antibodies that bind to gp41 and neutralize human immunodeficiency virus type 1 (hiv-1), its uses, n

专利摘要:
ANTIBODIES THAT BIND TO GP41 AND NEUTRALIZE TYPE 1 HUMAN IMMUNODEFICIENCY VIRUS (HIV-1), ITS USES IN THE PREVENTION OR TREATMENT OF HIV-1 INFECTION, PROTEIN, NUCLEIC ACID MOLECULE, EXPRESSION VECTOR, CELL, HONEY CELL KIT, AS WELL AS METHODS OF DETECTION OF HIV-1 INFECTION AND POTENTIAL IMMUNOGEN TEST. The present invention relates to monoclonal antibodies or antigen-binding fragment thereof that specifically binds to gp41 and neutralizes human immunodeficiency virus type 1 (HIV-1) and its use in the prevention or treatment of HIV-1 infection . In addition, the present invention relates to the archaea protein, nucleic acid molecule, expression vector, host cell, composition, kit, as well as methods for detecting an HIV-1 infection and testing for potential immunogen.
公开号:BR112014010823B1
申请号:R112014010823-4
申请日:2012-11-07
公开日:2021-02-17
发明作者:Mark Connors；Jinghe Huang；Leo B. Laub；Peter Kwong；Gary Nabel；John R. Mascola；Baoshan Zhang；Rebecca S. Rudicell；Ivelin Georgiev；YongPing Yang；Jiang Zhu；Gilad Ofek
申请人:The United States Of America, As Represented By The Secretary, Department Of Health And Human Services；
IPC主号:

专利说明:

[0001] [001] This Application Claims the benefit of U.S. Provisional Application No. 61 / 702,703, filed on September 18, 2012; U.S. Provisional Application No. 61 / 698,480, filed September 7, 2012; U.S. Provisional Application No. 61 / 672,708, filed on July 17, 2012; and U.S. Provisional Order No. 61 / 556,660, filed on November 7, 2011. Each of these previous Orders is incorporated by reference in its entirety. FIELD OF THE INVENTION
[0002] [002] This refers to the identification of neutralizing monoclonal antibodies, such as, but not limited to, antibodies that bind to the region close to the HIV-1 gp41 membrane. BACKGROUND
[0003] [003] An effective Human Immunodeficiency Virus type 1 (HIV-1) vaccine will likely need to induce neutralizing antibodies (NAbs) that block HIV-1 entry into human cells. To be effective, vaccine-induced antibodies must be active against most circulating strains of HIV-1. Unfortunately, current HIV-1 vaccines are unable to induce broadly reactive NAbs. A major obstacle to planning better vaccines is a limited understanding of which region of HIV-1 envelope glycoproteins, such as gp120 and gp41, is recognized by NAbs. Some neutralizing monoclonal antibodies (mAbs) have been isolated from HIV-1 infected individuals and these mAbs define specific regions (epitopes) in the virus that are vulnerable to NAbs.
[0004] [004] Although envelope glycoproteins are immunogenic and induce a variety of antibodies, the neutralizing antibodies that are induced are lineage specific, and most immune responses are shifted to non-neutralizing determinants (Weiss, RA, et al., Nature, 1985. 316 (6023): p. 69-72; Wyatt, R. and J. Sodroski, Science, 1998. 280 (5371): p. 1884-8). Widely neutralizing antibodies have been isolated only rarely from natural HIV infection. Three examples of widely neutralizing antibodies that bind gp41 are 2F5, 4E10 and Z13E1. These gp41 neutralizing antibodies recognize the membrane-close region (MPER) of the HIV-1 gp41 glycoprotein. Unfortunately, these antibodies are limited in their lineage cross-reactivity or potency and, therefore, do not provide a viable choice for therapeutic intervention. Thus, there is a need for methods to prepare broadly neutralizing monoclonal antibodies that can provide protection from an infectious agent, such as HIV. SUMMARY
[0005] [005] Isolated human monoclonal neutralizing antibodies that specifically bind gp41 are provided here. In certain examples, the ability to bind and / or neutralize these antibodies has been optimized. Also described here are compositions including the described antibodies that specifically bind gp41, nucleic acids encoding these antibodies, expression vectors including the nucleic acids, and isolated host cells that express the nucleic acids. Antigen binding fragments of the isolated antibodies are also provided.
[0006] [006] In some embodiments, an isolated human monoclonal antibody, or antigen binding fragment thereof, includes a heavy chain and a light chain, wherein the heavy chain includes an amino acid sequence at least about 80% identical to the sequence amino acid established as SEQ ID NO: 1. In several such embodiments, the antibody, or antigen binding fragment thereof, specifically binds to gp41 and contacts L, WF, LW and R in the amino acid sequence established as LWNWFDITNWLWYIR (SEQ ID NO: 26, residues 14 to 28), and is neutralizing. In additional embodiments, the antibody, or antigen binding fragment thereof, specifically binds to gp41 and contacts NWF, T, and R in the amino acid sequence established as NWFDITNWLWYIR (SEQ ID NO: 13, residues 7 to 19), and it's neutralizing. In additional embodiments, an isolated monoclonal antibody or antigen binding fragment is provided that includes a heavy chain and a light chain, where the heavy chain includes amino acids 26 to 33 (heavy chain complementarity determining region 1 (HCDR1)) , 51 to 60 (HCDR2), or 99 to 120 (HCDR3) of SEQ ID NO: 11, where X1 is Q or R, X2 is V or A, X3 is S or Y, and X4 is T or I. antibody or antigen binding fragment specifically binds HIV-1 gp41, and is neutralizing. In some such embodiments, the isolated human monoclonal antibody or antigen binding fragment includes a heavy chain including one or more of amino acids 26 to 33 (HCDR1), 51 to 60 (HCDR2), and 99 to 120 (HCDR3) from one of the SEQ ID NO: 1, 3, 5, 147-149, 189 to 192, or 200 to 204. In some such embodiments, the isolated human monoclonal antibody heavy chain includes an amino acid sequence at least 90% identical to the amino acid sequence established as one of SEQ ID NOs: 1, 3, 5, 147-149, 189-192, or 200-204. In other embodiments, the isolated human monoclonal antibody heavy chain or antigen binding fragment thereof, includes the amino acid sequence established as one of SEQ ID NOs: 1, 3, 5, 147-149, 189-192, or 200 -204.
[0007] [007] In additional embodiments, the isolated human monoclonal antibody or antigen binding fragment thereof includes a light chain at least about 80% identical to the amino acid sequence established as SEQ ID NO: 2. In other embodiments, the light chain includes amino acids 26 to 31 (light chain complementarity determining region 1 (LCDR1)), 49-51 (LCDR2), and 87-98 (LCDR3) of SEQ ID NO: 12, where X4 is E or D, X5 is Y or H, X6 is K or I, X7 is V or I, X8 is S or T, X9 is D or E, X10 is E or D, and X11 is T or I. In additional embodiments, the light chain includes amino acids 26 to 31 (LCDR1), 49 to 51 (LCDR2), or 87-98 (LCDR3) from one of SEQ ID NO: 2, 4, 6, 12, 150 to 152, or 164 to 186. In some embodiments, the isolated human monoclonal antibody light chain or antigen binding fragment includes an amino acid sequence at least 90% identical to the amino acid sequence established as one of SEQ ID NO: 2, 4, 6, 12, 150 to 152, or 164 to 186. In one embodiment, the Light weight of the isolated human monoclonal antibody or antigen binding fragment includes the amino acid sequence established as one of SEQ ID NO: 2, 4, 6, 12, 150 to 152, or 164 to 186.
[0008] [008] In some embodiments, an isolated human monoclonal antibody or antigen binding fragment thereof is provided, wherein the heavy chain includes the amino acid sequence established as SEQ ID NO: 1 and the light chain includes the established amino acid sequence as SEQ ID NO: 2. The antibody specifically binds HIV-1 gp41 and is neutralizing. In other embodiments, an isolated human monoclonal antibody or antigen binding fragment thereof is provided, wherein the heavy chain includes the amino acid sequence set to SEQ ID NO: 154 and the light chain includes the amino acid sequence set to SEQ ID NO: 152. The antibody specifically binds HIV-1 gp41 and is neutralizing. In other embodiments, an isolated human monoclonal antibody or antigen binding fragment thereof is provided, wherein the heavy chain includes the amino acid sequence set to SEQ ID NO: 192 and the light chain includes the amino acid sequence set to SEQ ID NO: 152. The antibody specifically binds HIV-1 gp41 and is neutralizing.
[0009] [009] The antibodies and compositions described here can be used for a variety of purposes, such as to detect the presence of HIV-1 in a biological sample or to diagnose AIDS. These methods can include a sample from an individual with a monoclonal antibody that specifically binds gp41, and detect the binding of the antibody to the sample. An increase in antibody binding to the sample, relative to antibody binding, for a control sample to identify the individual as an individual with HIV-1 infection and / or AIDS. In some non-limiting examples, an increase in antibody binding to the relative sample for a control sample to detect the presence of HIV-1.
[0010] [0010] Also described are methods for treating an individual with HIV infection, such as, but not limited to, an individual with AIDS. The methods include administering a therapeutically effective amount of a monoclonal antibody as described above to an individual.
[0011] [0011] The foregoing and other characteristics and advantages of this invention, will become more evident from the following detailed description of the various modalities that proceed with reference to the accompanying figures. BRIEF DESCRIPTION OF THE FIGURES
[0012] [0012] Figures 1A to 1C are a group of tables and a diagram illustrating neutralization and 10E8 antibody sequence analyzes. (A) Inferred germline genes encoding the variable regions of 10E8, 7H6 and 7N16. (B) Antibody neutralizing activity against an HIV-1 isolate 181. envelope protein (Env) pseudovirus panel. Dendrograms indicate the distance of primary HIV-1 Envs isolate gp160 protein. (C) The data below the dendrogram shows the number of viruses tested, the percentage of viruses neutralized, and the geometric mean IC50 for viruses neutralized with an IC50 <50 μg / ml. Average titers are based on all viruses tested, including those with IC50> 50ug / ml, which have been assigned a value of 100.
[0013] [0013] Figures 2A and 2B illustrate the binding specificity of 10E8. (A) Binding enzyme linked immunosorbent assay (ELISA) from mAb 10E8 or 4E10 to peptide gp140, gp120, gp41, or 4E10. The error bars indicate a standard error of the mean (SEM). (B) Inhibition of neutralization of 10A8 or 4E10 mAbs from C1 HIV2 / HIV-1 MPER virus by 4E10 alanine scan peptides. The peptide was incubated with mAb 4E10 or 10E8 for one hour before infection of TZM-bl cells. The Y axis shows the percentage of neutralization for each condition. Residues W672, F673, T676 and R683 were the positions so that the mutant alanine peptide would not block neutralization (R683 for antibody 10E8 only). Residues 16 to 28 of SEQ ID NO: 26 are shown.
[0014] [0014] Figures 3A and 3B are a group of graphs and a group of digital images illustrating the 10E8 autoreactivity analysis. (A) Surface Plasmon Resonance (SPR) analysis of 10E8 binding to anionic phospholipids. 10E8 was injected over PC-CLP liposomes or PC-PS liposomes immobilized on the BIACORE® L1 sensor chip. 4E10 and 2F5 were used as positive controls and 13H1, 17b, and the anti-RSV F protein as negative controls. (B) Reactivity of 10E8 with HEP-2 epithelial cells. Controls are as above with VRC01 added as an additional negative control. The antibody concentration was 25 μg / ml. All images are shown at 400x magnification.
[0015] [0015] Figures 4A-4H are a group of tape diagrams illustrating the crystalline structure of antibody 10E8 in complex with its MPER epitope of gp41. (A) 10E8 recognizes a highly conserved gp41 helix to neutralize HIV-1. Fab 10E8 is shown in representation on tapes (dark gray tones for heavy chain and light gray for light chain) in complex with a gp41 peptide (dark gray) that covers the MPER (Asn656-Arg683; NEQELLELDKWASLWNWFDITNWLWYIR (SEQ ID NO : 26)). (B) Interface between 10E8 and gp41 with select 10E8 side chains and gp41 side chains in stick representation. By analogy on the one hand, the joint can be seen as being held by a thumb (represented by CDR H2), the C-terminal helix as being suspended over a corresponding extended indicator (represented by CDR H3), and residues that start the C-terminal helix as being captured in the gap between the thumb and forefinger (represented by the junction of the CDR handles). (C-D) Blunted contact surfaces and epitope conservation. An examination of the blunted contact surface in gp41 (gray; C) reveals that epitope residues (labeled, D) that are directly contacted by 10E8 are highly conserved across 2870 strains examined (conservation percentages provided in parentheses; see also figures 26 to 28). E-H, alanine mutagenesis of parotope and epitope. The residues on the 10E8 CDR H3's outer edge and inside the hydrophobic slit are crucial for gp41 recognition and virus neutralization (figures 31 to 32), as mapped on the blunted 10E8 contact surface (E, G). These results reflect the effects of alanine scan mutations of the 10E8 epitope (figures 18-19), as mapped on the blunted gp41-b surface (F, H). A comparison of the effects of alanine mutagenesis of the parotope and epitope reveals that the residues of the epitope that are most crucial for the neutralization and recognition of 10E8, are also the most highly conserved (D).
[0016] [0016] Figures 5A and 5B are a table, a group of graphs and a schematic diagram illustrating a vulnerability site for gp41. (A) Impact of sequence variation under 10E8 neutralization. Predicted amino acid sequences within the 10E8 binding epitope for three 10E8-resistant viruses and the patient's virus are shown. The 10E8 binding region and differences in sequence compared to the JR2 virus are labeled in light gray. IC50 and IC80 values that are> 20 times that of JR2 wild type pseudovirus are highlighted in light gray. Error bars denote SEM. (B) Structural definition of a highly conserved region of gp41 recognized by neutralizing antibodies. Atoms of highly conserved residues that make direct contact with 10E8 are in medium gray and shown in stick representation, atoms blunted by 10E8 are in dark gray, and main chain contact atoms are in light gray. Semitransparent surfaces of the gp41 MPER are shaded according to the fundamental atoms. 90º views are shown, with 10E8 antibody binding on the right panel. The 10E8 CDR H3 interacts with highly conserved hydrophobic residues, while the CDR H2 contacts main chain atoms at the junction between the N and C terminal helices. Most of the unbound residues of the MPER (gray) are hydrophobic, especially those in the helix terminal C. In the structure of a late melting intermediate (Figure 16), these residues are opposite the outer side of a helical spiral roller; in the pre-fusion conformation of the viral spike, they can interact with the viral membrane or with other hydrophobic regions of Env.
[0017] [0017] Figures 6A and 6B describe a sequence alignment of the heavy and light chains of 10EB antibodies to gp41 (SEQ ID NO: 1 and 2), 7H6 (SEQ ID NO: 3 and 4), 7N16 (SEQ ID NO: 5 and 6), IGHV3-15 * 05 (SEQ ID NO: 7) and the germline sequence of IGLV3-19 * 01 (SEQ ID NO: 8). The light gray residues represent the substitutions of the germline sequence. The dot symbol means the deletion of residue. The Kabat and IMGT numbering is shown and is used to identify specific identity residues in the 10E8 heavy and light chains.
[0018] [0018] Figure 7 is a graph illustrating the correlation of neutralization powers between donor serum N152 and antibody 10E8. A plot of the donor serum neutralization ID50s N152 compared to 10E8 neutralization IC50s is shown, tested against a panel of 20 pseudoviruses. A non-parametric Spearman correlation was used to assess the correlation between IC50s of 10E8 and ID50s of N152.
[0019] [0019] Figures 8A-8C are a graph and a group of tables illustrating the binding specificity of 10E8. (A) ELISA binding of mAbs indicated to peptides MPER, 2F5, Z13e1, 4E10 and 4E10.19. Amino acid sequences of peptides are also shown (descendant, SEQ ID NO: 26 with N- and C- terminal lysine triplicates, residues 1 to 16 of SEQ ID NO: 26, residues 11 to 21 of SEQ ID NO: 26 with a C-terminal lysine triplicate, residues 16-24 of SEQ ID NO: 26 with a C-terminal lysine triplicate and residues 16-28 of SEQ ID NO: 26 with an N-terminal cysteine and a terminal lysine triplicate Ç). (B-C) Inhibition of neutralization of chimeric mAb virus of C1 HIV-2 / HIV-1 MPER by addition of MPER peptides, 2F5, Z13e1, 4E10 and 4E10.19. The doubling effect was calculated as the ratio of neutralization IC50 with simulation peptide / IC50 (B), or the ratio of neutralization IC80 to simulation peptide / IC80 (C), for the indicated peptide. Values> 5 are shaded in light gray.
[0020] [0020] Figures 9A and 9B illustrate surface plasmon resonance analysis of 10E8, 2F5 and 4E10 antibody binding surface to a gp41 MPER peptide. (A) A biotinylated MPER peptide comprised of residues 656 to 683 of gp41 was immobilized on a streptavidin SA chip (GE Healthcare) and fluid antibody Fabs over those analyzed in 2-fold increasing concentrations ranging from 3.9 to 125 nM (10E8), 0.49-31.25 nM (2F5), and 0.25 nM to 62.5 nM (4E10). Association and dissociation phases of three minutes and five minutes, respectively, were used, at a flow rate of 30 μl / minute, and each analyzed concentration was performed in triplicate. (B) Binding constants, listed, were obtained by adjusting sensograms with a 1: 1 Langmuir model. The amino acid sequence of SEQ ID NO: 26 is shown.
[0021] [0021] Figure 10 is a pie chart illustrating the frequency of HIV-1 + sera with a specific specificity. Sera from 78 healthy donors infected with HIV-1 was used in this assay. Frequency measured by the ability of the patient's sera to neutralize HIV-2 / HIV-1 chimeras containing portions of the MPER and confirmed with peptide block. Neutralization of serum ID50s is reported in figure 21. Changes in ID50 duplication after peptide block are reported in figure 22. The six patients with sera containing 10E8-like antibodies were no different from the remaining 72 patients with respect to viral load (6748 copies per ml with 10E8 vs 5446 without; p> 0.05), CD4 count (437 cells / μl vs. 557; p> 0.05), years since diagnosis (20 years vs. 13; p> 0.05), or average neutralization titer (302 vs. 156; p> 0.05).
[0022] [0022] Figures 11A-11C illustrate the accessibility of 10E8 to the MPER. (A) Binding of 10E8, 4E10 and 2F5 a spike of life-size HIVJR-FL envelopes, mutant 4E10 (Phe673Ser) or mutant 2F5 (Lys665Glu) expressed on the 293T cell surface when measured by flow cytometry. Serially diluted antibody was incubated with cells for one hour. 2G12 and b12 antibodies were used as positive controls and F105 was used as a negative control. VRC01 is used as an additional control in cells transfected by JR-FL. Percentage of relative binding is calculated as the mean fluorescence intensity (MFI) divided by the mean MFI of the positive control 2G12 X 100. (B) Accessibility of MPER was determined by washing the antibody-virion mixture before infection with TZM- cells. bl. Pseudoviruses were incubated with antibodies at 37 ° C for 30 minutes, and the antibody-virus mixture was washed or not before infection of target cells. (C) The impact of the wash on antibody neutralization was measured by the area under the curve (AUC) or in the IC80. For BaL and JRFl, the IC80 was not obtained in the condition without washing and the high inhibitory concentration (IC60 and IC75, respectively) was used.
[0023] [0023] Figures 12A and 12B are schematic diagrams illustrating comparison of two copies of the gp41 MPER in the crystalline asymmetric unit. (A) gp41 peptides from the two 10E8-gp41 complexes in the asymmetric crystalline unit are shown in stick representation (complex 1, dark gray; complex 2, medium gray), surrounded by their 2fo-fc electron density contoured at 1σ ( dark grey). Images shown are rotated 180 with respect to each other, and are in the same orientation as in figures 4C and 4D. (B) An alignment of the peptides in the two crystalline complexes. Shown, in 90º views, is the superposition of the two peptides in the asymmetric unit based on the alignment of all atoms of residues 671-683. The N-terminal helix in complex 2 is offset by 45 ° from that in complex 1 in this alignment. While the different orientations of the N-terminal helix in the two complexes suggest a degree of structural plasticity, joint residues and the C-terminal helix in the two complexes are highly conserved and are involved in the most critical interactions with the antibody.
[0024] [0024] Figures 13A-13C are a group of graphs illustrating the surface plasmon resonance analysis of 10E8 paratope alanine variants. MPER peptide binding sensograms are shown for 25 variants of the 10E8 paratope, as well as the wild type 10E8 (weight). Variant IgGs were captured on a bisensor chip coupled to anti-human IgG at surface densities of 1000 to 2000 response units and MPER peptide (listed) fluid over the analyte in two-fold serial dilutions starting at 500 nM (with the exception of HC D30A, W100bA, S100cA, P100fA, which started at 250 nM). Association and dissociation phases of three minutes and five minutes, respectively, were used at flow rates of 30 μl / minutes. Sensograms were adjusted with a 1: 1 Langmuir model using BIACORE® BIAEVALUATION® software (GE Healthcare). Binding constants are reported in figure 31. The amino acid sequence of SEQ ID NO: 26 is shown in figure 13C.
[0025] [0025] Figure 14 shows a graph illustrating the 10E8 variant binding and neutralization correlation. Binding KD's of the 10E8 alanine parotope variants plotted with respect to their mean neutralization IC50s and IC80s. A non-parametric Spearman correlation was used to assess the relationship between binding and neutralization. KD’s and neutralization IC50s and IC80s are reported in figures 31 to 32.
[0026] [0026] Figures 15A-15H illustrate the recognition of a C-terminal MPER helix structurally conserved by 10E8 and 4E10. 10E8 and 4E10 use substantially different recognition modes to attach a structurally conserved helix to the C-terminal of the gp41 MPER. (A) Conformation of MPER and blunt surface for neutralizing antibodies 10E8, 2F5 (Protein DataBank (PDB) ID No. 1TJI, incorporated by reference here as presented in the database on 22 October 2012), Z13e1 (PDB ID No. 3FN0, incorporated by reference here as presented in the database on 22 October 2012), and 4E10 (PDB ID No. 2FX7, incorporated by reference here as presented in the database on 22 October 2012). Cα tape representations of the MPER of gp41 bound by any antibody are shown, with bar graphs showing the amount of blunted surface per residue. The amino acid sequence of SEQ ID NO: 26 is shown below in each bar graph. The sequence of the crystallized epitope is shown in capital letters. (B) Superposition of the MPER C terminal helix, in conformations linked to 10E8 (dark gray) and 4E10 (light gray), displayed in 90 ° orientations. Representations of Cα tape are shown, with side chains displayed as sticks. (CF) Comparison of recognition of 10E8 and 4E10 of the C-terminal MPER helix, with molecules displayed in Cα tape representations. (C) 10E8 variable domains in complex with the MPER, shaded according to figure 4A. (D) 4E10 variable domains in complex with the MPER (PDB ID No. 2FX7, incorporated by reference here as presented in the database on 22 October 2012), in an orientation based on the alignment of C-terminal propellers of gp41 described in (B; left panel). gp41 is shaded from not entirely white, heavy chain of 4E10, dark gray, and light chain of 4E10, medium gray. (E-F) 90 ° views of (C) and (D), looking from terminal C to terminal N of the helix of terminal C of conserved MPER. (G, H) Helical steel representations of the C-terminal propellers of MPER connected to 10E8- and 4E10, reflecting the orientation shown in (B; right panel). Enhanced antibody contact faces are based on the direct contact observed between the antibodies and gp41, as described in figure 35.
[0027] [0027] Figure 16 shows a tape diagram illustrating the vulnerability gp41 site mapped in a "late intermediate" conformation of gp41. The vulnerability gp41 site as defined by the 10E8 antibody contact footprint in gp41 is shown, mapped to the 10E8-linked MPER peptide structure (left, similar orientation as figure 6B) and a six-helix beam conformation of intermediate gp41 (PDB ID No. 2XR7 (right), incorporated by reference here as presented in the database on 22 October 2012). The atoms recognized by the 10E8 antibody are shaded in dark gray and shown in stick representation. The contact of atom or residue exclusive to antibodies 2F5, Z13e1, and 4E10, is shown as rods, shaded in light or medium gray. The defined site of 10E8 of external vulnerability faces, outside the core axis of the beam, and appears to be widely accessible in this conformation. A potential N-linked glycosylation site at position 674 could not be compatible with the late intermediate conformation, since the side chain of residue 674 is externally facing the six helix bundle.
[0028] [0028] Figures 17A-17F are a group of tables illustrating the neutralizing properties of 10E8. (A) Neutralization of 10E8 and 7H6 compared to an isolated mini pseudovirus Env panel 5. IC50 values less than 1 μg / ml are highlighted in gray. (B) Ne152 serum neutralization profile of the patient and monoclonal antibodies. aData for N152 shows the serum ID50 (virus dose required for 50% infection) compared to each virus. ID50> 1000 is highlighted in dark gray, 500 <ID50 <1000 is in medium gray, and 100 <ID50 <500 is in light gray. Data for monoclonal antibodies show the IC50. IC50 <1 μg / ml is highlighted in medium gray; IC10 of 1-10 μg / ml is highlighted in light gray; and IC50 of 10-50 μg / ml is highlighted in dark gray. (C-F) Antibody neutralization data compared to 181 HIV-1 Env pseudovirus. IC50 <1 μg / ml is highlighted in medium gray; IC10 of 1-10 μg / ml is highlighted in light gray; and IC50 of 10-50 μg / ml is highlighted in dark gray.
[0029] [0029] Figure 18 is a table illustrating the binding of 10E8 and 4E10 to peptides analyzed by gp41 MPER analin, by ELISA. The duplication change was calculated as peptide IC50 / simulation peptide IC50. Duplication change values> 10 are highlighted in light gray.
[0030] [0030] Figure 19 is a table listing the neutralization data for 10E8 compared to the pseudotyped HIV-1JR2 MPER alanine mutants. The concentration is μg / ml. IC50 and IC90 values> 20 times compared to wild type JR2 for 10E8 or> 100 times for 4E10 are highlighted in light gray.
[0031] [0031] Figure 20 is a table listing sequences of HIV-2HIV-1 chimeras used for neutralization assays. The sequences of 7312A, C1, C1C, C3, C7, C6, C4, C4GW and C8 are shown (SEQ ID NOs: 15 to 22, respectively). Fragments of the MPER sequence that correspond to the HIV-1 MPER sequence are underlined.
[0032] [0032] Figure 21 is a table illustrating the mapping of neutralizing anti-MPER sera / antibodies with HIV-2 / HIV-1 chimeras. the IC50 (μg / ml) is shown. b ID50 values are shown. The numbers are in bold and highlighted in light gray, if the ID50 of the HIV-2 / HIV-1 chimeras was both 3 times greater than the wild-type HIV-2 control>> 100. "-" indicates no neutralization. "ND" indicates that the serum classification cannot be determined.
[0033] [0033] Figure 22 is a table illustrating the blocking of neutralization by mAb- and serum from HIV-2 / HIV-1 C1 chimeras using mutant MPER peptides. The sequence of the blocking peptides is shown in figure 8A. b The IC50 duplication change refers to (peptide IC50) / (simulation peptide IC50). cThe ID50 duplication change refers to (simulation peptide ID50) / (peptide ID50). The light gray highlight indicates a 3-fold change in the IC50 / ID50 with respect to the control peptide.
[0034] [0034] Figure 23 is a table illustrating the reactivity of 10E8 with autoantigens. The reactivity of 10E8 with autoantigens was detected by the Luminex assay. An antiRSV monoclonal antibody, Synagis (MedImmune, Gaithersburg, MD), was used as a negative control. Antibodies 4E10, 2F5, VRC01 and 17b were also tested for comparison. SSA refers to antigen A of Sjogren's syndrome; SSB refers to the B antigen of Sjogren's syndrome; Sm refers to the Smith antigen; RNP refers to ribonucleoprotein; Scl 70 refers to scleroderma 70; Jo1 refers to the antigen; CentrB refers to centromere B.
[0035] [0035] Figure 24 is a table listing the data collection and refinement statistics for the 10E8 crystalline structure studies. The high resolution invoculum is shown in parentheses. The data set shown was collected from a crystal.
[0036] [0036] Figure 25 is a table listing the Phi-Psi angles of antibody-bound gp41 peptides. aFor the 4E10: gp41 complex, the PDB ID No. 2FX7 structure was used (incorporated by reference here as presented in the database on 22 October 2012). b For the Z13e1: gp41 complex, the PDB ID No. 3FN0 structure was used (incorporated by reference here as presented in the database on 22 October 2012).
[0037] [0037] Figure 26 is a table listing the total blunt surface area in 10E8 and gp41. All interactions were performed using PISA (ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver). §BSA refers to the Dull Surface Area, Å2.
[0038] [0038] Figure 27 is a table listing the blunt surface areas at the interface between the heavy chain of 10E8 and gp41, by residue. All interactions were performed using PISA (ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver). £ Percentage of identity of this residue in an analysis of 2870 HIV strains deposited in the Los Alamos HIV sequence database (as of 12/2011). ‡ ASA refers to the Accessible Surface Area, Å2. §BSA refers to the Blunted Surface Area, Å2. §§Bars represent the percentage of blunt area, one bar by 10%. ΔiG refers to the effect of the solvation energy, kcal / mol.
[0039] [0039] Figure 28 is a table listing Dull Surface Areas at the interface between the 10E8 and gp41 light chains, by residue. All interactions were performed with PISA (ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver). £ Percentage of identity of this residue in an analysis of 2870 HIV strains deposited in the Los Alamos HIV sequence database (as of 12/2011). ASA refers to the Accessible Surface Area, Å2. BSA refers to the Blunted Surface Area, Å2. Bars represent the percentage of blunted area, one bar by 10%. ΔiG refers to the effect of the solvation energy, kcal / mol.
[0040] [0040] Figure 29 is a table listing the hydrogen bonds and salt bridges between 10E8 and gp41. Hydrogen bonds were determined using the Ligplot program (McDonald et al., J Mol Biol 238, 777-793, 1994). H chain refers to heavy chain complex 1; Chain B refers to heavy chain complex 2; P chain refers to gp41 peptide complex 1; Chain F refers to gp41 peptide complex 2.
[0041] [0041] Figure 30 is a table listing Van der Waals contact between 10E8 and gp41. Van der Waals contacts were determined using the Ligplot program (McDonald et al., J Mol Biol 238, 777- 793, 1994). Chain H and chain L refer to complex 1 heavy and light chains, respectively; Chain P refers to complex 1 gp41 peptide. Chain B and chain D refer to complex 2 heavy and light chains, respectively; Chain F refers to complex 2 gp41 peptide.
[0042] [0042] Figure 31 is a table illustrating binding affinities of 10E8 alanine variants to a soluble MPER peptide. SE refers to standard error; nb refers to the weak to undetectable binding in the used concentration range. ^ Duplication is defined as a change in duplication with respect to wild type 10E8 cycles performed in parallel with the variants. $ Average of the three cycles of 10E8 of the individual wild type. #Only those mutations that produce effects greater than 10 times were mapped on the blunted surface of 10E8 b in figure 4E (medium gray,> 100x; light gray, 50x <100x; dark gray, 10x <50x). Heavy chain residues Y99AHC and G100hAHC showed almost no binding to the soluble peptide, while mutations of additional CDR H3 residues (F100aAHC, G100dAHC, P100fAHC, P100gAHC, E100iAHC, and E100jAHC) decreased affinity 50 to 120 times ( Kabat numbering is used to identify specific residues in the 10E8 heavy and light chains). Loop mutations of CDR H1 and region 2 of structure W33AHC and R50AHC, respectively, which are present in the hydrophobic cleft, also knockout binding to the MPER peptide, and the CDR H2 E53AHC mutation in the cleft decreased the affinity for the MPER peptide in 60 times. Light chain residue R91LC, which is located at the base of the hydrophobic cleft and forms direct interactions with CDR H3 residues, knockout bond when mutated into alanine possibly destabilizing the cleft itself.
[0043] [0043] Figure 32 is a table listing the IC50s and IC80s for neutralizing 10E8 alanine scan variants. In cases where neutralizing IC50 or IC80 were not obtained at the highest concentration of antibody, the highest concentration was used in calculating the mean. ^ Average doubling effect is defined as the average of the individual doubling effects observed compared to each viral strain. & Y99AHC, F100aAHC, W100bAHC, and G100hAHC mutations, all had harmful effects on neutralization, decreasing the power more than 1000 times. Other mutations also had strong effects on neutralization, including P100gAHC and E100iAHC from CDR H3, and W33AHC and R50AHC at the hydrophobic slit. The R91ALC light chain mutation, similar to its effects on peptide binding, decreased the neutralization potency by more than 1000 times.
[0044] [0044] Figure 33 is a table illustrating deviations from the mean square root (RMSD) of gp41 structures linked to the antibody. Alignments were performed using the LSQKAB program (Winn, M. D. et al. Acta Crystallogr D Biol Crystallogr, 67, 235-242, 2011). For the 4E10: gp41 complex, the PDB ID No. 2FX7 structure was used (incorporated by reference here as presented in the database on 22 October 2012). For the Z13e1: gp41 complex, the PDB ID No. 3FN0 structure was used (incorporated by reference here as presented in the database on 22 October 2012). For the 2F5: gp41 complex, the PDB ID No. 1TJI structure was used).
[0045] [0045] Figure 34 is a table showing comparison of blunted surfaces of MPER specific antibody in gp41. Interaction studies were performed with PISA (ebi.ac.uk/msdsrv/prot_int/cgi-bin/piserver). The values in parentheses are for complex 2. For the 4E10 complex: gp41, the PDB ID No. 2FX7 structure was used (incorporated by reference here as presented in the database on 22 October 2012). For the Z13e1: gp41 complex, the PDB ID No. 3FN0 structure was used (incorporated by reference here as presented in the database on 22 October 2012). For the 2F5: gp41 complex, the PDB ID No. 1TJI structure was used (incorporated by reference here as presented in the database on 22 October 2012). Regarding the recognition of a more extensive residue range, 10E8 has a gp41 footprint less than 4E10. If the comparison is limited to the overlapping peptide range, residues 671 to 683, the difference in footprints will be even more pronounced, with 10E8 covering approximately 25% less of the gp41 surface area than 4E10.
[0046] [0046] Figure 35 is a table showing comparison of direct contact of antibodies 10E8 and 4E10 with gp41. Direct contacts were determined using the Ligplot program (McDonald et al., J Mol Biol 238, 777-793, 1994). H refers to the hydrogen bond; N refers to the van der Waals contact.
[0047] [0047] Figure 36 is a group of tables illustrating results of the neutralization of MPER mutants of COT6.15 envelope (clade C) pseudotyped with antibodies 10E8 and 4E10.
[0048] [0048] Figure 37 is a table illustrating the results of neutralization tests using the cross complementation of the heavy and light chains of 10E8, 7H6 and 7N16.
[0049] [0049] Figure 38 is a schematic diagram of the crystalline structure of the 10E8 antibody in complex with a gp41 peptide, showing the electrostatic surface charge of the antibody.
[0050] [0050] Figure 39 is a schematic diagram of the crystalline structure of the 10E8 antibody in complex with a gp41 peptide, showing the electrostatic surface charge of the peptide.
[0051] [0051] Figure 40 is a schematic diagram of the crystalline structure of the 10E8 antibody in complex with a gp41 peptide, showing the positions of the residue changes in the 10E8 7H6 and 7N16 variant antibodies.
[0052] [0052] Figure 41 is digital images of the crystalline structures of antibodies 2F5, 4E10 and Z13E1 in complex with peptides of gp41 and a schematic of gp41 illustrating the relative binding sites of antibodies 2F5, 4E10, Z13E1 and 10E8. The amino acid sequence of SEQ ID NO: 26 is shown.
[0053] [0053] Figures 42A and 42B are scattergram plots showing the results of FACS isolation of CD19 + IgAIgDIgM-B cells from a PBMC sample.
[0054] [0054] Figure 43 is a table and a group of tape diagrams illustrating an alanine scan of the indicated 10E8 residues (Kabat positions) as described in examples 1 and 8.
[0055] [0055] Figures 44A and 44B are a series of tape diagrams illustrating mutagenesis based on the 10E8 structure to enhance hydrophobic interactions with gp41.
[0056] [0056] Figure 45 is a table illustrating the results of neutralization assays in an HIV-1 virus panel using the 10E8 mutants based on the structure described in figures 44A and 44B and Example 8 (with reference to the Kabat numbering) .
[0057] [0057] Figure 46 is a schematic diagram illustrating the design of partially reversed antibody variants.
[0058] [0058] Figure 47 is a sequence alignment and a tape diagram illustrating partial inverter of germ lines of the heavy and light chain 10E8. The strings shown are SEQ ID NO: 7 (10E8_germ_H), SEQ ID NO: 147 (10E8gH01), SEQ ID NO: 148 (10E8gH02), SEQ ID NO: 149 (10E8gH03), SEQ ID NO: 1 (10E8_HEAVY), SEQ ID NO: 8 (10E8_germ_L), SEQ ID NO: 150 (10E8gL01), SEQ ID NO: 151 (10E8gL02), SEQ ID NO: 152 (10E8gL03), SEQ ID NO: 2 (10E8_LIGHT).
[0059] [0059] Figure 48 is a group of tables illustrating results of neutralization assays in an HIV-1 virus panel using the 10E8 antibody germline inverter as described in Figure 47 and Example 8. "10E8-R1" refers to the 10E8gH01 heavy chain (SEQ ID NO: 147) paired with the 10E8gL01 light chain (SEQ ID NO: 150). "10E8-R3" refers to the 10E8gH03 heavy chain (SEQ ID NO: 149) paired with the 10E8gL03 light chain (SEQ ID NO: 152).
[0060] [0060] Figure 49 is a group of tables illustrating the results of neutralization assays in an HIV-1 virus panel using a series of 10E8 mutants in the 10E8gH03 base heavy chain (SEQ ID NO: 149) or base 10E8gL03 (SEQ ID NO: 152) as shown in figure 47 and described in example 8.
[0061] [0061] Figures 50A to 50F are a series of graphs illustrating the identification of somatic 10E8 antibody variants by sequencing and grid sampling. (A) Identity / divergence plots of donor N152 light chain antibodyE 10E8 (left), with grid sampling (right). Identity to 10E8 is shown on the vertical axis, and divergence of the germline V gene origin is plotted on the horizontal axis, with antibody frequency shown as a heat map. Grid sampling is shown, with selected antibodies that do not express or bind to MPER in empty circles and with selected antibodies that do not bind to solid circles, shaded according to their phylogenetic distance of 10E8 in (C). (B) Identity / divergence plots of donor N152 light chain antibodyE 10E8 (left), with grid sampling (right). Axes and shading are the same as in (A). (C / D) Phylogenetic tree of variant identified by grid for heavy chain (C) and light chain (D). (EF) Neutralization of 10E8 and 10E8 variants, evaluated in duplicate in 6 HIV-1 isolates for heavy chain (E) and light chain (F) variants. The mean IC50s of gVRCH1dN152: 10E8L and gVRC-H11dN152: 10E8L were improved approximately 6 times more than the original model 10E8. Variants are arranged and named for their genetic distance of 10E8, and shaded in relation to their phylogenetic distance.
[0062] [0062] Figures 51A-51D are a series of sequence alignments and tape diagrams illustrating sequences and modeled structures of 10E8 variants that neutralize HIV-1. (A) Heavy chain sequences (SEQ ID NO: 1, and 153-163, descending; gVRC-H1dN152 (SEQ ID NO: 153); gVRC-H2dN152 (SEQ ID NO: 154); gVRC-H3dN152 (SEQ ID NO: : 155); gVRC-H4dN152 (SEQ ID NO: 156); gVRC-H5dN152 (SEQ ID NO: 157); gVRC-H6dN152 (SEQ ID NO: 158); gVRC-H7dN152 (SEQ ID NO: 159); gVRC- H8dN152 (SEQ ID NO: 160); gVRC-H9dN152 (SEQ ID NO: 161); gVRC-H10dN152 (SEQ ID NO: 162) gVRC-H11dN152 (SEQ ID NO: 163)). The sequences are arranged by genetic distance of 10E8, with sequence changes of 10E8 underlined. CDR structure and residues are highlighted, as they are residues that interact with the gp41 MPER epitope (open circle, main chain interaction; open circle with rays, side chain interactions; solid circle, both main chain and chain interactions side). (B) Modeled structures of complex heavy chain variants with gp41 epitope. Variants of 10E8 with enhanced recognition (with heavy chains shaded according to the phylogenetic distance as in 1B) were modeled on the structure of 10E8 with the complete MPER region of HIV-1 gp41 (not entirely white). The structures are displayed as Cα ribbons, with amino acid side chains ranging from 10E8 highlighted in stick representation, dark gray. (C) Light chain sequences (SEQ ID NO: 2 and 164-186, descending; gVRC-L1dN152 (SEQ ID NO: 164); gVRC-L2dN152 (SEQ ID NO: 165); gVRC-L3dN152 (SEQ ID NO: 166); gVRC-L4dN152 (SEQ ID NO: 167); gVRC-L5dN152 (SEQ ID NO: 168); gVRC-L6dN152 (SEQ ID NO: 169); gVRC-L7dN152 (SEQ ID NO: 170); gVRC-L8dN152 (SEQ ID NO: 171); gVRC-L9dN152 (SEQ ID NO: 172); gVRC-L10dN152 (SEQ ID NO: 173); gVRC-L11dN152 (SEQ ID NO: 174); gVRC-L12dN152 (SEQ ID NO: 175) ); gVRC-L13dN152 (SEQ ID NO: 176); gVRC-L14dN152 (SEQ ID NO: 177); gVRC-L15dN152 (SEQ ID NO: 178); gVRC-L16dN152 (SEQ ID NO: 179); gVRC-L17dN152 ( SEQ ID NO: 180); gVRC-L18dN152 (SEQ ID NO: 181); gVRC-L19dN152 (SEQ ID NO: 182); gVRC-L20dN152 (SEQ ID NO: 183); gVRC-L21dN152 (SEQ ID NO: 184) ; gVRC-L22dN152 (SEQ ID NO: 185); gVRC-L23dN152 (SEQ ID NO: 186)). The sequences are arranged by genetic distance of 10E8, with changes in 10E8 sequences underlined. CDR structure and residues are highlighted, as they are residues that interact with the MPER epitope of gp41 (as described in (A). (D) Modeled structures of light chain variants in complex with gp41 epitope. 10E8 variants with enhanced recognition (with light chains shaded according to the phylogenetic distance as in 1C) were modeled on the 10E8 structure with HIV-1 gp41 entire MPER region (not entirely white). Structures are displayed as Cα ribbons, with side chains amino acid ranging from 10E8 highlighted in stick representation, dark gray.
[0063] [0063] Figures 52A to 52D are a table and a group of graphs illustrating the phylogenetic branching of 10E8 heavy and light chain variants. (A) Matching phylogenetic branching. From the phylogenetic tree of antibodies identified by grid (Figure 1 C, D), the branches were named based on the distance of 10E8, b1-H for heavy and b1-L for light for the branch containing 10E8, and in descending order , b2-H (b2-L), b3-H (b3-L), and b4-H for the furthest branch. The variant of each branch that exhibited the most potent neutralization with a 10E8 wild-type partner was chosen, and a total array of 12 antibodies reconstituted. (B) Neutralization of HIV-1. Neutralization was evaluated in 5 isolates, for the 10E8 variants of paired and mismatched branching pairings. (C) Hep2 staining. The autoreactivity was accessed with Hep2 cell staining, for 10E8 variants of paired and mismatched branching pairings.
[0064] [0064] Figure 53 is a table illustrating 10E8 monoclonal antibodies composed of the heavy and light chain variants, indicated based on the neutralization potency of the indicated variants when complemented with the complementary wild type 10E8 chain.
[0065] [0065] Figure 54 is a table illustrating 10E8 antibodies composed of the phylogenetically indicated light and heavy chain variants.
[0066] [0066] Figures 55A to 55C show a group of tables illustrating results of neutralization assays on an HIV-1 virus panel using the series of 10E8 variants matched by neutralizing power or phylogenetically paired as shown in figures 53 and 54 and described in example 8.
[0067] [0067] Figure 56 is a group of tables illustrating results of autoreactivity assays in a panel of HIV-1 viruses using the series of 10E8 variants matched by neutralizing power or phylogenetically paired as shown in figures 53 and 54 and described in example 8.
[0068] [0068] Figure 57 is a group of tables illustrating results of neutralization assays in an HIV-1 virus panel using the series of 10E8 variants matched by neutralizing power or phylogenetically paired as shown in figures 53 and 54 and described in example 8.
[0069] [0069] Figures 58A and 58B are a group of graphs illustrating the results of neutralization assays testing the neutralization properties of the indicated series of antibodies, containing 10E8 light and heavy chains, or variants of 10E8 light and heavy chains, in a panel of 20 HIV viruses.
[0070] [0070] Figure 59 is a table illustrating the nomenclature and SEQ ID No. for the heavy and light chains of certain antibody variants 10E8.
[0071] [0071] Figures 60A and 60B are a group of tables illustrating a summary of 10E8 heavy chain variants. Mutants "Germline Inverters," "454," "Alanine Scan," "Based on the structure," and "additional" refer to the 10E8 heavy chain substitutions illustrated in example 8.
[0072] [0072] Figures 61A and 61B are a group of tables illustrating a summary of the 10E8 light chain variants. "Germ line inverters" and "454" mutants refer to the 10E8 light chain substitutions illustrated in example 8. SEQUENCE LISTING
[0073] [0073] The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter codes for amino acids, as defined in 37 CFR 1,822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. The sequence listing is submitted as an ASCII text file in the form of a file named "Sequence.txt" (~ 160 kb), which was created on 7 November 2012, which is incorporated by reference here. In the accompanying sequence listing: SEQ ID NO: 1 is the amino acid sequence of the heavy chain of the gp41 specific antibody 10E8. SEQ ID NO: 2 is the light chain amino acid sequence of the gp41 specific antibody 10E8. SEQ ID NO: 3 is the amino acid sequence of the gp41 specific antibody 7H6 heavy chain. SEQ ID NO: 4 is the amino acid sequence of the gp41 specific antibody 7H6. SEQ ID NO: 5 is the amino acid sequence of the 7N16 gp41 specific antibody heavy chain. SEQ ID NO: 6 is the amino acid sequence of the 7N16 gp41 specific antibody light chain. SEQ ID NO: 7 is the amino acid sequence of the IGHV3-15 * 05 heavy chain. SEQ ID NO: 8 is the amino acid sequence of the IGLV3-19 * 01 light chain. SEQ ID NO: 9 and 10 are epitopes in the MPER of gp41. SEQ ID NO: 11 is the amino acid sequence of the heavy chain of a specific gp41 antibody. SEQ ID NO: 12 is the amino acid sequence of the light chain of a specific gp41 antibody. SEQ ID NO: 13 is the amino acid sequence of a gp41 epitope that specifically binds 10E8 and 10E8-like antibodies. SEQ ID NOs: 14-25 are the amino acid sequence of MPER sequences of mutant gp41. SEQ ID NO: 26 is the amino acid sequence of an MPER region of gp41. SEQ ID NO: 27-34 are the nucleic acid sequences of sequencing primers. SEQ ID NO: 35-115 are nucleic acid sequences of exemplary 10E8-like antibody heavy chains. SEQ ID NOs: 116-145 are the nucleic acid sequences of exemplary 10E8-like antibody light chains. SEQ ID NO: 146 is the consensus amino acid sequence of the heavy chain of a specific gp41 antibody. SEQ ID NOs: 147-149 are the amino acid sequence of germline inverters heavy chains of monoclonal antibody 10E8. SEQ ID NO: 150-152 are the amino acid sequence of the variable regions of light chain of germline inverters of monoclonal antibody 10E8. SEQ ID NOs: 153-163 are the amino acid sequence of the heavy chain variable region of specific gp41 antibodies. SEQ ID NOs: 164-186 are the amino acid sequence of the variable regions of the light chain of specific gp41 antibodies. SEQ ID NO: 187 is the consensus amino acid sequence of the heavy chain variable region of a specific gp41 antibody. SEQ ID NO: 188 is the consensus amino acid sequence of the heavy chain variable region of a specific gp41 antibody. SEQ ID NOs: 189-192 are the amino acid sequence of the heavy chain variable region of specific gp41 antibodies. SEQ ID NO: 193-196 are the nucleic acid sequences of primers. SEQ ID NOs: 197-199 are the amino acid sequence of the variable regions of the light chain of specific gp41 antibodies. SEQ ID NO: 200-205 are the amino acid sequence of the heavy chain variable region of specific gp41 antibodies. SEQ ID NO: 205-209 are the amino acid sequence of the variable regions of the light chain of specific gp41 antibodies. DETAILED DESCRIPTION I. Summary of Terms
[0074] [0074] Unless otherwise noted, technical terms are used in accordance with conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 1999; Kendrew et al., (Eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; and other particular references.
[0075] [0075] As used here, the singular forms "one", "one", and "the" refer to both the singular and the plural, unless the context, clearly, indicate otherwise. For example, the term "an antigen" includes singular or plural antigens and can be considered equivalent to the phrase "at least one antigen".
[0076] [0076] As used here, the term "comprises" means "includes". Thus, "comprising an antigen" means "including an antigen" without excluding other elements.
[0077] [0077] It should also be understood that any and all base sizes or sizes of amino acid, and all values of molecular weight or molecular weight, provided for nucleic acids or polypeptides are approximate, and are provided for purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described here can be used, methods and materials particular to those described below. In the event of a conflict, this specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.
[0078] [0078] To facilitate the revision of the modalities, the following explanations of terms provided:
[0079] [0079] Administration: The introduction of a composition in an individual by a chosen routine. Administration can be local or systemic. For example, if the chosen routine is intravenous, the composition will be administered by introducing a composition into a vein in the individual. In some examples, a described antibody, or antigen binding fragment thereof, specific for an HIV gp41 polypeptide is administered to an individual.
[0080] [0080] Agent: any substance or any combination of substances that is useful to obtain a purpose or result; for example, a substance or combination of substances useful for inhibiting HIV infection in an individual. The agents include proteins, nucleic acid molecules, compounds, small molecules, organic compounds, inorganic compounds, or other molecules of interest. An agent can include a therapeutic agent (such as an antiretroviral agent), a diagnostic agent or a pharmaceutical agent. In some embodiments, the agent is a polypeptide agent (such as an HIV neutralizing antibody), or an antiviral agent. The skilled technician will understand that particular agents can be useful to obtain more than one result.
[0081] [0081] Amino acid substitution: The substitution of an amino acid in the polypeptide with a different amino acid.
[0082] [0082] Amplification: a technique that increases the number of copies of a nucleic acid molecule (such as an RNA or DNA). An example of amplification is the polymerase chain reaction, in which a biological sample is connected with a pair of oligonucleotide primers, under conditions that allow the primers to hybridize to a nucleic acid model in the sample. The primers are extended under suitable conditions, isolated from the model, and then rescanned, extended, and dissociated to amplify the nucleic acid copy number. The amplification product can be characterized by electrophoresis, restriction endonuclease cleavage and patterns, oligonucleotide hybridization or ligation, and / or nucleic acid sequencing using standard techniques. Other examples of amplification include standard displacement amplification, as described in U.S. Patent No. 5,744,311; transcription-free isothermal amplification, as described in U.S. Patent No. 6,033,881; amplification of repair chain reaction, as described in WO 90/01069; in ligase chain reaction amplification, as described in EP-A-320 308; amplification of gap filler ligase chain reaction, as described in U.S. Patent No. 5,427,930; and transcription-free amplification of NASBA ™ RNA, as described in U.S. Patent No. 6,025,134.
[0083] [0083] Animal: live multicellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human. Similarly, the term "individual" includes both human and veterinary patients.
[0084] [0084] Antibody: A polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or antigen binding fragments thereof, which specifically bind and recognize an analyte (antigen) such as gp41 or an immunogenic fragment of gp41, for example , the region close to the gp41 membrane. The immunoglobulin genes the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
[0085] [0085] Antibodies exist, for example, as intact immunoglobulins and as a number of well-characterized antigen-binding fragments produced by digestion with various peptidases. For example, Fabs, Fvs, scFvs that specifically bind to gp41 or fragments of gp41 would be specific gp41 binding agents. A scFv protein is a fusion protein in which an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region are found by a linker, the same time as in dsFvs, the chains have been changed to produce a disulfide bond to stabilize the chain association. The term also includes genetically engineered forms such as chimeric antibodies and heteroconjugate antibodies such as bispecific antibodies. See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, Immunology, 3rd Edition, W.H. Freeman & Co., New York, 1997.
[0086] [0086] Antibody fragments include, but are not limited to, the following: (1) Fab, the fragment containing a monovalent antigen binding fragment of an antibody molecule produced by digestion of integral antibody with the enzyme papain to produce an intact light chain and a portion of a heavy chain; (2) Fab ', the fragment of an antibody molecule obtained by treating integral antibody with pepsin, followed by reduction, to produce an intact light chain and a portion of the heavy chain; two Fab 'fragments are obtained per antibody molecule; (3) (Fab ') 2, the antibody fragment obtained by treating integral antibody with the enzyme pepsin without subsequent reduction; (4) F (ab ') 2, a dimer of two Fab' fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
[0087] [0087] The term "antibody", as used here, also includes antibody fragments produced by modifying whole antibodies or those newly synthesized using recombinant DNA methodologies. In some examples, the term antibody includes the amino acid sequence of one or more of the CDRs of the antibody grafted onto a scaffold.
[0088] [0088] Typically, a naturally occurring immunoglobulin has heavy chains (H) and light chains (L) interconnected by disulfide bonds. There are two types of light chain, lambda λ) and kappa (κ). There are five main classes of heavy chain (or isotopes) that determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. The described antibodies can be changed in class.
[0089] [0089] Each light and heavy chain contains a constant region and a variable region, (the regions are also known as "domains"). In various modalities, the light and heavy chain variable domains combine to specifically bind to the antigen. In additional embodiments, only the heavy chain variable domain is required. For example, naturally occurring camelid antibodies that consist of a heavy chain are only functional and stable in the absence of a light chain (see, for example, Hamers-Casterman, and another, Nature, 363: 446-448, 1993; Sheriff, and another, Nat. Struct. Biol., 3: 733-736, 1996). The light and heavy chain variable domains contain a "structure" region interrupted by three intervariable regions, also called "complementarity determining regions" or "CDRs" (see, for example, Kabat, and another, Sequences of proteins of Immunological Interest, US Department of Health and Human Services, 1991). The sequences of the structure regions of different heavy or light chains are relatively conserved within a species. The structure region of an antibody, which are the combined structure regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.
[0090] [0090] CDRs are primarily responsible for binding to an epitope on an antigen. The limits of the amino acid sequence of a given CDR can be easily determined using any of a number of well-known schemes, including those described by Kabat, and another, ("Sequences of proteins of Immunological Interest," 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991; "Kabat" numbering scheme), Al-Lazikani, and others, (JMB 273,927-948, 1997; "Chothia" numbering scheme), and Lefranc, and others, ("IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains, "Dev. Comp. Immunol., 27: 55-77, 2003;" IMGT "numbering scheme).
[0091] [0091] The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (from the N-terminal to the C-terminal), and are also typically identified by the chain in which the particular CDR is located. Thus, a CDR3 VH is located in the variable domain of the antibody heavy chain in which it is found, whereas a CDR1 VL is CDR1 in the variable domain of the antibody light chain in which it is found. Light chain CDRs are sometimes referred to as CDR L1, CDR L2, and CDR L3. Heavy chain CDRs are sometimes referred to as CDR H1, CDR H2, and CDR H3.
[0092] [0092] References to "VH" or "VH" refer to the variable region of an immunoglobulin heavy chain, including that of an antibody fragment, such as Fv, scFv, dsFv or Fab. References to "VL" or "VL" refer to the variable region of an immunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.
[0093] [0093] A "monoclonal antibody" is an antibody produced by a simple class of B-lymphocytes or by a cell in which the light and heavy chain genes of a single chain antibody have been transferred. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by preparing hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. These fused cells and their progeny are called "hybridomas". Monoclonal antibodies include humanized monoclonal antibodies. In some instances, monoclonal antibodies are isolated from an individual. The amino acid sequence of such isolated monoclonal antibodies can be determined.
[0094] A "humanized" immunoglobulin is an immunoglobulin including a human frame region and one or more CDRs of a non-human immunoglobulin (such as a mouse, rat, or synthetic). The non-human immunoglobulin that provides CDRs is called a "donor", and the human immunoglobulin that provides the structure is called an "acceptor". In one embodiment, all CDRs are from the donor immunoglobulin to a humanized immunoglobulin. The constant regions need not be present, but if they are, they must be substantially identical to the human immunoglobulin constant regions, such as at least about 85 to 90%, such as about 95% or more identical. As a result, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to the corresponding parts of natural human sequences. A "humanized antibody" is an antibody including a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that supplies the CDRs. The receptor structure of a humanized immunoglobulin or antibody can have a limited number of amino acid substitutions taken from the donor structure. Humanized monoclonal antibodies or other monoclonal antibodies may have additional conservative amino acid substitutions that have, substantially, no effect on antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed using genetic engineering (for example, see U.S. Patent No. 5,585,089).
[0095] [0095] Antibody autoreactivity: a property of an antibody, according to which the antibody reacts with autoepitopes, that is, epitopes of proteins and / or lipids that are produced by the individual. For example, an antibody, such as 10E8 that has no autoreactivity, does not bind to the lipids present in the membrane of an individual's cell, such as a cell infected with HIV and / or expressing gp41 on its surface. Methods of determining whether an antibody reacts with autoepitopes are known to the person skilled in the art and described here (for example, in examples 1 and 8). In one example, antibody autoreactivity is assessed using an anticardiolipin assay or an antinuclear antigen (ANA) assay.
[0096] [0096] Antibody scaffolding: refers to a protein that is grafted with one or more CDRs of an antibody of interest on its surface. The transplantation of CDRs can be performed computationally in a way that preserves their conformation and relevant structure. Mutations within the recipient scaffold are made to accommodate the CDR graft.
[0097] [0097] Antibody: The entire repertoire of the antibody light and heavy chain sequence expressed in an individual. The individual may be an individual infected with a pathogen, for example, HIV.
[0098] [0098] Antigen: A polypeptide that can stimulate the production of antibodies or a T cell response in an animal, including polypeptides that are injected or absorbed into an animal. An animal reacts with specific cellular or humoral immunity products, including those induced by heterologous antigens, such as the described antigens. "Epitope" or "antigenic determinant" refers to the region of an antigen to which B and / or T cells respond. In one embodiment, T cells respond to the epitope, when the epitope is presented together with an MHC molecule. Epitopes can be formed from either contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary duplication of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturation solvents whereas epitopes formed by tertiary duplication are typically lost in treatment with denaturation solvents. An epitope typically includes at least 3, and more generally, at least 5, about 9, or about 8 to 10 amino acids in a single spatial conformation. Methods of determining spatial epitope conformation include, for example, x-ray crystallography and nuclear magnetic resonance.
[0099] [0099] Immunogenic polypeptides and immunogenic properties are non-limiting examples of antigens. In some examples, antigens include polypeptides derived from a pathogen of interest, such as a virus. An antigen that can stimulate the production of antibodies or a T cell response in an individual to a polypeptide expressed by a virus is a viral antigen. An "HIV antigen" can stimulate the production of antibodies or a T cell response in an individual to an HIV expressed polypeptide. In some embodiments, an HIV antigen is an HIV-expressed polypeptide, such as gp160, or a fragment thereof, such as gp145, gp140, gp120 or gp41.
[0100] [00100] A "target epitope" is a specific epitope on an antigen that specifically binds an antibody of interest, such as a monoclonal antibody. In some examples, a target epitope includes amino acid residues that contact the antibody and interest, such that the target epitope can be selected by the determined amino acid residues in contact with the antibody and interest.
[0101] [00101] Antiretroviral agent: An agent that, in particular, inhibits a retrovirus from the duplication or infection of cells. Non-limiting examples of antiretroviral drugs include entry inhibitors (eg enfuvirtide), CCR5 receptor antagonists (eg aplaviroc, vicriviroc, maraviroc), reverse transcriptase inhibitors (eg lamivudine, zidovudine, abacavir, tenofovir, entric , efavirenz), protease inhibitors (eg, lopivar, ritonavir, raltegravir, darunavir, atazanavir), maturation inhibitors (eg, interferon alfa, bevirimat and vivecon).
[0102] [00102] Antiretroviral therapy (ART): A therapeutic treatment for HIV infection that involves the administration of at least one antiretroviral agent (for example, one, two, three or four antiretroviral agents) to an HIV-infected individual during a period of treatment. Non-limiting examples of antiretroviral agents include entry inhibitors (eg, enfuvirtide), CCR5 receptor antagonists (eg, aplaviroc, vicriviroc, maraviroc), reverse transcriptase inhibitors (eg, lamivudine, zidovudine, abacavir, tenofovir, entricitabine , efavirenz), protease inhibitors (eg, lopivar, ritonavir, raltegravir, darunavir, atazanavir), maturation inhibitors (eg, interferon alfa, bevirimat and vivecon). An example of an ART regimen includes treatment with a combination of tenofovir, emtricitabine and efavirenz. In some instances, ART includes Highly Active Antiretroviral Therapy (HAART).
[0103] [00103] Atomic coordinates or structure coordinates: the mathematical coordinates derived from mathematical equations related to the patterns obtained in diffraction of a monochromatic X-ray beam by the atoms (dispersion centers) such as an antigen, or an antigen in complex with an antibody . In some instances, that antigen may be gp41, a complex of gp41: antibody, or combinations of them in a crystal. Diffraction data is used to calculate an electron density map of the crystal's repeating unit. Electron density maps are used to stabilize the positions of individual atoms within the unit cell of the crystal. In one example, the term "structure coordinates" refers to Cartesian coordinates derived from mathematical equations related to the patterns obtained in the diffraction of a monochromatic X-ray beam, such as by the atoms of a gp41 in the form of a crystal.
[0104] [00104] Those skilled in the art understand that a set of structure coordinates determined by X-ray crystallography is not without standard error. For the purpose of this description, any set of structure coordinates having a root means square deviation of protein skeleton atoms (N, Cα, C and 0) less than about 1.0 angstroms when overlapping, such as about 0.75, or about 0.5, or about 0.25 angstrons, using skeleton atoms, (in the absence of an explicit specification to the contrary) will be considered identical.
[0105] [00105] B-cell and B-cell of memory: B-cells are a subset of lymphocytes, that is, white blood cells (leukocytes). Mature B cells differentiate into plasma cells, which produce antibodies, and memory B cells. A "B cell progenitor" is a cell that can grow into a mature B cell. B-cell progenitors include stem cells, initial pro-B cells, final pro-B cells, large pre-B cells, small pro-B cells, and immature B cells and transition B cells. Generally, the initial pro-B cells (which express, for example, CD43 or B220) undergo the immunoglobulin heavy chain redisposition to become pre B and pro B cells, and also undergo the light chain redisposition of immunoglobulin. immunoglobulin to become an immature B cell. In humans, immature B cells (e.g., immature peripheral transition B cells) include CD38hi, IgD +, CD10 +, CD24hi, CD44lo, CD23lo and CD1lo cells. In this way, immature B cells include B220 expression cells (CD45R) in which the heavy and heavy chain immunoglobulin genes are redisposed. In one embodiment, immature B cells express CD45R, class II, IgM, CD19 and CD40. Immature B cells can develop into mature B cells, which can produce immunoglobulins (for example, IgA, IgG or IgM). Mature B cells have acquired surface IgM and IgD, are capable of responding to the antigen, and express characteristic markers such as CD21 and CD23 cells (CD23hiCD21hi). Plasma cells are terminally differentiated Plasma B cells that are the predominant antibody secreting cells.
[0106] [00106] After a B cell parent (for example, a small pre-compromised lymphocyte) is stimulated by an antigen, it differentiates into an explosion cell, which differs into an immature plasma cell that can differentiate into one mature plasma cell or a memory B cell. A "mature plasma cell" secretes immunoglobulins in response to a specific antigen.
[0107] [00107] B cells can be activated by agents such as lipopolysaccharide (LPS) or IL-4 and antibodies to IgM. Common biological sources of B cells and B cell progenitors include bone marrow, peripheral blood, spleen and lymph nodes.
[0108] [00108] A "memory B cell" is a B cell that supports isotype switching and somatic hypermutation that are generally encountered during a secondary immune response (a subsequent antigen exposure after a primary exposure), but can also be detected during a primary antigen response. The generation of memory B cells requires helper T cells. The development of memory B cells occurs in germinal centers (GC) of lymphoid follicles where antigen-regulated lymphocytes undergo somatic hypermutation and affinity selection, presumably under the influence of helper T cells. Typically, memory B cells express high affinity antigen-specific immunoglobulin (B cell receptor) on their cell surface. In one embodiment, memory B cells include cells that express CD19, but do not express IgA, IgD or IgM (CD19 + IgA-IgD-IgM- cells).
[0109] [00109] Bispecific antibody: a recombinant molecule of two different antigen binding domains that, consequently, bind to two different antigenic epitopes. Bispecific antibodies include chemically or genetically linked molecules from two antigen binding domains. The antigen binding domains can be linked using a linker. The antigen binding domains can be monoclonal antibodies, antigen binding fragments (e.g., Fab, scFv), eAds, bispecific single chain antibodies or a combination thereof. A bispecific antibody can include one or more constant domains, but it does not necessarily include a constant domain. An example of a bispecific antibody is a bispecific single chain antibody including a scFv that specifically binds to the bound gp41 (via a peptide linker) to an scFv that specifically binds to an antigen other than gp41. Another example is a bispecific antibody including a Fab that specifically binds to gp41 bound to an scFv that specifically binds to an antigen other than gp41.
[0110] [00110] B cell repertoire: B cells in a sample or in a specific individual for the antigen of interest.
[0111] [00111] CD40: a co-stimulating protein has found an antigen that contains cells that are required for its activation. The binding of CD40 ligand (CD40L), also known as CD154, to CD40 activates antigen presenting cells. This receptor has been found to be essential in mediating a wide variety of immune and inflammatory responses including T-cell-dependent immunoglobulin change and class, B-cell development of memory, and formation of the germinal center. An exemplary amino acid sequence for CD40, and an exemplary mRNA sequence that encodes CD40 can be found in GENBANK® accession No. NM_001250, (June 10, 2012), which is incorporated by reference.
[0112] [00112] Ligand CD40 (CD40L): A ligand that is also called CD154, which is expressed in activated T cells and is a member of the molecule tumor necrosis superfamily. It binds to CD40 in antigen presenting cells, which leads to many effects that depend on the type of target cell. In general, CD40L plays the role of a co-stimulating molecule and induces activation in antigen presenting cells in association with T cell receptor stimulation by MHC molecules in antigen presenting cells. In total CD40L it has three binding patterns: CD40, α5β1 and αIIbβ3 integrin. CD154 is expressed on the T cell surface. It regulates B cell function by engaging CD40 on the B cell surface. An exemplary amino acid sequence for CD40, and an exemplary mRNA sequence encoding CD40 can be found in GENBANK® accession No. NM_000074 .2, (June 10, 2012), which is incorporated by reference.
[0113] [00113] Chimeric antibody: An antibody that includes sequences derived from two different antibodies, which are typically of different species. In some examples, a chimeric antibody includes one or more CDRs and / or framework regions of a human antibody and CDRs and / or framework regions of another human antibody. In some examples, a chimeric antibody is produced by grafting one or more CDRs onto an antibody scaffold.
[0114] [00114] Clonal variant: any sequence, which differs by one or more nucleotides or amino acids, in the presence of V region with identical mutations in comparison with the use of identical, germinating VJ or VDJ gene, and identical D and J length. The "germline" sequence is intended to be the sequence encoding the antibody / immunoglobulin (or any fragment thereof) deprivation of mutations, for example, somatic mutations. The percentage of homology represents an indication of the mutational events that any type of heavy chain portion undergoes after contact with an antigen.
[0115] [00115] Computer-Readable Storage Media: any medium or media, which can be read and accessed directly by a computer, so that the media are suitable for use in a computer system. Such media include, but are not limited to: magnetic storage media such as floppy disks, hard disk storage media and magnetic tape; optical storage media such as optical discs or CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as optical / magnetic storage media.
[0116] [00116] Computer system: The hardware that can be used to analyze atomic coordinate data and / or plan an antigen that uses atomic coordinate data or to analyze an amino acid or nucleic acid sequence, for example, to compare two or more sequences to a calculated sequence divergence and / or similarity. The minimum hardware in a computer-based system typically includes a central processing unit (CPU), an input device, for example, a mouse, keyboard, and the like, an output device, and a data storage device . Desirably, a monitor is provided to view the structure data. The data storage device can be RAM or other readable means of accessing the computer. Examples of such systems are microcomputer workstations available with Silicon Graphics Incorporated and Sun Microsystems running Unix based Windows NT or IBM OS / 2 operating systems.
[0117] [00117] Conjugate: A complex of two molecules linked to each other, for example, linked to each other by a covalent bond. In one embodiment, an antibody is bound to an effector molecule; for example, an antibody that specifically binds to gp41 covalently linked to an effector molecule or toxin. A bond can be by chemical or recombinant means. In one embodiment, the bond is chemical, in which a reaction between the antibody portion and the effector molecule produced a covalent bond formed between the two molecules to form a molecule. A peptide linker (short peptide sequence) can optionally be included between the antibody and the effector molecule. Because conjugates can be prepared from two molecules with separate functionalities, such as an antibody and an effector molecule, they are also sometimes referred to as "chimeric molecules". In one embodiment, an antibody attached to an effector molecule is also attached to a lipid or another molecule to a protein or peptide to increase its half-life in the body.
[0118] [00118] Contact: placement in direct physical association; includes both in solid and liquid form, which can occur in a vacuum or in vitro. Contact includes contact between a molecule and another molecule, for example, the amino acid on the surface of a polypeptide, such as an antigen, which contacts another polypeptide, such as an antibody. Polypeptide contact can include direct contact between amino acids of two or more polypeptides (for example, hydrogen bonding or Van der Waals force interactions between polypeptides), as well as other interactions between polypeptides that produce an interface between polypeptides with solvent accessibility reduced (without all the amino acids of the interface necessarily forming direct bonds). In some embodiments, direct contact refers to the formation of a hydrogen bond or Van der Waals interaction with, in particular, an identical residue or residues in a sequence, but not with the other residues in the sequence. The person skilled in the art is familiar with methods of determining contact between polypeptides (see, for example, Example 1). Contact may also include contacting a cell, for example, by placing an antibody in direct physical association with a cell. In some embodiments, an antibody (for example, 10E8) only contacts particular residues of an epitope on an antigen, such as the 10E8 epitope on gp41 as described here.
[0119] [00119] Control: a reference standard. In some modalities, control is a sample obtained from a healthy patient. In other modalities, the control is a sample of tissue obtained from a patient diagnosed with HIV infection. In still other modalities, control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of HIV patients with a known prognosis or outcome, or group of samples representing baseline or normal values).
[0120] [00120] A difference between a test sample and a control can be an increase or contrary to a decrease. The difference can be a qualitative difference or a quantitative difference, for example, a statistically significant difference. In some instances, a difference is an increase or decrease, compared to a control, of at least about 5%, such as at least about 10%, at least about 20%, at least about 30%, at least at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or more than than 500%.
[0121] [00121] Cytokine / Interleukin (IL): A generic name for a diverse group of peptides and soluble proteins that act as humoral regulators in nano- to picomolar concentrations and that, under normal or pathological conditions, modulate the functional activities of cells and tissues individual. These proteins also directly mediate interactions between cells and regulate the processes that occur in the extracellular environment. Many growth factors and cytokines act as cell survival factors to prevent programmed cell death. Cytokines and interleukins include both naturally occurring peptides and variants that retain partial total biological activity. Although specific cytokines / interleukins are described in the specification, they are not limited to the peptides specifically described.
[0122] [00122] Cytotoxicity: the toxicity of a molecule, such as an immunotoxin, to cells destined to be targeted, when opposed to cells in the rest of an organism. In one embodiment, in contrast, the term "toxicity" refers to the toxicity of an immunotoxin to cells other than those cells that are intended to be targeted by the bleaching portion of the immunotoxin, and the term "animal toxicity" refers to toxicity immunotoxin for an animal due to immunotoxin toxicity for cells targeted by the immunotoxin.
[0123] [00123] Detectable marker: A detectable molecule (also known as a label) that is conjugated directly or indirectly to a second molecule, such as an antibody, to facilitate the detection of the second molecule. For example, the detectable marker may be capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as CT scans, MRIs, ultrasound, fiber optic examination, and laparoscopic examination). Non-limiting, specific examples of detectable markers include fluorophores, fluorescent proteins, chemiluminescent agents, enzyme bonds, radioactive isotopes and heavy metals or compounds (for example, superparamagnetic iron oxide nanocrystals for MRI detection). In one example, a "labeled antibody" refers to the incorporation of another molecule into the antibody. For example, the label is a detectable marker, such as the incorporation of a radio-labeled amino acid or binding to a polypeptide of biotinyl moieties that can be detected by labeled avidin (for example, streptavidin containing a fluorescent marker or enzyme activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (such as 35S or 131I), fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide matches), enzyme labels (tai such as horseradish peroxidase, betagalactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinylated groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequence, linking sites for secondary antibodies, binding domain domains metal, epitope label), or magnetic agents, such as gadolinium chelates. In some embodiments, the labels are connected by spacer arms of various lengths to reduce potential steric impediment. Methods of using detectable markers and guides in choosing appropriate detectable markers for various purposes are discussed, for example, in Sambrook, and others, (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989) and Ausubel, and another, (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).
[0124] [00124] Detection: to identify the existence, presence, or fact of something. General detection methods are known to the skilled person and can be supplemented with the protocols and reagents described here. For example, included here are methods of detecting a cell that expresses gp41 in an individual.
[0125] [00125] DNA sequencing: The process of determining the nucleotide order of a given DNA molecule. The general characteristics of "deep sequencing" are that the genetic material is amplified, such as by polymerase chain reaction, and then the amplified products are attached to a solid surface. The sequence of the amplified target genetic material is then carried out in parallel and the sequence information is captured by a computer. Generally, sequencing can be performed using automated Sanger sequencing (AB13730xl genome analyzer), pyose sequencing on a solid support (454 sequencing, Roche), sequencing by synthesis with reversible terminations (ILLUMINA® Genome Analyzer), sequencing by ligation (ABI SOLiD ®) or sequencing by synthesis with virtual terminators (HELISCOPE®). In some embodiments, DNA sequencing is performed using a chain termination method developed by Frederick Sanger, and thus called "Sanger-based sequencing" or "SBS." This technique uses sequence-specific termination of a DNA synthesis reaction using modified nucleotide substrates. Extension is initiated at a specific site in the standard DNA using a short oligonucleotide primer complementarity to the standard in that region. The oligonucleotide primer is extended using DNA polymerase in the presence of the four deoxynucleotide bases (DNA building blocks), along with a low concentration of a chain-terminating nucleotide (most commonly a deoxynucleotide). The limited incorporation of the chain-terminating nucleotide by the DNA polymerase results in a series of related DNA fragments that are terminated only in positions where that particular nucleotide is present. The fragments are then separated by size by electrophoresis, a polyacrylamide gel, or a narrow glass (capillary) tube deposited with a viscous polymer. An alternative to using a labeled initiator is to use labeled terminators instead; this method is often called "dye terminator sequencing".
[0126] [00126] "Pyrosquencing" is a provision-based method, which has been marketed by 454 Life Sciences (Branford, CT). In some modalities of the disposition-based methods, the single-stranded DNA is annealed and amplified by means of EmPCR®. These DNA-linked beads are then placed in cavities on a fiber optic chip along with enzymes that produce light in the presence of ATP. When free nucleotides are washed on this chip, light is produced, since PCR amplification occurs and ATP is generated when the nucleotides bond with their complementary base pairs. The addition of one (or more) nucleotide (s) results in a reaction that generates a light signal that is recorded, such as by the charge-coupled device (CCD) camera, inside the instrument. The signal strength is proportional to the number of nucleotides, for example, homopolymer stretches, incorporated into a single nucleotide stream.
[0127] [00127] Effective amount: the amount of an agent (such as CD40L, IL-21 or IL-2) that alone, or together with one or more additional agents, induces the desired response.
[0128] [00128] Effector molecule: the portion of a chimeric molecule that is understood to have a desired effect on a cell to which the chimeric molecule is targeted. The effector molecule is also known as an effector moiety, therapeutic agent, or diagnostic agent, or similar term.
[0129] [00129] Epitope: an antigenic determinant. These are particular chemical groups or peptide sequences in a molecule that are antigenic, such that they elicit a specific immune response, for example, an epitope is the region of an antigen to which the B and / or T responses respond. In some examples, a specifically described antibody binds to an epitope on the surface of HIV gp41.
[0130] [00130] Epitopes can be formed from both contiguous amino acids and non-contiguous amino acids juxtaposed by tertiary duplication of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents while epitopes formed by tertiary duplication are typically lost in treatment with denaturing solvents. An epitope typically includes at least 3, and more generally, at least 5, about 9, or about 8 to 10 amino acids in a single spatial conformation. Methods of determining the spatial conformation of epitopes include, for example, x-ray crystallography and nuclear magnetic resonance.
[0131] [00131] Fc Polypeptide: The polypeptide that includes an antibody constant region excluding the first constant region immunoglobulin domain. The Fc region generally refers to the last two IgA and IgG constant region immunoglobulin domains, and the last three IgE and IgM constant region immunoglobulin domains. A Fc region can also include part or all of the flexible hinge N-terminal to these domains. For IgA and IgM, an Fc region may or may not include the cap, and may or may not be linked by the J chain. For IgG, the Fc region includes immunoglobulin domains Cgamma2 and Cgamma3 (Cγ2 and Cγ3) and the bottom of the hinge between Cgamma1 (Cγ1) and Cγ2. Across the limits of the Fc region may vary, the Fc region of human IgH heavy chain is generally defined by including C226 or P230 residues at its carboxyl terminus, where the numbering is according to the EU index as in Kabat . For IgA, the Fc region includes immunoglobulin domains Calfa2 and Calfa3 (Cα2 and Cα3) and the bottom of the hinge between Calpha1 (Cα1) and Cα2. Covered within the definition of the Fc region are analogues and functionally equivalent variants of the Fc region. A functionally equivalent analog of the Fc region can be a variant Fc region, including one or more amino acid modifications from the naturally occurring or wild-type Fc region. The variant Fc regions will have at least 50% homology with a naturally occurring Fc region, such as about 80%, and about 90%, or at least about 95% homology. Functionally equipped analogs of the Fc region can include one or more amino acid residues added to or deleted from the N or C terminus of the protein, such as no more than 30 or no more than 10 additions and / or deletions. The functionally equivalent analogs of the Fc region include the Fc regions operably linked to a fusion partner. The functionally equivalent analogs of the Fc region must include the majority of all Ig domains that make up the Fc region as defined above; for example, the IgG and IgA Fc regions as defined here should include the majority of the sequences encoding CH2 and the majority of the sequence encoding CH3. Thus, the CH2 domain itself, or the CH3 domain itself, are not considered the Fc region. The Fc region can refer to this region in isolation, or this region in relation to an Fc fusion polypeptide (immunoadhesin, see below).
[0132] [00132] Structure region: the amino acid sequences interposed between the CDRs. They include heavy variable and light variable structure regions. The framework regions serve to keep the CDRs in proper orientation for antigen binding.
[0133] [00133] gp41: a specific HIV protein. The HIV-1 envelope protein is initially synthesized as a longer precursor protein of 845 to 870 amino acids in size, called gp160. Gp160 forms a homotrimer and undergoes glycosylation within the Golgi apparatus. In vivo, it is then cleaved by a cell protease into gp120 and gp41. The amino acid sequence of an example of gp41 is set out in GENBANK® accession No. CAD20975 (as available on October 16, 2009) which is incorporated by reference here. It is understood that the sequence of gp41 may vary from that provided in GENBANK® accession No. CAD20975. Gp41 contains a transmembrane domain and typically remains in a trimester configuration; it interacts with gp120 in a non-covalent manner.
[0134] [00134] HIV envelope protein (Env): the HIV envelope protein is initially synthesized as a longer precursor protein of 845 to 870 amino acids in size, called gp160. Gp160 forms a homotrimer and undergoes glycosylation within the Golgi apparatus. In vivo, it is then cleaved by a cell protease into gp120 and gp41. Gp120 contains most of the external domains, exposed on surfaces, of the HIV envelope glycoprotein complex, and it is gp120 that binds to both cellular CD4 receptors and cellular chemokine receptors (such as CCR5). The gp41 contains a transmembrane domain and remains in a trimetric configuration; it interacts with gp120 in a non-covalent manner.
[0135] [00135] Host cells: Cells in which a vector can be propagated and its DNA expressed, for example, a described antibody can be expressed in a host cell. The cell can be prokaryotic or eukaryotic. The term also includes any progeny of the individual's host cell. It is understood that every progeny cannot be identical to the parental cell, since there are mutations that occur during replication. However, such progenies are included when the term "host cell" is used.
[0136] [00136] Human Immunodeficiency Virus (HIV): A retrovirus that causes immunosuppression in humans (HIV disease), and induces a disease complex known as acquired immunodeficiency syndrome (AIDS). "HIV disease" refers to a well-recognized constellation of signs and symptoms (including the development of opportunistic infections) in people who are infected with an HIV virus, as determined by antibody or western blot studies. Laboratory findings associated with this disease include a progressive decline in T cells. HIV includes HIV type 1 (HIV-1) and HIV type 2 (HIV-2). Related viruses that are used as animal models include simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV). Treatment of HIV-1 with HAART was effective in reducing viral load and improving the effects of HIV-1 infection in infected individuals.
[0137] [00137] HXB2 numbering system: A reference numbering system for HIV protein and nucleic acid sequences, using HIV-1 HXB2 lineage sequences as a reference for all other HIV lineage sequences. The person skilled in the art is familiar with the HXB2 numbering system, and this system is mentioned in "Numbering Positions in HIV Relative to HXB2CG," Bette Korber, et al, Human Retroviruses and AIDS 1998: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Korber B, Kuiken CL, Foley B, Hahn B, McCutchan F, Mellors JW, and Sodroski J, Eds. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM, which are hereby incorporated by reference here in their entirety. HXB2 is also known as: HXBc2, for clone 2 of HXB 2; HXB2R, in the Los Alamos HIV database, with the R for revised, when it was slightly revised with respect to the original HXB2 sequence; and HXB2CG in GENBANK ™, for the complete HXB2 genome. The numbering used in the gp41 polypeptides described here is relative to the HXB2 numbering scheme.
[0138] [00138] IgA: A polypeptide belonging to the class of antibodies that is substantially encoded by an immunoglobulin alpha gene. In humans, this class or isotype includes IgA1 and IgA2. IgAn antibodies can exist as monomers, polymers (referred to as pIgA) in a predominantly dimeric form, and secretory IgA. The wild-type IgA constant chain contains an extension of 18 amino acids at its C-terminus called the tail portion (tp). Polymeric IgA is secreted by plasma cells with a 15-kDa peptide called the J chain linking the two IgA monomers through the cysteine residue conserved in the tail.
[0139] [00139] IgG: A polypeptide belonging to the class or isotype of antibodies that are substantially encoded by a recognized gamma immunoglobulin gene. In humans, this class includes IgG1, IgG2, IgG3, and IgG4.
[0140] [00140] Immune complex: The binding of antibody to a soluble antigen forms an immune complex. The formation of an immune complex can be detected by conventional methods known to the skilled person, for example, immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (eg Western blot), magnetic resonance imaging, scans CT, X-ray and affinity chromatography. Immunological binding properties of selected antibodies can be quantified using methods well known in the art.
[0141] [00141] Immunoadhesin: A molecular fusion of a protein with the Fc region of an immunoglobulin, where the immunoglobulin maintains specific properties, such as Fc receptor binding and increased half-life. An Fc fusion combines the Fc region of an immunoglobulin with a fusion partner, which in general can be any protein, polypeptide, peptide, or small molecule. In one example, an immunoadhesin includes the articulation, CH2, and CH3 domains of the immunoglobulin gamma 1 heavy chain constant region. In another example, the immunoadhesin includes the CH2, and CH3 domains of an IgG.
[0142] [00142] Immunogen: A compound, composition, or substance (for example, a protein or a portion thereof) that is capable of inducing an immune response in a mammal, such as an infected mammal or at risk of infection with a pathogen. Administration of an immunogen can induce protective immunity and / or proactive immunity against a pathogen of interest. In some instances, an immunogen is an HIV antigen. Examples of immunogens include, but are not limited to, peptides, lipids, polysaccharides, combinations thereof, and nucleic acids containing antigenic determinants, such as those recognized by an immune cell. In some examples, immunogens include peptides derived from a pathogen of interest. Exemplary pathogens include bacteria, fungi, viruses and parasites. In specific examples, an immunogen is derived from HIV, such as an HIV-derived gp41 peptide or antigenic fragment thereof.
[0143] [00143] Immunological probe: A molecule that can be used to select antibodies from sera that are directed against a specific epitope, including sera from a human patient. Epitope scaffolding, along with related point mutants, can be used as immunological probes in both positive and negative selection of antibodies against the epitope graft. In some examples, immunological probes are planned variants of gp120.
[0144] [00144] Immunologically reactive conditions: Include reference to conditions that allow a high antibody against a particular epitope to bind to that epitope to a degree detectably greater than, and / or to the substantial exclusion of, binding to substantially all other epitopes. Immunologically reactive conditions are dependent on the format of the antibody binding reaction and are typically those used in immunoassay protocols or those conditions found in vivo. See, Harlow & Lane, below, for a description of immunoassay formats and conditions. The immunologically reactive conditions employed in the methods are "physiological conditions" that include reference to conditions (for example, temperature, osmolarity and pH) that are typical inside a living mammal or a mammalian cell. While it is recognized that some organs are subjected to extreme conditions, the intraorganismal and intracellular environment usually lies around pH 7 (for example, from pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), contain water as the predominant solvent, and exist at a temperature above 0 ° C and below 50 ° C. Osmolarity is in the range that supports cell viability and proliferation.
[0145] [00145] Inhibition or treatment of a disease: The inhibition of the complete development of a disease or condition, for example, in an individual who is at risk of a disease such as acquired immunodeficiency syndrome (AIDS). "Treatment" refers to a therapeutic intervention that improves a sign or symptom of a disease or pathological condition after it has started to develop. The term "improvement," with reference to a disease or pathological condition, refers to any observable beneficial effect of treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible individual, a reduction in the severity of some or all of the clinical symptoms of the disease, a slower progression of the disease, a reduction in viral load , an improvement in the overall health, or well-being of the individual, or by other parameters well known in the art that are specific to the particular disease. A "prophylactic" treatment is treatment given to an individual who shows no signs of an illness or shows only early signs for the purpose of decreasing the risk of developing the condition.
[0146] [00146] Interleukin-2 (IL-2): IL-2 is a cytokine that is necessary for the growth and function of T cells. Antigen binding to the T cell receptor (TCR) stimulates the secretion of IL-2, and the expression of IL-2R IL-2 receptors. The IL-2 / IL-2R interaction then stimulates the growth, differentiation and survival of antigen-selected cytotoxic T cells through the activation of specific genes. As such, IL-2 is necessary for the development of T cell immune memory, which depends on the expansion of the number and function of selected T cell clones of antigen. IL-2 is also needed during the development of T cell in the thymus for the maturation of a subset of T cells that are called regulatory T cells. An exemplary amino acid sequence of human IL-2 is provided in GENBANK® Accession No. NM_000586 (June 10, 2012), which is incorporated by reference.
[0147] [00147] Interleukin-21 (IL-21): The cloned cytokine of a cDNA library derived from activated CD3 + T cells (ParrishNovak, et al., Nature 408: 57-63, 2000). The IL-21 cDNA encodes a 131-amino acid-secreted protein most closely related to IL-2 and IL-15. The IL-21 gene was mapped to a human chromosome 4q26-q27 next to the IL-2 gene.
[0148] IL-21 mRNA has been shown to be expressed in activated CD4 + cells, but not in other T cells, B cells, or monocytes (Parrish-Novak, et al., Nature 408: 57-63, 2000). However, IL-21 has been shown to stimulate the proliferation of B cells that are stimulated by crosslinking the CD40 antigen and proliferation of B cells stimulated by IL-4 in addition to anti-IgM. IL-21 has also been shown to stimulate the proliferation of cells that have not received treatment (CD45RA (+)), mediated by CD3 impairment. IL21 has also been shown to stimulate the proliferation of progenitor bone marrow to cells and enhance the expression of the CD56 cell marker in the presence of IL-15. (For review, see Horst Ibelgaufts' COPE: Cytokines Online Pathfinder Encyclopedia, available on the internet). The amino acid sequence of an exemplary human IL-21 is shown as SEQ ID NO: 1 in U.S. Published Patent Application No. 2003/0003545, which is incorporated herein by reference. A representative clone containing all or most of the sequence for IL-21 (designated HTGED19) was deposited with the American Type Culture Collection ("ATCC") on March 5, 1998, and the ATCC deposit number was provided 209666 (see, for example, Published Patent Application US No. 2003/0003545).
[0149] [00149] The IL-21 receptor has been isolated and has been found to be expressed by CD23 + B cells, B cell lines, a T cell leukemia line, and NK cell lines. The receptor gene was mapped to the human chromosome 16p12 (see, Parrish-Novak, and another, Nature 408: 57-63, 2000; Ozaki, and another, Proc. Natl. Acad. Sci. USA 97: 11439-11444, 2000 ).
[0150] [00150] Interleukin 15 (IL-15): The cytokine with structural similarity to IL-2. IL-15 binds to and signals through the IL-2 / IL-15 beta chain (CD122) and the common gamma chain (gamma-C, CD132). IL-15 is secreted by mononuclear phagocytes (and some other cells) following virus infection. This cytokine induces cell proliferation of natural killer cells; cells of the innate immune system whose main role is to kill virally infected cells.
[0151] [00151] Isolated: An "isolated" biological component (such as an antibody, for example, an antibody that specifically binds gp41, a nucleic acid, peptide, protein or antibody) has been substantially separated, produced apart from, or purified away from other biological components in the organism's cell in which the component naturally occurs, such as other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins that have been "isolated" in this way include nucleic acids and proteins purified by standard purification methods. The term also encompasses nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids or polypeptides. In some examples, an antibody, such as a specific antibody such as gp41, can be isolated, for example, isolated from an HIV-infected individual.
[0152] [00152] An "isolated" cell is a cell that has been purified from other cellular components of a tissue. The cells can be isolated by mechanical (such as using FACS) and / or enzymatic methods. In various embodiments, an isolated cell population (such as a B cell repertoire) includes more than about 80%, about 85%, about 90%, about 95%, or more than about 99% of the cells of interest. In another embodiment, an isolated population of cells is one in which no other cell of a different phenotype can be detected. In another embodiment, an isolated cell population is a cell population that includes less than about 20%, about 15%, about 10%, about 5%, or less than about 1% of a cell of a different phenotype than the cells of interest.
[0153] [00153] KD: The dissociation constant for a given interaction, such as a polypeptide ligand interaction or an antigen-antibody interaction. For example, for the bimolecular interaction of an antibody (such as that described here) and an antigen (such as gp41) it is the concentration of the individual components of the bimolecular interaction divided by the concentration of the complex.
[0154] [00154] Linker: A bifunctional molecule that can be used to link two molecules into a contiguous molecule, for example, to link an effector molecule to an antibody. In some embodiments, a conjugate includes a linker between the detectable marker or effector molecule and an antibody. In some embodiments, the linker is cleavable under intracellular conditions, so that cleavage of the linker releases the effector molecule or detectable marker of the antibody in the intracellular environment. In still other embodiments, the linker is not cleavable and the effector molecule or detectable marker can be released, for example, by antibody degradation. In some cases, a linker is a peptide within an antibody binding fragment (such as an Fv fragment) that serves to indirectly link the variable heavy chain to the variable light chain.
[0155] [00155] The terms "conjugate," "join," "link" or "connect" refer to the production of two polypeptides in a contiguous polypeptide molecule, to covalently link a radionucleotide or other molecule to a polypeptide, such as an antibody that specifically binds gp41, or an antibody binding fragment thereof. In the specific context, the terms include reference to the binding of a linker, such as an antibody moiety, to an effector molecule. The ligation can be by chemical or recombinant methods. "Chemical methods" refers to a reaction between the antibody portion and the effector molecule so that there is a covalent bond formed between the two molecules to form a molecule.
[0156] [00156] External Membrane Outer Region (MPER) of gp41: A region that is immediately N-terminal of the gp41 transmembrane region. MPER is highly hydrophobic (50% of waste is hydrophobic) and is highly conserved through many HIV clades (Zwick, M.B., and another, J Virol, 75 (22): p. 10892- 905, 2001). The conserved MPER of HIV-1 gp41 is a target of two neutralizing human monoclonal antibodies, 2F5 and 4E10. The nucleus of the 2F5 epitope has been shown to be ELDKWAS (SEQ ID NO: 9). With this epitope, residues D, K, and W were found to be more critical for recognition by 2F5. The nucleus of the 4E10 epitope, NWFDIT (SEQ ID NO: 10), maps only the C terminal to the 2F5 epitope in the gp41 ectodomain.
[0157] [00157] Neutralizing antibody: an antibody that reduces the infectious titration of an infectious agent by binding to a specific antigen on the infectious agent. In some instances, the infectious agent is a virus. In some instances, an antibody that is specific for gp41, neutralizes infectious HIV titration. A "broadly neutralizing antibody" is an antibody that binds to and inhibits the function of related antigens, such as antigens that share at least 85%, 90%, 95%, 96%, 97%, 98% or 99% of the surface antigenic identity of antigen. With respect to an antigen of a pathogen, such as a virus, the antibody can bind to and inhibit the function of an antigen of more than one class and / or subclass of the pathogen. For example, with respect to a human immunodeficiency virus, the antibody can bind to and inhibit the function of an antigen, such as gp41 from more than one clade. In one embodiment, broadly neutralizing antibodies to HIV are distinguished from other antibodies to HIV in that they neutralize a high percentage of most circulating types of HIV.
[0158] [00158] Nucleic acid: A polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogues thereof) linked by means of phosphodiester bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogues thereof. Thus, the term includes nucleotide polymers in which the nucleotides and the bonds between them include synthetic non-naturally occurring analogs, such as, for example, and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-ribonucleotides -O-methyl, peptide nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "oligonucleotide" typically refers to the shortest polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it also includes an RNA sequence (i.e., A, U, G, C) where " U "replaces" T. "
[0159] [00159] Conventional notation is used here to describe nucleotide sequences: the left end of a single stranded nucleotide sequence is the 5 'end; the left direction of a double-stranded nucleotide sequence is referred to as the 5 'direction. The addition of 5 'to 3' direction of nucleotides to the nascent RNA transcripts is referred to as the transcription direction. The strand of DNA having the same sequence as an mRNA is referred to as "coding strand;" sequences in the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located at the 5 'to 5' ends of the RNA transcription are referred to as "upstream sequences;" sequences in the DNA strand having the same sequence as the RNA and which are 3 'to 3' end of the encoding RNA transcription are referred to as "downstream sequences."
[0160] [00160] "cDNA" refers to a DNA that is complementary or identical to an mRNA, in the form of single strand or double strand.
[0161] [00161] "Coding" refers to the inherent property of specific nucleotide sequences in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as a standard for the synthesis of other polymers and macromolecules in biological processes having a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the resulting biological properties therefrom. Thus, a gene encodes a protein if the transcription or translation of mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is generally provided in sequence listings, and the non-coding strand, used as a standard for transcription, of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of one another and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA can include introns.
[0162] [00162] "Recombinant nucleic acid" refers to a nucleic acid having nucleotide sequences that are not naturally linked together. This includes nucleic acid vectors including an amplified or assembled nucleic acid that can be used to transform a suitable host cell. A host cell that includes the recombinant nucleic acid is referred to as a "recombinant host cell." The gene is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide." A recombinant nucleic acid can also serve as a non-coding function (for example, promoter, origin of replication, ribosome binding site, etc.).
[0163] [00163] A first sequence is an "antisense" with respect to a second sequence if a polynucleotide whose sequence is the first sequence, specifically hybridizes to a polynucleotide whose sequence is the second sequence.
[0164] [00164] Operationally linked: A first nucleic acid sequence is operationally linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter, such as the CMV promoter, is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, sequences of operationally linked DNA are contiguous and, where necessary to link two protein coding regions, in the same reading frame.
[0165] [00165] Pharmaceutical agent: A compound or chemical composition capable of inducing a desired therapeutic or prophylactic effect, when properly administered to an individual or a cell. In some examples, a pharmaceutical agent includes one or more of the antibodies described herein.
[0166] [00166] Pharmaceutically acceptable vehicles: Pharmaceutically acceptable vehicles for use are conventional. Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical release of the antibodies described here.
[0167] [00167] In general, the nature of the vehicle will depend on the particular mode of administration being employed. For example, parenteral formulations generally include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a carrier. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral vehicles, pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example, sodium acetate or sorbitan monolaurate .
[0168] [00168] Polypeptide: Any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation). In one embodiment, the polypeptide is a gp41 polypeptide. In one embodiment, the polypeptide is a described antibody or a fragment thereof. A "residue" refers to an amino acid or amino acid mimetic incorporated into a polypeptide by an amide bond or amide bond mimetic. A polypeptide has an amino terminus (N-terminus) and a carboxy terminus (C-terminus).
[0169] [00169] Promoter: A promoter is an arrangement of nucleic acid control sequences that directs the transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences close to the transcription start site, for example, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal repressing or enhancing elements that can be located as many as several thousand base pairs from the transcriptional pinic site. Both constitutive and inducible promoters are included (see, for example, Bitter, et al., Methods in Enzymology 153: 516-544, 1987).
[0170] [00170] Non-limiting, specific examples of promoters include promoters derived from the mammalian cell genome (such as the metallothionein promoter) or from mammalian viruses (such as the retrovirus long terminal repeat; the adenovirus late promoter; the 7 promoter; 5K of the vaccinia virus) can be used. Promoters produced by recombinant DNA or synthetic techniques can also be used. A polynucleotide can be inserted into an expression vector that contains a promoter sequence that facilitates efficient transcription of the inserted genetic sequence from the host. The expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells.
[0171] [00171] Purified: The term purified does not require absolute purity; preferably, it is understood as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein (such as an antibody) is more enriched than the peptide or protein and is in its natural environment within the cell. In one embodiment, a preparation is purified so that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation.
[0172] [00172] Recombinant: A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separate segments of sequence. This artificial combination is often carried out by chemical synthesis or, more commonly, by artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques.
[0173] [00173] Sequence Identity: The similarity between the amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as the sequence identity. Sequence identity is often measured in terms of percentage of identity (or similarity or homology); the higher the percentage, the more similar the two strings are. Homologues or variants of a polypeptide will have a relatively high degree of sequence identity when aligned using standard methods.
[0174] [00174] Methods of aligning sequences for comparison are well known in the art. Various alignment programs and algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2: 482, 1981; Needleman and Wunsch, J. Mol. Biol. 48: 443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444, 1988; Higgins and Sharp, Gene 73: 237, 1988; Higgins and Sharp, CABIOS 5: 151, 1989; Corpet et al., Nucleic acids Research 16: 10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444, 1988. Altschul et al., Nature Genet. 6: 119, 1994, present a consideration of methods for aligning their salience and homology calculations.
[0175] [00175] The NCBI Basic Local Alignment Research Tool (BLAST) (Altschul et al., J. Mol. Biol. 215: 403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda , MD) and on the internet, for use in connection with the blastp, blastn, blastx, tblastn and tblastx sequence analysis programs. A description of how to determine the sequence identity using this program is available on the NCBI website on the internet.
[0176] [00176] Homologues and variants of a VL or VH of an antibody that specifically binds a polypeptide are typically characterized by possession of at least about 75%, for example, at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of sequence identity told about the full-size alignment with the amino acid sequence of interest. Proteins with even greater similarity to the reference sequences will show an increasing percentage of identities when evaluated by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared to the sequence identity, homologues and variants will typically have at least 80% sequence identity over short windows of 10 to 20 amino acids, and may have sequence identities of at least 85% or at least 90% or 95% depending on its similarity to the reference sequence. Methods for determining the sequence identity on such short windows are available on the NCBI website on the internet. Someone skilled in the art will appreciate that these sequence identity strips are provided for guidance only; it is entirely possible that strongly significant counterparts can be obtained, which fall outside the ranges provided.
[0177] [00177] Specifically bound: when referring to an antibody, it refers to a binding reaction that determines the presence of a target protein, peptide, or target polysaccharide in the presence of a heterogeneous population of proteins and other biologicals. Thus, under designated conditions, an antibody preferentially binds to a particular target protein, peptide or polysaccharide (such as a present antigen on the surface of a pathogen, for example, gp41) and does not bind in a significant amount to other proteins or polysaccharides present in the sample or individual. Specific binding can be determined by methods known in the art, such as by assessing the affinity of the antibody for an antigen. In one embodiment, affinity is calculated by modifying the Scatchard method described by Frankel et al., Mol. Immunol., 16: 101-106, 1979. In another embodiment, binding affinity is measured by an antigen dissociation rate /antibody. In yet another embodiment, a high binding affinity is measured by a competition radioimmunoassay. With reference to an antibody-antigen complex, specific binding of the antigen and antibody has a Kd less than about 10-6 Molar, such as less than about 10-6 Molar, 10-7 Molar, 10-8 Molar , 10-9, or even less than about 10-10 Molar.
[0178] [00178] Substantially purified: The term substantially purified indicates that the individual is substantially free of other molecular or cellular constituents with which he is naturally associated. Thus, a substantially purified population of cells (such as B cells, B cell progenitors, mature B cells, memory B cells, plasma cells, etc.) is substantially free of other cellular components of the tissue in which other cellular components of the tissue in which it is naturally found, such as bone marrow, peripheral blood, spleen, lymph node, etc. For example, a substantially pure population of B cells (for example, a B cell parent, an immature B cell, a mature B cell, a memory B cell, a plasma cell, etc.) is at least 50%, for example. example, at least about 80% or alternatively at least about 90% free of other cellular components. In one embodiment, the population of B cells is at least about 95% free of other cells. For example, a population of purified B cells, obtained from a tissue such as peripheral blood, is substantially free of red blood cells, T cells, platelets, and other cells typically found in peripheral blood.
[0179] [00179] T cell: A white globule critical for the immune response. T cells include, but are not limited to, CD4 + T cells and CD8 + T cells. A CD4 + T lymphocyte is an immune cell that carries a marker on its surface known as a "differentiation cluster 4" (CD4). These cells, also known as helper T cells, help to orchestrate the immune response, including antibody response as well as killer T cell responses. CD8 + T cells carry the "differentiation cluster 8" marker (CD8). In one embodiment, a CD8 T cell is a cytotoxic T lymphocyte. In another embodiment, a CD8 cell is a suppressor T cell.
[0180] [00180] Therapeutic Agent: used in a generic sense, it includes treatment agents, prophylactic agents, and substitution agents. A therapeutic agent is used to improve a specific group of conditions in an individual with a disease or disorder.
[0181] [00181] Therapeutically effective amount or effective amount: An amount of a specific substance, such as a described antibody, sufficient to obtain a desired effect on an individual being treated. For example, this may be the amount needed to inhibit HIV replication or to treat AIDS. In several embodiments, a therapeutically effective amount is the amount needed to reduce an AIDS sign or symptom, and / or to decrease an individual's viral titration. When administered to an individual, a dosage will generally be used that will obtain concentrations of target tissue that have been shown to obtain a desired in vitro effect.
[0182] [00182] Toxin: An effector molecule that induces cytoxicity when it contacts a cell. Non-limiting, specific examples of toxins include, but are not limited to, abrin, ricin, auristatins (such as monomethyl auristatin E (MMAE; see, for example, Francisco et al., Blood, 102: 1458-1465, 2003)) and monomethyl auristatin F (MMAF; see, for example, Doronina et al., BioConjugate Chem., 17: 114-124, 2006), maytansinoids (such as DM1; see, for example, Phillips et al., Cancer Res., 68 : 9280-9290, 2008), Pseudomonas exotoxin (PE, such as PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin, saporin, restrictocin or gelonin, or modified toxins do the same, or other toxic agents that directly or indirectly inhibit cell growth or kill cells. For example, PE and DT are highly toxic compounds that typically cause death through liver toxicity. PE and DT, however, can be modified into a form for use as an immunotoxin by removing the toxin's native targeting component (such as the PE domain Ia and DT B chain) and replacing it with a targeting portion. different, such as an antibody.
[0183] [00183] Under sufficient conditions for: the phrase that is used to describe any environment that allows a desired activity. In one example, the desired activity is the formation of an immune complex. In particular examples, the desired activity is the treatment of a tumor.
[0184] [00184] Vector: A nucleic acid molecule when introduced into a host cell, thereby producing a transformed host cell. A vector can include nucleic acid sequences that allow it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art.
[0185] [00185] Virus: Microscopic infectious organism that reproduces living cells inside. A virus essentially consists of a nucleus of a simple nucleic acid surrounded by a protein coating, and has the ability to replicate itself only within the living cell. "Viral replication" is the production of additional viruses by the occurrence of at least one viral life cycle. A virus can destroy the normal functions of the host cell, causing the cell to behave in a manner determined by the virus. For example, a viral infection can result in a cell producing a cytokine, or responding to a cytokine, when the uninfected cell normally does not.
[0186] [00186] "Retroviruses" are RNA viruses in which the viral genome is RNA. When a host cell is infected with a retrovirus, the genomic RNA is reverse transcribed into a DNA intermediate that is integrated very efficiently into the chromosomal DNA of the infected cells. The integrated DNA intermediary is referred to as a provirus. The term "lentivirus" is used in its conventional sense to describe a genus of viruses containing reverse transcriptase. Lentivirus includes the "immunodeficiency virus" which includes human immunodeficiency virus (HIV) type 1 and type 2 (HIV-I and HIV-II), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV) .
[0187] [00187] Methods and materials suitable for the practice or testing of this invention are described below. Such methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar or equivalent to those described here can be used. For example, conventional methods well known in the art to which this described invention belongs, are described in a number of general and more specific references, including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and supplements to 2012); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th edition, Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999. II. Description of Various Modalities A. Monoclonal Neutralizing Antibodies
[0188] [00188] Isolated human monoclonal antibodies that specifically bind gp41 are described here. The antibodies specifically described bind the extracellular region close to the membrane (MPER) of gp41. Also described herein are compositions including these human monoclonal antibodies and a pharmaceutically acceptable carrier. Nucleic acids encoding these antibodies, expression vectors including these nucleic acids, and isolated host cells that express nucleic acids are also provided.
[0189] [00189] Compositions including gp41 specific human monoclonal antibodies can be used for research, diagnostic and therapeutic purposes. For example, the human monoclonal antibodies described here can be used to detect HIV-1 in a biological sample or interfere with HIV-1 activity, for example, to diagnose or treat an individual having an HIV-1 infection and / or AIDS. For example, antibodies can be used to determine the titer of HIV-1 in an individual. The antibodies described here can also be used to study the biology of the human immunodeficiency virus.
[0190] [00190] The described antibodies that specifically bind gp41, bind the extracellular region close to the membrane (MPER) of gp41 in a previously uncharacterized epitope, which is designated here as the 10E8 epitope, for the first member of this class of antibodies discovered (similar antibodies to 10E8). The crystalline structure of the 10E8 antibody was resolved in complex with a gp41 peptide (see example 1), which allowed for detailed analysis of the binding of this class of the 10E8 and gp41 antibodies, and describes at the atomic level the binding of 10E8-like antibodies (such as such as 10E8) to the 10E8 epitope. This epitope, and thus antibodies in this class (10E8-like antibodies), can be distinguished from other antibodies that bind gp41 by virtue of their binding to the 10E8 epitope. In several modalities, the 10E8 epitope, for example, KWASLWNWFDITNWLWYIR (SEQ ID NO: 13), extends the C terminal to the 2F5 epitope (although there is some overlap) in the gp41 ectodomain and is distinguished from the 4E10 and Z13E1 epitope expanding to connection to C-terminal residues, previously believed to be inaccessible (for example, these residues were believed to be blunted in the lipid bilayer). The person skilled in the art will understand that 10E8 antibodies can specifically bind to gp41 MPER residues by extending the N-terminus to the above sequence. In some embodiments, the 10E8-like antibodies described specifically bind to a polypeptide including an amino acid sequence established as residues 1-28, 2-28, 3-28, 4-28, 5-28, 6-28, 7- 28, 8-28, 9-28, 10-28, 11-28, 12-28, 13-28 or 14-28 of SEQ ID NO: 26, which correspond to gp41 residues 656-683, 657-683, 658-683, 659-683, 660-683, 661-683, 662-683, 663-683, 664-683, 665-683, 666-683, 667-683, 668-683, or 669-683, respectively (HXB2 numbering system).
[0191] [00191] In some embodiments, residues believed to make contact with the 10E8 antibody include the K, SLWNWF, TN, LW, and IR shown in bold above. Thus, in some embodiments, an antibody similar to 10E8 specifically binds to one or more of the K, SLWNWF, TN, LW, and IR of SEQ ID NO: 13, such as at least 1, at least 2, at least 3 at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or all 13 of these residues. In some examples, an antibody similar to 10E8 binds to the NWF, T and R residues shown in bold in the following NWFDITNWLWYIR sequence (residues 7 to 19 of SEQ ID NO: 13).
[0192] [00192] In additional embodiments, the antibody or antigen binding fragment contacts L, WF, LW and R shown in bold in the amino acid sequence established as LWNWFDITNWLWYIR (SEQ ID NO: 26, residues 14 to 28). In additional embodiments, the described antibody contacts LW, WF, LW and R shown in bold in the amino acid sequence established as LWNWFDITNWLWYIR (SEQ ID NO: 26, residues 14 to 28). In additional embodiments, the described antibody contacts SLW, WF, LW and R shown in bold in the amino acid sequence established as SLWNWFDITNWLWYIR (SEQ ID NO: 26, residues 13 to 28). In additional embodiments, the antibody described contacts L, DK, SLWNWF, TN, LW and IR shown in bold in the amino acid sequence established as LELDKWASLWNWFDITNWLWYIR (SEQ ID NO: 26, residues 6-28). In additional embodiments, the described antibody contacts NWF, T, and R shown in bold in the amino acid sequence established as NWFDITNWLWYIR (SEQ ID NO: 13, residues 7 to 19). In additional embodiments, the described antibody contacts K, SLNWF, T, and IR shown in bold in the amino acid sequence established as KWASLWNWFDITNWLWYIR (SEQ ID NO: 13). In additional embodiments, the antibody described in the antibody specifically binds to the NWF, T, and R residues shown in bold in the amino acid sequence established as NWFDITNWLWYIR (SEQ ID NO: 13, residues 7 to 19). In additional embodiments, the antibody described specifically binds to the K, SLNWF, T, and IR residues shown in bold in the amino acid sequence established as KWASLWNWFDITNWLWYIR (SEQ ID NO: 13). In several such modalities, the antibody directly contacts the MPER of gp41 in the indicated residues when specifically bound to gp41, for example, through hydrogen bonding and / or Van der Waals contacts. In additional embodiments, the antibody contacts the gp41 MPER in the indicated residues when specifically bound to gp41, for example, through hydrogen bonding contacts, Van der Waals contacts, and / or interactions that cause reduced solvent access between the antibody and gp41 (that is, blunted surface area). As shown in figures 27 and 28, residues in 10E8 and 10E8-like antibodies that are important for binding to the 10E8 epitope include Kabat residues 28, 31, 33, 50, 52, 52B, 52C, 53, 56, 58, and 97-100J of the heavy chain and Kabat residues 91 and 95B of the light chain. These residues correspond to residues 28, 31, 33, 50, 52, 54, 55, 56, 59, 61, 103-116 of the heavy chain (residue numbers are provided with reference to SEQ ID NO: 1), and 91 and 97 of the light chain (residue numbers are given with respect to SEQ ID NO: 2). In some embodiments, an antibody similar to 10E8 specifically binds to gp41, and one or more of residues 28, 31, 33, 50, 52, 54, 55, 56, 59, 61, and / or 103-116 of the heavy chain (with respect to SEQ ID NO: 1), such as at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 17, at least 17, at least 19, at least 20, at least 21, at least 22 , at least 23, at least 24, at least 25, at least 26, or all 27 of these residues contact gp41. In some embodiments, an antibody similar to 10E8 specifically binds to gp41 and at least one of the light chain residues 91 and 97 (with respect to SEQ ID NO: 2) contacts gp41.
[0193] [00193] In some embodiments, the class of antibodies similar to 10E8 does not exhibit autoreactivity, that is, they do not bind autoantigens, such as human protein. Without being bound by theory, examination of the crystalline structure of 10E8 in complex with a gp41 peptide from MPER shows that 10E8 binds to MPER in a way that may not require any hydrophobic interaction with the membrane. Other known neutralizing antibodies that bind to the gp41 MPER, such as 2F5 and 4E10, include hydrophobic residues in the CDR H3 that do not contact the epitope and are believed to make specific contact with the lipid membrane on which gp41 is located.
[0194] [00194] While not being linked to theory, it is believed that the extent of neutralization of antibodies similar to 10E8 can tolerate conservative changes to the epitope while still maintaining the link. For example, while the C-terminal residue is shown as an arginine, antibodies of this class can tolerate a substitution of lysine at this site, and still maintain high binding affinity. In addition, someone skilled in the art can formulate a consensus sequence for the 10E8 epitope using the sequences of all variations of HIV gp41 for the HIV isolates listed in figure 17B or in figures 17C-17F. In some embodiments, antibodies in this class (10E8-like antibodies) can also be distinguished by amplitude of neutralization. In some embodiments, an antibody similar to 10E8 can neutralize at least 95% (such as at least 96%, at least 97%, at least 98% or at least 99%) of the HIV-1 isolates listed in figure 17B or in figures 17C-17F with an IC50 less than 50 µg / ml. In some embodiments, an antibody similar to 10E8 can neutralize at least 65% (such as at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, or at least 80%) of the HIV-1 isolates listed in figure 17B or in figures 17C-17F with an IC50 less than 1 µg / ml . In specific embodiments, an antibody similar to 10E8 is not the anti-prop Z13E1, 4E10 or 2F5.
[0195] [00195] The description of monoclonal antibodies below refers to isolated monoclonal antibodies that include heavy and light chain variable domains including a CDR1, CDR2 and CDR3. The person skilled in the art will understand that various CDR numbering schemes (such as Kabat, Chothia or IMGT numbering schemes) can be used to determine CDR positions. The heavy chain CDR positions of monoclonal antibody 10E8 according to the Kabat and IMGT numbering scheme are shown in figure 6A (Kabat) and figure 6B (IMGT). In several modalities, reference to the particular amino acid substitutions in the heavy and light chains of the described antibodies is made according to the Kabat or IMGT numbering schemes. For example, the substitution of amino acid S74W in 10E8 referenced here refers to the Kabat numbering scheme. Using the IMGT numbering scheme, this substitution can be referred to as S82W. In both cases, this substitution refers to the replacement of the serine residue at position 77 of SEQ ID NO: 1. The person skilled in the art will easily understand the use of various CDR numbering schemes when referencing the amino acids of the particular antibodies described here.
[0196] [00196] In some embodiments, an isolated antibody that specifically binds gp41, includes a heavy chain with one or more of amino acids 26 to 33 (complementarity determining region 1 (CDR1)), amino acids 51 to 60 (CDR2), and / or 99-120 (CDR3) of SEQ ID NO: 11: EVX1LX2ESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKG LEWVGRITGPGEGWSVDYAAPVEGRFTISRLNX3INFLYLEMNNLRM EDSGLYFCARTGKYYDFWSGYPPGEEYFQDWGRGTLVX4VSS (SEQ ID NO: 11), wherein X1 is Q or R, X2 is V or a, X3 is S or Y, X4 is T or I. In some embodiments, an isolated human monoclonal antibody that specifically binds gp41, includes a heavy chain with amino acids 26 to 33 (CDR1), 51 to 60 (CDR2), and 99 to 120 (CDR3) of SEQ ID NO: 11. In examples specific, the human monoclonal antibody heavy chain includes SEQ ID NO: 11.
[0197] [00197] In some embodiments, an isolated antibody that specifically binds gp41, includes a heavy chain with one or more of amino acids 26 to 33 (complementarity determining region 1 (CDR1)), amino acids 51-60 (CDR2), and / or 99 to 120 (CDR3) of SEQ ID NO: 146: EVX1LX2ESGGGLVKPGGSLRLSCSASGFX3FX4X5AWMTWVRQPPGKGLEWVGRITGPGEX6WSVDYAAPVEGRFTISRLNSINFLYLEMNNLRMEDSGLYFCARTGKYYDFWSGYPPGEEYFQDWGRGTLVX7VSS (SEQ ID NO: 146), wherein X1 is Q or R, X2 is V or A, X3 is D or W, X4 is D or W, X5 is N or W, X6 is G or W and X7 is T or I). In some embodiments, an isolated human monoclonal antibody that specifically binds gp41, includes a heavy chain with amino acids 26 to 33 (CDR1), 51 to 60 (CDR2), and 99 to 120 (CDR3) of SEQ ID NO: 146. In examples specific, the human monoclonal antibody heavy chain includes SEQ ID NO: 146.
[0198] [00198] In some embodiments, an isolated antibody that specifically binds gp41, includes a heavy chain with one or more of amino acids 26 to 33 (complementarity determining region 1 (CDR1)), amino acids 51 to 60 (CDR2), and / or 99 to 120 (CDR3) of SEQ ID NO: 187. SEQ ID NO: 187 is established as EX1X2LX3ESGGX4LVX5PGGSLRLSCX6ASGFX7FX8X9X10WMTWVRQX 11PGKGLEWVGRIX12GX13GX14X15WX16X17X18YAX19X20VX21GRFX22ISRX23X24X25X26X27X28X29YLX30MNX31X32X33X34X35DX36X37X38YX39CX40X41TX42KX43YX44FWX45GX46PPGEEYX47X48X49WGX50GTX51VX52VX53S, wherein X1 is V or I; X2 is Q or R; X3 is V or A; X4 is G, R, or D; X5 is K or R; X6 is S or A; X7 is D, N, S, A, or W; X8 is D, K, W or A; X9 is N, S, D, A, W, F, or Y; X10 is A, T, or Q; X11 is P, or A; X12 is T, S, or A; X13 is P or W; X14 is E, A, F, L, M, V, or W; X15 is G or W; X16 is S, T, A, or H; X17 is V or S; X18 is D, G, or A; X19 is A or E; X20 is P, S, or T; X21 is E, K or Q; X22 is T or I; X23 is L, D, M, I, or N; X24 is N or D; X25 is S, M, W, F, L, or M; X26 is I or K; X27 is N or D; X28 is F, T. or M; X29 is L or F; X30 is E or Q; X31 is N, S, or R; X32 is L or V; X33 is R, or K; X34 is M, T, I, or P; X35 is E or D; X36 is S, T or W; X37 is G, A, or W; X38 is L, V, S or Y; X39 is F, or Y; X40 is A, T or V; X41 is R, T, or H; X42 is G or E; X43 is Y or H; X44 is D, A, or N; X45 is S, G, or R; X46 is Y or A; X47 is F or L; X48 is Q or E; X49 is D or H; X50 is R or Q; X51 is L or Q; X52 is T or I; and X53 is S or P.) In some embodiments, an isolated human monoclonal antibody that specifically binds gp41, includes a heavy chain with amino acids 26 to 33 (CDR1), 51 to 60 (CDR2), and 99 to 120 (CDR3) of SEQ ID NO: 187. In additional embodiments, an isolated antibody that specifically binds gp41, includes a heavy chain variable region including the amino acid sequence established as SEQ ID NO: 187.
[0199] [00199] In several embodiments, the isolated antibody that specifically binds gp41, is neutralizing, and includes a heavy chain including one or more of the heavy chain complementarity determining regions (CDRs) of one of the heavy chain variable region sequences established as SEQ ID NO: 1, 3, 5, 11, 146, 147-149, 187, 189-192, and 200-204, according to the numbering systems of Kabat, IMGT, or Clothia. In some embodiments, an isolated antibody that specifically binds gp41 and is neutralizing, includes a heavy chain including CDR1, CDR2, and CDR3 from one of the variable regions of heavy chain sequences established as SEQ ID NOs: 1, 3, 5, 11 , 146, 147-149, 187, 189-192, or 200-204, according to the Kabat, IMGT, or Clothia numbering systems.
[0200] Thus, in some embodiments, an isolated antibody that specifically binds gp41, includes one or more of amino acids 26 to 33 (CDR1), amino acids 51 to 60 (CDR2), and / or 99 to 120 (CDR3) of a from SEQ ID NOs: 1, 3, 5, 11, 146, 147-149, 187, 189-192, and 200-204. In some embodiments, an isolated human monoclonal antibody that specifically binds gp41, includes a heavy chain with amino acids 26 to 33 (CDR1), 51 to 60 (CDR2), and 99 to 120 (CDR3) from one of SEQ ID NOs: 1, 3, 5, 11, 146, 147-149, 187, 189-192, and 200-204. In specific examples, the human monoclonal antibody heavy chain includes one of SEQ ID NOs: 1, 3, 5, 11, 146, 147-149, 187, 189-192, and 200-204.
[0201] [00201] For example, in some embodiments, an isolated antibody that specifically binds gp41, includes a heavy chain including one or more of the complementarity determining regions (CDRs) of gp41, 7H6 and / or 7N16 antibody 10E8 heavy chain. The 10E8 antibody heavy chain of gp41 is established as SEQ ID NO: 1. In some embodiments, an isolated antibody that specifically binds gp41, includes one or more of amino acids 26-33 (27-38 in figure 6B) (CDR1), amino acids 51-60 (56-65 in figure 6B) (CDR2), and / or 99-120 (105-126 in figure 6B) (CDR3) of SEQ ID NO: 1. In some embodiments, an isolated human monoclonal antibody that specifically binds gp41, includes a heavy chain with amino acids 26 to 33 (CDR1), 51 to 60 (CDR2), and 99 to 120 (CDR3) of SEQ ID NO: 1. In specific examples, the heavy chain of the human monoclonal antibody includes SEQ ID NO: 1. The 7H6 heavy chain of gp41 antibody is established as SEQ ID NO: 3. Thus, in some embodiments, an isolated antibody that specifically binds gp41, includes one or more of amino acids 26 to 33 (CDR1) , 51 to 60 (CDR2), and / or 99 to 120 (CDR3) of SEQ ID NO: 3. In some embodiments, an isolated human monoclonal antibody that specifically binds gp41, includes a chain heavy with amino acids 26 to 33 (CDR1), 51 to 60 (CDR2), and 99 to 120 (CDR3) of SEQ ID NO: 3. In specific examples, the human monoclonal antibody heavy chain includes SEQ ID NO: 3. A heavy chain of antibody 7N16 of gp41 is established as SEQ ID NO: 5. Thus, in some embodiments, an isolated antibody that specifically binds gp41, includes one or more of amino acids 26 to 33 (CDR1), 51 to 60 (CDR2) , and / or 99 to 120 (CDR3) of SEQ ID NO: 5. In some embodiments, an isolated human monoclonal antibody that specifically binds gp41, includes a heavy chain with amino acids 26 to 33 (CDR1), 51 to 60 (CDR2) , and 99 to 120 (CDR3) of SEQ ID NO: 5. In specific examples, the human monoclonal antibody heavy chain includes SEQ ID NO: 5.
[0202] [00202] In additional embodiments, an isolated antibody that specifically binds gp41 includes a heavy chain including the amino acid sequence of any of the 10E8-like heavy chains described here and also including an amino acid substitution at position 77 (position 74 of according to a Kabat numbering), such as a replacement for S77Y (S74Y according to a Kabat numbering). In additional embodiments, an isolated antibody that specifically binds gp41, includes a heavy chain including one or more of the heavy chain complementarity determining regions (CDRs) of any of the 10E8-like heavy chains described herein and also including an amino acid substitution in position 77 (position 74 according to a Kabat numbering), as well as a replacement for S77Y (S74Y according to a Kabat numbering). In additional embodiments, an isolated antibody that specifically binds gp41 includes a heavy chain including one or more of the heavy chain complementarity determining regions (CDRs) of one of the gVRC-H2dN152 or gVRC-H2dN152 antibodies to gp41 with an amino acid substitution in position 77 (position 74 according to Kabat numbering). In some instances, the amino acid substitution is a substitution of serine for tyrosine. The heavy chain of gpCR-H2dN152 antibody of gp41 is established as SEQ ID NO: 154. Thus, in some embodiments, an isolated antibody that specifically binds gp41, includes one or more of amino acids 26 to 33 (CDR1), 51 to 60 (CDR2), and / or 99 to 120 (CDR3) of SEQ ID NO: 154. In some embodiments, an isolated human monoclonal antibody that specifically binds gp41, includes a heavy chain with amino acids 26 to 33 (CDR1), 51 to 60 (CDR2), and 99 to 120 (CDR3) of SEQ ID NO: 154. In specific examples, the human monoclonal antibody heavy chain includes SEQ ID NO: 154. The gp41 antibody heavy chain of gp41 with serine substitution tyrosine at position 77 (position 74 using Kabat numbering) is established as SEQ ID NO: 192. Thus, in some embodiments, an isolated antibody that specifically binds gp41, includes one or more of amino acids 26 to 33 (CDR1), 51 to 60 (CDR2), and / or 99 to 120 (CDR3) of SEQ ID NO: 192. In some embodiments, a human monoclonal antibody in the isolate that specifically binds gp41, it includes a heavy chain with amino acids 26 to 33 (CDR1), 51 to 60 (CDR2), and 99 to 120 (CDR3) of SEQ ID NO: 192. In specific examples, the antibody heavy chain human monoclonal includes SEQ ID NO: 192.
[0203] [00203] In some embodiments, an isolated antibody that specifically binds gp41, includes one or more of the complementary determining regions (CDRs) of light chain amino acids 26 to 31 (CDR1), 49 to 51 (CDR2), and / or 87 to 98 (CDR3) of SEQ ID NO: 12: SYELTQX1TGVSVALGRTVVTITCRGDSLRSHX2ASWYQKKPGQAPX3LLFYGKNNRPSGX4PDRFSGSASGNRASLTIX5GAQAEDX6AX7YYCSSRDKSGSRLSVFGGGTKLX8VL (SEQ ID NO: 12), wherein X1 is E or D, X2 is Y or H, X3 is V or I, X4 is T or I, X5 is S or T, X6 is D or E, X7 is E or D, and X8 is T or I. In some embodiments, an isolated human monoclonal antibody that specifically binds gp41, includes a light chain with amino acids 26 to 31 (CDR1), 49 to 51 (CDR2), and 87 to 98 (CDR3) of SEQ ID NO: 12. In specific examples, the human monoclonal antibody light chain includes SEQ ID NO: 12.
[0204] [00204] In some embodiments, an isolated antibody that specifically binds gp41, includes one or more of the complementary determining regions (CDRs) of light chain amino acids 26 to 31 (CDR1), 49 to 51 (CDR2), and / or 87 to 98 (CDR3) of SEQ ID NO: 188: X1X2X3LTQX4X5X6VSVAX7X8X9TVX10ITCX11GDSLRX12X13YX14X15WYQX16X17X18X19QAPX20LX21X22YX23X24X25X26RPSX27X28X29DRFSX30X31X32SGNX33ASLTIX34GAX35X36X37DX38AX39YYCSSRDKSGSRLX40X41FGX42GTX43X44X45X46X4 7, wherein X1 is S or A; X2 is Y or S; X3 is E or D; X4 is E or D; X5 is T or P; X6 is G, A, or T; X7 is L or F; X8 is G, K, or E; X9 is R, Q, or K; X10 is T or R; X11 is R or Q; X12 is S, R, or N; X13 is H or Y; X14 is A, V, or T; X15 is S or G; X16 is K, E, or Q; X17 is K or R; X18 is P or T; X19 is G or R; X20 is I, V, or K; X21 is L or V; X22 is F, V, or I; X23 is G or P; X24 is K or R; X25 is N, D, or H; X26 is N or I; X27 is G, or P; X28 is V or I; X29 is P, H, or S; X30 is G or A; X31 is S or F; X32 is A, T, or S; X33 is R or T; X34 is S, A, or T; X35 is Q or E; X36 is A or G; X37 is E or D; X38 is D, E, or I; X39 is E or D; X40 is S, V; X41 is V, T; X42 is G, R; X43 is K or E; X44 is L, V, or R; X45 is T, S, or A; X46 is V, T, or G; and X47 is L, V, or P. In some embodiments, an isolated human monoclonal antibody that specifically binds gp41, includes a light chain with amino acids 26 to 31 (CDR1), 49 to 51 (CDR2), and 87 to 98 (CDR3 ) of SEQ ID NO: 188. In additional embodiments, an isolated antibody that specifically binds gp41, includes a light chain variable region including the amino acid sequence established as SEQ ID NO: 188.
[0205] [00205] Several modalities include an isolated antibody that specifically binds gp41, is neutralizing, and includes a light chain including one or more of the light chain complementarity determining regions (CDRs) of one of the variable region light chain sequences established as SEQ ID Nºs: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199, according to the numbering systems Kabat, IMGT, or Clothia. In some embodiments, an isolated antibody that specifically binds gp41 and is neutralizing includes a light chain including CDR1, CDR2, and CDR3 from one of the variable region light chain sequences established as SEQ ID NOs: 2, 4, 6, 12 , 150-152, 164-186, 188, or 197-199, according to the Kabat, IMGT, or Clothia numbering systems. In additional embodiments, an isolated antibody that specifically binds gp41, includes one or more of amino acids 26-31 (27-38 in figure 6B) (CDR1), 49- 51 (56-65 in figure 6B) (CDR2), and / or 87-98 (105-116 in figure 6B) (CDR3) from one of SEQ ID NOs: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199. In other embodiments, an isolated human monoclonal antibody that specifically binds gp41, includes a light chain with amino acids 26 to 31 (CDR1), 49 to 51 (CDR2), and 87 to 98 (CDR3) from one of SEQ ID NOs: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199. In specific examples, the human monoclonal antibody light chain includes one of SEQ ID NOs: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199.
[0206] [00206] For example, in some embodiments, an isolated antibody that specifically binds gp41, includes one or more of the complementarity determining regions (CDRs) of 10E8 gp41, 7H6 and / or 7N16 antibody. The 10E8 antibody light chain of gp41 is established as SEQ ID NO: 2. Thus, in some embodiments, an isolated antibody that specifically binds gp41, includes one or more of amino acids 26 to 31 (27-38 in figure 6B) ( CDR1), 49 to 51 (56 to 65 in figure 6B) (CDR2), and / or 87 to 98 (105 to 116 in figure 6B) (CDR3) of SEQ ID NO: 2. In some embodiments, a human monoclonal antibody isolate that specifically binds gp41, includes a light chain with amino acids 26 to 31 (CDR1), 49 to 51 (CDR2), and 87 to 98 (CDR3) of SEQ ID NO: 2. In specific examples, the light chain of the monoclonal antibody human includes SEQ ID NO: 2. The 7H6 antibody light chain of gp41 is established as SEQ ID NO: 4. Thus, in some embodiments, an isolated antibody that specifically binds gp41, includes one or more of amino acids 26 to 31 ( CDR1), 49 to 51 (CDR2), and / or 87 to 98 (CDR3) of SEQ ID NO: 4. In some embodiments, an isolated human monoclonal antibody that specifically binds gp41, includes a chain light with amino acids 26 to 31 (CDR1), 49 to 51 (CDR2), and 87 to 98 (CDR3) of SEQ ID NO: 4. In specific examples, the human monoclonal antibody light chain includes SEQ ID NO: 4. A 7N16 antibody light chain of gp41 is established as SEQ ID NO: 6. Thus, in some embodiments, an isolated antibody that specifically binds gp41, includes one or more of amino acids 26-31 (CDR1), 49-51 (CDR2) , and / or 87 to 98 (CDR3) of SEQ ID NO: 6. In some embodiments, an isolated human monoclonal antibody that specifically binds gp41, includes a heavy chain with amino acids 26-31 (CDR1), 49-51 (CDR2) , and 87 to 98 (CDR3) of SEQ ID NO: 6. In specific examples, the human monoclonal antibody heavy chain includes SEQ ID NO: 6.
[0207] [00207] In additional embodiments, an isolated antibody that specifically binds gp41, includes a heavy chain including one or more of the complementarity determining regions (CDRs) of gp41gL03 antibody 10E8 heavy chain. The 10E8 antibody light chain of gp41gH03 is established as SEQ ID NO: 152. Thus, in some embodiments, an isolated antibody that specifically binds gp41, includes one or more of amino acids 26 to 31 (CDR1), 49-51 (CDR2 ), and / or 87 to 98 (CDR3) of SEQ ID NO: 152. In some embodiments, an isolated human monoclonal antibody that specifically binds gp41, includes a heavy chain with amino acids 26-31 (CDR1), 49-51 (CDR2 ), and 87 to 98 (CDR3) of SEQ ID NO: 152. In specific examples, the human monoclonal antibody heavy chain includes SEQ ID NO: 152.
[0208] [00208] Additional modalities include an isolated antibody that specifically binds gp41 and is neutralizing, and includes a heavy chain including one or more of the heavy chain complementarity determining regions (CDRs) from one of the variable regions of heavy chain sequences established as SEQ ID Nºs: 1, 3, 5, 11, 146, 147-149, 187, 189-192, and 200-204, according to the numbering systems Kabat, IMGT, or Clothia, and one or more of the determining regions of light chain complementarity (CDRs) of one of the variable region light chain sequences established as SEQ ID NO: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199, according to numbering systems Kabat, IMGT, or Clothia, respectively. Additional modalities include an isolated antibody that specifically binds gp41 and is neutralizing, and includes a heavy chain including heavy chain complementarity determining region 1 (HCRD1), HCRD2, and HCDR3 from one of the variable regions of heavy chain sequences established as SEQ ID Nºs: 1, 3, 5, 11, 146, 147-149, 187, 189-192, and 200-204, according to the numbering systems Kabat, IMGT, or Clothia, and the region 1 determinant of complementarity of light chain (HCRD1), HCRD2, and HCDR3 of one of the variable region light chain sequences established as SEQ ID NO: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199, of according to the Kabat, IMGT, or Clothia numbering systems, respectively.
[0209] Thus, in some embodiments, an isolated antibody that specifically binds gp41, includes a heavy chain including amino acids 26 to 33 (27 to 38 in figure 6B) (CDR1), amino acids 51 to 60 (56 to 65 in figure 6B ) (CDR2), and / or 99 to 120 (105 to 126 in figure 6B) (CDR3) of one of SEQ ID NOs: 1, 3, 5, 11, 146, 147-149, 187, 189-192, and 200-204, and a light chain including amino acids 26 to 31 (27 to 38 in figure 6B) (CDR1), 49 to 51 (56 to 65 in figure 6B) (CDR2), and / or 87-98 (105-116 in figure 6B) (CDR3) of one of SEQ ID NOs: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199. In additional embodiments, an isolated antibody that specifically binds gp41, includes a heavy chain including amino acids 26 to 33 (27 to 38 in figure 6B) (CDR1), amino acids 51 to 60 (56 to 65 in figure 6B) (CDR2), and 99 to 120 (105 to 126 in figure 6B) (CDR3) of one of SEQ ID NOs: 1, 3, 5, 11, 146, 147-149, 187, 189-192, and 200-204, and a light chain including amino acids 26-31 (27-38 in figure 6B) (CDR1), 49-51 (56-65 in figure 6B) (CDR2), and 87-98 (105-116 in figure 6B) (CDR3) from one of SEQ ID NO: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199.
[0210] [00210] In some embodiments, an isolated antibody that specifically binds gp41, includes a heavy chain including amino acids 26 to 33 (27 to 38 in figure 6B) (CDR1), amino acids 51- 60 (56 to 65 in figure 6B) (CDR2 ), and / or 99 to 120 (105 to 126 in figure 6B) (CDR3) of SEQ ID NO: 1, and a light chain including amino acids 26 to 31 (27 to 38 in figure 6B) (CDR1), 49 to 51 (56 to 65 in figure 6B) (CDR2), and / or 87 to 98 (105 to 116 in figure 6B) (CDR3) of SEQ ID NO: 2. In some embodiments, an isolated antibody that specifically binds gp41, includes a heavy chain including amino acids 26 to 33 (27 to 38 in figure 6B) (CDR1), amino acids 51 to 60 (56 to 65 in figure 6B) (CDR2), and / or 99 to 120 (105 to 126 in figure 6B) ( CDR3) of SEQ ID NO: 154, and a light chain including amino acids 26 to 31 (27 to 38 in figure 6B) (CDR1), 49 to 51 (56 to 65 in figure 6B) (CDR2), and / or 87 to 98 (105 to 116 in figure 6B) (CDR3) of SEQ ID NO: 152. In some embodiments, an isolated antibody that specifically binds gp41, i includes a heavy chain including amino acids 26 to 33 (27 to 38 in figure 6B) (CDR1), amino acids 51 to 60 (56 to 65 in figure 6B) (CDR2), and / or 99 to 120 (105 to 126 in figure 6B ) (CDR3) of SEQ ID NO: 192, and a light chain including amino acids 26 to 31 (27 to 38 in figure 6B) (CDR1), 49 to 51 (56 to 65 in figure 6B) (CDR2), and / or 87 to 98 (105 to 116 in figure 6B) (CDR3) of SEQ ID NO: 152.
[0211] [00211] In additional examples, an isolated antibody that specifically binds gp41 and is neutralizing includes a heavy chain variable region and a light chain variable region, where the heavy chain variable region includes the amino acid sequence established as one of the SEQ ID NO: 1, 3, 5, 11, 146, 147-149, 187, 189-192, or 200-204, and the light chain variable region includes the amino acid sequence established as one of SEQ ID NO: 2 , 4, 6, 12, 150-152, 164-186, 188, or 197-199. In one example, the heavy chain variable region includes the amino acid sequence set to SEQ ID NO: 1, and the light chain variable region includes the amino acid sequence set to SEQ ID NO: 2. In another example, the variable region heavy chain includes the amino acid sequence established as SEQ ID NO: 192, and the light chain variable region includes the amino acid sequence established as SEQ ID NO: 152. In another example, the heavy chain variable region includes the sequence amino acid established as SEQ ID NO: 154, and the light chain variable region includes the amino acid sequence established as SEQ ID NO: 152.
[0212] [00212] In other embodiments, an isolated antibody that specifically binds gp41, includes a heavy chain variable region including an amino acid sequence including no more than 10 (such as more than 1, 2, 3, 4, 5, 6 , 7, 8, or no more than 9) amino acid substitutions compared to one of SEQ ID NOs: 1, 3, 5, 11, 146, 147-149, 187, 189-192, and 200-204. In additional embodiments, an isolated antibody that specifically binds gp41, includes a light chain variable region including an amino acid sequence including no more than 10 (such as more than 1, 2, 3, 4, 5, 6, 7, 8, or no more than 9) amino acid substitutions compared to one of SEQ ID NOs: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199. In other embodiments, an isolated antibody that specifically binds gp41, includes a heavy chain variable region including an amino acid sequence including no more than 10 (such as more than 1, 2, 3, 4, 5, 6, 7, 8, or no more than 9) amino acid substitutions compared to one of SEQ ID Nos: 1, 3, 5, 11, 146, 147-149, 187, 189-192, and 200-204, and a variable region light chain including an amino acid sequence including no more than 10 (such as more than 1, 2, 3, 4, 5, 6, 7, 8, or no more than 9) amino acid substitutions compared to SEQ ID NOs: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199.
[0213] [00213] In other embodiments, an isolated antibody that specifically binds gp41, includes a heavy chain variable region including an amino acid sequence including no more than 10 (such as more than 1, 2, 3, 4, 5, 6 , 7, 8, or no more than 9) amino acid substitutions compared to one of SEQ ID NO: 1, and a light chain variable region including an amino acid sequence including no more than 10 (such as more than 1, 2, 3, 4, 5, 6, 7, 8, or no more than 9) amino acid substitutions compared to SEQ ID NO: 2.
[0214] [00214] In other embodiments, an isolated antibody that specifically binds gp41 includes a heavy chain variable region including the amino acid sequence established as SEQ ID NO: 187, wherein the amino acid sequence includes no more than 25 (as more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or no more than 24) amino acid substitutions compared to SEQ ID NO: 1. In additional embodiments, an isolated antibody that specifically binds gp41, includes a light chain variable region including the amino acid sequence established as SEQ ID NO: 188, wherein the amino acid sequence includes no more than 33 (such as more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 35, 36, 37, 38, 39, 30, 31, 32 or no more than 33) amino acid substitutions compared to SEQ ID NO: 2. In other embodiments, an isolated antibody that specifically binds gp41, includes u a heavy chain including the amino acid sequence established as SEQ ID NO: 187, wherein the amino acid sequence includes no more than 25 (such as more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or no more than 24) amino acid substitutions compared to SEQ ID NO: 1, and a light chain including the amino acid sequence established as SEQ ID NO: 188, wherein the amino acid sequence includes no more than 33 (such as more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 35, 36, 37, 38, 39, 30, 31, 32 or no more than 33) amino acid substitutions compared to SEQ ID NO: 2.
[0215] [00215] In other embodiments, an isolated antibody that specifically binds gp41, includes a heavy chain variable region including an amino acid sequence having no more than 10 (such as more than 1, 2, 3, 4, 5, 6 , 7, 8, or no more than 9) amino acid substitutions compared to one of SEQ ID Nos: 1, 3, 5, 11, 146, 147-149, 187, 189-192, and 200-204, and wherein substitutions are selected from the substitution of amino acids listed in figures 60A and 60B. In additional embodiments, an isolated antibody that specifically binds gp41, includes a light chain variable region including an amino acid sequence including no more than 10 (such as more than 1, 2, 3, 4, 5, 6, 7, 8, or no more than 9) amino acid substitutions compared to one of the amino acid sequence established as one of SEQ ID NOs: 2, 4, 6, 12, 150-152, 164-186, 188, or 197- 199, where substitutions are selected from the amino acid substitutions shown in figures 61A and 61B. In other embodiments, an isolated antibody that specifically binds gp41, includes a heavy chain variable region including an amino acid sequence having no more than 10 (such as more than 1, 2, 3, 4, 5, 6, 7, 8, or no more than 9) amino acid substitutions compared to one of SEQ ID Nos: 1, 3, 5, 11, 146, 147-149, 187, 189-192, and 200-204, where the substitutions are selected from the amino acid substitutions shown in figures 60A and 60B, and a light chain variable region including an amino acid sequence including no more than 10 (such as more than 1, 2, 3, 4, 5, 6, 7 , 8, or no more than 9) amino acid substitutions compared to one of the amino acid sequence established as one of SEQ ID NOs: 2, 4, 6, 12, 150-152, 164-186, 188, or 197 -199, and where substitutions are selected from the substitution of amino acids listed in figures 61A and 61B. In other embodiments, an isolated antibody that specifically binds gp41, includes a heavy chain variable region including an amino acid sequence having no more than 10 (such as more than 1, 2, 3, 4, 5, 6, 7, 8, or no more than 9) amino acid substitutions compared to SEQ ID NO: 1, where the substitutions are selected from the amino acid substitution listed in figures 60A and 60B, and a light chain variable region including an amino acid sequence having no more than 10 (such as more than 1, 2, 3, 4, 5, 6, 7, 8, or no more than 9) amino acid substitutions compared to SEQ ID NO: 2, and in which substitutions are selected from the amino acid substitution listed in figures 61A and 61B.
[0216] [00216] In other embodiments, an isolated antibody that specifically binds gp41, includes a heavy chain with a maximum of one, a maximum of two, a maximum of three or a maximum of four amino acid substitutions at amino acids 26 to 31 (CDR1), 49 to 51 (CDR2), and 87 to 98 (CDR3) of SEQ ID NO: 11, and a light chain. In some embodiments, an isolated antibody that specifically binds gp41 includes a heavy chain with a maximum of one, a maximum of two, a maximum of three, a maximum of five or a maximum of five amino acid substitutions at amino acids 26 to 33 (CDR1), 51 to 60 (CDR2), and 99 to 120 (CDR3) of SEQ ID NO: 1.
[0217] [00217] In some embodiments, the antibody may include a heavy chain with a maximum of one, a maximum of two, a maximum of three, a maximum of five amino acid substitutions at amino acids 26 to 33 (CDR1), 51 to 60 ( CDR2), and 99 to 120 (CDR3) of SEQ ID NO: 3. In some embodiments, the antibody may include a heavy chain with a maximum of one, a maximum of two, a maximum of three, a maximum of four or a maximum of five substitutions of amino acid at amino acids 26 to 33 (CDR1), 51 to 60 (CDR2), and 99 to 120 (CDR3) of SEQ ID NO: 5. In some embodiments, the antibody may include a heavy chain with a maximum of one, a maximum of two , a maximum of three, a maximum of four or a maximum of five amino acid substitutions at amino acids 26 to 33 (CDR1), 51 to 60 (CDR2), and 99 to 120 (CDR3) of SEQ ID NO: 154.
[0218] [00218] In other embodiments, an isolated antibody that specifically binds gp41, and includes a light chain with a maximum of one, a maximum of two, a maximum of three or a maximum of four amino acid substitutions at amino acids 26 to 31 (CDR1), 49 to 51 (CDR2), and 87 to 98 (CDR3) of SEQ ID NO: 12. In some embodiments, an isolated antibody that specifically binds gp41, and includes a light chain with a maximum of one, a maximum of two, a maximum of three or more maximum four amino acid substitutions at amino acids 26 to 31 (CDR1), 49 to 51 (CDR2), and 87 to 98 (CDR3) of SEQ ID NO: 2. In some embodiments, the antibody may include a light chain with a maximum of one , at most two, at most three or at most four amino acid substitutions at amino acids 26 to 31 (CDR1), 49-51 (CDR2), and 87-98 (CDR3) of SEQ ID NO: 4. In some embodiments, the antibody may include a light chain with a maximum of one, a maximum of two, a maximum of three, or a maximum of four amino acid substitutions amino acids 26 to 31 (CDR1) , 49 to 51 (CDR2), and 87 to 98 (CDR3) of SEQ ID NO: 6. In some embodiments, the antibody may include a light chain with a maximum of one, a maximum of two, a maximum of three or a maximum of four substitutions amino acid amino acids 26 to 31 (CDR1), 49 to 51 (CDR2), and 87 to 98 (CDR3) of SEQ ID NO: 152.
[0219] [00219] In some embodiments, an isolated antibody that specifically binds gp41 as described here includes up to 10 amino acid substitutions (such as up to 1, 2, 3, 4, 5, 6, 7, 8, or up to 9 amino acid substitutions) in the framework regions (for example, according to the Kabt, Clothia or IMGT numbering systems) of the antibody heavy chain, the antibody light chain, or the heavy and light chains of the antibody.
[0220] [00220] In some embodiments, an isolated antibody that specifically binds gp41, includes a heavy chain variable region including no more than 10 (such as 1, 2, 3, 4, 5, 6, 7, 8 or 9) substitutions of amino acid in the structure regions of one of SEQ ID NOs: 1, 3, 5, 11, 146, 147-149, 187, 189-192, and 200-204. Structure regions of SEQ ID NOs: 1, 3, 5, 11, 146, 147-149, 187, 189-192, and 200-204 include amino acids 1-25 (FR1), 34-50 (FR2), 61- 66 (FR3) and 121-131 (FR4) of SEQ ID NO: 1, 3, 5, 11, 146, 147-149, 187, 189-192, and 200-204, respectively (according to a Kabat numbering) . In some embodiments, an isolated antibody that specifically binds gp41, includes a light chain variable region including no more than 10 (such as 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions in structure regions of SEQ ID NOs: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199, respectively. Structure regions of SEQ ID NO: 2 include amino acids 1-25 (LFR2), 32-48 (LFR2), 52-86 (LFR3) and 99-108 (OFR4) of SEQ ID NO: 2, 4, 6, 12 , 150-152, 164-1886, 188, or 197-199, respectively (according to a Kabat numbering).
[0221] [00221] In other embodiments, an isolated antibody that specifically binds gp41, includes a heavy chain variable region including no more than 10 (such as 1, 2, 3, 4, 5, 6, 7, 8 or 9) substitutions of amino acid in the structure regions of SEQ ID NO: 1 and a variable light chain region including no more than 10 (such as 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions in structure regions of SEQ ID NOs: 2. In other embodiments, an isolated antibody that specifically binds gp41, includes a heavy chain variable region including no more than 10 (such as 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions in the structure regions of SEQ ID NO: 154 and a light chain variable region including no more than 10 (such as 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions in the structure regions of SEQ ID NO: 152. In other embodiments, an isolated antibody that specifically binds gp41, includes a heavy chain variable region includes n not more than 10 (such as 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions in the structure regions of SEQ ID NO: 192 and a light chain variable region including no more than 10 (such as 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions in the structure regions of SEQ ID NO: 152.
[0222] [00222] In some embodiments, the human monoclonal antibody heavy chain includes an amino acid sequence having at least 80% (such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%) sequence identity with the amino acid sequence established as one of SEQ ID NOs: 1, 3, 5, 11, 146, 147-149, 187, 189- 192, or 200-204. In additional examples, the heavy chain includes the amino acid sequence established as one of SEQ ID NOs: 1, 3, 5, 11, 146, 147-149, 187, 189-192, or 200-204. In some examples, the human monoclonal antibody light chain includes the amino acid sequence having at least 80% (such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%) sequence identity with the amino acid sequence established as one of SEQ ID NOs: 2, 4, 6, 12, 150-152, 164-186, 188, or 197- 199. In other embodiments, an isolated antibody that specifically binds gp41 includes a heavy chain variable region and a light chain variable region, where the heavy chain variable region includes the amino acid sequence having at least 80% (such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%) sequence identity with the amino acid sequence established as a SEQ ID NOs: 1, 3, 5, 11, 146, 147-149, 187, 189-192, or 200-204, and the light chain variable region includes the amino acid sequence having at least 80% (such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 8%, or at least 99%) sequence identity with the amino acid sequence established as one of SEQ ID NOs: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199.
[0223] [00223] As described here, depth sequencing was used to identify additional antibodies that bind to substantially similar epitopes on the surface of gp41 in orientation substantially equal to 10E8, 7H6, and / or 7N16 binding. Exemplary nucleic acid sequences encoding antibody heavy chains are established as SEQ ID NOs: 35-115 in the accompanying sequence listing. These encode variable heavy chain regions, at least about 80% identical to the antibody heavy chain variable region 10E8 (SEQ ID NO: 1). Thus, described here are amino acid molecules encoding antibody heavy chain variable regions that are at least about 80% identical to the heavy chain variable region established as SEQ Id NO: 1, such as at least about 80%, at least at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85% at least about 86%, at least about 87%, at least about 88%, or at least about 89% at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 1. Exemplary nucleic acid sequences encoding antibody light chains similar to 10E8 are established as SEQ ID NOs: 116-145 in the accompanying sequence listing. Exemplary nucleic acid sequences encoding antibody light chains are established as SEQ ID NOs: 116-145 in the accompanying sequence listing. These encode variable light chain regions of antibody at least about 80% identical to the variable light chain region of antibody 10E8 (SEQ ID NO: 2). Thus, described here are amino acid molecules encoding antibody light chain variable regions that are at least about 80% identical to the heavy chain variable region established as SEQ ID NO: 2, such as at least about 80%, at least at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85% at least about 86%, at least about 87%, at least about 88%, or at least about 89% at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95% at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 2.
[0224] [00224] In some embodiments, an isolated antibody that specifically binds gp41 includes one or more of the heavy chain complementarity determining regions (CDRs) encoded by the nucleic acid sequence established as one of SEQ ID NOs: 35-115. In some embodiments, an isolated human monoclonal antibody that specifically binds gp41, includes a heavy chain with all CDRs encoded by the nucleic acid sequence, established as one of SEQ ID NOs: 35-115. In specific examples, the human monoclonal antibody heavy chain includes the amino acid sequence encoded by the nucleic acid sequence, established as one of SEQ ID NOs: 35-115.
[0225] [00225] In some embodiments, an isolated antibody that specifically binds gp41 includes one or more of the light chain complementarity determining regions (CDRs) encoded by the nucleic acid sequence, established as one of SEQ ID NOs: 116-145. In some embodiments, an isolated human monoclonal antibody that specifically binds gp41, includes a light chain with all antibody CDRs, encoded by the nucleic acid sequence, established as one of SEQ ID NOs: 116-145. In specific examples, the human monoclonal antibody light chain includes the amino acid sequence encoded by the nucleic acid sequence, established as one of SEQ ID NOs: 116-145.
[0226] [00226] In some embodiments, an isolated antibody that specifically binds gp41, includes a heavy chain variable region encoded by a nucleic acid derived from the IGHV3-15 germline allele origin, for example, the IGHV3-15 germline allele origin * 01, IGHV3-15 * 02, IGHV3-15 * 03, IGHV3-15 * 04, IGHV3-15 * 05, IGHV3-15 * 06, IGHV3-15 * 07, IGHV3-15 * 08, IGHV3-15 * 09 , IGHV3-15 * 10, IGHV3-15 * 11, IGHV3-15 * 12, IGHV3-15 * 13, IGHV3-15 * 14, or IGHV3-15 * 15. In some embodiments, the heavy chain variable region is encoded by a nucleic acid derived from the germline allele origin IGHV3-15, for example, germline allele origin IGHV3-15 * 01, IGHV3-15 * 02, IGHV3-15 * 03, IGHV3-15 * 04, IGHV3-15 * 05, IGHV3-15 * 06, IGHV3-15 * 07, IGHV3-15 * 08, IGHV3-15 * 09, IGHV3-15 * 10, IGHV3-15 * 11 , IGHV3-15 * 12, IGHV3-15 * 13, IGHV3-15 * 14, or IGHV3-15 * 15, and it is about 10%, 15%, 20%, 25%, 30%, 35% or 40% , such as about 15% to 40% divergent from the respective heavy chain germline sequence.
[0227] [00227] In some embodiments, an isolated antibody that specifically binds gp41 includes a light chain variable region encoded by a nucleic acid derived from the IGLV3-19 germline allele origin, such as an IGLV3-19 germline allele origin * 01. In some embodiments, the light chain is encoded by a nucleic acid derived from the IGLV3-19 germline allele origin, such as an IGLV3-19 * 01 germline allele origin, and is about 10%, 15%, 20% , 25%, 30%, 35% or 40%, as well as about 15% to 40% respective light chain germline sequence.
[0228] [00228] In some examples, the heavy chain variable domain is a clonal variant of the N152 donor, with a heavy chain encoded by the VH3-15 gene and VJ-1J genes. In other examples, the light chain variable domain is a clonal variant of the N152 donor, with a light chain encoded by an LV3-19 V gene and an LJ-3 J gene. The isolated monoclonal antibody may include a heavy chain and a chain light, where the heavy chain variable region is a clonal variant of the N152 donor with a heavy chain variable region amino acid sequence established as SEQ ID NO: 1. The heavy chain is derived from a VH3-15 and LJ-3 gene J genes. The light chain variable domain is a clonal variant of the N152 donor with a light chain amino acid sequence variable region established as SEQ ID NO: 2. The light chain is derived from an LV3-19 V gene and an LJ-3 gene J, the monoclonal antibody specifically binds gp41, competes with 10E8 for binding to gp41, and is neutralizing.
[0229] [00229] In some embodiments, the heavy chain of an antibody similar to 10E8 can be complemented by the light chain of the antibody 10E8, 7H6, and / or 7N16 and still maintains the binding for gp41, for example, maintains the specific binding for the epitope of 10E8. In some embodiments, the light chain of an antibody similar to 10E8 can be complemented by the heavy chain of antibody 10E8, 7H6, and / or 7N16 and still maintains the binding for gp41, for example, maintains the specific binding for the 10E8 epitope . Thus, antibodies similar to 10E8 are described here, which can be identified by complementing the heavy or light chains of 10E8, 7H6, and / or 7N16.
[0230] [00230] Since a heavy or light chain variable domain of interest is identified, binding to gp41 or an epitope of interest (such as the 10E8 epitope) can be determined using a cross-complement analysis. Briefly, if the variable domain of interest is a heavy chain variable domain, the amino acid sequence of this heavy chain variable domain is produced. The heavy chain variable domain is then paired with a reference sequence light chain variable domain, such as 10E8 (SEQ ID NO: 2), 7H6 (SEQ ID NO: 4), and / or 7N16 (SEQ ID NO : 6), light chain variable domain, and it is determined whether the antibody specifically binds the antigen (the epitope) with a specific affinity, such as a KD of 10-8, 10-9 or 10-10. Similarly, if the variable domain of interest is a light chain variable domain, this amino acid sequence is produced. The variable light chain variable domain is then paired with a reference sequence heavy chain variable domain, such as variable domain is then paired with a reference sequence light chain variable domain, such as heavy chain variable domain 10E8 (SEQ ID NO: 1), 7H6 (SEQ ID NO: 3), and / or 7N16 (SEQ ID NO: 5), and it is determined whether the antibody specifically binds the antigen (the epitope) with a specific affinity, such as a 10-8, 10-9 or 10-10 KD.
[0231] [00231] Fully human monoclonal antibodies include regions of human structure. Thus, any of the antibodies that specifically binds gp41 here, can include the human framework region and can include the framework regions of the amino acid sequence established as one of SEQ ID NO; 1-6, 11, 12, and / or 146-192 or encoded by one of SEQ ID NO: 35-145. However, the structure regions can be from another source. Additional examples of structure sequences that can be used include the structure amino acid sequences of the heavy and light chains described in PCT Publication No. WO 2006/074071 (see, for example, SEQ ID NOs: 1-16), which is incorporated here by reference.
[0232] [00232] In some embodiments, one or more of the gp41 antibody heavy and / or light chain complementarity determining regions (CDRs) established as one of 1-6, 11, 12, and / or 146-192 or encoded by SEQ ID Nos: 35-145 are expressed on the surface of another protein, such as a supporting protein. The expression of antibody domains on the surface of a supporting protein is known in the art (see, for example, Liu et al., J. Virology 85 (17): 8467-8476, 2011). Such expression creates a chimeric protein that maintains binding to gp41, such as specific binding to the 10E8 epitope. As described in example 1, the 10E8 class of antibodies makes most of their contacts through heavy chain CDRs (see, for example, the tables provided in figures 27-30, and the molecular models shown in figures 4, 5, 12, 15, 16 and 39-41A). Thus, in some specific embodiments, one or more of the heavy chain CDRs are grafted onto a support protein, such as one or more of the heavy chain CDR1, CDR2, and / or CDR3 established as one of SEQ ID NO; 1,3, 5, 11, 146-149, 153-163, 187, 189-192, or 200-204 or encoded by one of SEQ ID NOs: 35-115.
[0233] [00233] The monoclonal antibody can be of any isotype. The monoclonal antibody can be, for example, an IgM or IgG antibody, such as IgG1, IgG2, IgG3, or IgG4. The class of an antibody that specifically binds gp41 can be switched with another. In one aspect, a nucleic acid molecule encoding VL or VH is isolated using methods well known in the art, so that it does not include any nucleic acid sequences encoding the light or heavy chain constant region, respectively.
[0234] [00234] In particular examples, the VH amino acid sequence is established as one of SEQ ID NO: 1, 3, 5, 11, 146-149, 153-163, 187, 189-192, or 200-204 or encoded by one of SEQ ID NOs: 35-115. In other examples, the VL amino acid sequence is established as one of SEQ ID NO: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199 or encoded by one of SEQ ID NOs: 116 -145. The nucleic acid molecule encoding VL or VH is then operationally linked to a nucleic acid sequence encoding the CL or CH of a different class of immunoglobulin molecule. This can be achieved using a vector or nucleic acid molecule that includes a CL or CH chain, as known in the art. For example, an antibody that specifically binds gp41, which was originally IgM, can be a class changed to an IgG. The class change can be used to convert one IgG subclass to another, such as from IgG1 to IgG2, IgG3, or IgG4.
[0235] [00235] In some examples, the antibodies described are antibody oligomers, such as dimers, trimers, tetramers, pentamers, hexameters, septamers, octomers and so on. In some examples, antibodies are pentamers.
[0236] [00236] In some examples, the antibodies, or an antibody binding fragment thereof, are modified so that they are directly cytotoxic to infected cells, or use natural defenses such as complement, antibody-dependent cell cytoxicity (ADCC), or macrophage phagocytosis.
[0237] (1) Fab, o fragmento que contém um fragemento de ligação de antígeno monovalente de uma molécula de anticorpo, pode ser produzido por digestão de anticorpo inteiro com nezima papaína para produzir uma cadeia leve intacta e uma porção de uma cadeia pesada; (2) Fab', o fragmento de uma molécula de anticorpo pode ser obtido por tratamento de anticorpo inteiro com pepsina, seguido por redução, para produzir uma cadeia leve intacta e uma porção da cadeia pesada; dois fragmentos de Fab' são obtidos por molécula de anticorpo; (3) (Fab')2, o fragmento do anticorpo que pode ser obtido por tratamento de anticorpo inteiro com a enzima pepsina sem redução subsequente; F(ab')2 é um dímero de dois fragmentos de Fab' mantidos juntamente por duas ligações de dissulfeto; (4) Fv, um fragmento geneticamente projetado contendo a região variável da cadeia leve e a região variável da cadeia pesada expressa como duas cadeias; e (5) Anticorpo de cadeia simples (tal como scFv), definido como uma molécula geneticamente projetada contendo a região variável da cadeia leve, a região variável da cadeia pesada, ligada por um ligador de polipeptídeo adequado como uma molécula de cadeia simples geneticamente fundida. (6) Um dímero de um anticorpo de cadeia simples (scFV2), definido como um dímero de um scFV. Este foi também denominado um "minianticorpo." [00237] Antibody fragments are encompassed by the present invention, such as Fab, F (ab ') 2, and Fv which include a heavy and light chain variable region and specifically bind gp41. These antibody fragments maintain the ability to selectively bind to the antigen and are "antigen binding" fragments. These fragments: (1) Fab, the fragment containing a monovalent antigen binding fraction of an antibody molecule, can be produced by digesting whole antibody with papain ninety to produce an intact light chain and a portion of a heavy chain; (2) Fab ', the fragment of an antibody molecule can be obtained by treating the entire antibody with pepsin, followed by reduction, to produce an intact light chain and a portion of the heavy chain; two Fab 'fragments are obtained per antibody molecule; (3) (Fab ') 2, the antibody fragment that can be obtained by treating the entire antibody with the enzyme pepsin without subsequent reduction; F (ab ') 2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody (such as scFv), defined as a genetically engineered molecule containing the light chain variable region, the heavy chain variable region, linked by a suitable polypeptide linker as a genetically fused single chain molecule. (6) A dimer of a single chain antibody (scFV2), defined as a dimer of a scFV. This was also called a "mini-antibody."
[0238] [00238] Methods of preparing these fragments are known in the art (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988).
[0239] [00239] In another group of modalities, the antibodies are Fv antibodies, which are typically about 25 kDa and contain a complete antigen binding site with three CDRs for each heavy chain and each light chain. To produce these antibodies, VH and VL can be expressed from two individual nucleic acid constructions in a host cell. In particular examples, the VH amino acid sequence includes the CDRs of one of SEQ ID NOs: 1, 3, 5, 11, 146-149, 153-163, 187 or 189-192 or encoded by one of SEQ ID NOs: 35-115. In other examples, the VL amino acid sequence includes the CDRs of SEQ ID No.: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199 or encoded by one of SEQ ID No.: 116 -145. In additional examples, the VH amino acid sequence includes the amino acid sequence established as one of SEQ ID NOs: 1, 3, 5, 11, 146-149, 153-163, 187 or 189-192 or encoded by one of SEQ ID NOs: 35-115. In other examples, the VL amino acid sequence includes the amino acid sequence established as SEQ ID NO: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199 or encoded by one of SEQ ID No. 116-145.
[0240] [00240] If VH and VL are expressed non-contiguously, Fv antibody chains are typically held together by non-covalent interactions. However, these chains tend to dissociate under dilution, so methods have been developed to cross-link the chains through glutaraldehyde, intermolecular disulfides, or a peptide linker. Thus, in one example, the Fv can be disulfide stabilized Fv (dsFv), in which the heavy chain variable region and the light chain variable region are chemically linked by disulfide bonds.
[0241] [00241] In a further example, the Fv fragments include VH and VL chains connected by a peptide linker. These single chain antigen (scFv) binding proteins are prepared by constructing a structural gene including DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. Recombinant host cells synthesize a single polypeptide chain with a linker peptide linking the two V domains. Methods for producing scFvs are known in the art (see, Whitlow et al., Methods: a Companion to Methods in Enzymology, Volume 2, page 97, 1991; Bird et al., Science 242: 423, 1988; US Patent No. 4,946,778; Pack et al., Bio / Technology 11: 1271, 1993; and Sandhu, supra). Dimers of a single chain antibody (scFV2) are also contemplated.
[0242] [00242] Antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by E. coli expression of DNA encoding the fragment. Antibody fragments can be obtained by digesting whole antibody pepsin or papain by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F (ab ') 2. This fragment can also be cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from the cleavage of disulfide bonds, to produce monovalent 3.5 'Fab' fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab 'fragments and one Fc fragment directly (see, US Patent No. 4,036,945 and US Patent No. 4,331,647, and references contained therein; Nisonhoff et al., Arch Biochem. Biophys. 89: 230, 1960; Porter, Biochem. J. 73: 119, 1959; Edelman et al., Methods in Enzymology, Volume 1, page 422, Academic Press, 1967; and Coligan et al. In the sections 2.8.1-2.8.10 and 2.10.1-2.10.4).
[0243] [00243] Other methods of cleavage antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, other fragment cleavage, or other enzymatic, chemical, or genetic techniques can also be used, provided the fragments are bind to the antigen, which is recognized by the intact antibody.
[0244] 1) Alanina (A), Serina (S), Treonina (T); 2) Ácido aspártico (D), Ácido Glutâmico (E); 3) Asparagina (N), Glutamina (Q); 4) Arginina (R), Lisina (K); 5) Isoleucina (I), Leucina (L), Metionina (M), Valina (V); e 6) Fenilalanina (F), Tirosina (Y), Triptofan (W). [00244] Someone skilled will realize that conservative variants of antibodies can be produced. Such conservative variants employed in antibody fragments, such as dsFv fragments or scFv fragments, will maintain the critical amino acid residues necessary for correct duplication and stabilization between the VH and VL regions, and maintain the charge characteristics of the residues in order to preserve the low pI and low toxicity of the molecules. Amino acid substitutions (such as a maximum of one, a maximum of two, a maximum of three, a maximum of four, or a maximum of five amino acid substitutions) can be made in the VH and VL regions to increase production. In particular examples, the VH sequence is SEQ ID NO: 1, 3, 5, 11, 146-149, 153-163, 187 or 189-192 or encoded by one of SEQ ID NO: 35-115. In other examples, the VL sequence is SEQ ID No.: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199 or encoded by one of SEQ ID No.: 116-145. Conservative amino acid substitution tables providing functionally similar amino acids are well known to someone skilled in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0245] [00245] The antibodies described here can be isolated using masked antigens, as described in PCT Publication No. WO 2009/100376. Briefly, the antigens are masked for the antigen's target antigenicity for a specific epitope that specifically binds to the antibody of interest, such as a neutralizing antibody.
[0246] [00246] Additional recombinant human neutralizing antibodies that specifically bind the same epitope of gp41 bound by the antibodies described here, which specifically bind gp41, can be isolated by analysis of a recombinant combinatorial antibody library, such as a Fab phage display library. (see, for example, US Patent Application Publication No. 2005/0123900). In some cases, phage display libraries are prepared using cDNAs from the variable regions of heavy and light chains prepared from mRNA derived from human lymphocytes. Methodologies for preparing and analyzing such libraries are well known in the art. There are commercially available kits for generating phage display libraries (for example, the Pharmacy Recombinant Phage Antibody System, Catalog No. 27-9400- 01; and the Stratagene SurfZAP ™ phage display kit, catalog No. 240612) . There are also other methods and reagents that can be used in the generation and analysis of antibody display libraries (see, for example, US Patent 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; Fuchs et al., Bio / Technology 9: 1370-1372, 1991; Hay et al., Hum. Antibod. Hybridomas 3: 81-85, 1992; Huse et al., Science 246: 1275-1281, 1989; McCafferty et al., Nature 348 : 552-554,1990; Griffiths et al., EMBO J. 12: 725-734, 1993)
[0247] [00247] In one embodiment, to isolate additional human antibodies that specifically bind gp41, a neutralizing antibody that specifically binds gp41, as described here, is first used to select human heavy and light chain sequences having gp41-like binding activity, such as as using the epitope printing methods described in PCT Publication No. WO 93/06213. The antibody libraries used in this method are the scFv libraries prepared and analyzed, using methods such as those described in PCT Publication No. WO 92/01047, McCafferty et al., Nature 348: 552-554, 1990; and / or Griffiths et al., EMBO J. 12: 725-734, 1993 using gp120.
[0248] [00248] Since variable light chain (VL) and human variable heavy chain (VH) segments are selected, "mixing and matching" experiments, in which different pairs of the initially selected VL and VH segments are analyzed for the binding of gp41, are performed to select VL / VH pair combinations of interest. In addition, to increase the binding affinity of the antibody, the VL and VH segments can be randomly mutated, such as within the H-CDR3 region or the L-CDR3 region, in a process analogous to the in vivo somatic mutation process responsible for maturation of antibody affinity during a natural immune response. In this way, affinity maturation in vitro can be performed by expanding the VH and VL regions using complementary PCR primers for H-CDR3 or L-CDR3, respectively. In this process, the primers were "reinforced" with a random mixture of the four nucleotide bases at certain positions, so that the resulting PCR products encode the VH and VL segments in which random mutations were introduced in the CDR3 regions of VH and / or VL. These randomly mutated VH and VL segments can be tested to determine binding affinity for gp41. In particular examples, the VH amino acid sequence is SEQ ID NO: 1, 3, 5, or 11, 146-149, 153-163, 187, 189-192, or 200-204 or encoded by one of SEQ ID NO. : 35-115. In other examples, the VL amino acid sequence is SEQ ID NO: 2, 4, 6, 12, 150-152, 164-186, 188, or 197-199 or encoded by one of SEQ ID NO: 116-145.
[0249] [00249] Following analysis and isolation of an antibody that binds gp41 from a recombinant immunoglobulin display library, nucleic acid encoding the selected antibody can be retrieved from the display package (for example, from the phage genome) and subcloned into other vectors of expression by standard recombinant DNA techniques, as described here. If desired, the nucleic acid can also be manipulated to create other antibody fragments, also as described herein. To express an isolated recombinant antibody by analyzing a combinatorial library, the DNA encoding the antibody is cloned into a recombinant expression vector and introduced into a mammalian host cell, as described here.
[0250] [00250] Effector molecules, such as therapeutic, diagnostic, or detection moieties can be linked to an antibody of interest, using any number of methods known to those skilled in the art. Both covalent and non-covalent bonding methods can be used. The procedure for binding an effector molecule to an antibody varies according to the chemical structure of the effector. Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH2) or sulfhydryl (-SH) groups, which are available with a suitable functional group to result in binding of the effector molecule. Alternatively, the antibody is derived to expose or link additional reactive functional groups. The derivation may involve the attachment of any number of binding molecules such as those available from Pierce Chemical Company, Rockford, IL. The linker can be any molecule used to attach the antibody to the effector molecule. The linker is capable of forming covalent bonds to both the antibody and the effector molecule. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight or branched chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody and the effector molecule are polypeptides, the linkers can be linked to the constituent amino acids through their side groups (such as through a disulfide bond to cysteine) or to the alpha amino carbon and carboxyl groups of the terminal amino acids.
[0251] [00251] In some circumstances, it is desirable to release the antibody's effector molecule when the immunoconjugate has reached its target site. Therefore, in these circumstances, immunoconjugates will include bonds that are cleavable in the vicinity of the target site. Cleavage of the linker to release the antibody's effector molecule can be induced by enzymatic activity or conditions to which the immunoconjugate is individualized within the target cell or in the vicinity of the target site.
[0252] [00252] In view of the large number of methods that have been reported for binding a variety of radiodiagnostic compounds, radiotherapeutic compounds, label drugs (such as enzymes or fluorescent molecules), toxins, and other agents to antibodies someone skilled in the art will be able to determine a suitable method for binding a particular agent to an antibody or other polypeptide.
[0253] [00253] The antibodies or antibody fragments described here can be derived from or linked to another molecule (such as another peptide or protein). In general, the antibody or portion thereof is derived so that binding to gp41 is not adversely affected by the derivation or labeling. For example, the antibody can be functionally linked (by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more molecular entities, such as another antibody (for example, a bispecific antibody or a diabody), an agent detection agent, a pharmaceutical agent, and / or a protein or peptide that can mediate the association of the antibody or antibody moiety with another molecule (such as a streptavidin core region or a polyhistidine label).
[0254] [00254] A type of derived antibody is produced by cross-linking two or more antibodies (of the same or different types, such as to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (such as disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company (Rockford, IL).
[0255] [00255] An antibody that specifically binds gp41 can be labeled with a detectable portion. Useful detection agents include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide matches and the like. Bioluminescent markers are also in use, such as luciferase, green fluorescent protein, yellow fluorescent protein. An antibody can also be labeled with enzymes that are useful for detection, such as spicy horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. When an antibody is labeled with a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the spicy rhodium peroxidase agent is present, the addition of hydrogen peroxide and diaminobenzidine induces a colored reaction product, which is visually detectable. An antibody can also be labeled with biotin, and detected through indirect assessment of avidin or streptavidin binding. It should be noted that avidin itself can be labeled with an enzyme or a fluorescent label.
[0256] [00256] An antibody can be labeled with a magnetic agent, such as gadolinium. Antibodies can also be labeled with lanthanides (such as europium and dysprosium), and manganese. Paramagnetic particles such as superparamagnetic iron oxide are also used as labels. An antibody can also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (such as leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, epitope labels). In some embodiments, labels are linked by spacer branches of varying lengths to reduce a potential steric impediment.
[0257] [00257] An antibody can also be labeled with a radiolabeled amino acid. The radio label can be used for both diagnostic and therapeutic purposes. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I.
[0258] [00258] An antibody can also be derived with a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These groups can be useful for improving the biological characteristics of the antibody, such as for increasing the serum half-life or for increasing tissue binding.
[0259] [00259] Methods for detecting such labels are well known to those skilled in the art. In this way, for example, radio labels can be detected using photographic film or scintillation counters, fluorescent markers can be detected using a photodetector to detect emitted lighting. Enzyme labels are typically detected by supplying the enzyme with a substrate and detecting the reaction product produced by the enzyme's action on the substrate, and colorimetric labels are detected simply by viewing the colored label.
[0260] The present invention also relates to crystals obtained from antibody 10E8, 7H6, and / or 7N16, or portions thereof in complex with gp41 (or peptides from gp41), the crystalline structures of antibody 10E8, 7H6, and / or 7N16 or portions thereof in complex with gp41 (or gp41 peptides), the three-dimensional coordinates of the antibody 10E8, 7H6, and / or 7N16 or portions thereof in complex with gp41 (or gp41 peptides) and three-dimensional structures of antibody models 10E8, 7H6, and / or 7N16 or portions thereof in complex with gp41 (or gp41 peptides).
[0261] [00261] Those skilled in the art will understand that a group of structure coordinates for antibody 10E8, 7H6, and / or 7N16 or portions thereof in complex with gp41 or a portion thereof, is a relative group of points that defines the shape in three dimensions. In this way, it is possible that an entirely different group of coordinates can define a similar or identical shape. However, slight variations in the individual coordinates will have little effect on the overall shape. The variations in the coordinates described above can be generated because of mathematical manipulations of the structure coordinates.
[0262] [00262] The invention also provides systems, such as computer systems, designed to generate structures and / or carry out rational compound or drug planning for an antigenic compound capable of eliciting an immune response in an individual. The system may contain one or more or all of: atomic coordinate data according to an antibody complex 10E8, 7H6, and / or 7N16 or a subgroup thereof, and the figures derived from this by homology modeling, the data defining the three-dimensional structure of an antibody complex 10E8, 7H6, and / or 7N16 or at least one subdomain of it, or structural factor data such as gp41, the structural factor data being derived from the atomic coordinate data of the antibody complex 10E8, 7H6, and / or 7N16 or a subgroup of the same and the figures. B. Polynucleotides and Expression
[0263] [00263] Amino acid molecules (also referred to as polynucleotides) encoding the polypeptides provided here (including, but not limited to antibodies) can easily be produced by someone skilled in the art. For example, these nucleic acids can be produced using the amino acid sequence provided here (such as the CDR sequences, heavy chain and light chain sequences).
[0264] [00264] Someone skilled in the art can easily use the genetic code to construct a variety of functionally equivalent nucleic acids, such as nucleic acids that differ in sequence, but encode the same antibody sequence, or encode a conjugate or fusion protein including the VL and / or VH nucleic acid sequence.
[0265] [00265] Nucleic acid sequences encoding antibodies that specifically bind gp41 can be prepared by any suitable method including, for example, cloning appropriate sequences or by direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth . Enzymol. 68: 90-99, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68: 109-151, 1979; the diethylphosphoramidite method by Beaucage et al., Tetra. Lett. 22: 1859-1862, 1981; the solid phase triester phosphoramidite method by Beaucage & Caruthers, Tetra. Letts. 22 (20): 1859-1862, 1981, for example, using an automated synthesizer as described in, for example, Needham-VanDevanter et al., Nucl. Acids Res. 12: 6159-6168, 1984; and, the solid support method of U.S. Patent No. 4,458,066. Chemical synthesis produces a single-stranded oligonucleotide. This can be converted to double-stranded DNA by hybridization to a complementary sequence or by polymerization with a DNA polymerase using the single strand as a standard. Someone skilled in the art may recognize that, while chemical synthesis of DNA is generally limited to sequences of about 100 bases, longer sequences can be obtained by linking shorter sequences.
[0266] [00266] Exemplary nucleic acids can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques, and sufficient instructions to target people with knowledge through many cloning exercises are found in Sambrook et al., Supra, Berger and Kimmel (eds.), Supra, and Ausubel, supra. Product information from manufacturers of biological reagents and experimental equipment also provides useful information. Such manufacturers include SIGMA Chemical Company (Saint Louis, MO), R&D Systems (Minneapolis, MN), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (Carlsbad, CA), and Applied Biosystems (Foster City, CA), as well as many other commercial sources known to someone knowledgeable.
[0267] [00267] Nucleic acids can also be prepared by amplification methods. Amplification methods include the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustaining sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to skilled people.
[0268] [00268] Any of the nucleic acids encoding any of the antibodies, VH and / or VL, described here (or fragment thereof) can be expressed in a recombinantly designed cell such as mammalian cells, bacteria, plant, yeast, and insect. These antibodies can be expressed as an individual VH and / or VL chain, or they can be expressed as a fusion protein. An immunoadhesin can also be expressed. Thus, in some examples, nucleic acids encoding VH and VL, and immunoadhesin are provided. Nucleic acid sequences can optionally encode a leader sequence.
[0269] [00269] To create a single chain antibody, (scFv) DNA fragments encoding VH- and VL are operationally linked to another fragment encoding a flexible linker, for example, encoding the amino acid sequence (Gly4-Ser) 3, from so that the VH and VL sequences can be expressed as a contiguous single chain protein, with the VL and VH domains linked by the flexible linker (see, for example, Bird et al., Science 242: 423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883, 1988; McCafferty et al., Nature 348: 552-554, 1990). Optionally, a cleavage site can be included in a linker, such as a furin cleavage site.
[0270] [00270] The nucleic acid encoding the VH and / or the VL can optionally encode an Fc domain (immunoadhesin). The Fc domain can be an IgA, IgM or IgG Fc domain. The Fc domain can be an optimized Fc domain, as described in U.S. Published Patent Application No. 20100/093979, incorporated herein by reference. In one example, immunoadhesin is an IgG1 Fc. In one example, immunoadhesin is an IgG3 Fc.
[0271] [00271] The single chain antibody can be monovalent, if only one VH and VL is used, bivalent, if two VH and VL are used, or polyvalent, if more than two VH and VL are used. Bispecific or polyvalent antibodies can be generated that specifically bind to gp120 and another molecule, such as gp41. The optionally encoded VH and VL can include a purine cleavage site between the VH and VL domains.
[0272] [00272] Those skilled in the art are expected to be understood in the numerous expression systems available for protein expression including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO, HeLa and myeloma.
[0273] [00273] The host cell can be a gram positive bacterium including, but not limited to Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, and Oceanobacillus. Methods for expressing protein in gram positive bacteria, such as Lactobaccillus are well known in the art, see, for example, Published U.S. Patent Application No. 20100/080774. Expression vectors for lactobacillus are described, for example, in U.S. Patent 6,100,388, and U.S. Patent No. 5,728,571. Leader strings can be included for expression in Lactobacillus. Gram positive bacteria include, but are not limited to E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, and Ureaplasma.
[0274] [00274] One or more DNA sequences encoding the antibody or fragment thereof can be expressed in vitro by transferring DNA into a suitable host cell. The cell can be prokaryotic or eukaryotic. The term also includes any progeny of the individual's host cell. It is understood that the entire progeny may not be identical to the parental cell, since there may be mutations that occur during replication. Proper transfer methods, meaning that foreign DNA is continuously maintained in the host, are known in the art. Hybridomas expressing the antibodies of interest are also encompassed by this invention.
[0275] [00275] The expression of nucleic acids encoding the isolated proteins described here can be obtained by operationally binding DNA or cDNA to a promoter (which is constitutive or inducible), followed by incorporation into an expression cassette. The promoter can be any promoter of interest, including a cytomegalovirus promoter and a human T cell lymphotrophic virus (HTLV) -1 promoter. Optionally, a enhancer, such as a cytomegalovirus enhancer, is included in the construct. The cassettes may be suitable for replication and integration in prokaryotes or eukaryotes. Typical expression cassettes contain specific sequences useful for regulating the expression of the DNA encoding the protein. For example, expression cassettes may include appropriate promoters, enhancers, transcription and translation terminators, initiation sequences, a start codon (ie, ATG) in front of a protein-encoding gene, intron interlacing signal, sequences for maintaining the correct reading structure for that gene to allow for appropriate mRNA translation, and interruption codons. The vector can encode a selectable marker, such as a marker encoding drug resistance (for example, resistance to ampicillin or tetracycline).
[0276] [00276] To obtain high-level expression of a cloned gene, it is desirable to construct expression cassettes that contain at least a strong promoter to target transcription, a ribosome binding site for translational initiation (internal ribosomal binding sequences) , and a transcription / translation terminator. For E. coli, this includes a promoter such as the T7, trp, lac, or lambda promoters, a ribosome binding site, and preferably a transcription termination signal. For eukaryotic cells, control sequences can include a promoter and / or a enhancer derived from, for example, an immunoglobulin, HTLV, SV40 or cytomegalovirus gene, and a polyadenylation sequence, and can also include donor and / or recipient sequences binding (for example, CMV and / or HTLV binding receptor and donor sequences). The cassettes can be transferred into the chosen host cell by well-known methods such as transformation and electroporation for E. coli and treatment with calcium phosphate, electroporation or lipofection for mammalian cells. Cells transformed by the cassettes can be selected by antibiotic resistance conferred by genes contained in the cassettes, such as the amp, gpt, neo and hyg genes.
[0277] [00277] When the host is a eukaryote, such methods of DNA transfection as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a wrapped plasmid and, liposomes, or virus vectors can be used. Eukaryotic cells can also be co-transformed with polynucleotide sequences encoding the antibody, labeled antibody, or antibody binding fragment thereof, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see, for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). Someone skilled in the art can easily use an expression system such as plasmids and vectors for use in producing proteins in cells including higher eukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines.
[0278] [00278] Modifications can be made to the nucleic acid encoding a polypeptide described here without decreasing its biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the target molecule into a fusion protein. Such modifications are well known to those skilled in the art and include, for example, termination codons, a methionine added at the amino terminal to provide an initiation, site, additional amino acids placed at the terminal to create conveniently located restriction sites, or additional amino acids ( such as poly His) to assist the purification steps. In addition to the recombinant methods, the immunoconjugates, effector moieties, and antibodies of the present invention can also be constructed in whole or in part using standard peptide synthesis well known in the art.
[0279] [00279] Once expressed, recombinant immunoconjugates, antibodies, and / or effector molecules can be purified according to standard procedures in the art, including precipitation of ammonium sulfate, affinity columns, column chromatography, and the like (see, generally , R. Scopes, PROTEIN PURIFICATION, Springer-Verlag, NY, 1982). Antibodies, immunoconjugates and effector molecules need not be 100% pure. Once purified, partially or even homogeneously as desired, if to be used therapeutically, the polypeptides must be substantially free of endotoxin.
[0280] [00280] Methods for expressing antibodies and / or reduplicating to an appropriate active form, including single chain antibodies, from bacteria such as E. coli have been described and are well known and are applicable to the antibodies described here. See, Buchner et al., Anal. Biochem. 205: 263-270, 1992; Pluckthun, Biotechnology 9: 545, 1991; Huse et al., Science 246: 1275, 1989 and Ward et al., Nature 341: 544, 1989.
[0281] [00281] Often, functional heterologous proteins from E. coli or other bacteria are isolated from inclusion bodies and require solubilization using strong denaturants, and subsequent reduplication. During the solubilization step, as is well known in the art, a reducing agent must be present to separate the disulfide bonds. An exemplary buffer with a reducing agent is: 0.1 M tris, pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol). Reoxidation of disulfide bonds can occur in the presence of low molecular weight thiol reagents in reduced and oxidized form, as described in Saxena et al., Biochemistry 9: 5015-5021, 1970, and especially as described by Buchner et al., supra.
[0282] [00282] Renaturation is typically carried out by diluting (for example, 100 times) the denatured protein and reduced in reduplication buffer. An exemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M Larginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.
[0283] [00283] As a modification to the two-chain antibody purification protocol, the heavy and light chain regions are separately solubilized and reduced, and then combined in the reduplication solution. Exemplary production is achieved when these two proteins are mixed in a molar ratio, so that a 5-fold molar excess of one protein over the other is not exceeded. Excessive oxidized glutathione or other oxidizing low molecular weight compounds can be added to the reduplication solution after redox shuffling is completed.
[0284] [00284] In addition to the recombinant methods, the antibodies, labeled antibodies and antibody-binding fragment thereof that are described here can also be constructed in whole or in part, using standard peptide synthesis. Solid phase synthesis of the polypeptides less than about 50 amino acids in length can be accomplished by linking the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by Barany & Merrifield, The Peptides: Analysis, Synthesis, Biology. Volume 2: Special Methods in Peptide Synthesis, Part A. pages 3 to 284; Merrifield et al., J. Am. Chem. Soc. 85: 2149-2156, 1963, and Stewart et al., Solid Phase Peptide Synthesis, 2nd edition., Pierce Chem. Co., Rockford, Ill., 1984. Longer-length proteins can be synthesized by condensation of the amino and carboxyl termini of shorter fragments. Methods of forming peptide bonds by activating a carboxyl terminal end (such as by using the N, N'-dicylexylcarbodimide coupling reagent) are well known in the art. C. Compositions and Therapeutic Methods
[0285] [00285] Methods are described here for the prevention or treatment of an HIV infection, such as an HIV-1 infection. Prevention can include inhibiting HIV-1 infection. The methods include contacting a cell with an effective amount of the human monoclonal antibodies described herein that specifically bind gp41, or an antibody binding fragment thereof or a nucleic acid encoding such antibodies or antibody binding fragment thereof. The method may also include administering to a subject a therapeutically effective amount of human monoclonal antibodies to an individual, or an antibody binding fragment thereof or a nucleic acid encoding such antibodies or antibody binding fragment thereof, for example, the antibody binding fragment can be one or more of the CDRs grafted onto a supporting protein. In some examples, the antibodies, or an antibody-binding fragment thereof or a nucleic acid encoding such antibodies or antibody-binding fragment thereof, can be used in post-exposure prophylaxis. In some examples, antibodies, or an antibody binding fragment thereof or a nucleic acid encoding such antibodies or antibody binding fragment thereof, can be used to eliminate the viral repertoire. For example, a therapeutically effective amount of the antibodies, or an antibody binding fragment thereof or a nucleic acid encoding such antibodies or antibody binding fragment thereof, can be administered to an individual being treated with antiviral therapy. In some examples, the antibodies, or an antibody binding fragment thereof, are modified so that they are directly cytotoxic to infected cells, or use natural defenses such as complement, antibody-dependent cell cytoxicity (ADCC), or macrophage phagocytosis .
[0286] [00286] Methods for assaying neutralization activity include, but are not limited to, a single cycle infection assay as described in Martin et al. (2003) Nature Biotechnology 21: 71-76. In this trial, the level of viral activity is measured using a selectable marker whose activity is reflective of the amount of viable virus in the sample, and the IC50 is determined. In other assays, acute infection can be monitored in the PM1 cell line or in primary cells (normal PBMC). In this assay, the level of viral activity can be monitored by determining p24 concentrations using ELISA. See, for example, Martin et al. (2003) Nature Biotechnology 21: 71-76.
[0287] [00287] HIV infection does not need to be completely eliminated for the composition to be effective. For example, a composition can decrease HIV infection by a desired amount, for example, by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least at least 90%, at least 95%, at least 98%, or at least 100% (elimination of detectable HIV-infected cells), when compared to HIV infection in the absence of the composition. For example, the cell is also contacted with an effective amount of an additional agent, such as an antiviral agent. The cell can be in vivo or in vitro. The methods may include administration of one or more additional agents known in the art. In additional examples, HIV replication can be reduced or inhibited by similar methods. HIV replication does not need to be completely eliminated for the composition to be effective. For example, a composition can decrease HIV replication by a desired amount, for example, by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 100% (detectable HIV elimination), when compared to HIV replication in the absence of the composition. In one example, the cell is also contacted with an effective amount of an additional agent, such as an antiviral agent. The cell can be in vivo or in vitro.
[0288] [00288] Compositions are provided that include one or more of the antibodies that specifically bind gp41, or an antibody binding fragment thereof or a nucleic acid encoding such antibodies or antibody binding fragment thereof, which are described herein in a vehicle . The compositions can be prepared in unit dosage forms for administration to an individual. The amount and time of administration are at the discretion of the treating physician to achieve the desired purposes. The antibody can be formulated for systemic or local administration. In one example, the antibody that specifically binds gp41, or an antibody binding fragment thereof or a nucleic acid encoding such antibodies or antibody binding fragment thereof, is formulated for parenteral administration, such as intravenous administration.
[0289] [00289] Compositions for administration may include an antibody solution that specifically binds gp41, or an antibody binding fragment thereof or a nucleic acid encoding such antibodies or antibody binding fragment thereof, dissolved in a pharmaceutically acceptable carrier, such as an aqueous variety. A variety of aqueous vehicles can be used, for example, buffered saline and the like. These solutions are sterile and generally free of unwanted substance. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions can contain pharmaceutically acceptable auxiliary substances when required to approximate physiological conditions such as buffering and pH-adjusting agents, toxicity-adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride , sodium lactate and the like. The concentration of antibody in these formulations can vary widely, and will be selected primarily based on fluid volumes, body weight and the like according to the particular mode of administration selected and the individual's needs.
[0290] [00290] A typical pharmaceutical composition for intravenous administration includes about 0.1 to 10 mg of antibody per individual per day. Dosages of 0.1 to about 100 mg per individual per day can be used, particularly if the agent is administered to an isolated site and not in the circulatory or lymphatic system, such as in a body cavity or an organ lumen. Current methods for preparing manageable compositions will be known or evident to those skilled in the art and are described in greater detail in such publications as Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA (1995).
[0291] [00291] Antibodies, or an antibody binding fragment thereof or a nucleic acid encoding such antibodies or antibody binding fragment thereof, may be supplied in lyophilized form and rehydrated with sterile water prior to administration, although they are also provided sterile solutions of known concentration. The antibody solution, or an antibody binding fragment thereof or a nucleic acid encoding such antibodies or antibody binding fragment thereof, is then added to an infusion bag containing 0.9% sodium chloride, USP , and typically administered in a dosage of 0.5 to 15 mg / kg body weight. Considerable experience is available in the art in the administration of antibody drugs, which have been marketed in the United States since the approval of RITUXAN® in 1997. Antibodies, or an antibody binding fragment thereof or a nucleic acid encoding such antibodies or binding fragment antibodies, they can be administered by slow infusion, rather than in an intravenous pulse or bolus. In one example, a high loading dose is administered, with subsequent maintenance doses being administered at a lower level. For example, an initial loading dose of 4 mg / kg can be infused over a period of 90 minutes, followed by weekly maintenance doses for 4 to 8 weeks of 2 mg / kg infused over a period of 30 minutes if the previous dose was well tolerated.
[0292] [00292] A therapeutically effective amount of a gp41 specific human antibody, or an antibody binding fragment thereof or a nucleic acid encoding such antibodies or antibody binding fragment thereof, will depend on the severity of the disease and / or infection and the general state of the patient's health. A therapeutically effective amount of the antibody is that which provides subjective relief from a symptom (s) or an objectively identifiable improvement as observed by the physician or other qualified observer. These compositions can be administered in conjunction with another therapeutic agent, either simultaneously or sequentially.
[0293] [00293] In one embodiment, administration of the antibody, or an antibody binding fragment thereof or a nucleic acid encoding such antibodies or antibody binding fragment thereof, results in a reduction in the establishment of HIV infection and / or a reduction in progression of subsequent HIV disease in an individual. A reduction in the onset of HIV infection and / or a reduction in the progression of subsequent HIV disease encompasses any statistically significant reduction in HIV activity. In some embodiments, methods are described for treating an individual with an HIV-1 infection. These methods include administering to the individual a therapeutically effective amount of an antibody, or a nucleic acid encoding the antibody, thereby preventing or treating HIV-1 infection.
[0294] [00294] Studies have shown that the rate of HIV transmission from mother to child is significantly reduced when zidovudine is administered to HIV-infected women during pregnancy, childbirth and to the child after birth (Connor et al., 1994 Pediatr Infect Dis J 14: 536-541). Several studies of mother-to-child transmission of HIV have demonstrated a correlation between maternal viral load on release and risk of HIV transmission to the child. The present invention provides isolated human monoclonal antibodies that are of use in decreasing mother-to-child HIV transmission. Thus, in some examples, a therapeutically effective amount of a gp41 specific human antibody, or an antibody binding fragment thereof or a nucleic acid encoding such antibodies or antibody binding fragment thereof, is administered in order to prevent HIV transmission, or decrease the risk of HIV transmission, from a mother to a child. In some examples, a therapeutically effective amount of the antibody, or an antibody binding fragment thereof or a nucleic acid encoding such antibodies or antibody binding fragment thereof, is administered to the mother and / or the child during delivery. In other examples, a therapeutically effective amount of the antibody is administered to the mother and / or baby prior to breastfeeding in order to prevent viral transmission to the baby or to decrease the risk of viral transmission to the baby. In some embodiments, both a therapeutically effective amount of the antibody and a therapeutically effective amount of another agent, such as zidovudine, are administered to the mother and / or baby.
[0295] [00295] For any application, the antibody, or an antibody binding fragment thereof or a nucleic acid encoding such antibodies or antibody binding fragment thereof, can be combined with antiretroviral therapy. Antiretroviral drugs are broadly classified by the phase of the retrovirus life cycle that the drug inhibits. The described antibodies can be administered in conjunction with nucleoside analogue reverse transcriptase inhibitors (such as zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine, entecavir, and apricitabine), nucleotide reverse transcriptase inhibitors (such as tenofovir and adefovir), non-nucleotide reverse transcriptase inhibitors (such as efavirenz, nevirapine, delavirdine, etravirine, and rilpivirine), protease inhibitors (such as saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir, tiprenavir, fosamprenzan, levamprenavir, fosamprenzan darunavir), entry or fusion inhibitors (such as maraviroc and enfuvirtide), maturation inhibitors, (such as bevirimat and vivecon), or broad-spectrum inhibitors, such as natural antivirals. In some examples, a described antibody or active fragment thereof or nucleic acids encoding such, is administered in conjunction with IL-15, or conjugated to IL-15.
[0296] [00296] In some examples, an individual is also administered one or more additional antibodies that bind HIV glycoproteins, such as gp120 and gp41. Examples of neutralizing antibodies that can be administered in conjunction with the described antibodies can be found in International Patent Publication No. WO 2011/038290, published on March 31, 2011, which is specifically incorporated herein by reference in its entirety.
[0297] [00297] Single or multiple administrations of the compositions including the antibodies described herein are administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition must provide a sufficient amount of at least one of the antibodies described here to effectively treat the patient. The dosage can be administered once, but it can be applied periodically until a therapeutic result is obtained or until the side effects justify discontinuing the therapy. In one example, the dose of the antibody is infused for thirty minutes every two days. In this example, about one to about ten doses can be administered, just as three or six doses can be administered every two days. In another example, a continuous infusion is administered for about five to ten days. The individual can be treated at regular intervals, such as monthly, until a desired therapeutic result is achieved. Usually, the dose is sufficient to treat or ameliorate symptoms or signs of illness, without producing unacceptable toxicity to the patient.
[0298] [00298] Parenteral controlled release formulations can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems see, Banga, A.J., Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. The microcapsules contain the therapeutic protein, such as a cytotoxin or a drug, as a central nucleus. In microspheres, the therapeutic is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 µm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 µm, so that only nanoparticles are administered intravenously. Microparticles are typically around 100 µm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pages 315 to 339, (1992).
[0299] [00299] Polymers can be used for ion-controlled release of the antibody compositions described here. Several degradable and non-degradable polymeric matrices for use in controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26: 537-542, 1993). For example, the blocking copolymer, polaxamer 407, exists as a viscous liquid, but mobile at low temperatures, but forms a semi-solid gel at body temperature. It has been shown to be an effective vehicle for the formulation and prolonged release of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9: 425-434, 1992; and Pec et al., J. Parent. Sci. Tech 44 (2): 58-65, 1990). Alternatively, hydroxyapatite was used as a microcarrier for controlled release of protein (Ijntema et al., Int. J. Pharm.112: 215-224, 1994). In yet another aspect, liposomes are used for controlled delivery as well as lipid-encapsulated drug drug targeting (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous additional systems for controlled release of therapeutic proteins are known (see, US Patent No. 5,055,303; US Patent No. 5,188,837; US Patent No. 4,235,871; US Patent No. 4,501,728; US Patent No. 4,837,028; Patent US No. 4,957,735; US Patent No. 5,019,369; US Patent No. 5,055,303; US Patent No. 5,514,670; US Patent No. 5,413,797; US Patent No. 5,268,164; US Patent No. 5,004,697; US Patent No. 4,902,505; ; US Patent No. 5,506,206; US Patent No. 5,271,961; US Patent No. 5,254,342 and US Patent No. 5,534,496).
[0300] [00300] In some examples, the individual is administered DNA encoding the antibody or antibody-binding fragment thereof, for example, the antibody-binding fragment can be one or more of the CDRs grafted onto a supporting protein, to provide antibody production in vivo, for example, using the individual's cellular machinery. Immunization by nucleic acid constructs is well known in the art and taught, for example, in U.S. Patent No. 5,643,578, and U.S. Patent No. 5,593,972 and U.S. Patent No. 5,817,637. U.S. Patent No. 5,880,103 describes several methods of releasing nucleic acids encoding an organism. The methods include liposomal release of nucleic acids. Such methods can be applied to the production of an antibody, or antibody-binding fragment thereof, by someone skilled in the art.
[0301] [00301] A method of administering nucleic acids is direct administration with plasmid DNA, such as with a mammalian expression plasmid. The nucleotide sequence encoding the described antibody, or antibody binding fragment thereof, can be placed under the control of a promoter to increase expression.
[0302] [00302] In another method for using nucleic acids, a described antibody, or antibody-binding fragment thereof, can also be expressed by attenuated viral vectors or bacterial vectors. Recombinant vaccinia virus, adeno-associated virus (AAV), herpes virus, retrovirus, cytomegalovirus or other viral vectors can be used to express the antibody. For example, vaccinia vectors and useful method protocols are described in U.S. Patent No. 4,722,848. BCG (Bacillus Calmette Guerin) provides another vector for expression of the described antibodies (see, Stover, Nature 351: 456-460, 1991).
[0303] [00303] In one embodiment, a nucleic acid encoding a described antibody, or antibody-binding fragment thereof, is introduced directly into cells. For example, the nucleic acid can be loaded into gold microspheres by standard methods and introduced into the skin by a device such as Bio-Rad’s HELIOS Gene Gun. Nucleic acids can be "naked," which consist of plasmids under the control of a strong promoter.
[0304] [00304] Typically, DNA is injected into the muscle, although it can also be injected directly into other sites. Injection dosages are generally around 0.5 µg / kg to about 50 mg / kg, and typically are about 0.005 mg / kg to about 5 mg / kg (see, for example, US Patent No. 5,589,466 ). D. Diagnostic Methods and Kits
[0305] [00305] A method is provided here for the detection of gp41 expression in vitro or in vivo. In one example, gp41 expression is detected in a biological sample, and can be used to detect HIV-1 infection as the presence of HIV-1 in a sample. The sample can be any sample, including, but not limited to, tissue from biopsies, autopsies and pathology specimens. Biological samples also include sections of tissue, for example, frozen sections taken for histological purposes. Biological samples also include body fluids, such as blood, serum, plasma, sputum, spinal fluid or urine.
[0306] [00306] In several modalities, a method is provided for detecting AIDS and / or an HIV-1 infection in an individual. The invention provides a method for detecting HIV-1 in a biological sample, wherein the method includes contacting a biological sample with the antibody under conditions conducive to the formation of an immune complex, and detecting the immune complex, to detect gp41 in the sample. biological. In one example, the detection of gp41 in the sample indicates that the individual has an HIV infection. In another example, the detection of gp41 in the sample indicates that the individual has AIDS. In another example, detection of gp41 in the sample confirms a diagnosis of AIDS and / or an HIV-1 infection in an individual.
[0307] [00307] In some embodiments, the antibodies described are used to test vaccines. For example, to test whether a vaccine composition assumes the same confirmation as a gp41 peptide. Thus, a method for testing a vaccine is provided, wherein the method includes contacting a sample containing the vaccine, such as a gp41 immunogen, with the antibody under conditions conducive to the formation of an immune complex, and detecting an immune complex , to detect the vaccine in the sample. In one example, the detection of the immune complex in the sample indicates that the vaccine component, such as a gp41 immunogen, assumes a conformation capable of binding the antibody.
[0308] [00308] In one embodiment, the antibody is directly labeled with a detectable label. In another embodiment, the antibody that binds gp41 (the first antibody) is unlabeled and a second antibody or other molecule that can bind the antibody that binds gp41 is used. As is well known to someone skilled in the art, a second antibody is chosen that is capable of specifically binding the specific species and class of the first antibody. For example, if the first antibody is a human IgG, then the secondary antibody can be an anti-human-IgG. Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are commercially available.
[0309] [00309] Suitable labels for the antibody or secondary antibodies are described above, and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials include umbeliferone, fluorescein, fluorescein isothiocyanate, rhodamine, fluorescein dichlorotriazinylamine, dansyl chloride or phycoerythrin. An exemplary non-limiting luminescent material is luminol; an exemplary non-limiting magnetic agent is gadolinium, and exemplary non-limiting radioactive labels include 125I, 131I, 35S or 3H.
[0310] [00310] The immunoassays and methods described here can be used for several purposes. Kits for detecting a polypeptide will typically include an antibody that binds gp41, just like any of the antibodies described here. In some embodiments, an antibody fragment, such as an Fv fragment or Fab is included in the kit. In another embodiment, the antibody is labeled (for example, with a fluorescent, radioactive, or enzymatic label).
[0311] [00311] In one embodiment, a kit includes instructional materials describing methods of use. Instructional materials can be written in electronic form (such as a computer diskette or compact disc) or they can be visual (such as video files). Kits can also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit may additionally contain methods for detecting a label (such as enzyme substrates for enzyme labels, filter sets for detecting fluorescent labels, appropriate secondary labels such as secondary antibodies, or the like). Kits can additionally include buffers and other reagents routinely used to practice a particular method. Such kits and appropriate grades are well known to those skilled in the art.
[0312] [00312] In one embodiment, the diagnostic kit includes an immunoassay. Although the details of the immunoassays may vary with the particular format used, the method of detecting gp41 in a biological sample generally includes the steps of contacting the biological sample with an antibody that specifically reacts, under immunologically reactive conditions, to gp41. The antibody is specifically allowed to bind under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (antibody bound) is detected directly or indirectly. E. Methods of Identifying Antibodies of Interest
[0313] [00313] Methods are provided for producing a monoclonal antibody that specifically binds to a target antigen. These methods include isolating a population of memory B cells from an individual who has been exposed to the target antigen, where the memory B cells are CD19 + IgA-IgD-IgM-B cells. The population of isolated memory B cells is contacted with an effective amount of IL-21, IL-2 and CD40 ligand (CD40L), and mRNA is isolated from the population of isolated memory B cells. Nucleic acids encoding the variable heavy chains and the variable light chains of antibodies are isolated from the cells, and the variable heavy chains and the variable light chains are expressed. A monoclonal antibody including a variable heavy chain and a variable light chain that specifically binds to the target antigen is then selected from combinations of the variable heavy chains and the variable light chains.
[0314] [00314] In some embodiments, a population of memory B cells is isolated from a biological sample from an individual that was previously exposed to the antigen of interest. The population of memory B cells is divided into subpopulations that are contacted with an effective amount of CD40L, IL-2 and IL-21 for a sufficient amount of time for the memory B cells to undergo cell division and produce antibodies. The presence or absence of antibodies that specifically bind to the antigen of interest is determined for the subpopulations of memory B cells. It is determined that the subpopulation of memory B cells produced antibodies that specifically bind to the antigen of interest, after which the subpopulation is selected. The nucleic acid sequence encoding the variable heavy and light chain domain of antibodies produced by memory B cells of the selected subpopulation can be determined, and monoclonal antibodies containing the variable regions of heavy and light chain antibodies produced by the selected B cell subpopulation of memory produced. Monoclonal antibodies are assayed for specific binding to the antigen of interest, and antibodies that specifically bind to the antigen of interest are selected, thereby identifying an antibody that specifically binds to the antigen of interest.
[0315] [00315] Methods are also provided for isolating the specific B cell repertoire for an individual target antigen. These methods include isolating a population of memory B cells from an individual who has been exposed to the target antigen, where the memory B cells are CD19 + IgA-IgD-IgMB cells. The population of isolated memory B cells is contacted with an effective amount of IL-21, IL-2 and CD40, and B cells are selected from the population that expresses antibodies that specifically bind the target antigen. These methods may also include isolation of the nucleic acid library encoding the variable heavy chains and the immunoglobulin variable light chains are isolated from nucleic acids. The library of variable heavy chains and variable light chains is then expressed to isolate the repertoire of B cells specific to the individual's target antigen.
[0316] [00316] A humoral repertoire including, but not limited to the complete humoral repertoire, for an entity, such as a pathogen or vaccine, can provide multi-dimensional information (e.g., specificity, affinities, stabilities, gene segment sequence preferences , etc.) that can be considered a "profile" of an individual's humoral response. The quantification of these parameters (Story et al., 2008 PNAS 105 (46): 17902-17907) can be used to correlate with protection from a pathogen or failure to protect. This information can then inform vaccine planning in an iterative model, provide the basis for a multi-parameter diagnostic assay for specific antigens, or be used directly to identify single or multiple neutralizing antibodies against a given pathogen.
[0317] [00317] In some embodiments, antibodies can be characterized. For example, groups of multiparametric data can be collected that describe the characteristics, for example, specificities, affinities, stabilities, isotypes, gene segment sequence preferences, etc. (Story et al., 2008 PNAS 105 (46): 17902-17907. In some representative, non-limiting modalities, the multiparametric profile or data set can be used to inform vaccine planning in an iterative model, provide the basis for a multi-parameter diagnostic assay for specific antigens, or be used directly to identify single or multiple neutralizing antibodies against a given pathogen.
[0318] [00318] Thus, the methods described here can be used to isolate one or more monoclonal antibodies that specifically bind a target antigen, and / or can be used to isolate the B cell repertoire that binds the target antigen in a sample or in an individual. The target antigen may be from a pathogen, including viruses, parasites, fungi and bacteria. In some embodiments, the pathogen is a virus, such as, but not limited to, a virus from one of the following families: Retroviridae (for example, human immunodeficiency virus (HIV); T-cell leukemia virus (HTLV); Picornaviridae (for example, polio virus, hepatitis A virus; hepatitis C virus; enterovirus, human coxsackie virus, rhinovirus, ecovirus; foot-mouth disease virus); Calciviridae (such as strains that cause gastroenteritis); Togaviridae (for example, virus equine encephalitis, rubella virus); Flaviridae (eg dengue virus; yellow fever virus; West Nile virus; St. Louis encephalitis virus; Japanese encephalitis virus; and other encephalitis viruses); Coronaviridae (for example, coronavirus; severe acute respiratory syndrome virus (SARS); Rhabdoviridae (for example, vesicular stomatitis virus, rabies virus); Filoviridae (for example, Ebola virus); Paramyxoviridae (for example, parainfluenza virus, mumps, virus d measles, respiratory syncytial virus (RSV)); Orthomyxoviridae (for example, influenza virus); Bunyaviridae (for example, Hantaan virus; Sin Nombre virus, Rift Valley fever virus; bunya virus, phlebovirus and Nairo virus); Arena viridae (hemorrhagic fever virus; Machupo virus; Junin virus); Reoviridae (for example, reovirus, orbivirus and rotavirus); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvovirus); Papovaviridae (papilloma virus, polyoma virus; BK virus); Adenoviridae (plus adenoviruses); Herpesviridae (herpes simplex virus (HSV) -1 and HSV-2; cytomegalovirus (CMV); Epstein-Barr virus (EBV); varicella zoster virus (VZV); and other herpes viruses, including HSV-6); Poxviridae (smallpox virus, vaccinia virus, syphilis virus); and Iridoviridae (such as African swine fever virus); Filoviridae (for example, Ebola virus; Marburg virus); Caliciviridae (for example, Norwalk virus) and unclassified viruses (for example, the etiological agents of spongiform encephalopathy, the agent of delta hepatitis (thought to be a defective hepatitis B virus satellite); and astrovirus.
[0319] [00319] In other embodiments, the target antigen is an antigen of a bacterium, such as, but not limited to Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (such as M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Streptococcus agalactia, group B), Streptococcus virocis Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus anthracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridia, pneumoniae, Clostridani Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pert enue, Leptospira, or Actinomyces israelli.
[0320] [00320] In other modalities, the antigen is from a fungus, such as Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, or Candida albicans. In other modalities, the antigen is from a parasite, such as, but not limited to, Plasmodium falciparum or Toxoplasma gondii.
[0321] [00321] In some modalities, the antigen is a cancer antigen. The cancer can be a solid tumor or a hematogenous cancer. In particular examples, the solid tumor is a sarcoma or a carcinoma, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, or other sarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, malignancy, colon cancer, malignancy pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary carcinoma, papillary carcinoma, papillary carcinoma , bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumoe, cervical cancer, testicular tumor, bladder carcinoma, or a CNS tumor (such as a glioma, astrocytoma, medulloblastoma, craniofariogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma or r ethinoblastoma).
[0322] [00322] In some instances, hematogenous cancer is leukemia, such as acute leukemia (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous and myeloblastic leukemia, promyelocytic, myelomonocytic, monocytic and erythroleukemia); a chronic leukemia (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (high-grade and indolent forms), multiple myeloma, macroglobulinemia of Waldenstrom, heavy chain disease, myelodysplastic syndrome, hair cell leukemia or myelodysplasia.
[0323] [00323] Tumor antigens are well known in the art and include, for example, carcinoembryonic antigen (CEA), human chorionic gonadotropin (HCG), alpha-fetoprotein (AFP), reactive lectin AFP, (AFP-L3), thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase (hTERT), RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate carcinoma tumor antigen 1 (PCTA-1), melanoma-associated antigen (MAGE), ELF2M, neutrophil elastase , efrinB2 and CD22. The CH2 or CH3 domain molecules can also bind any cancer-related proteins, such as IGF-I, IGF-II, IGR-IR or mesothelin. Antigens associated with additional tumor are provided in the table below: Exemplary tumors and their tumor antigens
[0324] [00324] In some modalities, the antigen is autoantigen. The antigen may be an antigen associated with an autoimmune disease, such as rheumatoid arthritis, juvnil oligoarthritis, collagen-induced arthritis, adjuvant-induced arthritis, Sjögren's syndrome, multiple sclerosis, experimental autoimmune encephalomyelitis, inflammatory bowel disease (for example, inflammatory bowel disease Crohn's disease, ulcerative colitis), autoimmune gastric atrophy, pemphigus vulgaris, psoriasis, vitiligo, type 1 diabetes, non-obese diabetes, myasthenia gravis, Grave's disease, Hashimoto's thyroiditis, sclerosing cholangitis, sclerosing sialodenitis, systemic lupus erythematosus autoimmune, Goodpasture's syndrome, Addison's disease, systemic sclerosis, polymyositis, dermatomyositis, autoimmune hemolytic anemia or pernicious anemia. B cell isolation
[0325] [00325] In several embodiments, a population of cells including memory B cells is obtained from an individual. Typically, a substantially pure population of memory B cells (such as CD19 + IgA-, IgD-, IgM-B cells) is isolated. Typically, the isolated population of cells is enriched by memory B cells.
[0326] [00326] The population of cells including memory B cells can be isolated from a biological sample obtained from an individual of interest. Exemplary biological samples for use with the present methods include bone marrow, spleen, lymph node, blood, for example, peripheral blood. However, the biological sample can also include any other source from which memory B cells can be isolated, including: tissue, biopsy, surgical specimens, fine needle aspirates, autopsy material, and the like. In several modalities, the biological sample is obtained from an individual who has been exposed to an antigen of interest. The individual can be any animal, preferably a mammal or a human. The individual may have a disease or condition including a tumor, an infectious disease, or an autoimmune disease, or have been immunized. In certain aspects, the individual may recover or survive from a disease or condition such as a tumor, an infectious disease, or an immune disease. In other respects, the individual may be under or after prevention and treatment for a disease or condition, such as therapy or therapy for disease by infection, or vaccination. For example, the individual has or has been exposed to an antigen that is an infectious agent, a tumor antigen, a tumor cell, an allergen or an autoantigen. Such an infectious agent can be any pathogenic viruses, pathogenic bacteria, fungi, protozoa, multicellular parasites, and aberrant proteins such as prions, as well as nucleic acids or antigens derived therefrom. An allergen can be any non-parasitic antigen capable of stimulating a type I hypersensitivity reaction in individuals, such as many common environmental antigens.
[0327] [00327] Fluorescence-activated cell classification (FACS) can be used to classify (isolate) cells, such as memory B cell populations, by contacting the cells with an appropriately labeled antibody and classifying the cells based on the binding of the labeled antibody to the cell. In one embodiment, several antibodies (such as antibodies that bind CD19, IgA, IgD, and / or IgM) and FACS classification can be used to produce substantially purified populations of memory B cells. These methods are known in the art, and exemplary protocols are described here.
[0328] [00328] FACS employs a plurality of color channels, and low angle and obtuse light scattering detection channels, and impedance channels, among other more sophisticated levels of detection, to separate or classify labeled or unlabeled cells with a detectable marker. Any FACS technique can be employed as long as it is not detrimental to the viability of the desired cells. (For exemplary FACS methods, see U.S. Patent No. 5,061,620). In one example, a FACSARIA III® cell classifier (Becton Dickinson, Franklin Lakes, NJ) is used. Antibodies can be conjugated to biotin, which can then be removed with avidin or streptavidin attached to a support, or fluorochromes, which can be used with a FACS, to enable cell separation. However, other techniques for differentiating efficacy can be employed to purify and isolate desired populations of cells. The separation techniques must maximize the viability retention of the fraction of the cells to be collected. The particular technique employed, in fact, will depend on the efficiency of separation, cytotoxicity of the method, the ease and speed of separation, and what equipment and / or technical experience is required.
[0329] [00329] Separation procedures include magnetic separation, using magnetic beads coated with antibody, affinity chromatography, cytotoxic agents, or attached to a monoclonal antibody or used in conjunction with complement, and "paniculation," which uses a monoclonal antibody attached to a solid matrix, or other convenient technique. Antibodies attached to magnetic beads and other solid matrices, such as agarose beads, polystyrene beads, hollow fiber membranes and plastic Petri dishes, provide direct separation. Cells that are bound by the antibody can be removed from a cell suspension simply by physically separating the solid support from a cell suspension. The exact conditions and duration of incubation of the cells with the antibodies bound to the solid phase will depend on several factors specific to the system employed. The selection of appropriate conditions, however, is well included in the experience in the technique.
[0330] [00330] The loose cells can then be collected (when negative selection for immunocounting is used) or removed with physiological buffer (when positive selection is benign for immunocounting) after sufficient time has been allowed for the cells expressing a marker of interest (for example, CD19) to bind to antibodies bound to the solid phase. The bound cells are then separated from the solid phase by any appropriate method, depending mainly on the nature of the solid phase and the antibody employed. After a first selection cycle using magnet beads, a second cycle (and additional cycles) can be used to also isolate a cell population of interest.
[0331] [00331] In some embodiments, cells expressing CD19 are separated from other cells by positive selection for cell surface CD19 expression. In one specific, non-limiting example, CD19 + cells are positively selected using FACS by labeling CD19 + cells with a specific CD19 antibody conjugated to a detectable marker, and then using FACS to select cells labeled with the antibody conjugated to the detectable marker. Specific antibodies to CD19 conjugated to detectable markers are known and are commercially available, for example, from BD Bioscience, Franklin Lakes, NJ. In another specific, non-limiting example, CD19 + cells are positively selected by magnetic bead separation, in which magnetic beads are coated with CD19 reactive monoclonal antibodies, and cells that are captured by CD19 reactive immunocounts are collected. The CD19 + cells are then removed from the magnetic beads.
[0332] [00332] In other embodiments, cells that do not express IgA on the cell surface are separated from other cells by the absence of cell surface IgA expression. In a specific, non-limiting example, IgA- cells are negatively selected using FACS by labeling IgA + cells with a specific IgA antibody conjugated to a detectable marker, and then using FACS to select cells that are not labeled with the specific antibody of IgA conjugated to the detectable marker. Specific IgA antibodies conjugated to detectable markers are known and are commercially available, for example, from Jackson ImmunoResearch Laboratories, Inc. West Grove, PA. In another specific non-limiting example, IgA- cells are negatively selected by magnetic bead separation, in which the magnetic beads are coated with monoclonal antibody reactive to IgA and cells that are not captured by immunocounts are collected.
[0333] [00333] In other embodiments, cells that do not express IgD on the cell surface are separated from other cells by the absence of IgD cell surface expression. In a specific, non-limiting example, IgD- cells are negatively selected using FACS by labeling IgD + cells with a specific IgD antibody conjugated to a detectable marker, and then using FACS to select cells that are not labeled with the specific antibody IgD conjugated to the detectable marker. Specific IgD antibodies conjugated to detectable markers are known and are commercially available, for example, from BD Pharmingen, Franklin Lakes, NJ. In another specific non-limiting example, IgD- cells are negatively selected by magnetic bead separation, in which the magnetic beads are coated with monoclonal antibody reactive to IgD and cells that are not captured by the immunocounts are collected.
[0334] [00334] In other embodiments, cells that do not express IgM on the cell surface are separated from other cells by the absence of IgM cell surface expression. In a specific, non-limiting example, IgM- cells are negatively selected using FACS by labeling IgM + cells with a specific IgM antibody conjugated to a detectable marker, and then using FACS to select cells that are not labeled with the specific antibody of IgM conjugated to the detectable marker. IgM specific antibodies conjugated to detectable markers are known and are commercially available, for example, from Jackson ImmunoResearch Laboratories, Inc. West Grove, PA. In another specific non-limiting example, IgM- cells are negatively selected by magnetic bead separation, in which the magnetic beads are coated with monoclonal antibody reactive to IgM and cells that are not captured by immunocounts are collected.
[0335] [00335] In other modalities, cells that express CD19, but do not express IgA, IgD or IgM on the cell surface are separated from other cells by positive selection as to the expression of CD19 cell surface and negative selection as to the expression of IgA, IgD and IgM on the cell surface. Using such methods, CD19 + IgA-IgD-IgM- cells can be collected. In a specific, non-limiting example, CD19 + IgA-IgD-IgM- cells are selected using FACS by labeling cells with four antibodies specific for CD19, IgA, IgD, and IgM, each of which is conjugated to a detectable marker that can be differentially detected using FACS analysis. FACS is then used to classify CD19 + IgA-IgD-IgM- cells. Someone skilled in the art can easily use FACS and establish appropriate ports to isolate CD19 + IgA-IgD-IgM- cells. Contact B cells with CD40L, IL-2 and IL-21
[0336] [00336] In several modalities, isolated memory B cells are contacted with CD40L, IL-2 and IL-21 for a sufficient period of time for the memory B cells to undergo cell division and produce antibodies. In some isolated memory B cell population it is contacted with CD40L, IL-2 and IL-21 by incubating the isolated memory B cell population with CD40L, IL-2 and IL-21 for about 10 to about 15 days. In additional modalities, the isolated population of memory B cells is incubated with CD40L, IL-2 and IL-21 for about 13 days. In several embodiments, B cells are contacted with CD40L, IL-2 and IL-21 for a sufficient period of time for memory B cells to undergo cell division and produce antibodies. In this context, a sufficient period of time can be at least 5 days, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 days, for example, 5-22, 5-21, 10-20, 10-15, 11-16, or 13-15 days. B cells can be contacted with CD40L, IL-2 and IL-21 in the presence of growth medium, such as Iscove's Modified Dulbecco's Medium, (IMDM) with 10% Fetal Bovine Serum, or other growth medium of tissue culture for use in B cell culture. The person skilled in the art is familiar with such a medium.
[0337] [00337] CD40L (also known as CD40 or CD154 ligand) is a protein that is primarily expressed, for example, on activated T cells, but is also found in soluble form. CD40L binds to CD40 on the cell surface of B cells, a binding event that can result in the activation of B cells, differentiation of mature B cells into plasma cells and memory cells, and production of antibodies. The person skilled in the art is familiar with CD40L, and CD40L is commercially available (see, for example, Life Technologies Cat. No. PHP0024). In some embodiments, B cells are contacted with CD40L by culturing the B cells with a cell line that expresses CD40L, such as a CD40L feeder cell line. CD40L feeder cell lines are known in the art (see, for example, Kershaw et al., Cancer Res., 61: 7920-7924, 2001). Exemplary concentrations of CD40L for use in the described methods include 1-2000 international units per milliliter, as well as 100 international units per milliliter. Exemplary nucleic acid and polypeptide sequences for human CD40L are available on the NCBI website as the GENBANK® Accession Number NM_000074.2 and GENBANK® Accession Number NP_000065.1, respectively, (as available on June 13, 2012) which are incorporated here by reference.
[0338] [00338] IL-2 is commercially available (see, for example, Life Technologies, Grand Island, NY, Cat. No. PHP0021). Exemplary nucleic acid and polypeptide sequences for human IL-2 are available on the NCBI website as the GENBANK® Accession Number NM_000586.3 and GENBANK® Accession Number NP_000577.2, respectively (as available on June 13, 2012) which are incorporated here by reference. Exemplary concentrations of IL-2 for use in the described methods include 10-200 international units per milliliter, as well as 100 international units per milliliter.
[0339] [00339] IL-21 is also commercially available (see, for example, Life Technologies, Grand Island, NY, Cat. No. PHC0211). Exemplary nucleic acid and polypeptide sequences for human IL-21 are available on the NCBI website as GENBANK® Accession No. NM021803 and GENBANK® Accession No. AF254069, respectively. These GENBANK® entries are incorporated by reference here. In several embodiments, isolated B cells are contacted with about 10 to 100 ng / ml of IL-21, such as about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ng / ml IL-21, for example, memory B cells can be contacted with 10-100, 20-90, 30-80, 40-70, 40-60 or 45-55 ng / ml IL-21. Exemplary concentrations of IL-2 for use in the described methods include 5 to 500 ng / ml IL-21, such as 50 ng / ml IL-21.
[0340] [00340] In several modalities, contacting the memory B cells with the combination of CD40L, IL-2 and IL21 provides synergy, that is, the amount of cell division and antibody production by the memory B cells contacted with these three molecules is greater in combination than the sum of the effect of that result of using the molecules separately. Selection of B cells that produce the antibody of interest
[0341] [00341] In some embodiments, subpopulations of B cells that have been contacted with CD40L, IL-2 and IL-21 for a period of time sufficient for memory B cells to undergo cell division and produce antibodies are tested for antibody expression that specifically bind to the antigen of interest. Methods for determining whether an antibody binds to an antigen of interest are familiar to the person skilled in the art, and include ELISA and neutralization assays.
[0342] [00342] Three general classes representative of methods of analysis that can be employed (a) antibody capture assays; (b) antigen capture assays; and (c) functional analysis. Combinations can also be used. In antibody capture assays, the antigen can be bound to a solid phase, monoclonal antibodies to be tested are allowed to bind to the antigen, unbound antibodies are removed by washing, and then the bound antibodies are detected, for example, by a secondary reagent such as a labeled antibody that specifically recognizes the antibody. For an antigen capture assay, the antigen can be directly labeled. In one embodiment, monoclonal antibodies to be tested can be bound to a solid phase and then reacted with the optionally labeled antigen. Alternatively, the antibody-antigen complex can be allowed to form by immunoprecipitation before binding the monoclonal antibody to be tested for a solid phase. Since the antibody-antigen complexes are bound to the solid phase, unbound antigen can be washed away and positives can be identified by antigen detection.
[0343] [00343] Several functional analyzes exist to identify monoclonal antibodies with desired activities. Examples include a virus neutralization assay; the agonistic activity assay and blocking assay; keratinocyte monolayer adhesion assay and mixed lymphocyte response (MLR) assay (Werther et al. J. Immunol. 157: 4986-4995 (1996)); tumor cell growth inhibition assays (as described in PCT Publication No. WO 89/06692, for example); antibody-dependent cellular-1-cytoxicity (ADCC) and complement-mediated cytoxicity (CDC) assays (U.S.5,500,362 patent); and hematopoiesis assays (see, WO 95/27062). The class / subclass of the antibodies can be determined, for example, by double diffusion assays; antibody capture on antigen-coated plates; and / or antibody capture on anti-IgG antibodies.
[0344] [00344] To analyze antibodies that bind to a particular epitope on the antigen of interest, a cross-block assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be carried out. Alternatively, epitope mapping, for example, as described in Champe et al. (J. Biol. Chem. 270: 1388-1394 (1995)) can be performed to determine whether the antibody binds an epitope of interest.
[0345] [00345] If it is determined that the subpopulation of memory B cells produces antibodies that specifically bind to the antigen of interest, then additional steps can be considered to isolate a monoclonal antibody that specifically binds to the antigen of interest from the subpopulation. For example, the variable region of the heavy chain and the light chain of the B cell immunoglobulin genes in the subpopulation are isolated, monoclonal antibodies containing the variable region of the heavy chain and the light chains are generated, and the specific binding acidity of the monoclonal antibodies as the antigen of interest is tested (for example, by ELISA or neutralization assay).
[0346] [00346] In certain respects, nucleic acid sequence information can be obtained. Obtaining the nucleic acid sequence information may include determining the nucleic acid sequence. For determining nucleic acid sequences, any nucleic acid sequencing methods known in the art can be used, including high throughput DNA sequencing. Non-limiting examples of high-throughput sequencing methods include sequencing-by-synthesis (for example, 454-sequencing), sequencing-by-ligation, sequencing-by-hybridization, single-molecule DNA sequencing, multiple polony sequencing, nanoporous sequencing, or a combination thereof.
[0347] [00347] In another embodiment the method may include (a) isolating RNA from a subpopulation of B cells; (b) transcribing said RNA into cDNA; (c) amplifying said cDNA said first lake of DNA molecules using a first mixture of oligonucleotides including at least two oligonucleotides capable of amplifying heavy chain variable domain coding regions; (d) amplifying said cDNA according to said lake of DNA molecules using a second mixture of oligonucleotides including at least two oligonucleotides capable of amplifying the light chain variable domain coding regions; and optionally (e) linking specimens from the first and second side of DNA molecules to each other by a DNA encoding said linker region (LR).
[0348] [00348] The cloning of variable regions is a standard procedure generally known in the art and has been described for several species, including humans, non-humans, primates, mice, rabbits, and hens. For review, see Barbas III et al. (eds.), Phage Display - A Laboratory manual, Cold Spring Harbor Press, 2001, in particular, the chapter Andris-Widhopf et al., Generation of Antibody Libraries: PCR Amplification and Assembly of Light- and Heavy-chain Coding Sequences , in this regard. Andris-Widhopf et al. describe oligonucleotide sequences capable of amplifying variable region coding regions (VR coding regions), preferably have heavy chain variable domain coding regions or light chain variable domain coding regions. In addition, oligonucleotides capable of amplifying heavy chain variable domain coding regions or light chain variable domain coding regions, preferably human heavy chain variable domain coding regions or light chain variable domain coding regions, can be designated by the skilled person by comparing known sequences of antibody coding regions that are available from databases such as, for example, Immunogenetics (imgt.cines.fr/), Kabat (Kabatdatabase.com), and Vbase (vbase. mrc-cpe.cam.ac.uk/), and identifying the appropriate consensus frameworks for initiator planning. Oligonucleotides capable of amplifying heavy chain variable domain coding regions or light chain variable domain coding regions, where primers may include restriction sites suitable for cloning amplified products and where oligonucleotides also encode a linker region are known in the art. Additional strategies for amplifying and cloning variable domains are described in Sblattero and Bradbury (1998) Immunotechnology 3: 271- 278 and Weitkamp et al. (2003), J. Immunol. Meth. 275: 223-237.
[0349] [00349] In some examples, the variable region of the heavy chain and the light chain of the immunoglobulin genes can be amplified by RT-PCR using known methods (see, for example, Tiller et al., J. Immunological Methods, 329: 112- 124, 2008). The PCR product including VH or VL region DNA can be cloned into the corresponding Igγ1, Igκ and Igλ expression vectors, which can be used to cotransfect into a permissive cell line (such as 293T cells) for expression and production of monoclonal antibody. In some instances, full-size IgG1 can be purified using standard procedures, and then tested for binding to the antigen of interest (for example, using ELISA or neutralization assay). The person skilled in the art will understand that expression vectors can be expressed in any permissive cell line or individual for testing (for example, in a mammalian cell line, a plant cell line, or using a viral expression vector for expression in a cell line or organism, and proteins including the sequence encoded by VH or VL region DNA can be produced synthetically for testing. Various Modalities Involving Methods for Identifying Antibodies of Interest
[0350] [00350] Several modalities include a method for producing a monoclonal antibody that specifically binds to a target antigen, wherein the method includes: (a) isolating a memory B cell population from an individual who has been exposed to the target antigen , where memory B cells are CD19 + IgA-IgD-IgM-B cells; (b) contact of the isolated memory B-cell population with an effective amount of IL-21, IL-2 and CD40; (c) isolation of amino acid molecules from the isolated population of B-cell memory; (d) amplification of nucleic acids encoding the variable heavy chains and the variable light chains of the nucleic acids; (e) expression of the variable heavy chains and the variable light chains of the nucleic acids to produce antibody molecules of the variable heavy chains and the variable light chains; and (f) selecting the monoclonal antibody that specifically binds to the target antigen.
[0351] [00351] Some modalities include a method for isolating the specific B cell repertoire from an individual's target antigen, including: (a) isolating a memory B cell population from an individual who has been exposed to the target antigen, in that memory B cells are CD19 + IgA-IgD-IgM-B cells; (b) contact of the isolated memory B-cell population with an effective amount of IL-21, IL-2 and CD40; and (c) selection of B cells from the population that expresses antibodies that specifically bind the target antigen, thereby isolating the repertoire of B cells specific to the individual's target antigen.
[0352] [00352] In several modalities, the target antigen is an antigen of a pathogen, such as a virus, a fungus, a parasite, or a bacterium. The antigen may be from a virus, for example, human immunodeficiency virus (HIV), such as HIV-1. In some embodiments, the antigen may be HIV-1 gp41. In some modalities where the target antigen is an HIV antigen, the individual is that individual infected with HIV, such as HIV-1. In other modalities, the target antigen is a cancer antigen. In some such modalities, the individual is an individual with cancer. In other modalities, the target antigen is a self-antigen. For example, in some embodiments, the target antigen is a toxin, such as a bacterial toxin, for example, anthrax toxin. In additional embodiments, the target antigen is an antigen included in a vaccine. The person skilled in the art will appreciate that the target antigen can be any antigen to which an individual is capable of producing a B cell response that results in the production of memory B cells that produce antibody that specifically binds to the target antigen.
[0353] [00353] In some embodiments, the isolated population of B-cells in memory is representative of the B-cell repertoire in the individual that is specific for the target antigen.
[0354] [00354] In some modalities of the described methods, contact of the isolated population of memory B cells with CD40L includes incubating the isolated memory B cells with a feeder cell that expresses the CD40L.
[0355] [00355] In additional modalities of the described methods, contacting the isolated memory B cell population isolated with CD40L, IL-2 and IL-21 includes incubating the isolated memory B cell population with CD40L, IL-2 and IL-21 for about 10 to about 15 days, such as about 13 days. EXAMPLES
[0356] [00356] The following examples are provided to illustrate the particular characteristics of certain modalities, however the scope of the claims should not be limited by those exemplified aspects. Example 1 Isolation and Characterization of Monoclonal Antibodies Specific to MPER Widely Neutralizing
[0357] [00357] This example illustrates the isolation of HIV-1 gp41-specific antibody characterization from an HIV-1 infected individual.
[0358] [00358] Summary. Characterization of human monoclonal antibodies provided considerable insight into mechanisms of broad neutralization of HIV-1. This example described the isolation of an antibody specific for (MPER) from the proximal outer region of the HIV-1 gp41 membrane, called 10E8, which neutralizes ~ 98% of the viruses tested. An analysis of sera from 78 healthy HIV-1-infected donors showed that 27% contained MPER-specific antibodies and 8% contained 10E8-like specificities. Unlike other neutralizing MPER antibodies, 10E8 did not bind to phospholipids, was not autoreactive, and bound to cell surface Env. The 10E8 structure in complex with the complete MPER revealed a site of vulnerability including a narrow extent of highly conserved hydrophobic gp41 residues and a critical Arg / Lys just before the transmembrane region. Analysis of resistant HIV-1 variants confirmed the importance of these residues for neutralization. Highly conserved MPER is a target for potent, non-autoreactive neutralizing antibodies, which suggest that HIV-1 vaccines should aim to induce antibodies to this HIV-1 Env region.
[0359] [00359] Introduction. Inducing an antibody response capable of neutralizing several HIV-1 isolates is a critical goal for vaccines that protect against HIV-1 infection. Potentially the biggest obstacle to achieving this goal is the extraordinary diversity that develops in the target of antibody neutralization, the envelope glycoprotein (Env). Although vaccines have thus failed to induce broadly neutralizing antibody responses, there are examples of patients chronically infected with sera that neutralize highly diverse HIV-1 isolates. These individuals provide evidence that it is possible for the human antibody response to neutralize highly diverse strains of HIV-1, although the mechanisms by which such responses are induced or mediated remain incompletely understood (Haynes et al., Nat Biotechnol 30, 423-433 , 2012; Walker et al., Curr Opin Immunol 22, 358-366, 2010).
[0360] [00360] Recently, isolation and characterization of human monoclonal antibodies from cells of chronically infected patients has provided considerable advances in understanding the specificities and mechanisms underlying the widely neutralizing responses to HIV-1. Env exists on the virion and infected cell surface as a trimer of heterodimers prepared from subunits of gp120 and gp41s. For a while, only a small number of broadly neutralizing monoclonal antibodies (mAbs) were isolated, consisting of an antibody that binds to the CD4 binding site in gp120 (b12), the one that binds a glycan configuration on the outer domain of gp120 (2G12) and three that connect the proximal outer region to the membrane (MPER) in gp41 (2F5, Z13e1, and 4E10; Zwick et al., J Virol 75, 10892-10905, 2001; Burton et al., Science 266, 1024-1027, 1994; Muster et al., J Virol 67, 6642-6647, 1993). More recently, considerably broader and more potent antibodies have been discovered, which target the envelope protein's CD4 binding site (for which VRC01 is a prototype; Bonsignori et al., J Virol 86, 4688-4692, 2012; Wu et al, Science 333, 1593-1602, 2011; Scheid et al., Science 333, 1633-1637, 2011; Wu et al., Science 329, 856-861, 2010) and glycan containing regions from the V1 / V2 regions and gp120 V3 (for which PG9 and PGT128 are prototypes; Walker et al., PLoS Pathog 6, e1001028, 2010; Walker et al., Nature 477, 466-470, 2011; Bonsignori et al., J Virol 85, 9998-10009, 2011; Walker et al., Science 326, 285-289, 2009). The specifics of these new antibodies are the provision of important information with respect to the antigen targets in the Env to which the humoral immune response can be directed to mediate wide and potent neutralization. However, evidence for these specificities in many chronically infected patients within the cohort is deficient, suggesting that wide and potent neutralization can be mediated by other specificities.
[0361] [00361] This example describes the isolation of a broad and potent MPER from gp41 specific human mAb, 10E8, from an HIV-1 infected individual with high neutralization titers. 10E8 is among the broadest and most potent antibodies, therefore, well described, and lacks many of the features previously thought to limit the usefulness of specific MPER antibodies in vaccines or passive therapies, including lipid binding and autoreactivity. In addition, the crystalline structure of 10E8, together with biochemical linkage studies, demonstrate that the amplitude of 10E8 is mediated by its unique way of recognizing a structurally conserved site of vulnerability within the gp41 MPER.
[0362] [00362] Isolation and neutralization properties of 10E8. To understand the specificities and binding characteristics that underlie a broadly neutralizing antibody response, techniques have been developed that have enabled the isolation of human monoclonal antibodies without prior knowledge of specificity (Walker et al., Science 326, 285-289, 2009). Donor serum, N152, exhibited neutralizing amplitude and potency at the top of 1% of the cohort against a panel of 20 hybrid clade pseudoviruses (FIG. 17; Doria-Rose et al., J Virol 84, 1631-1636, 2010) . Peripheral blood CD19 + IgM-IgD-IgA memory B cells from this patient were classified and expanded for 13 days with IL-2, IL-21, and feeder cells expressing CD40 ligand. Supernatants from ~ 16,500 B cell cultures were assayed and IgG genes from wells with neutralizing activity were cloned and restated (Tiller et al., Journal of immunological methods 329, 112-124, 2008) and two new antibodies (10E8 and 7H6) were isolated.
[0363] [00363] Nucleotide sequence analysis of DNA encoding 10E8 and 7H6 revealed that both were IgG3 antibodies and were somatic variants of the same IgG clone. These antibodies were derived from germline genes of IGHV3-15 * 05 and IGLV3-19 * 01, and were highly somatically mutated into variable genes from both heavy chain (21%) and lambda light chain (14%) compared to the germline. These antibodies also had a heavy chain complementarity determining region loop (CDR H3) composed of 22 amino acids (FIG. 1A). The heavy chains of 10E8 and 7H6 were identical and there were only two residue differences in the light chain (FIG. 6).
[0364] [00364] To assess the neutralization activity of the clonal variants, they were initially tested against 5 Env pseudoviruses (FIG. 17A), and 10E8 of mAb was selected for another study. To determine whether the neutralization activity of 10E8 was representative of the total neutralization specificity present in patient N152 donor serum, the neutralization panel was expanded to 20 Env pseudoviruses, and 10E8 was tested in parallel with N152 donor serum. Although there are some similarities in the pattern of neutralization of highly resistant variants, a correlation of the IC50 of neutralization of 10E8 of mAb and ID50 of serum of N152 was not statistically significant (p = 0.11; figures 7 and 17B). This finding suggests that although 10E8 may play an important role, the total amplitude of neutralization by N152 serum is likely to be mediated by an amalgam of antibodies similar to 10E8 or others.
[0365] [00365] To compare the potency and amplitude of neutralization of 10E8 with other widely neutralizing anti-HIV-1 antibodies, 10E8 was then tested on a panel of 181 Env pseudoviruses isolated in parallel with 4E10, 2F5, VRC01, NIH45- 46, 3BNC117, PG9, and PG16 (Figures 1B and Figures 17C-17F). At an IC50 below 50 µg / ml, 10E8 neutralized 98% of the tested pseudoviruses compared to 98% for 4E10 and 89% for VRC01. However, at an IC50 below 1 µg / ml, 10E8 neutralized 72% of the viruses tested compared to 37% for 4E10. The IC50 values of the mean and geometric mean for 10E8 were below 1 µg / ml. In this way, 10E8 mediates broad and potent neutralization against a wide range of viruses and the potency is comparable to some of the best monoclonal antibodies available.
[0366] [00366] Specificity and epitope binding of 10E8. To map the epitope of the 10E8 antibody, binding to different Env sub-regions by enzyme-linked immunosorbent assay was tested (ELISA). 10E8 strongly linked to gp140, gp41, and the specific 4E10 MPER peptide, but not to gp120 (FIG. 2A). To also map the 10E8 epitope within the MPER, the binding of 10E8 to overlapping peptides that match the specificities of 2F5 (656-671), Z13e1 (666-677), and 4E10 (671-683) was examined. 10E8 linked to total MPER and specific 4E10 peptides, but not specific 2F5 or Z13e1 peptides. Within the 4E10 epitope, when a peptide with a truncated C-termination was tested, 4E10.19 (671-680), 10E8 binding was weakened considerably, suggesting that the three terminal amino acids of the MPER, Tyr681, Ile682, and Arg683, were crucial for the binding of 10E8 (FIG. 8A). Consistent with these results, only the total MPER and specific 4E10 peptides blocked the 10E8-mediated neutralization of the chimeric C1 virus, which contains the HIV-2 Env with the HIV-1 MPER (FIG. 8B). Used together, these data suggest that the minimum 10E8 epitope is located within residues 671-683 of the MPER, although additional contact to the amino termination of the MPER cannot be excluded.
[0367] [00367] To more accurately map the 10E8 epitope, a panel of mutant alanine peptides examining residues of MPER 671-683 was used to block the 10E8 neutralization of the chimeric C1 virus in a TZM-bl assay (FIG. 2B ; Brunel et al., J Virol 80, 1680-1687, 2006). For these assays, the base peptide was the MPE peptide of 4E10.22 (CNWFDITNWLWYIRKKK; SEQ ID NO: 14) with the indicated alanine substitutions. MPER peptides with alanine substitutions to Trp672, Phe673 or Thr676 did not block the neutralization of 4E10 or 10E8, suggesting that these residues are critical for binding to both 4E10 and 10E8. Residue Asn671 and residue Arg683, both of which are not required for binding to 4E10, have been found to be critical for the binding and neutralization of 10E8 (Figures 2B and 18). The ability of 10E8 to neutralize HIV-1JR2 pseudoviruses with alanine substitutions in MPER 660-683 residues was also tested (FIG. 19). Consistent with the effects of alanine substitutions on peptide binding, residues Asn671 and Arg683 were critical for neutralization of 10E8, but not 4E10. Individual alanine substitutions at residues 671-673, 680 and 683 resulted in reduced neutralization sensitivity to 10E8 more evident at the IC90 level instead of the IC50 level. Although the mechanism for this phenomenon is not clear, a similar effect has been observed previously when mutations in MPER cause partial resistance to 4E10 (Zwick et al., J Virol 79, 1252-1261, 2005). Employed together, these results suggested that 10E8 recognized a new epitope, which overlaps with the well-known epitopes of 4E10 and Z13e1, but differs in a critical dependence on binding to Asn671 and Arg683, the last residue of the RERM.
[0368] [00368] Whether the neutralizing potency greater than 10E8 compared to other antibodies of MPER was a result of greater affinity of binding to MPER, it was then investigated. Capture of a biotinylated peptide that covers the total MPER (656-683) for a surface plasmon resonance chip, allowed the Fabs binding kinetics 10E8, 2F5 and 4E10 to be examined. In contrast to its higher neutralizing potency, the KD of 10E8 for this MPER peptide was weaker than that of 2F5 and 4E10; 17 nM for 10E8 versus 3.8 nM for 2F5 and 0.74 nM for 4E10 (FIG. 9). Therefore, the affinity of 10E8 for MPER in a soluble peptide format did not explain its greater neutralization potency compared to other specific MPER antibodies.
[0369] [00369] Prevalence of antibodies similar to 10E8. The prevalence of MPE-specific and 10E8-like neutralizing antibodies in the HIV-1-infected donor cohort was then investigated. Next, 78 sera from the cohorts with an ID50 neutralization> 100 against at least one pseudovirus in a mini-panel of 5 viruses were selected (Doria-Rose et al., J Virol 84, 1631-1636, 2010). The median time since diagnosis of these donors was 13.5 years, the median CD4 count was 557 cells / l, median plasma HIV RNA - 5573 copies / ml, and they were not receiving antiretrovirals. Neutralization against the HIV-2 / HIV-1 chimera C1 was tested (FIG. 20). Of 78 sera, 21 exhibited neutralizing activity against the HIV-2 / HIV-1 C1 virus (FIG. 21). To map the region that was targeted by these sera, neutralization was measured using 7 HIV-2 / HIV-1 chimeras containing subdomains of the MPER (FIG. 20; Gray et al., J Virol 81, 6187-6196, 2007) . Of the 21 sera with neutralization activity against the entire MPER, 8 exhibited a neutralization pattern similar to that observed for 10E8, which linked the neutralization of only those chimeric HIV-2 / HIV-1 viruses that contained the terminal residue of the MPER Arg683 ( C4, C4GW and C8; FIG. 21). To also confirm these results, peptides corresponding to the different portions of the MPER to block neutralization of sera from the HIV2 / HIV-1 C1 chimera were used (FIG. 22). Of the 8 sera found to have a similar pattern to 10E8 based on the neutralization of the chimeras, 3 were blocked by peptides consistent with 10E8-like activity. An additional 3 of 8 10E8-like sera were blocked by peptides in a pattern consistent with a combination of 10E8 and Z13e1-like antibodies. The 6 patients whose sera had activity similar to 10E8 did not differ from the remaining 72 patients with respect to the clinical course or HIV neutralization (legend; FIG. 10, legend). In general, 27% of the tested patient sera exhibited antiMPER neutralizing activity. This prevalence is considerably higher than that observed in previous work, possibly related to the selection of donors with known neutralization activity (Gray et al., J Virol 83, 8925-8937, 2009; Tomaras et al ,. J Virol 85, 11502- 11519, 2011; Morris et al., PLoS ONE 6, e23532, 2011; Gray et al., J Virol 83, 11265-11274, 2009). In addition, 8% of the tested sera had antibodies similar to 10E8 (FIG. 10), suggesting that antibodies similar to 10E8 are not uncommon.
[0370] [00370] 10E8 autoreactivity analysis. A common property of MPER mAbs previously characterized 2F5 and 4E10 is that they interact with autoantigens (Haynes et al., Science 308, 1906-1908, 2005). In addition, binding to both the cell membrane and the Env trimer is believed to be important for optimal neutralization by these antibodies and this autoreactivity can be an obstacle to the elicitation of similar antibodies by a vaccine (Haynes et al., Science 308, 1906 -1908, 2005; Alam et al., Proceedings of the National Academy of Sciences of the United States of America 106, 20234-20239, 2009). Surface plasmon resonance analysis showed that 10E8 does not bind to anionic phospholipids, such as phosphatidyl choline-cardiolipin liposomes (PC-CLP) and phosphatidyl choline-phosphatidyl serine (PC-PS) (FIG. 3A). 10E8 also did not bind to HEP-2 epithelial cells, unlike 2F5 and 4E10 which bound in a cytoplasmic and nuclear pattern (FIG. 3B). In addition, 10E8 did not bind to autoantigens, such as Sjogren's syndrome A and B antigens, Smith's antigen, ribonucleoprotein, scleroderma antigen 70, Jo1 antigen, centromere B and histone (FIG. 23). Used together, these results suggest that 10E8, unlike other MPER antibodies, is not autoreactive.
[0371] [00371] 10E8 virion accessibility. The 2F5 and 4E10 antibodies have been shown to bind relatively poorly to the peak of HIV-1 Env on the surface of infected cells or to free cell virions, and react more efficiently after CD4 receptor Env compromise (Chakrabarti et al., J Virol 85, 8217-8226, 2011). Binding to the life-sized Env peaks cleaved in HIVJRFL transfected cells was measured (FIG. 11A). Although 10E8 binds less efficiently than other antibodies such as VRC01 or F105, where accessibility is not a result, it does bind more efficiently than 2F5 or 4E10. Contrary to the results of alanine substitution, a mutation in the region of 4E10 (F673S) in life-sized HIVJRFL Env peaks enhanced the binding of 10E8 although the mechanism remains unclear. A mutation in the 2F5 region (K665E) had no influence on the binding of 10E8. These data suggest that 10E8 has modestly greater access to the MPER epitope on the cell surface than 2F5 or 4E10.
[0372] [00372] To assess cell-free virus binding, virions were incubated with antibody, the loose antibody removed, and neutralization tested (Chakrabarti et al., J Virol 85, 8217-8226, 2011; Frey et al., Proceedings of the National Academy of Sciences of the United States of America 105, 3739-3744, 2008; Rathinakumar et al., J Virol 86, 1820-1831, 2012). During washing, antibodies that cannot access their Env target over free virions will be largely removed and, therefore, neutralization will be decreased. As a control, the neutralization of the HXBc2 isolate was not diminished by washing, as the MPER region is accessible in this isolate adapted in the laboratory (Chakrabarti et al., J Virol 85, 8217-8226, 2011). Washing also had little impact on the neutralization of JRFL by VRC01. Consistent with previous work, neutralization of 2F5 and 4E10 from most of the virus isolates tested was substantially decreased after washing (FIG. 11B). Unlike 2F5 and 4E10, the wash had a lesser effect on the 10E8 neutralization of most tested viruses, as measured by the area under the curve or analysis of the amount of change in neutralization at a fixed inhibitory concentration (FIG. 11C). Although 10E8 is not fully able to access its epitope at the native viral peak similarly to VRC01, under the most experimental conditions tested it was more able to access its epitope than 2F5 or 4E10.
[0373] [00373] 10E8-gp41 complex structure. To provide an atomic level understanding of the interaction of 10E8 with HIV-1, the 10E8 antigen (Fab) binding fragment in complex with the peptide spanning the entire 28 residue gp41 MPER (residues 656-683) was crystallized. Diffracted monocyclic crystals for 2.1 Å resolution, and structure and refinement solution for Rcryst = 18.01% (Rfree = 21.76%) revealed two complexes in the asymmetric unit (until now referred to as complexes 1 and 2) (FIG. 24). In general, 10E8 attached to one side of the MPER peptide, which formed two helices, each 15-20 Å in length and oriented 100⁰ with respect to each other (FIG. 4A). Electronic density was observed for the entire MPER, ranging from Asn656-Arg683 (Leu660-Arg683 for complex 2), with the highest degree of ordered density observed from the Trp666 residue within an N terminal helix through Arg683 of the terminal helix C (FIG. 12). The analysis of main chain dihedral angles (FIG. 25) indicated that the N-terminal α-helix extends from the Asn657 to Ala667 residue, is retained in a 310 helix between the Ser668 and Leu669 residues, before returning to the residues Trp670 and Asn671. The C-terminal α-helix, buffered by Asn671, starts at the Trp672 residue and extends to the Arg683 residue, the final MPER residue (FIG. 4A, B).
[0374] [00374] The 10E8 antibody contacts the gp41 MPER primarily through its heavy chain, although the crucial contacts are also mediated by the light chain CDR L3 (Figures 4C and 26-28). Three predominant loci of interaction are observed between the antibody and gp41 (Figures 29-30): One between the residues of the CDR H3 loop tip and the tip of a C-terminal helix of the peptide, a second between the residues of the CDR loop H3 CDR H2 and residues from the peptide articulation region, and a third at the connection of the three heavy chain CDR loops and the light chain CDR L3, which form a hydrophobic slit that retains residues from the beginning of the MPER C-terminal helix ( FIG 4B).
[0375] [00375] 10E8-gp41 interface. To complement the results observed for the highly conserved 10E8 epitope mutagenesis (Figures 4D and 18), each residue of the 10E8 paratope, as determined from the crystal structure, was individually mutated to alanine and the resulting 25 variants of 10E8 evaluated for affinity for a soluble MPER peptide. In general, the most pronounced effects of alanine mutations on the binding affinity of 10E8 for a soluble MPER peptide occurred within the residues of the H3 CDR loop, although mutations within the hydrophobic slit also showed substantial effect (Figures 4E, 13 and 31). 10E8 residues identified by alanine scan as critical for interaction with gp41 extended from the crack all the way to the tip of the CDR H3 (FIG. 4E) and were reflected by a corresponding stretch of gp41 residues that substantially affected the binding of 10E8 when mutated to alanine (FIG. 4F).
[0376] [00376] The same 10E8 alanine mutation panel was tested for neutralization potency against a panel of five pseudoviruses-Env that included Tier 1 and Tier 2 viruses (FIG. 32). Similar to the binding data, 10E8 CDR H3 residues had dramatic effects on neutralization, as did residues from the hydrophobic slit (FIG. 4g). Generally, KDs of paratope mutants correlated with neutralization (FIG. 14). Main chain interactions (on both 10E8 and gp41) also contribute to the interface, especially between the 10E8 CDR H2 and the MPER hinge region, although these are silent in alanine scan analyzes. In general, 10E8 uses a narrow strip of residues (~ 20 x 5 Å) that extends from CDR H1 and H2 and extends across most of CDR H3 to recognize a series of highly conserved hydrophobic gp41 residues, and a critical charged residue, Arg / Lys683, which occurs just before the transmembrane region (FIG. 4F, H).
[0377] [00377] A determinant of neutralization of conserved gp41. Several structures of neutralizing antibody complexes with the gp41 MPER have been reported previously, including those for antibodies 2F5, Z13e1 and 4E10 (FIG. 15A; Julien et al., J Mol Biol 384, 377-392, 2008; Cardoso et al ., J Mol Biol 365, 1533-1544, 2007; Cardoso et al., Immunity 22, 163-173, 2005; Ofek et al., J Virol 78, 10724-10737, 2004; Pejchal et al., J Virol 83 , 8451-8462, 2009). The MPER adopts divergent loop conformations when connected by 2F5 and Z13e1 and an α-helix when connected by 4E10. Comparison of 2F5, Z13e1, and 4E10 epitopes with gp41 bound to 10E8 revealed that only the 4E10 epitope has a similar secondary structure, with overlap producing an RMSD of 2.49 Å for all atoms of residues 671-683 and 0.98 Å for the main chain atoms (Figures 15B and 33).
[0378] [00378] To compare the recognition of 10E8 and 4E10, their epitope angles methods were examined. As shown in figures 15C-15F, the recognized MPER propeller alignment places 10E8 and 4E10 in similar general spatial positions. The relative orientations of the recognized helix and the heavy and light chains of the two antibodies, however, differ dramatically. With 10E8, the C terminal helix is perpendicular to the plane, cutting heavy and light chains (Figures 15C, E); with 4E10, the recognized propeller is at the interface between heavy and light chains (FIG. 15D, F). Perhaps relevant to this, 10E8 uses CDR loops almost exclusively in its gp41 recognition, while 4E10 incorporates substantial β-filament interactions with gp41 at the interface between heavy and light chains.
[0379] [00379] The divergent modes of recognition of 10E8 and 4E10 of the conserved C-terminal MPER helix result in a substantial difference in the proportion of the recognized helical face: 10E8 contacts slightly one third of the helical face, at the same time that 4E10 contacts above the half (FIG. 15G, 15H and 34-35). The smaller 10E8 contact surface may provide an explanation for the reduced recognition of lipid surfaces by 10E8 versus 4E10 - providing an explanation based on the potential structure for reduced 10E8 autoreactivity.
[0380] [00380] Sequence variation and neutralization of 10E8. To place the specificity and structural data in the context of known variation of the MPER, viral sequences with resistance to neutralization by 10E8 were analyzed (FIG. 5A). Of the 183 viruses tested, only three were highly resistant to 10E8 with IC50> 50 μg / ml. Each of these viruses had substitutions in the positions found to perform the neutralization by alanine scan (Asn671, Trp672, Phe673, and Trp680). Plasma virus from patient N152, from whom 10E8 has been cloned, is also likely to be resistant to 10E8-mediated neutralization (Wu et al., J Virol 86, 5844-5856, 2012). Sequence analysis of plasma viral RNA revealed rare substitutions at the Trp680 and Lys / Arg683 positions (FIG. 5A). These residues are typically highly conserved with variation occurring only in 1.17% of 3,730 HIV Env strings in the Los Alamos Database (hiv.lanl.gov). When substitutions for the 3 resistant viruses and patient viruses were placed at the base of the sensitive JR2 virus, substitutions in Asn671Thr, Trp672Leu, and Phe673Leu had a modest effect on the IC50 but raised the IC80 above 20 g / ml. In structural analysis, direct contacts with 10E8 were not observed at position 671 suggesting that the effects on the neutralization of Thr or Ala substitutions at this position are mediated by conformational or other effects within gp41. The combination of Trp672Leu and Phe673Leu provided high level resistance at IC50 and IC80 levels. Changes that correspond to the patient's dominant circulating virus had a similar effect. Although Lys / Arg683Gln alone conferred resistance at the IC80 level, together Trp680Arg and Lys / Arg683Gln resulted in greater resistance to 10E8 (FIG. 5A). When used in conjunction with the 10E8 parotope analysis, these data suggest that in addition to Trp672, Phe673, and Trp680 found in the 4E10 epitope, the residue bound to additional 10E8 Lys / Arg683 is critical for neutralization. In addition to other differences in binding based on the structural analyzes mentioned above, it is possible that the additional potency of 10E8 compared to 4E10 along with naturally occurring viral variants can be mediated by linking highly conserved residues Trp680 and Lys / Arg683 that interact directly with the 10E8 CDRH3.
[0381] [00381] Discussion. 10E8 is a broad and potent neutralizing antibody with important implications for efforts to stimulate such antibodies with vaccines. Previous MPER antibodies were somewhat limited in potency, and had a more limited ability to access MPER in Env from primary isolates. In addition, lipid binding and autoreactivity were believed to be characteristics of MPER antibodies and important obstacles to their elicitation by vaccines. However, 10E8 lacks each of these characteristics. In addition, antibodies with similar specificity were not uncommon in the chronically infected cohort. This suggests that antibodies similar to 10E8 were not deleted from the repertoire because of autoreactivity. These results also suggest that antibodies similar to 10E8 may be elevated in a greater fraction of people not infected with HIV receiving a vaccine designed to elicit these antibodies without the chronic HIV infection B cell defects. Planning for such a vaccine will likely require not only the presentation of an intact 10E8 epitope, but also the use of a sufficiently immunogenic platform to control the evolution of antibodies similar to 10E8.
[0382] [00382] The extraordinary amplitude and power of 10E8 seem to be mediated by its ability to bind to highly conserved residues within MPER. Although the 10E8 epitope overlaps with those of known mAbs, such as 4E10, it differs in surface recognition, approach angle, lipid binding, and autoreactivity. Alanine scanning, structural analysis, and paratope analysis, each indicating that 10E8 makes crucial contact with highly conserved residues Trp672, Phe673, Trp676 and Lys / Arg683. The extraordinary breadth of some potent mAbs, for example, that bind to the CD4 binding site, is believed to be conferred by blocking a functionally important site that is critical for viral entry. Whether 10E8 impairs the Env function or simply acts by binding highly conserved waste remains to be determined. However, the breadth and potency of 10E8 demonstrates a conserved site of vulnerability of gp41 (FIG. 5B) that is an important target antigen for HIV neutralization and that probably will revive interest in HIV vaccine planning based on MPER. Methods Summary of Methods.
[0383] [00383] Peripheral blood CD19 + IgM-IgD-IgA-B cells were classified by flow cytometry, seeded into 4 cells per well, and expanded with cytokines and feeder cells. B cell culture supernatants were selected by microneutralization against pseudoviruses HIVMN.03 and HIVBal.26. IgG genes from wells with neutralizing activity were cloned and expressed again in 293T cells. Range of neutralization activity was confirmed against a panel of isolated 181 Env pseudovirus. Specificity was determined by scanning peptides of alanine and mutant Env pseudovirus. Lipid binding and 10E8 autoreactivity were measured by surface plasmon resonance, indirect HEp-2 cell immunofluorescence and bead arrangements. Binding of HIV envelopes to 293 transfected cells was detected by flow cytometry. Following preincubation with antibody, the impact of washing virions before infecting TZM-bl cells was used to measure access to viral MPER. The frequency of HIV-1 + sera with a given specificity was measured by the ability to neutralize HIV-2 / HIV-1 chimeras containing portions of the MPER. Successful cocrystallization of 10E8 with gp41 was obtained when a peptide comprised the entire residue 28 MPER gp41 (residues 656-683). The structure determination revealed two complexes in the crystal asymmetric unit. Analysis of differences between the two complexes allowed essential interactions to be discerned. The paratope, as defined by residues in the antibody that showed reduced solvent accessibility when complexed by gp41, was subjected to a comprehensive alanine scan, with each of the 25 alanine mutants 10E8 evaluated by SPR for gp41 recognition and by neutralization on a panel. 5 pseudotyped viruses. The patient's plasma viral RNA sequence was derived using limiting dilution RT-PCR.
[0384] [00384] Study patients. Peripheral blood and plasma mononuclear cells (PBMC) were selected from HIV-1 infected patients enrolled at the National Health Institute under a clinical protocol approved by the Investigational Inspection Examining Board at the National Institute of Allergy and Infectious Diseases (NIAID-IRB). All signed participants informed consent approved by the NIAID-IRB. The enrollment criteria were as follows: having a detectable viral load, a stable CD4 T cell count above 400 cells / μl, being diagnosed with HIV infection for at least 4 years, and treating off ARV for at least 5 days . Based on current locations and previous residences, all patients were presumed to be infected with clade B virus. Donor N152 was selected for B cell classification and antibody generation because its serum neutralization activity is among the most potent and broad in the group. He is a slow progressor based on the criteria described above (Migueles et al., Immunity 29, 1009-1021, 2008). At the time of leukapheresis, he was infected with HIV-1 for 20 years, with CD4 T cell counts of 325 cells / μl, plasma HIV-1 RNA values of 3,811 copies / ml and was not on antiretroviral treatment.
[0385] [00385] Memory B cell staining, antibody classification and cloning. Staining and simple cell classification of memory B cells were performed as follows. HIV-1-donor N152 PBMCs were stained with antibody cocktail consisting of anti-CD19-PE-Cy7 (BD Bioscience), IgA-APC (Jackson ImmunoResearch Laboratories Inc.), IgD-FITC (BD Pharmingen), and IgM -PE (Jackson ImmunoResearch Laboratories Inc.) at 4 ° C in the dark for 30 min. The cells were then washed with 10 ml of PBS-BSA buffer and resuspended in 500 μl of PBS-BSA. 66,000 CD19 + IgA-IgD-IgM memory B cells were sorted using a FACSAria III cell sorter (Becton Dickinson) and resuspended in IMDM medium with 10% FBS containing 100 U / ml IL-2, 50 ng / ml of IL-21 and 1x105 / ml of irradiated 3T3-msCD40L feeder cells (Kershaw et al., Cancer Res 61, 7920-7924, 2001). B cells were seeded in 384-well microtiter plates at a density of 4 cells / well in a final volume of 50 μl. After 13 days of incubation, 40 μl of culture supernatants from each well were collected and selected for neutralization activity using a high-yield microneutralization assay against HIV-1MN.03 and HIV-1Bal.26. B cells in each well were lysed with 20 μl of lysis buffer containing 0.25 μl of RNase inhibitor (New England Biolabs Inc.), 0.3 μl of 1 M Tris, pH8 (Quality Biological Inc.) and 19 , 45 µl of H2O treated by DEPC. B cell plates were stored at -80 ° C.
[0386] [00386] The variable region of the heavy chain and the light chain of the immunoglobulin gene were amplified by RT-PCR of the wells that were positive in both the HIV-1MN.03 and HIV-1Bal.26 neutralization assays. The cDNA product was used as a template in the PCR reaction. In order to amplify the highly somatically mutated gene immunoglobulin, two sets of primers as described previously (Tiller and another, Journal of immunological methods 329, 112-124, 2008) were used in two independent PCRs. A set of primers consisted of the forward and reverse primers specific for the leader region and constant region of IgH, Igκ or Igλ, respectively. The other set of primers consisted of the front primer mixes specific for FWR1 and respective reverse primers specific for the IgH, Igκ and Igλ J. genes. All PCRs were performed on 96-well PCR plates in a total volume of 50 μl containing 20 nM each primer or primer mixture, 10 nM each of dNTP (Invitrogen), 10 µl of 5x Q solution (Qiagen) and 1.2 U of HotStar Taq DNA polymerase (Qiagen). From the positive PCR reaction, DNA lakes from the VH or VL region were linked to a pCR2.1-Topo-TA (Invitrogen) vector for sequencing before cloning into the corresponding expression vector Igγ1, Igκ and Igλ. 10 µg of light and heavy chain plasmids, cloned from the same well and combined in all possible light and heavy chain pairs, were mixed with 40 µl of FuGENE 6 (Roche) in 1500 µl of DMEM (Gibco) and cotransfected in 293 T cells. The full-size IgG was purified using a recombinant protein A column (GE Healthcare).
[0387] [00387] Neutralization tests. Neutralization of monoclonal antibodies was measured using infection by HIV pseudovirus Env of single cycle TZM-bl cells (Li et al., J Virol 79, 10108-10125, 2005). HIV-1 Env pseudoviruses were generated by cotransfection of 293T cells with a pSG3ΔEnv backbone containing a luciferase reporter gene and a second plasmid that expressed HIV1 Env. At 72 hours post-transfection, supernatants containing pseudoviruses were harvested and frozen at -80 ° C until further use. In the neutralization assay, 10 µl of 5-fold serially diluted patient serum or mAb were incubated with 40 µl of pseudovirus in a 96-well plate at 37 ° C for 30 minutes before adding TZM-bl cells. After 2 days of incubation, the cells were lysed and viral infectivity was quantified by measuring luciferase activity with a Victor Light luminometer (Perkin Elmer). The 50% inhibitory concentration (IC50) was calculated as the antibody concentration that reduced the infection by 50%. Antibody epitopes were mapped using mutant alanine pseudovirus JR2 MPER from HIV-1 in a TZM-bl assay.
[0388] [00388] Chimera neutralization of HIV-2 / HIV-1. Chimera C1 of HIV-2 / HIV-1 (HIV-2 virus 7312A with MPER of HIV-1 gp41; Gray et al., J Virol 81, 6187-6196, 2007) was used in the competition trial. A fixed concentration of MPER peptide was incubated with antibody 2F5, 4E10, Z13e1 or 10E8 serially diluted at 37 ° C for 30 minutes before incubation with HIV-2 / HIV-1 C1 chimera. Wild-type HIV-2 7312A virus was used as a control. Antibody epitope mapping was completed by adding 10 µl of 10E8 mAb to 5 µl of serial dilutions of 4E10 peptide or its alanine mutants at 37 ° C for 30 minutes before the addition of HIV-2 / HIV-1 C1 chimera. The degree to which peptide-blocked antibody-mediated neutralization was calculated as the amount of change in the IC50 value of the antibody in the presence of 4E10 alanine mutants compared to the wild-type peptide. The precise binding region on the antibody-targeted or serum-treated MPER was determined using HIV-2 / HIV-1 chimeras containing different portions of HIV-1 MPER, such as C1 (Env-HIV-2 with HIV-MPER -1), C1C (Env of HIV-2 with MPER of clade C), C3 (Env of HIV-2 with epitope of 2F5), C4 (Env of HIV-2 with epitope of 4E10), C6 (Env of HIV- 2 with short 4E10 epitope NWFDIT), C7 (HIV-2 env with short 2F5 epitope ALDKWA) and C8 (HIV-2 env with both Z13 and 4E10 epitopes). Patient serum diluted 5 times or mAb was incubated with chimera in a 96-well plate at 37 ° C for 30 minutes before adding TZM-bl cells. The specificities in patient sera were confirmed by blocking the neutralization of the C1 chimera with 25 g / ml of 2F5, 4E10, MPER, Bal.V3, control peptide, or 50 g / ml of Z13 peptide.
[0389] [00389] ELISA assays. Each antigen at 2 μg / ml was coated on 96 well plates overnight at 4 ° C. Plates were blocked with BLOTTO buffer (PBS, 1% FBS, 5% nonfat milk) for one hour at room temperature (RT), followed by incubation with antibody serially diluted in burst buffer (PBS, 5% FBS , 2% BSA, 1% Tween-20) for one hour at room temperature. 1: 10,000 dilution of goat anti-human IgG antibody conjugated to horseradish peroxidase (HRP) was added over one hour at room temperature. Plates were washed between each step with 0.2% Tween 20 in PBS. Plates were developed using 3.3´, 5.5´- tetramethylbenzidine (TMB) (Sigma) and read at 450 nm.
[0390] [00390] Self-reactivity tests. Binding of 10E8 to the phospholipid was measured by SPR conducted on a BIACORE® 3000 instrument and data analysis was performed using the BIAevaluation® 4.1 software (BIACORE®) as previously described (Alam et al. Proc. Natl. Acad. Sci. USA, 106, 20234-20239, 2009). Liposomes containing phospholipids were captured on a BIACORE® L1 sensor chip, which uses an alkyl linker for anchoring lipids. Before capturing the lipids, the L1 chip surface was cleaned with a 60-s injection of 40 mM octyl-β-Dglucopyranoside, at 100 µl / minute, and the chip and fluids were washed with excess buffer to remove any traces of detergent. mAbs were then injected at 100 µg / ml at a flow rate of 30 µl / min. After each injection of Ab, the surface was again cleaned with octyl β-D-glucopyranoside, and 5-s injections of each 5 mM HCl, then 5 mM NaOH, to clean any adherent protein from the chip.
[0391] [00391] Reactivity to HIV-1 negative human epithelial cells (HEp-2) was determined by indirect immunofluorescence on slides using Evans Blue as a counter spot and FITC-conjugated goat anti-human OgG (Zeus Scientific, Raritan NJ; Haynes and others , Science 308, 1906-1908, 2005). Slides were photographed using a Nikon Optiphot fluorescence microscope. With reference to FIG. 3B, codachrome slides were taken from each MAb binding to Hep-2 cells in a 32-second exposure, and the slides were analyzed in digital format. The Luminex AtheNA MultiLyte ANA test (Wampole Laboratories, Princeton, NJ) was used to test for MAb reactivity to SSA / Ro, SS-B / La, Sm, ribonucleoprotein (RNP), Jo-1, double-stranded DNA (dsDNA ), centromere B, and histone and was performed according to the manufacturer's specifications and as previously described (Haynes et al., Science 308, 1906-1908, 2005). MAb concentrations tested were 50, 25, 12.5 and 6.25 µg / ml. 10 µl of each concentration were incubated with Luminex fluorescent beads and the test performed according to the manufacturer's specifications.
[0392] [00392] Staining of fluorescence-activated cell classification (FACS) of cell surface HIV-1 Env. FACS staining was performed as previously described (Chakrabarti et al, J Virol 85, 8217-8226, 2011; Koch et al, Virology 313, 387-400, 2003). 48 hours after transfection, cells were harvested and washed in FACS buffer (PBS, 5% HIFBS, 0.02% azide) and stained with monoclonal antibodies. The transfected cells were suspended in FACS buffer and incubated with the antibodies for one hour at room temperature. The monoclonal antibody cell mixture was washed extensively in FACS buffer and secondary goat anti-human phycoerythrin (PE) antibody (Sigma) was added over one hour in a 1: 200 dilution, followed by extensive washing to remove secondary antibody. Off. The cells stained with the PE antibody were acquired on a BD LSRII instrument and analyzed by FlowJo.
[0393] [00393] Antibody-virus removal experiments. From an initial concentration of 2 mg / ml, 12.5 μl of antibodies serially diluted 5 times in PBS were added to 487.5 μl of DMEM containing 10% HIFCS and 15 μl of pseudovirus in such a way that the final concentrations of antibodies 50 μg / ml to 0.08 μg / ml in a total volume of 500 μl. In the "no inhibitor" control, the same volume of PBS was added instead of antibody. The reaction mixture was incubated for 30 minutes at 37 ° C. The 250 μl reaction mixture was diluted to 10 ml with complete DMEM, centrifuged at 25,000 rpm in a SW41 rotor, for 2 hours at 4 ° C. The virus pellet was then washed two more times with 10 ml of PBS. During the washing steps, the virus-antibody complex was centrifuged at 40,000 rpm for 20 minutes at 4 ° C. After the final wash, 250 μl of DMEM was added to the washed virus pellet and it was resuspended by gentle agitation at 4 ° C for 30 min. 100 μl of the suspended viruses were used to infect 100 μl of TZM-bl cells (0.2 million / ml), in duplicate. Of the remaining 250 μl of reaction mixture, an equal volume of the antibody-virus mixture was used as a "no elimination" control. Plates were incubated at 37 ° C in a CO2 incubator for 2 days. After 2 days, the luciferase assay was performed as previously described (Mascola and another J Virol 76, 4810-4821, 2002). The data was then plotted to determine antibody-mediated neutralization under "wash" or "no wash" conditions.
[0394] [00394] Analysis and determination of structure. The 10E8 antigen binding fragment (Fab) was prepared using LysC digestion, as previously described (Ofek et al., Proc. Natl. Aca. Sci. .U.S.A., 107, 17880-17887, 2010). IgG was first reduced with 100 mM DTT for one hour at 37 ° C, followed by one hour of dialysis in Hepes, pH 7.6, to reduce the DTT concentration to 1 mM. Antibodies were then dialyzed against 2 mM iodoacetamide for 48 hours at 4 ° C, and subjected to final dialysis against Hepes, pH 7.6, for 2 h. After reduction and alkylation, the antibodies were cleaved with Lys-C (Roche), run on a Protein A column to secrete the Fc fragment, and then subjected to ion exchange (Mono S) and size exclusion chromatography ( S200). Purified 10E8 Fab was incubated with peptide in excess of 10 times RRR-NEQELLELDKWASLWNWFDITNWLWYIR (SEQ ID NO: 26) -RRR (American Peptide, CA) and the complex then established for 20 ° vapor diffusion settlement drop crystallizations C in the Honeybee 963 robot. 576 initial conditions adapted from commercially available Hampton (Hampton Research), Precipitant Synergy (Emerald Biosystems), and Wizard crystallization assessments (Emerald Biosystems) were established and worked on using Rockimager (Formulatrix), followed by manual optimization of crystal hits. The crystals were developed in 40% PEG 400, 0.1 M Na Citrate, 0.1 M Tris, pH 7.5 diffracted for 2.1 Å resolution in a mother liquid cryoprotectant compound supplemented with 15% 2R- 3R-butanediol and excess peptide. After mounting the crystals on a loop, they were instantly cooled and data were collected at 1.00 Å length in SER CAT ID-22 or BM-22 (APS) beamlines and processed using HKL-2000 (Otwinowski and another, Macromolecular Crystallography, Pt A 276, 307-326, 1997). The structures were resolved by molecular substitution with Phaser (McCoy et al, J Appl Crystallogr 40, 658-674, 2007; Winn et al, Acta Crystallogr D Biol Crystallogr 67, 235-242, 2011), using a free structure previously obtained of 10E8 as a research model. Refinement of the structure was undertaken with Phenix (Adams and others, Acta Crystallogr D Biol Crystallogr 58, 1948-1954, 2002), with the construction of an iterative model using Coot (Emsley, P. & Cowtan, K. Acta Crystallogr D Biol Crystallogr 60, 2126-2132 (2004) The structure was validated with MolProbity (Davis et al., Nucleic Acids Res 35, W375-383, 2007), producing 97% and 99.8% of waste covering the most favored regions of Ramachandran and regions of Ramachandran The structure was analyzed with APBS (Baker et al., Proceedings of the National Academy of Sciences of the United States of America 98, 10037-10041, 2001) for electrostatics (Ligplot; McDonald et al., J Mol Biol 238, 777-793, 1994), for direct contacts, PISA (Krissinel et al., J Mol Biol 372, 774-797, 2007), for hidden surface areas, and LSQKAB (ccp4 Package; Winn, MD and other Acta Crystallogr D Biol Crystallogr, 67, 235-242, 2011) for RMSD alignments. s using the Pepwheel program (150.185.138.86/cgibin/emboss/pepwheel). All graphics were prepared with Pymol (PyMOL Molecular Graphics System).
[0395] [00395] Evaluation of binding affinities of variants 10E8 and 10E8 to gp41 MPER. Surface plasmon resonance (SPR) (BIACORE® T200, GE Healthcare) was used to assess binding affinity of wild-type 10E8 to a gp41 MPER peptide. A biotinylated peptide composed of residues 656-683 of gp41 MPER (RRR-NEQELLELDKWASLWNWFDITNWLWYIR (SEQ ID NO: 26) - RRK-biotin; American Peptide, CA) was coupled to a BIACORE® SA chip at a surface density of 20-50 Response Units (UK). The 10E8 antigen binding (Fab) fragment was then flowed over as analyzed in concentrations ranging from 0.25 nM to 125 nM, in serial dilutions 2 times, with association or dissociation phases of up to five minutes, at a rate flow rate 30 ml ∕ min. The binding of the 2F5 and 4E10 Fab controls to the same peptide was examined under identical conditions.
[0396] [00396] Binding affinities of the 10E8 paratope alanine mutants to the MPER were also evaluated with SPR, but using an antibody capture method. A BIACORE® CM5 chip was coupled by amine with anti-human Fc antibody at high surface densities of ~ 10,000 RU. The 10E8 paratope variant IgGs were then captured for between 1500-2500 RU and a peptide composed of residues 656-683 of the gp41 MPER (RRR-NEQELLELDKWASLWNWFDITNWLWYIR (SEQ ID NO: 26) - RRR) flowed over as analyzed in dilutions serial 2 times starting at 500 nM (with the exception of HC D30A, W100bA, S100cA, P100fA, which started at 250 nM). Association or dissociation phases lasted three minutes and five minutes, respectively, at an EEEEE flow rate of 30 µl ∕ min. Binding sensograms were fit with 1∶ 1 Langmuir models using BIACORE® BiaEvaluation® Software (GE Healthcare). In all cases, BIACORE® HBSEP + buffer was used (10 mM Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.1% P-20).
[0397] [00397] PCR sequencing and amplification. Extraction of viral RNA from plasma and cDNA synthesis was performed as previously described (Imamichi et al., J Infect Dis 183, 36-50, 2001). Simple molecules of a 588bp fragment, spanning the MPER region of the HIV-1 envelope gene, obtained by limiting dilution, were amplified by PCR with the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) using the following primer sets: +7789 (sense) 5'- TCTTAGGAGCAGCAGGAAGCACTATGGG-3 '(SEQI ID NO: 193) and - 8524 (antisense) 5'-GTAAGTCTCTCAAGCGGTGGTAGC-3' (SEQI ID NO: 194) in a first series reaction; +7850 (sense) 5'- ACAATTATTGTCTGGTATAGTGCAACAGCA-3 '(SEQI ID NO: 195) and - 8413 (antisense) 5'-CCACCTTCTTCTTCGATTCCTTCGG-3' (SEQI ID NO: 196) in a second series reaction. Each PCR series consisted of 25 cycles, with the initial denaturation at 94 ° C for two minutes, followed by 25 cycles of denaturation at 94 ° C for 15 seconds, annealing at 50 ° C for 30 seconds, and extension at 72 ° C for 1 minute, with the final extension at 72 ° C for 7 min. The PCR products were purified with the QIA rapid PCR purification kit (QIAGEN, Valencia, CA), and then cloned into pCR2.1-TOPO vector (TOPO TA Cloning it, Invitrogen, Carlsbad, CA) for analysis sequence of individual molecular clones. The DNAs of 18 independent clones were sequenced with the ABI BigDye Terminator v3.1 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and analyzed with the ABI PRISM 3130xl genetic analyzer (Applied Biosystems, Foster City, CA).
[0398] [00398] Statistical analyzes. The relationship between patient serum potency of N152 and 10E8, and the relationship between 10E8 variant binding and neutralization were assessed using the Spearman rank method.
[0399] [00399] Deposits. The 10E8 light and heavy chain nucleotide sequence was submitted to GenBank under membership numbers JX645769 and JX645770, each of which is incorporated by reference here as presented in GenBank on September 18, 2012. Structure and coordinate factors for Fab de 10E8 in complex with the gp41 MPER were deposited with the Protein Database under the accession code 4G6F which is incorporated by reference here as presented on GenBank on September 18, 2012. Example 2 Neutralization tests
[0400] [00400] This example describes a method for testing the amplitude of neutralization and potency of the antibodies described here.
[0401] [00401] Neutralization of monoclonal antibodies was measured using a single-cycle infection by HIV-1 Pseudovirus Env and TZM-bl target cells. HIV-1 Env pseudoviruses were generated by cotransfection of 293T cells with a pSG3ΔEnv backbone containing a luciferase reporter gene and a second plasmid that expressed HIV-1 Env. Within 72 hours after transfection, the supernatant containing pseudovirus was harvested and frozen at -80 ° C until further use. Specific activity of the proximal outer region of anti-gp41 membrane (MPER) of patient serum and antibodies was measured using the HIV-2 / HIV-1 MPER chimeras. 7312A of wild-type HIV2 was used as a control. The 50% inhibitory concentration (IC50) was calculated as the antibody / inhibitor concentrations causing a 50% reduction in infection. For the competition assay, the fixed peptide concentration was incubated with 2F5, 4E10, Z13E1 or 10E8 Ab serially diluted at 37 ° C for 30 minutes before 7312A-C1 chimera incubation. Epitope mapping assay was evaluated by adding 0.5 μg / ml of 10E8 Ab to the serial dilutions of peptide 4E10 or its alanine mutants at 37 ° C for 30 minutes prior to the addition of 7312A-C1 chimera. The neutralizing blocking effect of the peptides was calculated as an amount of change in the IC50 value of the antibody in the presence of 4E10 alanine mutants compared to the wild type 4E10 peptide. Neutralization of 10E8 against COT6.15 mutant alanine pseudoviruses of HIV-1 was also measured using a TZM-bl assay (figure 36). Another neutralization of several strains of HIV-1 was tested with 10E8 antibody as well as antibodies containing 10E8, 7H6, and 7N16 intercomplemented light and heavy chains (figure 37). Example 3 ELISA assays
[0402] [00402] Each antigen at 2 μg / ml was used to coat 96 well plates overnight at 4 ° C. The coated plates were blocked with BLOTTO buffer (PBS, 1% FBS, 5% nonfat milk) for one hour at room temperature, followed by incubation with antibody serially diluted in burst buffer (PBS, 5% FBS, 2% BSA, 1% Tween-20) for one hour at room temperature. Goat anti-human IgG antibody conjugated to spicy horseradish peroxidase (HRP) at 1: 10,000 was added over one hour at room temperature. Plates were washed between each step with 0.2% Tween 20 in PBS. Plates were developed using 3.3´, 5.5´-tetramethylbenzidine (TMB) (Sigma) and read at 450 nm. Example 4 Gp41-specific HIV-1 monoclonal neutralizing antibodies to detect HIV-1 in a sample or a patient
[0403] [00403] This example describes the use of HIV-1 specific neutralizing antibodies to gp41 for the detection of HIV-1 in a sample or a patient. This example also describes the use of these antibodies to confirm the diagnosis of HIV-1 in a patient.
[0404] [00404] A biological sample, such as a blood sample, is obtained from the patient diagnosed with, undergoing screening for, or suspected of having an HIV-1 infection. A blood sample taken from a patient who is not infected is used as a control, although a standard result can also be used as a control. An ELISA is performed to detect the presence of HIV-1 in the blood sample. Proteins present in blood samples (the patient sample and the control sample) are immobilized on a solid support, such as a 96-well plate, according to methods well known in the art (see, for example, Robinson et al., Lancet 362: 1612-1616, 2003, incorporated herein by reference). Following immobilization, neutralizing HIV-1 monoclonal antibodies specific for gp41 that are directly labeled with a fluorescent marker are applied to the immobilized protein plate. The plate is washed in an appropriate buffer, such as PBS, to remove any unbound antibody and to minimize non-specific antibody binding. Fluorescence can be detected using a fluorometric plate reader according to standard methods. An increase in fluorescence intensity of the patient sample relative to the control sample indicates proteins specifically bound by the gp41 antibody in the blood sample, thereby detecting the presence of HIV-1 protein in the sample. Detection of HIV-1 protein in the patient sample indicates that the patient has HIV-1, or confirms a diagnosis of HIV-1 in the patient. Example 5 HIV-1 specific neutralizing antibodies to gp41 for the treatment of HIV-1
[0405] [00405] This example describes a particular method that can be used to treat HIV in a human patient by administering one or more human neutralizing mAbs specific for gp41. Although particular methods, dosages, and modes of administration are provided, one skilled in the art will appreciate that variations can be made without substantially affecting treatment.
[0406] [00406] Based on the teachings described here, HIV-1 can be treated by administering a therapeutically effective amount of one or more of the neutralizing mAbs described here, thereby reducing or eliminating HIV infection.
[0407] [00407] Evaluation of Patients. In particular examples, the patient is first selected to determine whether they have an HIV infection. Examples of methods that can be used to assess for HIV infection include a combination of assessing a patient's CD4 + T cell count and the level of HIV in serum blood levels. More methods using the gp41-specific mAbs described here can also be used to assess for HIV.
[0408] [00408] In some examples, HIV testing consists of an initial evaluation with an enzyme-linked immunosorbent assay (ELISA) to detect antibodies to HIV, such as to HIV-1. Specimens with a nonreactive result from the initial ELISA are considered HIV negative unless new exposure to an infected partner or partner of unknown HIV status has occurred. Specimens with a reactive ELISA result are retested in duplicate. If the result of any duplicate test is reactive, the specimen is reported as repeatedly reactive and undergoes a confirmatory test with a more specific supplementary test (eg Western blot or an immunofluorescence assay (IFA)). Specimens that are repeatedly reactive by ELISA and positive by IFA or reactive by Western blot are considered HIV-positive and indicative of HIV infection. Specimens that are repeatedly reactive by ELISA occasionally provide an indeterminate Western blot result, which may be an incomplete antibody response to HIV in an infected person, or non-specific reactions in an uninfected person. IFA can be used to confirm infection in these ambiguous cases. In some instances, a second specimen will be collected more than a month later and tested again for patients with indeterminate Western blot results. In more examples, nucleic acid testing (for example, method of amplifying proviral DNA or viral RNA) can also aid diagnostics in certain situations.
[0409] [00409] The detection of HIV in a patient's blood is indicative that the patient is infected with HIV and is a candidate to receive the therapeutic compositions described here. In addition, detection of a CD4 + T cell count below 350 per microliter, such as 200 cells per microliter, is also indicative that the patient is likely to have an HIV infection.
[0410] [00410] Pre-assessment is not required prior to administration of the therapeutic compositions described here.
[0411] [00411] Pre-treatment of patients. In particular examples, the patient is treated prior to administration of a therapeutic agent that includes one or more antiretroviral therapies known to those skilled in the art. However, such pre-treatment is not always required, and can be determined by a trained clinician.
[0412] [00412] Administration of therapeutic compositions. Following patient selection, a therapeutically effective dose of a gp41-specific neutralizing mAb described here is administered to the patient (such as an adult human or a newborn at risk of contracting HIV or known to be infected with HIV). More agents, such as antiviral agents, can also be administered to the patient simultaneously, either before, or after administration of the described agents. Administration can be activated by any method known in the art, such as oral, inhalation, intravenous, intramuscular, intraperitoneal, or subcutaneous administration.
[0413] [00413] The amount of the composition administered to prevent, reduce, inhibit, and / or treat HIV or a condition associated with it depends on the patient being treated, the severity of the disorder, and the manner of administration of the therapeutic composition. Ideally, a therapeutically effective amount of an agent is the amount sufficient to prevent, reduce, and / or inhibit, and / or treat the condition (e.g., HIV) in a patient without causing a substantial cytotoxic effect on the patient. An effective amount can be readily determined by someone skilled in the art, for example, using stabilizing dose response curves from routine experiments. As such, these compositions can be formulated with an inert diluent or a pharmaceutically acceptable carrier.
[0414] [00414] In a specific example, antibodies are administered at 5 mg per kg every two weeks or 10 mg per kg every two weeks depending on the particular stage of HIV. In one example, antibodies are administered continuously. In another example, antibodies or antibody fragments are administered at 50 µg per kg given twice a week for 2 to 3 weeks.
[0415] [00415] Administration of the therapeutic compositions can be taken in the long term (for example over a period of months or years).
[0416] [00416] Evaluation. Following administration of one or more therapies, HIV patients can be monitored for reductions in HIV levels, increases in a patient's CD4 + T cell count, or reductions in one or more clinical symptoms associated with HIV. In particular examples, patients are analyzed one or more times, starting 7 days after treatment. Patients can be monitored using any method known in the art. For example, the patient's biological samples, including blood, can be obtained and changes in CD4 + or HIV T cell levels assessed.
[0417] [00417] Additional treatments. In particular examples, if patients are stable or have a minor, mixed or partial response to treatment, they can be treated again after reevaluation with the same planning and preparation of agents that they previously received for the desired period of time, including duration of a patient's lifetime. A partial response is a reduction, such as at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 70% in HIV infection, HIV replication or combination of these. A partial response can also be an increase in CD4 + T cell count such as at least 350 T cells per microliter. Example 6 PCR for 454 sequencing of patient N152 cells
[0418] [00418] This example describes a PCR assay to amplify sample DNA for deep sequencing. The process included cDNA generation from patient cells using RT PCR followed by amplification of VH3 and VL3 genes from the generated cDNA.
[0419] [00419] Patient sample N152 had 33-36 million PBMCs / ml.
[0420] [00420] mRNA was prepared using Qiagen Oligotex Kit (as described in the manufacturer's instructions).
[0421] [00421] RT PCR was performed on mRNA using Invitrogen reagents (which included oligo dT as a primer, and Superscript II RT).
[0422] [00422] After RT reaction of PCRs, cDNA was then subjected to amplification using specific VH3 and VL3 gene primers designed to amplify the leading sequences of the IGHV-3- 15 * 05 and IGLV3-19 * 01 alleles, which are the putative 10E8 precursors.
[0423] [00423] The initiators used were as follows: 5 'Heavy chain > XLR-A_5L-VH3 CCATCTCATCCCTGCGTGTCTCCGACTCAGAAGGTGTCCAGTGTGARGTGCAG (SEQ ID NO: 28) > XLR-A_VH3-L1-MP CCATCTCATCCCTGCGTGTCTCCGACTCAGGCTATTTTAAAAGGTGTCCAATGT (SEQ ID NO: 29) > XLR-A_VH3 / 4_L1_MP CCATCTCATCCCTGCGTGTCTCCGACTCAGGTGGCAGCTCCCAGATGGGTCCTGTC (SEQ ID NO: 30) > XLR-A_VH3 / 4_L3_MP CCATCTCATCCCTGCGTGTCTCCGACTCAGGTTGCAGTTTTAAAAGGTGTCCAGTG (SEQ ID NO: 31) 5 'Light Chain > XLR-A_5L- VL3CCATCTCATCCCTGCGTGTCTCCGACTCAGGCTCTGTGACCTCCTATGAGCTG (SEQ ID NO: 32) > XLR-A_5MP-VL3-1 CCATCTCATCCCTGCGTGTCTCCGACTCAGGCTTACTGCACAGGATCCGTGGCC (SEQ ID NO: 33) > XLR-A_5MP-VL3-19 CCATCTCATCCCTGCGTGTCTCCGACTCAGACTCTTTGCATAGGTTCTGTGGTT (SEQ ID NO: 34) > XLR-A_5MP-VL3-21 CCATCTCATCCCTGCGTGTCTCCGACTCAGTCTCACTGCACAGGCTCTGTGACC (SEQ ID NO: 35)
[0424] [00424] The samples were extracted by gel and purified by phenolchloroform.
[0425] [00425] Final Production: 1.15 µg of heavy chain; 0.9 µl light chain
[0426] [00426] 0.5 µg of each chain was sent for 454 sequencing.
[0427] [00427] The total number of sequences obtained from a total 454 chip for each chain: Heavy chain: 843084 raw sequences, 37669 sequences belonging to the IGHV3-15 family; Light Chain: 1219214 raw strings, 91951 strings belonging to the IGKV3-15 family. Example 7 Method for isolating and producing monoclonal antibodies without prior knowledge of antigen specificity.
[0428] [00428] This example illustrates a method for isolating and producing all the antibodies that an organism produces against a target antigen, such as virions, cancers, or toxins. Current methods for the isolation and production of monoclonal antibodies rely on known, specific epitopes to isolate and produce new monoclonal antibodies. Using these methods, B cells are classified and immunoglobulin genes are isolated based on the specificity of the epitope. These methodologies, however, can be polarized because they have previously known antibody specificities or epitopes. Additionally, because these methodologies have previously known antibody specificities or epitopes, these methodologies do not allow the discovery of new monoclonal antibodies with unknown epitope specificities. Unlike current methodologies, this methodology begins with a population of B cells in an organism that is representative of all B cells produced by the organism. This population is expanded and selected for activity against a feature or antigen of interest, such as a virion, toxin, protein or cancer. This allows for the isolation of a complete repertoire of memory B cells that an organism produces specific for an antigen without prior knowledge of specificity or binding characteristics. For example, memory B cells in this repertoire can be selected for functional activity (such as neutralization activity) and used to produce monoclonal antibodies specific for the antigen. Methods
[0429] [00429] Simple cell staining and classification of memory B cells are performed as follows. B cell isolation
[0430] a. Ligar, esquentar, e conduzir CST; conduzir Auto Drop delay (seguir as etapas no manual); b. Preparar um frasco de PBS-BSA estéril (ou refiltrar o frasco existente) c. Esterilizar o tubo: (1) carregar a amostra de 15% de contrad e conduzir durante cinco minutes na taxa de fluxo 8; (2) carregar uma amostra de 10% de branqueamento e conduzir durante cinco minutos em taxa de fluxo 8; (3) carregar uma amostra de PBSBSA estéril (em tubo estéril) e conduzir durante minutos em taxa de fluxo 8; (4) retornar para a taxa de fluxo 1 d. Auxiliar a corrente de classificação: (1) colocar um tubo guava vazio em bloco de classificação e ligar o bloco; (2) abrir a porta da corrente; (3) Diva: na janela de corrente lateral (chamada "70 mícron"), abrir a gaveta de resíduo; verifique se os escorregadores 1,3,4 estão estabelecidos em zero, escorregador 2 deve estar em torno de 48; (4) clique em Voltagem em seguida Classificação de Teste; (5) olhe na porta de corrente – é a corrente de classificação que atinge o centro do tubo ; (6) uso escorregadores na janela de corrente lateral para ajustar a corrente então em seu centro; (7) clique Voltagem para desligar; feche a gaveta de resíduos e. DIVA (software Aria): copiar um padrão antigo escolhendo uma classificação anterior, em seguida "Duplicar sem dados" ou estabelecer novo experimento com fechamento como mostrado abaixo f. Ligar tubo resfriador ao bloco de classificação; ligar o resfriador 2. Preparar as células: a. cuidadosamente limpar o capuz e pipetas com etanol antes de iniciar b. preparar pelo menos 400 ml de IMDM (com glutamax)/10% de FBS/micozap c. aquecer dois tubos cônicos de 15 ml de 7,5 ml de IMDM / 10% de FBS com 15 µl de benzonase d. preparar a mistura mestre de manchamento em tubo eppindorf: Centrifugar os anticorpos IgM e IgA 1 minuto antes da aliquotagem.Preparar mistura mestre: 50 µl por 50 milhões de células de anticorpo
[0431] [00431] turn for 20 minutes at 4ºC in microfuge; FACS pigments (PE: Phycoerythrin; Cy7: a cyanine pigment; APC: Allophicocyanin; FITC: fluorescein isothiocyanate; CD19-PE-Cy7 (anti-CD19 mAb conjugated to B-cell specific pigments (except plasma b cells); IgA: anti-IgA mAb; IgD: anti-IgD mAb; IgM: anti-IgM mAb and. thaw 2 patient cell vials: (1) heat the vials in a water bath until the floating ice is visible; (2) add 1 ml of preheated medium / benzonase to the vial, allow to set for 15 seconds; (3) move the entire contents of the vial to a conical tube of 15 ml of heating medium; (4) pellet 1200 rpm 10 minutes; (5) resuspend each in 1 ml of PBS-BSA, combine, move 50 µl to each of 5 conical tubes; (6) pelletize all 6 conical tubes; (7) NOTE - if only one vial is needed, you can skip steps v. and saw. f. Resuspend cell pellets: (1) main sample: in 100 µl of master mix; (2) comps. in 50 µl of PBS-BSA, add simple dyes (0.5 µl of CD19-PE-Cy7 etc) g. 30 min 4ºC covered in thin metal sheet H. washing: add 1000 µl of PBS-BSA with P1000, mix well, add another 2 ml of PBS-BSA i. pelletize, meanwhile, open a new bag of hooded filter cap tubes and label them j. resuspend in 500 µl of PBS-BSA and transfer to sterile filter cap tubes by injecting cells through the cap (touch the tip to the cap while injecting) k. keep cold until they are ready to flow 3. Sort the cells: (1) press Aria, close as shown in the example; use old compensation if the voltages seem OK, otherwise adjust the voltages and conduct the compensation; (2) adjust the flow rate between 1-2 so that the event rate is as high as possible, but not more than 13,000 evt / second. Monitor this during classifications; (3) establish the classification (Click on Classification> New classification layout; Purity 1.5; Classification: continuous; Choose port P6 for classification on the Left); (4) Place 250 µl of medium into a 2 ml o-ring tube, load into the classification block e. Sort by Test 500 cells; (5) Unload the sample, return to cold; (6) Conduct the reflux of the sample line for 1 to 2 minutes, then load the fresh tube of PBS-BSA into the stream; (7) Evaluate 8 for two minutes and empty the main sample; (8) Remove the classification tube from the classification block, use P1000 to gently wash the sides using tube medium; (9) Move everything to a flow tube; (10) Flow and record post-classification purity; (12) Load the main sample into Aria and classify 20,000 to 30,000 B cells; (13) Repeat steps 5-7; (14) Take 10 µl, add to 100 µl of PBSBSA in a flow tube, flow and record the post-classification purity; (15) Place 270 µl of Guava ViaCount in a guava tube, add 30 µl of classified cells, two minutes, then count in Guava (record the concentration only; 0.25 ml as volume will not be accepted); (16) Calculate the number of cells needed to sow at the desired density.
[0432] [00432] Treat cells with growth factors to induce cell division
[0433] a. Concentrações (Mistura Alimentadora): 3T3-msCD40L: 5000 células/ 50 ul / cavidade = 105 células/ml; IL2 100 u/ml; IL21 50 u/ml; Em meio de cultua de célula IMDM /10% de FBS (veja acima); Para 20 placas = 350 ml: 336 ml IMDM/10%FBS; 3.5 ml IL2 (10000 u/ml); 175 µl IL21; 10 ml 3T3msCD40L b. Preparar a mistura alimentadora como calculado; Para 10 placas: pôr de parte 12,5 ml para nenhum controle de célula B. 2. Semear células: (1) Usar placas brancas de 384 cavidades; rotulá-las sobre a tampa e sobre o lado da placa; (2) Usar pipetas de 12 canais; (3) Portadores de placa angulada podem ser úteis; (4) Colocar 100 µl de dI-H2O estéril com gent em cavidades externas (necessários 7,7 ml/placa); (5) Antes adicionar células b para misturar, semear 50 µl/cavidade de mistura de alimentadores na série D de todas as placas (isto é; (6) controle de nenhum anticorpo); (7) Adicionar a quantidade apropriada de células B à mistura de alimentadores restante (Para 10 placas, 2,5 células B/cavidade = 50 células/ml, necessárias 8125 células B); (8) Mover a mistura celular para bacias estéreis grandes, e semear 50 µl/cavidade em 308 cavidades internas (exceto a série D); (9) Tenha certeza de misturar as células na bacia frequentemente; (10) Semear um quarto de placa extra para ELISA posteriormente; (11) Mover novamente da incubadora, deixar não incomodada durante 13 dias3. Fornecedores: IL2 – Roche- 11147528001 (50 ml); IL21 – Invitrogen – PHC0215 (25ug); IMDM + Glutamax – Invitrogen – 31980-097 (1x10btls)[00433] CD19 + IgA-IgD-IgM memory B cells are resuspended in Iscove IMDM Modified Dulbecco's Medium with 10% FBS containing 100 U / ml IL-2, 50 ng / ml IL-21 and 1x105 / ml of irradiated 3T3-msCD40L feeder cells produced as previously described (Kershaw et al., Cancer Res., 61: 7920-7924, 2001). B cells are seeded in 384-well microtiter plates at a density of 4 cells / well in a final volume of 50 μl. Additional description of treatment of cells with growth factors 1. Prepare the sowing conditions: (1) Defrost 1 irradiated 35x106 vial of irradiated 3T3-msCD40L cells (benzonase method), resuspended in 10 ml of IMDM / 10% FBS; (2) Calculate the reagent quantities. The. Concentrations (Feed Mixer): 3T3-msCD40L: 5000 cells / 50 ul / well = 105 cells / ml; IL2 100 u / ml; IL21 50 u / ml; In cell culture medium IMDM / 10% FBS (see above); For 20 plates = 350 ml: 336 ml IMDM / 10% FBS; 3.5 ml IL2 (10,000 u / ml); 175 µl IL21; 10 ml 3T3msCD40L B. Prepare the feed mixture as calculated; For 10 plates: set aside 12.5 ml for no B cell control. 2. Seed cells: (1) Use 384-well white plates; label them on the lid and on the side of the plate; (2) Use 12-channel pipettes; (3) Angled plate holders can be useful; (4) Place 100 µl of sterile dI-H2O with gent in external cavities (7.7 ml / plate required); (5) Before adding b cells to mix, seed 50 µl / well of mixer feeders in the D series of all plates (ie; (6) no antibody control); (7) Add the appropriate amount of B cells to the remaining feeder mixture (For 10 plates, 2.5 B cells / well = 50 cells / ml, 8125 B cells required); (8) Move the cell mixture to large sterile basins, and sow 50 µl / well in 308 internal wells (except for the D series); (9) Be sure to mix the cells in the bowl frequently; (10) Sow a quarter of an extra ELISA plate afterwards; (11) Move again from the incubator, leave undisturbed for 13 days 3. Suppliers: IL2 - Roche-11147528001 (50 ml); IL21 - Invitrogen - PHC0215 (25ug); IMDM + Glutamax - Invitrogen - 31980-097 (1x10btls)
[0434] a. Preparar uma placa ELISA antecipadamente (revestir uma placa Maxisorp™ com anti-IgG); Limpar o capuz e pipetas com etanol e em seguida RNaseAway™; rotular tantas placas brancas de 384 cavidades quantas você tiver no dia 11 de placas. b. Mover 40 µl de cada cavidade da placa no dia 11 para a correspondente cavidade de placa fresca: (1) usar uma pipeta de 12 canais; (2) Pegar a ponta mais distante para baixo em cavidade quanto possível – Ok para tocar a base ligeiramente, mover a ponta para cima ligeiramente, em seguida puxar para cima; (3) dispensar para a nova placa; (4) cobrir sup. das placas com adesivo de folha delgada de metal e colocar a tampa sobre; a quarta parte da placa vai para 4ºC; (5) armazenar as sup. das placas a -80 e usar como necessário para ensaio de neutralização. c. Fazer tampão de captura de lise – suficiente para 308 cavidades * 1,15 * número de placas: para 1 cavidade (0,3 µl de Tris, pH 8, 1M; 0,25 µl de Inibidor de RNase; 19,45 µl de H2O tratada com depc); colocar 20 µl de tampão de captura de lise sobre todas as cavidades de célula B (dispensar com pipeta de múltiplos canais; cobrir com adesivo de folha delgada de metal e colocar a tampa sobre; armazenar a -80°C); realizar ELISA usando 10 µl de sup da quarta parte da placa para medir a concentração de IgG e avaliar o impacto. [00434] After 13 days of incubation, 40 μl of culture supernatants from each well are collected and selected for neutralization activity using a functional target of the antigen of interest (for example, if an HIV-1 antigen is the target antigen, the neutralization assay can be a high yield microneutralization assay against HIV-1MN.03 and HIV-1Bal.26). Additional description of supernatant collection for neutralization assay 1. Collection of supernatant for neutralization assay The. Prepare an ELISA plate in advance (coat a Maxisorp ™ plate with anti-IgG); Clean the hood and pipettes with ethanol and then RNaseAway ™; label as many 384-well white plates as you have on the 11th plate. B. Move 40 µl from each plate well on day 11 to the corresponding fresh plate well: (1) use a 12-channel pipette; (2) Take the tip as far down in the cavity as possible - Ok to touch the base slightly, move the tip up slightly, then pull up; (3) dispense to the new plate; (4) cover sup. the plates with thin metal foil adhesive and place the lid on; the fourth part of the plate goes to 4ºC; (5) store the sup. plates at -80 ° C and use as needed for neutralization testing. ç. Make lysis capture buffer - sufficient for 308 wells * 1.15 * number of plates: for 1 well (0.3 µl of Tris, pH 8, 1M; 0.25 µl of RNase Inhibitor; 19.45 µl of H2O treated with depc); place 20 µl of lysis capture buffer over all B cell wells (dispense with multi-channel pipette; cover with foil adhesive and place the lid on; store at -80 ° C); perform ELISA using 10 µl of sup from the fourth part of the plate to measure the concentration of IgG and assess the impact.
[0435] [00435] B cells in each well are lysed with 20 μl lysis buffer containing 0.25 μl RNase inhibitor (New England Biolabs Inc.), 0.3 μl 1M Tris, pH8 (Quality Biological Inc.) and 19.45 μl of H2O treated with DEPC. B cell plates are stored at -80 ° C. The variable region of the heavy chain and the light chain of the immunoglobulin gene are amplified by RT-PCR of the wells that were classified positive in the neutralization assay. The cDNA product is used as a template in the PCR reaction. In order to amplify the highly somatically mutated gene immunoglobulin, two sets of primers as described previously (Tiller and another, J. Immunological Methods, 329: 112-124, 2008) are used in two independent PCRs. A set of primers includes the forward and reverse primers specific for the leader region and constant region of IgH, Igκ or Igλ, respectively. The other set of primers includes the FWR1-specific forward primer mixes and respective reverse primers specific for the J IgH, Igκ and Igλ genes. All PCRs are performed on 96-well PCR plates in a total volume of 50 μl containing 20 nM of each primer or primer mixture, 10 nM of each dNTP (Invitrogen), 10 μl of 5x Q-solution (Qiagen) and 1.2 U HotStar Taq DNA polymerase (Qiagen). From the positive reaction of PCRs, DNA lakes from the VH or VL region are linked to a vector pCR2.1-Topo-TA (Invitrogen) for sequencing before cloning into the corresponding expression vector Igγ1, Igκ and Igλ. 10 μg of light and heavy chain plasmids, cloned from the same well and combined in all possible light and heavy chain pairs, are mixed with 40 μl of FUGENE® 6 (Roche) in 1500 μl of DMEM (Gibco) and cotransfected into 293T cells. The full-size IgG is purified using a recombinant protein A column (GE Healthcare). Neutralization assays for monoclonal antibodies
[0436] [00436] Following the purification of natural-sized IgG, the antibodies produced are tested for neutralization activity.
[0437] [00437] For example, if the antigen of interest is an HIV-1 antigen, the neutralization activity of monoclonal antibodies can be measured using an infection by HIV-1 environs of single-cycle TZM-bl cells. HIV-1 Env. Pseudoviruses can be generated by cotransfection of 293T cells with pSG3 Env backbone and a second plasmid that expressed HIV-1 Env. Within 72 hours after transfection, supernatants containing pseudoviruses are harvested and frozen at - 80 ° C until further use. In the neutralization assay, 10 μl of patient serum serially diluted 5 times or mAb are incubated with 40 μl of pseudovirus in a 96-well plate at 37 ° C for 30 minutes before adding TZMbl cells. After 2 days of incubation, cells are lysed and viral infectivity quantified by measuring luciferase activity with a VICTOR® Light luminometer (Perkin Elmer). The 50% inhibitory concentration (IC50) is calculated as the antibody concentration that reduces the infection by 50%. Example 8 10E8 modifications
[0438] [00438] This example illustrates modifications of the 10E8 antibody to increase affinity for gp120, without increasing autoreactivity. As described below, several methods have been undertaken to identify and plan developments for the 10E8 antibody, including alanine scan mutagenesis based on the 10E8 paratope structure; Structure-based mutagenesis to enhance hydrophobic interactions; Partial germline reversal; combination of these methods, and deep pyrosequencing of 454 to identify phylogenetic variants of 10E8 with improved potency. In addition, combinations of these four methods were also undertaken, combining the best elements and the results of each one. Below is a summary of each of the four methods with the rationale behind each. (1) Alanine scan mutagenesis based on the 10E8 paratope structure
[0439] [00439] Based on the 10E8 Fab crystal structure with the gp41 MPER (described above), 10E8 paratope heavy chain residues were mutated to alanine. The mutant antibody constructs were generated using standard techniques, and the antibodies were expressed and purified as described in Example 1. All 10E8 paratope residues were mutated to alanine (see FIG. 43), and several residue positions led to neutralization enhanced 10E8-mediated virus activity (see figures. 32 and 49). These include residues of 10E8 D28, D30, N31, T52, E53, S56, D58, and Y100e (Kabat numbering). Neutralization tests were performed as described in example 2. (2) Structure-based mutagenesis to enhance hydrophobic interactions
[0440] [00440] Based on the 10E8 Fab crystal structure with the gp41 MPER, residues that were supposed to be coplanar with the 10E8e epitope have been mutated to hydrophobic residues (see Figures 44A and 44B). The mutant antibody constructs were generated using standard techniques, and the antibodies were expressed and purified as described in example 1. As shown in FIG. 45, several residue positions, both singly and in combination, resulted in an increased neutralization power of 10E8. Of the mutants listed in FIG. 45, mutation of residue S74 to tryptophan generated the largest increase in neutralization, followed by the double mutant D30-N31 and the single mutant D28. Neutralization tests were performed as described in example 2. (3) Reversal of partial germline
[0441] [00441] CDR graft was used to revert a fraction of the maturation changes from 10E8 back to the germline sequence. CDR grafting is a procedure that is typically used for humanizing antibodies from a non-human source (for example, mouse): the procedure uses a sequence alignment of the non-human antibody with a human counterpart (typically a human germline gene) and retains the original antibody CDR's, however, it mutates selected structure residues for the respective human entities. Structure residues are selected for mutation based on structural and sequence analysis. In certain cases, not all of the maturation changes seen in an antibody can be beneficial, and partial reversion to germline can help improve certain antibody properties, such as increased half-life, decreased immunogenicity, or improved potency. By applying 10R8 CDR graft, three different heavy chain inverters and three different partially reversed light chains were designated (see FIG. 47). At the amino acid level, mutation rates were decreased from 28% to 12-20% for the heavy chain and from 21% to 7-14% for the light chain. The mutated antibodies were constructed and produced using standard methods, described in example 1. Different combinations of heavy and light chain inverters were tested for binding to MPER and for neutralization (see FIG. 48). The greatest improvement in neutralization (<2-fold) was seen for the inverter which included the heavy and light chain reversals closest to the mature antibody ("10E8-R3," which includes the 10E8gH03 heavy chain (SEQ ID NO: 149 ) and the 10E8gL03 light chain (SEQ ID NO: 152)). Additional improvements were seen when the inverters were paired with heavy / light chains from other sources (such as deep sequencing, described below). (4) Additional Mutations
[0442] [00442] Based on neutralization data, inverter analysis, paratope alanine analysis, and enhanced hydrophobicity plans described above, the following additional mutations were introduced in the 10E8gH03 10E8 heavy chain variant and the 10E8 light chain variant 10E8gL03 of 10E8, antibodies were constructed and produced according to standard methods, as described in example 1, and tested for neutralization activity according to methods described in example 2 (figure 49)
[0443] [00443] This example illustrates the identification and functional pairing of 10E8-like heavy and light chain sequences determined in separate 454 pyrosequencing reactions. Starting from the 10E8 neutralizing antibody wild type sequence, clonal variants for the heavy and light chain were identified and evaluated for function by pairing with the complementary 10E8 wild type chain. The phylogenetic trees of the light and heavy chains revealed similar branching topologies around the wild type 10E8 sequences, allowing branches of the heavy and light chain phylogenetic trees to be compared based on their relative 10E8 distances. By evaluating a reconstituted antibody matrix of compared and erroneously compared branches for neutralizing HIV-1 and reactivity with autoantigens, the impact of phylogenetic matching on function was quantified.
[0444] [00444] The widely neutralizing antibody 10E8 has been identified in the HIV-1 infected N152 donor, and recognizes a helix in the proximal outer membrane region (MPER) just before the transmembrane joining region of the HIV-1 gp41 glycoprotein, and neutralizes 98% of diverse HIV-1 isolates at a 50% inhibitory concentration (IC 50) of 0.32 µg / ml. The 10E8 antibody heavy chain is derived from IgHV3-15 and IgHJ1, has a third complementarity determining region (CDR H3) of 22 amino acids, and has a somatic mutation rate of 21%. The 10E8 antibody light chain is derived from IgVL3-19 and IgLJ3, has a CDR L3 of 12 amino acids, and has a somatic mutation rate of 14%. Deep sequencing of donor B cell transcripts using polymerase chain reaction (PCR) to amplify IgG heavy chain sequences from the IgHV3 family and to amplify IgG light chain sequences from the IgVL3 family was performed as described in example 6. mRNA from a An estimated 5 million peripheral blood mononuclear cells (PBMCs) were used for reverse transcription to produce standard cDNA, and in both cases, primers that were upstream of the start of the V gene leader sequences and downstream of the chain end Have already been used.
[0445] [00445] Pyro sequencing of 454 Roche provided 843,084 heavy chain readings and 1,219,214 light chain readings for N152 donor (see FIG. 50). After primary analysis using a bioinformatics information channel 36,318 heavy chain sequences were assigned to the allele family of IgHV1-3 and 54,583 light chain sequences were assigned to the allele families of IgVL3-IgJ3 (see, for example, Wu and another , Science, 333, 1593-1602, 2011, and Zhu et al., Frontiers in microbiology, 3, 315-315, 2012). Readings were analyzed for identity for 10E8 and divergence of unmutated V genes, and their frequencies plotted on identity / divergence grids (figures 50A and 50B, left panels). Notably with the heavy chain several well-separated islands of high identity and about 25% divergence were observed, and with the light chain, a single well-separated island with high identity and about 15% divergence were observed. Sampling based on the grid of the high identity divergence region, selected 61 heavy chain sequences and 48 light chain sequences. The phylogenetic link of these sequences to 10E8 was analyzed (figures 50C and 50D) and also synthesized, reconstituted with the complementary wild-type 10E8 chain, and expressed by transient transfection in a 96-well format. Enzyme linked immunosorbent assays (ELISAs) of the expressed 10E8 variants identified 11 heavy chains and 24 light chains (the nomenclature and sequences of these light and heavy chains are shown in FIG. 51 and FIG. 59) that when paired with 10E8 chains partners linked to a peptide corresponding to the entire MPER of HIV-1 gp41 (figures 50A and 50B, right panels). In a panel of 6-HIV-1 isolates, up to ~ 5-fold increases in neutralizing power were observed (figures 50E and 50F).
[0446] [00446] The functional 10E8 heavy chains derived from three distinct islands in the identity / divergence plots (figure 50A), and displayed sequences and mutational patterns consistent with a common clonal origin (figure 51A). Mutations grouped in CDR H1 and H3, and also in the first, third and fourth structure regions (FR1, FR3 and FR4). The most divergent sequence, gVRC-H11dN152, had 25 amino acid changes, corresponding to a 19.1% difference in the wild-type 10E8 heavy chain. The functional 10E8 light chains derived from different regions of the identity / divergence plots, including a single distinct island and several regions overlapping the primary light chain population (figure 50B). Like the heavy chain, functional light chains exhibited mutational patterns consistent with a common clonal origin (figure 51C). Mutations grouped in the CDR L1 and CDR L2 regions, and all structure regions. The most divergent sequence, gVRCL23dN152, had 33 amino acid changes, corresponding to a 30.3% difference from the wild-type 10E8 light chain.
[0447] [00447] Although functional, 10E8 variants reconstituted with 10E8 wild-type complementarity chains do not represent natural pairs. A known disadvantage of the deep sequencing method for antibody characterization is that separate sequencing regions are required for light and heavy chains, and critical information related to natural ontogeny and functional phenotype is lost. Therefore, an evolution-based analysis was performed to provide sufficient information to recap approximate natural pairings. The maturation / evolution of light and heavy chains must be linked because of their physical association as proteins, the presence of their evolving genes in the same object cells for the same enzymatic mutation processes, and the requirement for cooperative structural change in response to even immunogen. In addition, sampling of light and heavy chains paired in a single mRNA population cDNA library of antibody transcripts that must be highly correlated because they originate from the same cells. In principle, this similarity in sampling should lead to correlations in frequencies of corresponding branches of heavy and light chain of phylogenetic trees.
[0448] [00448] Phylogenetic analyzes of the experimentally tested 10E8 heavy and light chains selected from the grid found in most neutralizing antibodies to popular three branches, close to and including the standard 10E8 (figures 50C and 50D). The b1-H branch for the heavy chain (or b1-L for the light chain) contained 10E8, the b2-H branch (b2-L) was the closest branch to b1-H, and the b3-H branch (b3 -L) was the next closest branch. A single neutralizing sequence (gVRCH11dN152) occupied a more distant branch (b4-H). Because the pyrosequencing of 454 produces an average of about 5 errors per variable antibody domain, the most potent antibody of each branch was selected as representative, when the most functional antibody is likely to have almost a non-paired function-error of 454. Twelve antibodies were reconstituted, including a complete heavy / light chain matching matrix from the 4 heavy chain branches and the 3 light chain branches (FIG 52A). 11 of the 12 reconstituted antibodies expressed sufficient levels of IgG to assess neutralization, which was done on a panel of five HIV-1 isolates. All 11 expressed antibodies were neutralizing. Heavy and light chain pairings that compared the phylogenetic distance of 10E8 (for example, b1-H to b1- L, b2-H to b2-L, and b3-H to b3-L), were slightly more potent on average than that the poorly compared pairings, but the different was not statistically significant (figure 52B).
[0449] [00449] The pairing reactivity compared and poorly compared with autoantigens was also tested. Notably, the paired comparison showed significantly less Hep2 staining (p = 0.049) (figure 52C).
[0450] [00450] The results show with donor 10E8 and N152 (i) how sampling of identity / divergence grid can be used to identify somatic variants, (ii) how the phylogenetic tree architecture can be used to approximate natural pairings, and (iii) that the antibodies paired by phylogenetic comparison show less autoreactivity. Such reduced autoreactivity is probably related to the in vivo selection that natural antibodies undergo. With an antibody such as 10E8, which may mechanically be more prone to autoreactivity than other antibodies, such a natural matching recap may be useful. Although natural pairings are lost with deep sequencing, the aforementioned suggests that it is possible to approximate them using topological similarities between heavy and light chain phylogenetic trees. Thus, it may be possible to use phylogenetic similarity or lineage analysis as screening methods, as these provide an exclusively computational method for identifying somatic variants. (5) Additional variants and combinations
[0451] [00451] Additional combinations of the identified light and heavy chains, as well as the identified heavy and light chain variants were generated to test for neutralization and autoreactivity. Combinations of certain light and heavy chains are indicated in FIGs. 53-54, including neutralization-based combinations in the context of the wild-type 10E8 complement chain, as well as combinations based on phylogenetic matching. The nomenclature and sequence information for these pairings is shown in FIG. 59, except for "rL3" which corresponds to the 10E8gL03 light chain germline inverter shown in FIG. 47 and listed as SEQ ID NO: 149. Neutralization values (IC50 in µg / ml) against a panel of six viruses are shown in FIGs. 55A-55C, and the autoreactivity values are shown in FIG. 56. The neutralization values (IC50 in µg / ml) against a panel of twenty viruses are shown in FIG. 57. The results indicate several heavy and light chain pairings that show increased neutralization activity compared to wild type 10E8, but do not have increased autoactivity, including HC6 heavy chain pairing (gVRC-H2dn152; SEQ ID NO: 154) and light chain 10E8gL03 (rL3; SEQ ID NO: 152).
[0452] [00452] Other heavy chain variants have been developed and supplemented with 10E8 light chain or 10E8 light chain variants and tested for neutralization activity (see FIGS. 58A and 58B). The serine residue at position 74 (Kabat numbering) of the heavy chain HC6 of the 10E8 variant (gVRC-H2dn152; SEQ ID NO: 154) was additionally mutated to alanine, arginine, valine or tyrosine. The sequences of these variants are provided as SEQ ID NO: 189 (HC6 S74A), SEQ ID NO: 190 (HC6 S74R), SEQ ID NO: 191 (HC6 S74V), SEQ ID NO: 192 (HC6 S74Y). These 10E8 variant heavy chains were complemented with the 10E8 "rL3" light chain variant (10E8gL03; SEQ ID NO: 149) and the resulting antibody was tested for neutralization activity against a panel of 20 HIV viruses, including 6, 8, 4, 1 and 1 viruses of clades A, B, C, AG, and G, respectfully. Neutralization tests were performed as described in example 1, and a graph summarizing the results of these tests is shown in FIG. 58A (showing the IC50 values) and FIG. 58B (showing IC80 values).
[0453] [00453] Additional heavy chain and light chain mutants have been developed to produce gp41 binding antibodies with increased solubility, reducing the number of hydrophobic residues exposed to solvent. Residues that are not required for epitope recognition (based on 454 deep sequencing data, as well as structure-based protein replanning) were selected for substitution with polar or charged residues. The following 10E8-like light and heavy chains were constructed (Kabat amino acid substitutions with reference to HC6 light and heavy chains (SEQ ID NO: 154) and rL3 (SEQ ID NO: 152), respectively) Light chain: 10E8gL03_hp_L01 (SEQ ID NO: 197; I44K) 10E8gL03_hp_L02 (SEQ ID NO: 198; S2Y, V10T, L14A, I44V, L106P) 10E8gL03_hp_L03 (SEQ ID NO: 199; V10T, L14A, I44K, L106P) Heavy chain: HC6_S77Y_hp_H01 (SEQ ID NO: 200; L18Q, S74Y) HC6_S77Y_hp_H02 (SEQ ID NO: 201; L72D, S74Y, I75K, F77T, M84T) HC6_S77Y_hp_H03 (SEQ ID NO: 202; L18Q, W55K, S74Y, M84T) HC6_S77Y_hp_H04 (SEQ ID NO: 203; L18Q, W55K, L72D, S74Y, I75K, F77T, M84T)
[0454] [00454] An inverting construction of HC6 partial germline was also built: HC6rH03S77Y (SEQ ID NO: 204; S23A, N73D, S74Y, E81Q, N82bS, R83K, M84T, S87T, L89V, F91Y, R105Q)
[0455] [00455] Additional 10E8 light chain variants were built. These variants include 10E8 wild-type light chain variants, the 10E8 light chain partial germline rL3 inverter, and the three light chain solubility mutants mentioned above. These constructs contain a single mutation R23Q that removes a potential integrin binding site. Q is the germline identity at position 23 in the 10E8 light chain. Additional mutants are mentioned as: 10E8_L_R23Q (SEQ ID NO: 205); 10E8gL03_R23Q (SEQ ID NO: 206); 10E8gL03_hp_L01_R23Q (SEQ ID NO: 207); 10E8gL03_hp_L02_R23Q (SEQ ID NO: 208); 10E8gL03_hp_L03_R23Q (SEQ ID NO: 209). The person skilled in the art will appreciate that 10E8 or any of the 10E8 variant light chain variable regions described here can optionally include the substitution of R23Q. (6) Summary of 10E8 mutations.
[0456] [00456] The 10E8 substitutions described above are summarized in the tables given as FIGs. 60A and 60B (heavy chain substitutions) and FIGS 61A and 61B (light chain substitutions).
[0457] [00457] It will be evident that the precise details of the described methods or compositions can be varied or modified without departing from the spirit of the described modalities. All modifications and variations that fall within the scope and spirit of the claims below have been claimed.

权利要求:
Claims (27)
[0001]
Isolated monoclonal antibody, characterized by the fact that it comprises: a heavy chain variable region comprising heavy chain complementarity determining regions (HCDR) 1, (HCDR) 2 and (HCDR) 3 comprising amino acids 26 to 33, 51 to 60 and 99 to 120, respectively, of SEQ ID NO: 154; and a light chain variable region comprising light chain complementarity determining regions (LCDR) 1, (LCDR) 2 and (LCDR) 3 comprising amino acids 26 to 31, 49 to 51 and 88 to 99, respectively, of SEQ ID NO: 152, a (FR) 2 and a (FR) 3 structure comprising amino acids 32 to 48 and 52 to 86, respectively, of SEQ ID NO: 152; where the antibody specifically binds to gp41 and neutralizes HIV-1.
[0002]
Isolated human monoclonal antibody according to claim 1, characterized in that the variable heavy chain region of the antibody comprises the amino acid sequence determined as one of SEQ ID NOs: 1, 3, 5, 149, 189 to 192, 200 to 201 or 204.
[0003]
Isolated human monoclonal antibody according to claim 1, characterized by the fact that: the heavy chain variable region comprises the amino acid sequence determined in SEQ ID NO: 154, and the light chain variable region comprises the amino acid sequence determined in SEQ ID NO: 152.
[0004]
Isolated human monoclonal antibody according to claim 1, characterized by the fact that the variable region of the heavy chain comprises the amino acid sequence established as SEQ ID NO: 192, and the variable region of the light chain comprises the amino acid sequence established as SEQ ID NO: 152.
[0005]
Isolated human monoclonal antibody according to claim 1, characterized by the fact that the variable region of the light chain comprises the amino acid sequence established as SEQ ID NO: 152.
[0006]
Isolated human monoclonal antibody according to claim 1, characterized by the fact that the variable region of the heavy chain comprises the amino acid sequence established as SEQ ID NO: 154.
[0007]
Isolated human monoclonal antibody according to claim 1, characterized by the fact that the variable region of the heavy chain comprises the amino acid sequence established as SEQ ID NO: 192.
[0008]
Isolated human monoclonal antibody according to any one of claims 1 to 7, characterized in that the antibody is an IgG, IgM or IgA antibody.
[0009]
Isolated human monoclonal antibody according to claim 8, characterized by the fact that the antibody is an IgG1 antibody.
[0010]
Isolated human monoclonal antibody according to any one of claims 1 to 9, characterized in that the antibody comprises a variant Fc region.
[0011]
Isolated human monoclonal antibody according to any one of claims 1 to 10, characterized in that the antibody neutralizes at least 98% of the HIV-1 isolates listed in figures 17C-17F with an inhibitory concentration (IC50) less than 50 µg / mL.
[0012]
Isolated human monoclonal antibody according to any one of claims 1 to 10, characterized in that the antibody neutralizes at least 72% of the HIV-1 isolates listed in figures 17C-17F with an inhibitory concentration (IC50) less than 1 µg / mL.
[0013]
Bispecific antibody, characterized in that it comprises isolated human monoclonal antibody, as defined in any one of claims 1 to 12.
[0014]
Isolated human monoclonal antibody according to any one of claims 1 to 13, characterized by the fact that it is linked to an effector portion.
[0015]
Isolated human monoclonal antibody according to claim 14, characterized by the fact that the effector portion is a toxin or a detectable label.
[0016]
Isolated nucleic acid molecule, characterized by the fact that it encodes the antibody, as defined in any one of claims 1 to 13.
[0017]
Isolated nucleic acid molecule according to claim 16, characterized by the fact that it is operationally linked to a heterologous promoter.
[0018]
Expression vector, characterized by the fact that it comprises the isolated nucleic acid molecule, as defined in claim 16 or 17.
[0019]
Composition, characterized by the fact that it comprises: (a) the antibody, as defined in any one of claims 1 to 15, the nucleic acid molecule, as defined in claim 16 or 17, the expression vector, as defined in claim 18, or a combination of two or more of the themselves; and (b) a pharmaceutically acceptable vehicle.
[0020]
Method of detecting an infection by the human immunodeficiency virus (HIV) -1 in an individual, characterized by the fact that it comprises: contacting, in vitro, a biological sample from the individual with the isolated human monoclonal antibody, as defined in any one of claims 1 to 15, under conditions sufficient to form an immune complex; and detecting the presence of the immune complex in the individual's sample, in which the presence of the immune complex in the individual's sample indicates that the individual has an HIV-1 infection; and optionally, wherein the isolated human monoclonal antibody is directly labeled.
[0021]
Method, according to claim 20, characterized by the fact that it further comprises: contacting the sample in vitro with a second antibody that specifically binds to the isolated human monoclonal antibody; and detecting the binding of the second antibody to the sample; wherein an increase in the binding of the second antibody to the sample compared to the binding of the second antibody to a control sample, detects the presence of an HIV-1 infection in the individual.
[0022]
Use of the antibody, as defined in any one of claims 1 to 15, the nucleic acid molecule as defined in claim 16 or 17, the expression vector as defined in claim 18, or a combination of two or more of the same, characterized by the fact that it is for the preparation of a composition for the prevention or treatment of infection by the human immunodeficiency virus (HIV) -1 in an individual.
[0023]
Use, according to claim 22, characterized by the fact that it is a use for the treatment of an HIV infection1, and in which the individual has acquired immune deficiency syndrome (AIDS).
[0024]
Use according to claim 22 or 23, characterized in that it further comprises an additional antiviral agent, optionally wherein the additional antiviral agent comprises a nucleoside analog reverse transcriptase inhibitor, a nucleotide reverse transcriptase inhibitor, a non-nucleoside reverse transcriptase inhibitor, a protease inhibitor, an entry or fusion inhibitor, a maturation inhibitor, or a broad spectrum inhibitor or a combination thereof.
[0025]
Use according to any one of claims 22 to 24, characterized in that it further comprises one or more additional antibodies, or nucleic acids encoding such antibodies, wherein the additional antibodies specifically bind gp120 and / or gp41; and / or it further comprises an effective amount of IL-15.
[0026]
Method for testing a potential immunogen, characterized in that it comprises contacting the potential immunogen with an antibody, as defined in any one of claims 1 to 15; and detecting the binding of the antibody to the immunogen, thereby determining that the immunogen is in use to produce an immune response to a human immunodeficiency virus.
[0027]
Kit, characterized by the fact that it comprises: (a) the antibody, as defined in any one of claims 1 to 15, the nucleic acid molecule, as defined in claim 16 or 17, the expression vector, as defined in claim 18, the composition, as defined in claim 19 , a combination of two or more of the same; and (b) instructions for using the kit.

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同族专利:

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CN104080805A|2014-10-01|

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US20160333076A1|2016-11-17|

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US10047148B2|2018-08-14|

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BR112014010823A2|2017-09-12|

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US10047147B2|2018-08-14|

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US20140348785A1|2014-11-27|

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WO2013070776A8|2014-06-12|

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EP2776463A4|2015-03-25|

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RU2014118462A|2015-12-20|

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US20140342407A1|2014-11-20|

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US20180002406A1|2018-01-04|

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ZA201403264B|2017-08-30|

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RU2624046C2|2017-06-30|

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EP2776463A1|2014-09-17|

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WO2013070776A1|2013-05-16|

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CN104080805B|2017-02-22|

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law|

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2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|

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2019-08-13| B07E| Notice of approval relating to section 229 industrial property law|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-10-01| B06U| Preliminary requirement: requests with searches performed by other patent offices: suspension of the patent application procedure|

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2020-07-28| B07A| Technical examination (opinion): publication of technical examination (opinion)|

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2020-11-17| B09A| Decision: intention to grant|

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2021-02-17| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 07/11/2012, OBSERVADAS AS CONDICOES LEGAIS. |

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