppression period, but gradually decreased to pre-treatment levels as viremia became detectable again.

[0186] The plasma concentrations of each mAb were determined by measuring their plasma neutralizing activity against a select HIV-1 pseudovirus strain that was sensitive to one or the other antibody but not to both (Figure 13A). In all treated animals, suppression of SHIVAD8EO viremia was maintained 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, where a modest transient decrease in plasma viral RNA levels was observed. Interestingly, mAbs administered to clinically symptomatic macaque DCM8 and DCF1 either had shortened half-lives or were undetectable. As previously mentioned, macaque DCM8 had extremely low CD4+ T cell levels (43 cells / μL plasma), and macaque DCF1 had to be euthanized on day 56 after the start of treatment due to its deteriorating clinical condition. Autopsy 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, which has been previously shown to affect susceptibility to 10-1074 or 3BNC117 mAbs. In each case, rebound virus present in the plasma after immunotherapy was unchanged. To further test the susceptibility of the reappearing virus, combination therapy with 10-1074 and 3BNC117 (10 mg / kg each) was re-administered to two clinically asymptomatic monkeys (DBZ3 and DC99A). The viral load in each animal again rapidly decreased and became undetectable by day 7 of the second immunotherapy cycle. Viremia was suppressed for 7 days in the macaque DBZ3 and for 21 days or longer in the monkey DC99A. Taken together, these results suggest that the reappearance of the virus after the first treatment cycle in these two animals represented insufficient mAb levels in vivo, rather than antibody-selective 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-described examples and descriptions of preferred embodiments should be considered illustrative, not limiting, the invention as defined by the claims. For ease of understanding, a great many variations and combinations of the above features can be used without departing from the claimed invention. Such variations are not considered departures from the scope of the invention, and all such variations are construed to fall within the scope of the subsequent claims. All references cited herein are incorporated herein by reference in their entirety.

Claims

1. A pharmaceutical composition used to prevent or treat HIV infection or HIV-related disease, The pharmaceutical composition comprises (i) an anti-HIV antibody or its antigen-binding portion and (ii) an antiviral agent. The anti-HIV antibody or its antigen-binding portion is (1) Complementarity-determining regions (CDRs) H1 containing the amino acid sequence of SEQ ID NO: 69, CDRH2 containing the amino acid sequence of SEQ ID NO: 70, CDRH3 containing the amino acid sequence of SEQ ID NO: 71, CDRL1 containing the amino acid sequence of SEQ ID NO: 72, CDRL2 containing the amino acid sequence of SEQ ID NO: 73, and CDRL3 containing the amino acid sequence of SEQ ID NO: 74; (2) CDRH1 containing the amino acid sequence of SEQ ID NO: 39, CDRH2 containing the amino acid sequence of SEQ ID NO: 40, CDRH3 containing the amino acid sequence of SEQ ID NO: 41, CDRL1 containing the amino acid sequence of SEQ ID NO: 42, CDRL2 containing the amino acid sequence of SEQ ID NO: 43, and CDRL3 containing the amino acid sequence of SEQ ID NO: 44; (3) CDRH1 containing the amino acid sequence of SEQ ID NO: 51, CDRH2 containing the amino acid sequence of SEQ ID NO: 52, CDRH3 containing the amino acid sequence of SEQ ID NO: 53, CDRL1 containing the amino acid sequence of SEQ ID NO: 54, CDRL2 containing the amino acid sequence of SEQ ID NO: 55, and CDRL3 containing the amino acid sequence of SEQ ID NO: 56; (4) CDRH1 containing the amino acid sequence of SEQ ID NO: 57, CDRH2 containing the amino acid sequence of SEQ ID NO: 58, CDRH3 containing the amino acid sequence of SEQ ID NO: 59, CDRL1 containing the amino acid sequence of SEQ ID NO: 60, CDRL2 containing the amino acid sequence of SEQ ID NO: 61, and CDRL3 containing the amino acid sequence of SEQ ID NO: 62; (5) CDRH1 containing the amino acid sequence of SEQ ID NO: 63, CDRH2 containing the amino acid sequence of SEQ ID NO: 64, CDRH3 containing the amino acid sequence of SEQ ID NO: 65, CDRL1 containing the amino acid sequence of SEQ ID NO: 66, CDRL2 containing the amino acid sequence of SEQ ID NO: 67, and CDRL3 containing the amino acid sequence of SEQ ID NO: 68; (6) CDRH1 containing the amino acid sequence of SEQ ID NO: 75, CDRH2 containing the amino acid sequence of SEQ ID NO: 76, CDRH3 containing the amino acid sequence of SEQ ID NO: 77, CDRL1 containing the amino acid sequence of SEQ ID NO: 78, CDRL2 containing the amino acid sequence of SEQ ID NO: 79, and CDRL3 containing the amino acid sequence of SEQ ID NO: 80; (7) CDRH1 containing the amino acid sequence of SEQ ID NO: 81, CDRH2 containing the amino acid sequence of SEQ ID NO: 82, CDRH3 containing the amino acid sequence of SEQ ID NO: 83, CDRL1 containing the amino acid sequence of SEQ ID NO: 84, CDRL2 containing the amino acid sequence of SEQ ID NO: 85, and CDRL3 containing the amino acid sequence of SEQ ID NO: 86; (8) CDRH1 containing the amino acid sequence of SEQ ID NO: 87, CDRH2 containing the amino acid sequence of SEQ ID NO: 88, CDRH3 containing the amino acid sequence of SEQ ID NO: 89, CDRL1 containing the amino acid sequence of SEQ ID NO: 90, CDRL2 containing the amino acid sequence of SEQ ID NO: 91, and CDRL3 containing the amino acid sequence of SEQ ID NO: 92; (9) CDRH1 containing the amino acid sequence of SEQ ID NO: 93, CDRH2 containing the amino acid sequence of SEQ ID NO: 94, CDRH3 containing the amino acid sequence of SEQ ID NO: 95, CDRL1 containing the amino acid sequence of SEQ ID NO: 96, CDRL2 containing the amino acid sequence of SEQ ID NO: 97, and CDRL3 containing the amino acid sequence of SEQ ID NO: 98; (10) CDRH1 containing the amino acid sequence of SEQ ID NO: 99, CDRH2 containing the amino acid sequence of SEQ ID NO: 100, CDRH3 containing the amino acid sequence of SEQ ID NO: 101, CDRL1 containing the amino acid sequence of SEQ ID NO: 102, CDRL2 containing the amino acid sequence of SEQ ID NO: 103, and CDRL3 containing the amino acid sequence of SEQ ID NO: 104; and (11) CDRH1 containing the amino acid sequence of SEQ ID NO: 131, CDRH2 containing the amino acid sequence of SEQ ID NO: 132, CDRH3 containing the amino acid sequence of SEQ ID NO: 133, CDRL1 containing the amino acid sequence of SEQ ID NO: 134, CDRL2 containing the amino acid sequence of SEQ ID NO: 135, and CDRL3 containing the amino acid sequence of SEQ ID NO: 136; A pharmaceutical composition comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 selected from the group consisting of the above.

2. The pharmaceutical composition according to claim 1, wherein the antiviral agent includes an anti-HIV agent.

3. The pharmaceutical composition according to claim 2, wherein the anti-HIV drug comprises a non-nucleoside reverse transcriptase inhibitor, a protease inhibitor, an entry or fusion inhibitor, or an integrase inhibitor.

4. The pharmaceutical composition according to claim 2 or 3, wherein the anti-HIV antibody or its antigen-binding portion comprises CDRH1 containing the amino acid sequence of SEQ ID NO: 69, CDRH2 containing the amino acid sequence of SEQ ID NO: 70, CDRH3 containing the amino acid sequence of SEQ ID NO: 71, CDRL1 containing the amino acid sequence of SEQ ID NO: 72, CDRL2 containing the amino acid sequence of SEQ ID NO: 73, and CDRL3 containing the amino acid sequence of SEQ ID NO:

74.

5. The use of (i) an anti-HIV antibody or its antigen-binding portion and (ii) an antiviral agent in the manufacture of a drug used to prevent or treat HIV infection or HIV-related disease, The anti-HIV antibody or its antigen-binding portion is (1) Complementarity-determining regions (CDRs) H1 containing the amino acid sequence of SEQ ID NO: 69, CDRH2 containing the amino acid sequence of SEQ ID NO: 70, CDRH3 containing the amino acid sequence of SEQ ID NO: 71, CDRL1 containing the amino acid sequence of SEQ ID NO: 72, CDRL2 containing the amino acid sequence of SEQ ID NO: 73, and CDRL3 containing the amino acid sequence of SEQ ID NO: 74; (2) CDRH1 containing the amino acid sequence of SEQ ID NO: 39, CDRH2 containing the amino acid sequence of SEQ ID NO: 40, CDRH3 containing the amino acid sequence of SEQ ID NO: 41, CDRL1 containing the amino acid sequence of SEQ ID NO: 42, CDRL2 containing the amino acid sequence of SEQ ID NO: 43, and CDRL3 containing the amino acid sequence of SEQ ID NO: 44; (3) CDRH1 containing the amino acid sequence of SEQ ID NO: 51, CDRH2 containing the amino acid sequence of SEQ ID NO: 52, CDRH3 containing the amino acid sequence of SEQ ID NO: 53, CDRL1 containing the amino acid sequence of SEQ ID NO: 54, CDRL2 containing the amino acid sequence of SEQ ID NO: 55, and CDRL3 containing the amino acid sequence of SEQ ID NO: 56; (4) CDRH1 containing the amino acid sequence of SEQ ID NO: 57, CDRH2 containing the amino acid sequence of SEQ ID NO: 58, CDRH3 containing the amino acid sequence of SEQ ID NO: 59, CDRL1 containing the amino acid sequence of SEQ ID NO: 60, CDRL2 containing the amino acid sequence of SEQ ID NO: 61, and CDRL3 containing the amino acid sequence of SEQ ID NO: 62; (5) CDRH1 containing the amino acid sequence of SEQ ID NO: 63, CDRH2 containing the amino acid sequence of SEQ ID NO: 64, CDRH3 containing the amino acid sequence of SEQ ID NO: 65, CDRL1 containing the amino acid sequence of SEQ ID NO: 66, CDRL2 containing the amino acid sequence of SEQ ID NO: 67, and CDRL3 containing the amino acid sequence of SEQ ID NO: 68; (6) CDRH1 containing the amino acid sequence of SEQ ID NO: 75, CDRH2 containing the amino acid sequence of SEQ ID NO: 76, CDRH3 containing the amino acid sequence of SEQ ID NO: 77, CDRL1 containing the amino acid sequence of SEQ ID NO: 78, CDRL2 containing the amino acid sequence of SEQ ID NO: 79, and CDRL3 containing the amino acid sequence of SEQ ID NO: 80; (7) CDRH1 containing the amino acid sequence of SEQ ID NO: 81, CDRH2 containing the amino acid sequence of SEQ ID NO: 82, CDRH3 containing the amino acid sequence of SEQ ID NO: 83, CDRL1 containing the amino acid sequence of SEQ ID NO: 84, CDRL2 containing the amino acid sequence of SEQ ID NO: 85, and CDRL3 containing the amino acid sequence of SEQ ID NO: 86; (8) CDRH1 containing the amino acid sequence of SEQ ID NO: 87, CDRH2 containing the amino acid sequence of SEQ ID NO: 88, CDRH3 containing the amino acid sequence of SEQ ID NO: 89, CDRL1 containing the amino acid sequence of SEQ ID NO: 90, CDRL2 containing the amino acid sequence of SEQ ID NO: 91, and CDRL3 containing the amino acid sequence of SEQ ID NO: 92; (9) CDRH1 containing the amino acid sequence of SEQ ID NO: 93, CDRH2 containing the amino acid sequence of SEQ ID NO: 94, CDRH3 containing the amino acid sequence of SEQ ID NO: 95, CDRL1 containing the amino acid sequence of SEQ ID NO: 96, CDRL2 containing the amino acid sequence of SEQ ID NO: 97, and CDRL3 containing the amino acid sequence of SEQ ID NO: 98; (10) CDRH1 containing the amino acid sequence of SEQ ID NO: 99, CDRH2 containing the amino acid sequence of SEQ ID NO: 100, CDRH3 containing the amino acid sequence of SEQ ID NO: 101, CDRL1 containing the amino acid sequence of SEQ ID NO: 102, CDRL2 containing the amino acid sequence of SEQ ID NO: 103, and CDRL3 containing the amino acid sequence of SEQ ID NO: 104; and (11) CDRH1 containing the amino acid sequence of SEQ ID NO: 131, CDRH2 containing the amino acid sequence of SEQ ID NO: 132, CDRH3 containing the amino acid sequence of SEQ ID NO: 133, CDRL1 containing the amino acid sequence of SEQ ID NO: 134, CDRL2 containing the amino acid sequence of SEQ ID NO: 135, and CDRL3 containing the amino acid sequence of SEQ ID NO: 136; Use including CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 selected from the group consisting of the above.

6. The use according to claim 5, wherein the antiviral agent includes an anti-HIV agent.

7. The use according to claim 6, wherein the anti-HIV drug comprises a non-nucleoside reverse transcriptase inhibitor, a protease inhibitor, an entry or fusion inhibitor, or an integrase inhibitor.

8. The use according to claim 6 or 7, wherein the anti-HIV antibody or its antigen-binding portion comprises CDRH1 containing the amino acid sequence of SEQ ID NO: 69, CDRH2 containing the amino acid sequence of SEQ ID NO: 70, CDRH3 containing the amino acid sequence of SEQ ID NO: 71, CDRL1 containing the amino acid sequence of SEQ ID NO: 72, CDRL2 containing the amino acid sequence of SEQ ID NO: 73, and CDRL3 containing the amino acid sequence of SEQ ID NO: 74.

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