Human immunodeficiency virus neutralizing antibodies and methods of use thereof

Abstract

To provide broadly neutralizing antibodies against HIV.SOLUTION: The invention provides broadly neutralizing antibodies directed to epitopes of Human Immunodeficiency Virus, or HIV. The invention further provides compositions containing antibodies used for prophylaxis, and methods for diagnosis and treatment of HIV infection.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a related application to U.S. Provisional Patent Application No. 61 / 486,960, filed May 17, 2011, and claims priority under 35 U.S.C. § 119 to that provisional application.

[0002] (Statement Regarding Federally Sponsored Research) Research leading to this invention was supported in part by National Institutes of Health Grant No. P01 AI08677-01. Accordingly, the United States Government may have certain rights in this invention.

[0003] FIELD OF THEINVENTION This invention relates to antibodies that bind to epitopes of the human immunodeficiency virus (HIV). This invention further relates to methods for making and using such antibodies to broadly neutralize antibodies that bind to the HIV gp120 envelope protein for the prevention and treatment of HIV infection. [Background technology]

[0004] 2. Background of the Invention HIV causes acquired immune deficiency syndrome (AIDS). Immune responses to HIV infection in long-term non-progressors suggest that specific viral immunity may limit infection and disease manifestations. Some HIV-infected individuals display widespread neutralizing IgG antibodies in serum. Despite their potential importance in the design of effective vaccines, little is known about the specificity and activity of these antibodies, and none have been found to correlate with protective immunity. In animal models, passive transfer of neutralizing antibodies can contribute to protection against viral challenge. Neutralizing antibodies can develop in HIV-infected individuals, but the detailed composition of the serological response has not yet been fully elucidated.

[0005] Several immunological abnormalities are described in AIDS, including, but not limited to, abnormalities in B-cell function, abnormal antibody responses, defective monocyte function, impaired cytokine production, suppressed natural killer cell and cytotoxic cell function, a defective ability of lymphocytes to recognize and respond to soluble antigens, and reduced numbers of T4 helper / attractant lymphocytes.

[0006] The amino acid and RNA sequences encoding the HIV envelope are known from several HIV strains (Modrow, S. et al., J. Virology 61(2): 570 (1987)). HIV virions (virions) are surrounded by a membrane or envelope that is derived from the surface membrane of the host cell. This membrane contains a group of envelope glycoproteins (gp160) that are anchored to the membrane bilayer at their C-terminal regions. Each glycoprotein contains two parts, an N-terminal part and a C-terminal part. The N-terminal part, called gp120 because of its relative molecular weight of about 120 kD, projects into the aqueous environment surrounding the virion. The C-terminal region, called gp41, spans the membrane. The N-terminal gp120 and the C-terminal gp41 are covalently linked by a peptide bond that is particularly susceptible to proteolytic cleavage. European Patent Application Publication No. 0 335 635, McCune et al., and the references therein. Each of the cited citations is incorporated herein by reference in its entirety.

[0007] Various approaches to AIDS vaccines have been proposed, including, but not limited to, inactivated and attenuated virus vaccines, subunit vaccines from virus-infected cells, recombinantly produced viral antigens, synthetic peptide-based vaccines, anti-idiotype vaccines, and viral carrier-based vaccines. Further approaches to therapeutic and prophylactic treatment of HIV include generating highly potent, broadly neutralizing monoclonal antibodies. Several studies have reported cloning and generation of monoclonal antibodies by various techniques to target the CD4 binding site as well as other parts of the virion spike and to neutralize HIV. Usually these techniques include autofusion or phage display technology. Typically, in generating HIV neutralizing antibodies using phage display technology, random combinations of heavy and light chains are combined and random pairs are selected. Studies have reported a limited number of monoclonal antibodies (e.g., phage display antibody b12) that are broadly highly potent and broadly neutralizing (meaning antibodies that neutralize multiple strains of HIV in serum) against HIV. Monoclonal antibody b12 is a broadly neutralizing antibody that has been reported to prevent HIV infection in macaques. Other broadly neutralizing antibodies include 2G12, which is atypical and has a structure not seen in other antibodies with three binding sites.

[0008] VRC01 is a recently discovered broadly neutralizing antibody that targets the CD4 binding site (CD4bs) on the HIV spike. VRCO1 was isolated by purifying single B cells binding to a soluble, biotin-labeled, stabilized, and resurfaced core fragment of HIV gp120 (X. Wu et al., Science 329, 856 (Aug 13, 2010)). Despite its success, the isolation was inefficient, requiring 25 million terminal antibodies from a single individual. Only three closely related HIV-binding antibodies were produced from peripheral blood mononuclear cells. Like other anti-HIV antibodies obtained by single-cell antigen capture methods, VRCO1-3 showed very high levels of somatic mutations that are essential for efficacy and spread. The high mutation frequency is a potential obstacle to cloning of antibodies, since the mutated sequences may not be complementary to the primers used for cloning.

[0009] Several studies have reported that some patients develop broadly neutralizing antibodies against HIV. Studies have shown that antibodies can protect against initial HIV infection in passive transfer experiments in non-human primates and can modulate viral load during infection. See, e.g., Mascola, 2000; Shibata, 1999; Veazey, 2003; Parren, 2001; Mascola, 1999; Trkola, 2005; Wei, 2003; Frost, 2005; Burton, 2004; Mascola, 2007; Karlsson Hedestam, 2008; McMichael, 2006; Zolla-Pazner, 2004. Summary of the Invention

[0010] Summary of the Invention The invention provides, in one aspect, a broadly neutralizing antibody to HIV. In one aspect, the invention provides an isolated HIV antibody comprising a heavy chain comprising the following consensus amino acid sequence: QXXLXQSGGXVKKPGXSVXVSCXASGYXXFXXYXIHWXRQAPGXGXXWVGXIXPRXGXXXXAXXFQGRLSLTRDXXXXXXTXXXFMDLXGLRXDDTAVYFCARXXXXXXXXXXXXXXXDX (SEQ ID NO: 1), where X represents any amino acid or the absence of an amino acid.

[0011] In another aspect, the invention provides an isolated HIV antibody comprising a light chain comprising the following consensus amino acid sequence: EIXLTQSPXSLSXSXGEXXTISCXXXQXXXXXXXLXWYQQRXGXAPRLLIXXXSXXXXGVPXRFSGXXXGXXYXLXISXLXXDDXAXYFCXXYEXXXXXXX (SEQ ID NO: 2), where X represents any amino acid or the absence of an amino acid.

[0012] In another aspect, the invention provides an isolated HIV antibody comprising a heavy chain comprising a highly conserved consensus sequence and a light chain comprising a highly conserved consensus sequence. A method for producing an isolated HIV antibody comprising a light chain that includes a conserved consensus sequence is provided.

[0013] In another aspect, the invention provides an isolated HIV antibody comprising a heavy chain comprising the consensus sequence of SEQ ID NO: 1, and a light chain comprising the consensus sequence of SEQ ID NO: 2. In a further aspect, the invention provides an isolated HIV antibody comprising a heavy chain comprising the consensus sequence of SEQ ID NO: 1, and one or both of a light chain comprising the sequence of SEQ ID NO: 2, or a sequence having at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identity thereto, provided that the antibody does not have the amino acid sequence of VRC01.

[0014] In another aspect, the invention provides an isolated HIV antibody comprising one or both of a heavy chain comprising the consensus sequence of SEQ ID NO:1 and a light chain comprising the consensus sequence of SEQ ID NO:2, wherein the antibody has an IC of less than 1.0 μg / ml. 50 Neutralizes HIV virus ZM53M.PB12 at concentrations less than 1.0 μg / ml 50 Neutralizes HIV virus R1166.c1 at concentrations below IC of 30 μg / ml 50 In another aspect, the invention provides an isolated HIV antibody comprising one or both of a heavy chain comprising the consensus sequence of SEQ ID NO:1 and a light chain comprising the consensus sequence of SEQ ID NO:2, wherein the antibody has an IC of 30 μg / ml or less. 50 Neutralizes VRC01-resistant HIV virus at concentrations

[0015] In another aspect of the invention there is provided an isolated HIV antibody selected from the group consisting of 3BNC117, 3BNC60, 12A12, 12A21, NIH45-46, 8ANC131, 8ANC134, IB2530, INC9 and 8ANC196.

[0016] In another aspect, the invention provides an isolated HIV antibody comprising heavy chain CDR1, CDR2 and CDR3 regions, and light chain CDR1, CDR2 and CDR3 regions, which comprise the amino acid sequences of the matching regions of an HIV antibody selected from the group consisting of 3BNC117, 3BNC60, 12A12, 12A21, NIH45-46, bANC131, 8ANC134, IB2530, INC9 and 8ANC196.

[0017] In another aspect, the invention provides an isolated HIV antibody comprising a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:5-438.

[0018] In another aspect, the invention provides an isolated HIV antibody comprising a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 439-583. In another aspect, the invention provides an isolated HIV antibody comprising a heavy chain and a light chain comprising an amino acid sequence as shown in Table A or Table B.

[0019] In another aspect, the invention provides an isolated HIV antibody comprising an insert sequence comprising the amino acid sequence: ASWDFDF (SEQ ID NO:3).

[0020] In another aspect, the invention provides an isolated HIV antibody comprising an insert sequence comprising the amino acid sequence: TARDY (SEQ ID NO:4).

[0021] In another aspect, the invention provides an isolated HIV antibody comprising the insert sequence of SEQ ID NO:3 and SEQ ID NO:4.

[0022] In another embodiment, the invention relates to at least one of the insert sequences of SEQ ID NO:3 and SEQ ID NO:4. The present invention provides a method for improving the HIV neutralization potency and breadth of isolated HIV antibodies, comprising inserting one

[0023] In another aspect, the invention provides a composition comprising an isolated HIV of the invention.

[0024] According to another aspect, the invention provides a pharmaceutical composition comprising an antibody according to the invention and a pharma- ceutical acceptable carrier.

[0025] In another aspect, the invention provides a nucleic acid molecule encoding an isolated HIV antibody according to the invention.

[0026] According to another aspect, there is provided a vector comprising a nucleic acid molecule encoding an isolated HIV antibody according to the invention, and a cell comprising such a vector.

[0027] According to another aspect, the invention provides a method for preventing or treating HIV infection or an HIV-associated disease, comprising identifying a mammalian subject in need of such prevention or treatment, and administering to said subject a therapeutically effective amount of at least one HIV antibody according to the invention.

[0028] According to another embodiment, the method further comprises administering a second therapeutic agent. According to another embodiment, the second therapeutic agent is an antiviral agent.

[0029] Another aspect of the invention provides a method for reducing viral replication or the spread of infection to additional host cells or tissues comprising contacting a mammalian cell with at least one of the antibodies of the invention. According to another aspect, the invention provides a method for treating a mammalian subject infected with HIV, the method comprising administering to the subject a pharmaceutical composition comprising at least one of the antibodies of the invention.

[0030] According to another aspect, the invention provides methods for making and administering HIV antibody formulations suitable for administration to a mammalian subject infected with or at risk for HIV in an amount and according to a schedule sufficient to induce a protective immune response against HIV or a reduction in HIV virus in the mammalian subject. In another aspect, the invention provides a method for detecting an HIV antibody comprising a heavy chain that contains a highly conserved consensus sequence and a light chain that contains a highly conserved consensus sequence.

[0031] In another aspect, the invention provides an isolated antibody of the invention for use in the treatment of HIV.

[0032] In another aspect, the invention provides a kit comprising at least one pharmacologically effective amount of a pharmacologically acceptable dosage unit of an isolated HIV antibody of the invention, and a pharmacologically effective amount of a pharmacologically acceptable dosage unit of an HIV agent selected from the group consisting of a non-nucleoside reverse transcriptase inhibitor, a protease inhibitor, an entry or fusion inhibitor, and an integrase inhibitor, wherein the two pharmacologically acceptable dosage units may be in the form of a single pharmacologically acceptable dosage unit.

[0033] In another embodiment, the invention provides a kit for the diagnosis, prognosis, or monitoring of treatment of HIV in a patient, comprising one or more detection reagents that specifically bind to anti-HIV neutralizing antibodies in a biological sample from a subject, said kit further comprising reagents for performing PCR or mass spectrometry. [Brief description of the drawings]

[0034] [Figure 1A-B]Figure 1A-D show the neutralization activity IC50 of HIV antibodies. (A) Limited panel. Toplines indicate donor number, clone or antibody (Table 4). Viruses are shown on the left. Colors indicate concentrations at IC50: red < 0.1 μg / ml, orange 0.1-1 μg / ml, yellow 1-10 μg / ml, green > 10 μg / ml, white indicates no neutralization at any concentration tested. (B) Expanded panel. (C) Neutralization summary graph comparing VRCO1, NIH45-46 and 3BNC117. Line length and circle size inversely proportional to IC50. Colors indicate virus clades: red for A, blue for B, green for C, magenta for D, black for AE and gold for AG. (D) The sequences of the heavy chains of 3BNC60 (SEQ ID NO: 893), 1B2530 and 8ANC134 with coverage by peptides discovered by mass spectrometry are shown in light grey. Red dots represent differences from the respective germline sequences. [Figure 1C-D] Figure 1A-D show the neutralization activity IC50 of HIV antibodies. (A) Limited panel. Toplines indicate donor number, clone or antibody (Table 4). Viruses are shown on the left. Colors indicate concentrations at IC50: red < 0.1 μg / ml, orange 0.1-1 μg / ml, yellow 1-10 μg / ml, green > 10 μg / ml, white indicates no neutralization at any concentration tested. (B) Expanded panel. (C) Neutralization summary graph comparing VRCO1, NIH45-46 and 3BNC117. Line length and circle size inversely proportional to IC50. Colors indicate virus clades: red for A, blue for B, green for C, magenta for D, black for AE and gold for AG. (D) The sequences of the heavy chains of 3BNC60 (SEQ ID NO: 893), 1B2530 and 8ANC134 with coverage by peptides discovered by mass spectrometry are shown in light grey. Red dots represent differences from the respective germline sequences. [Diagram 2]Figure 2A-C shows the binding characteristics of HIV antibodies. (A) Representative surface plasmon resonance (SPR) sensograms for binding to YU2-gpl40 and 2CC-core by 12A12, 12A21 and 12A-germline (GL) reverted antibodies. (B) K A for representative antibodies is shown. (C) Graph shows mean fluorescence intensity of anti-CD4i antibodies bound to Bal.26 expressed in 293T cells following incubation with the indicated antibodies. Table shows whether the antibody induces accessibility of CD4i sites. [Figure 3A-1] Figures 3A and B show the consensus sequence of HIV antibodies, and the amino acid sequences of HIV antibodies. (A) Alignment of amino acids associated with the framework (FR) and CDR regions for the consensus, germline gene, 10 selected antibodies, and 8ANC195 (SEQ ID NOs: 1 and 890-902, respectively, in order of appearance). Residues are numbered according to the structure of 3BNC60. (B) Light chain (SEQ ID NOs: 2 and 903-916, respectively, in order of appearance) represented as in (A). (C, D, and E) Crystal structure of the antigen-binding fragment of 3BNC60. [Figure 3A-2] Figures 3A and B show the consensus sequence of HIV antibodies, and the amino acid sequences of HIV antibodies. (A) Alignment of amino acids associated with the framework (FR) and CDR regions for the consensus, germline gene, 10 selected antibodies, and 8ANC195 (SEQ ID NOs: 1 and 890-902, respectively, in order of appearance). Residues are numbered according to the structure of 3BNC60. (B) Light chain (SEQ ID NOs: 2 and 903-916, respectively, in order of appearance) represented as in (A). (C, D, and E) Crystal structure of the antigen-binding fragment of 3BNC60. [Figure 3B]Figures 3A and B show the consensus sequence of HIV antibodies, and the amino acid sequences of HIV antibodies. (A) Alignment of amino acids associated with the framework (FR) and CDR regions for the consensus, germline gene, 10 selected antibodies, and 8ANC195 (SEQ ID NOs: 1 and 890-902, respectively, in order of appearance). Residues are numbered according to the structure of 3BNC60. (B) Light chain (SEQ ID NOs: 2 and 903-916, respectively, in order of appearance) represented as in (A). (C, D, and E) Crystal structure of the antigen-binding fragment of 3BNC60. [Figure 4A-1] 4A and B show restoration of a highly mutated immunoglobulin heavy chain using specific primers. (A) Side-by-side comparison of new and old primer sets. Red boxes indicate successful amplification of IgVH genes. 4A shows SEQ ID NOs: 917-979 (in order of appearance, respectively). (B) HIV antibodies binding to 2CC-core from Pt 8. Clonal families are shown by the differently extended slices. Two highly mutated clones that were not amplified with the old primer set are shown as striped pie slices. [Figure 4A-2] 4A and B show restoration of a highly mutated immunoglobulin heavy chain using specific primers. (A) Side-by-side comparison of new and old primer sets. Red boxes indicate successful amplification of IgVH genes. 4A shows SEQ ID NOs: 917-979 (in order of appearance, respectively). (B) HIV antibodies binding to 2CC-core from Pt 8. Clonal families are shown by the differently extended slices. Two highly mutated clones that were not amplified with the old primer set are shown as striped pie slices. [Figure 4B]4A and B show restoration of a highly mutated immunoglobulin heavy chain using specific primers. (A) Side-by-side comparison of new and old primer sets. Red boxes indicate successful amplification of IgVH genes. 4A shows SEQ ID NOs: 917-979 (in order of appearance, respectively). (B) HIV antibodies binding to 2CC-core from Pt 8. Clonal families are shown by the differently extended slices. Two highly mutated clones that were not amplified with the old primer set are shown as striped pie slices. [Figure 5A] FIG. 5 shows the sequences of the heavy chain (A) (SEQ ID NOs: 980 to 984, in order of appearance) and light chain (B) (SEQ ID NOs: 985 to 989, in order of appearance) of IgV of the new VRC01 clone members. [Figure 5B] FIG. 5 shows the sequences of the heavy chain (A) (SEQ ID NOs: 980 to 984, in order of appearance) and light chain (B) (SEQ ID NOs: 985 to 989, in order of appearance) of IgV of the new VRC01 clone members. [Figure 6A] Figure 6 shows the neutralizing activity of patient sera. (A) The table presents an overview of the neutralizing activity of purified serum IgG against a panel of Tier 2 viruses in the Tzm-bl assay. Dark red boxes indicate IC50 values ​​lower than 10 μg / ml, orange indicates values ​​between 10 and 100 μg / ml, and yellow indicates values ​​higher than 100 μg / ml. (B) The dot plot shows an overview of the patient IC50s tested in A that are higher than 4. [Figure 6B] Figure 6 shows the neutralizing activity of patient sera. (A) The table presents an overview of the neutralizing activity of purified serum IgG against a panel of Tier 2 viruses in the Tzm-bl assay. Dark red boxes indicate IC50 values ​​lower than 10 μg / ml, orange indicates values ​​between 10 and 100 μg / ml, and yellow indicates values ​​higher than 100 μg / ml. (B) The dot plot shows an overview of the patient IC50s tested in A that are higher than 4. [Figure 7A-B]FIG. 7 shows detection of antibodies by mass spectrometry. Collision activated doubly charged peptides HSDYCDFDVWGSGSQVIVSSASTK (SEQ ID NO: 888) (A) from 3BNC153HC and resolved MS / MS spectra recorded on DGLGEVAPAYLYGIDAWGQGTTVIVTSASTK (SEQ ID NO: 889) (B) from 8ANC134HC. Observed b-type fragment ions (including the N-terminus) and y-type fragment ions (including the C-terminus) are labeled in the spectra. Loss of water from fragment ions is indicated by *. Ions corresponding to loss of water from the original ion are labeled in the spectra. Observed backbone breaks are indicated in the sequence using (" symbol) for b-type ions and (" symbol) for y-type ions. [Figure 8A] Figures 8A and B show the affinity of HIV antibodies. (A) Antibody binding to gp140 and 2CC-core was measured by surface plasmon resonance (SPR). SPR sensograms for antibody binding over time for selected 3BNC antibody clones are shown. (B) Bar graphs show the binding affinity (KA) for gp140 and 2CC-core antigens for selected IgG antibodies shown in A. [Figure 8B] Figures 8A and B show the affinity of HIV antibodies. (A) Antibody binding to gp140 and 2CC-core was measured by surface plasmon resonance (SPR). SPR sensograms for antibody binding over time for selected 3BNC antibody clones are shown. (B) Bar graphs show the binding affinity (KA) for gp140 and 2CC-core antigens for selected IgG antibodies shown in A. [Figure 9A-1]Figures 9A-C illustrate somatic hypermutation analysis of selected HIV antibodies for (A) immunoglobulin heavy chain gene, (B) light chain kappa, and (C) light chain lambda gene sequences. Sequences are aligned with the respective germline nucleotide sequences. Somatic mutations are represented in red, plus grey boxes indicate substitution mutations. Germline amino acid sequences are shown above the nucleotide alignment with * indicating consensus residues. Figure 9A-1 shows SEQ ID NOs: 991, 990, and 992-997; Figure 9A-2 shows SEQ ID NOs: 991, 990, and 992-997 (continued) and SEQ ID NOs: 999, 998, and 1000-1003; Figure 9A-3 shows SEQ ID NOs: 999, 998, and 1000-1003 (continued), and Figure 9B-1 shows SEQ ID NOs: 1005, 1004, and 1006-1008 (continued). 9B-1 shows sequence numbers 1005, 1004, and 1006-1009 (continued) and sequence numbers 1011, 1010, and 1012-1015; FIG. 9B-2 shows sequence numbers 1005, 1004, and 1006-1009 (continued) and sequence numbers 1011, 1010, and 1012-1015; FIG. 9B-3 shows sequence numbers 1011, 1010, and 1012-1015 (continued); FIG. 9C shows sequence numbers 1017, 1016, and 1018-1019; all in order of appearance, respectively. [Figure 9A-2]Figures 9A-C illustrate somatic hypermutation analysis of selected HIV antibodies for (A) immunoglobulin heavy chain gene, (B) light chain kappa, and (C) light chain lambda gene sequences. Sequences are aligned with the respective germline nucleotide sequences. Somatic mutations are represented in red, plus grey boxes indicate substitution mutations. Germline amino acid sequences are shown above the nucleotide alignment with * indicating consensus residues. Figure 9A-1 shows SEQ ID NOs: 991, 990, and 992-997; Figure 9A-2 shows SEQ ID NOs: 991, 990, and 992-997 (continued) and SEQ ID NOs: 999, 998, and 1000-1003; Figure 9A-3 shows SEQ ID NOs: 999, 998, and 1000-1003 (continued), and Figure 9B-1 shows SEQ ID NOs: 1005, 1004, and 1006-1008 (continued). 9B-1 shows sequence numbers 1005, 1004, and 1006-1009 (continued) and sequence numbers 1011, 1010, and 1012-1015; FIG. 9B-2 shows sequence numbers 1005, 1004, and 1006-1009 (continued) and sequence numbers 1011, 1010, and 1012-1015; FIG. 9B-3 shows sequence numbers 1011, 1010, and 1012-1015 (continued); FIG. 9C shows sequence numbers 1017, 1016, and 1018-1019; all in order of appearance, respectively. [Figure 9A-3]Figures 9A-C illustrate somatic hypermutation analysis of selected HIV antibodies for (A) immunoglobulin heavy chain gene, (B) light chain kappa, and (C) light chain lambda gene sequences. Sequences are aligned with the respective germline nucleotide sequences. Somatic mutations are represented in red, plus grey boxes indicate substitution mutations. Germline amino acid sequences are shown above the nucleotide alignment with * indicating consensus residues. Figure 9A-1 shows SEQ ID NOs: 991, 990, and 992-997; Figure 9A-2 shows SEQ ID NOs: 991, 990, and 992-997 (continued) and SEQ ID NOs: 999, 998, and 1000-1003; Figure 9A-3 shows SEQ ID NOs: 999, 998, and 1000-1003 (continued), and Figure 9B-1 shows SEQ ID NOs: 1005, 1004, and 1006-1008 (continued). 9B-1 shows sequence numbers 1005, 1004, and 1006-1009 (continued) and sequence numbers 1011, 1010, and 1012-1015; FIG. 9B-2 shows sequence numbers 1005, 1004, and 1006-1009 (continued) and sequence numbers 1011, 1010, and 1012-1015; FIG. 9B-3 shows sequence numbers 1011, 1010, and 1012-1015 (continued); FIG. 9C shows sequence numbers 1017, 1016, and 1018-1019; all in order of appearance, respectively. [Figure 9B-1]Figures 9A-C illustrate somatic hypermutation analysis of selected HIV antibodies for (A) immunoglobulin heavy chain gene, (B) light chain kappa, and (C) light chain lambda gene sequences. Sequences are aligned with the respective germline nucleotide sequences. Somatic mutations are represented in red, plus grey boxes indicate substitution mutations. Germline amino acid sequences are shown above the nucleotide alignment with * indicating consensus residues. Figure 9A-1 shows SEQ ID NOs: 991, 990, and 992-997; Figure 9A-2 shows SEQ ID NOs: 991, 990, and 992-997 (continued) and SEQ ID NOs: 999, 998, and 1000-1003; Figure 9A-3 shows SEQ ID NOs: 999, 998, and 1000-1003 (continued), and Figure 9B-1 shows SEQ ID NOs: 1005, 1004, and 1006-1008 (continued). 9B-1 shows sequence numbers 1005, 1004, and 1006-1009 (continued) and sequence numbers 1011, 1010, and 1012-1015; FIG. 9B-2 shows sequence numbers 1005, 1004, and 1006-1009 (continued) and sequence numbers 1011, 1010, and 1012-1015; FIG. 9B-3 shows sequence numbers 1011, 1010, and 1012-1015 (continued); FIG. 9C shows sequence numbers 1017, 1016, and 1018-1019; all in order of appearance, respectively. [Figure 9B-2]Figures 9A-C illustrate somatic hypermutation analysis of selected HIV antibodies for (A) immunoglobulin heavy chain gene, (B) light chain kappa, and (C) light chain lambda gene sequences. Sequences are aligned with the respective germline nucleotide sequences. Somatic mutations are represented in red, plus grey boxes indicate substitution mutations. Germline amino acid sequences are shown above the nucleotide alignment with * indicating consensus residues. Figure 9A-1 shows SEQ ID NOs: 991, 990, and 992-997; Figure 9A-2 shows SEQ ID NOs: 991, 990, and 992-997 (continued) and SEQ ID NOs: 999, 998, and 1000-1003; Figure 9A-3 shows SEQ ID NOs: 999, 998, and 1000-1003 (continued), and Figure 9B-1 shows SEQ ID NOs: 1005, 1004, and 1006-1008 (continued). 9B-1 shows sequence numbers 1005, 1004, and 1006-1009 (continued) and sequence numbers 1011, 1010, and 1012-1015; FIG. 9B-2 shows sequence numbers 1005, 1004, and 1006-1009 (continued) and sequence numbers 1011, 1010, and 1012-1015; FIG. 9B-3 shows sequence numbers 1011, 1010, and 1012-1015 (continued); FIG. 9C shows sequence numbers 1017, 1016, and 1018-1019; all in order of appearance, respectively. [Figure 9B-3]Figures 9A-C illustrate somatic hypermutation analysis of selected HIV antibodies for (A) immunoglobulin heavy chain gene, (B) light chain kappa, and (C) light chain lambda gene sequences. Sequences are aligned with the respective germline nucleotide sequences. Somatic mutations are represented in red, plus grey boxes indicate substitution mutations. Germline amino acid sequences are shown above the nucleotide alignment with * indicating consensus residues. Figure 9A-1 shows SEQ ID NOs: 991, 990, and 992-997; Figure 9A-2 shows SEQ ID NOs: 991, 990, and 992-997 (continued) and SEQ ID NOs: 999, 998, and 1000-1003; Figure 9A-3 shows SEQ ID NOs: 999, 998, and 1000-1003 (continued), and Figure 9B-1 shows SEQ ID NOs: 1005, 1004, and 1006-1008 (continued). 9B-1 shows sequence numbers 1005, 1004, and 1006-1009 (continued) and sequence numbers 1011, 1010, and 1012-1015; FIG. 9B-2 shows sequence numbers 1005, 1004, and 1006-1009 (continued) and sequence numbers 1011, 1010, and 1012-1015; FIG. 9B-3 shows sequence numbers 1011, 1010, and 1012-1015 (continued); FIG. 9C shows sequence numbers 1017, 1016, and 1018-1019; all in order of appearance, respectively. [Figure 9C]Figures 9A-C illustrate somatic hypermutation analysis of selected HIV antibodies for (A) immunoglobulin heavy chain gene, (B) light chain kappa, and (C) light chain lambda gene sequences. Sequences are aligned with the respective germline nucleotide sequences. Somatic mutations are represented in red, plus grey boxes indicate substitution mutations. Germline amino acid sequences are shown above the nucleotide alignment with * indicating consensus residues. Figure 9A-1 shows SEQ ID NOs: 991, 990, and 992-997; Figure 9A-2 shows SEQ ID NOs: 991, 990, and 992-997 (continued) and SEQ ID NOs: 999, 998, and 1000-1003; Figure 9A-3 shows SEQ ID NOs: 999, 998, and 1000-1003 (continued), and Figure 9B-1 shows SEQ ID NOs: 1005, 1004, and 1006-1008 (continued). 9B-1 shows sequence numbers 1005, 1004, and 1006-1009 (continued) and sequence numbers 1011, 1010, and 1012-1015; FIG. 9B-2 shows sequence numbers 1005, 1004, and 1006-1009 (continued) and sequence numbers 1011, 1010, and 1012-1015; FIG. 9B-3 shows sequence numbers 1011, 1010, and 1012-1015 (continued); FIG. 9C shows sequence numbers 1017, 1016, and 1018-1019; all in order of appearance, respectively. [Figure 10A-1] Figures 10A-C show antibody sequences from one expanded neutralizing clone in (A) patient (Pt) 1, (B) Pt3, and (C) Pt8, respectively. Peptides identified by mass spectrometry are shown in color. Variants marked with an asterisk are uniquely identified by one or more mass spectrometry observed peptides (shown in light grey). The remaining mass spectrometry observed peptides map non-uniquely to multiple variants shown in dark grey. Amino acids shown underlined indicate non-trypsin cleavage sites in the indicated variants. Cleavage is presumed to occur at these sites through chymotrypsin cleavage or additional mutations (not observed among the cloned variants) that place lysine or arginine residues. Figure 10A shows SEQ ID NOs: 1020-1061; Figure 10B shows SEQ ID NOs: 1062-1113, and Figure 10c shows SEQ ID NOs: 1114-1138; all in order of appearance, respectively. [Figure 10A-2]Figures 10A-C show antibody sequences from one expanded neutralizing clone in (A) patient (Pt) 1, (B) Pt3, and (C) Pt8, respectively. Peptides identified by mass spectrometry are shown in color. Variants marked with an asterisk are uniquely identified by one or more mass spectrometry observed peptides (shown in light grey). The remaining mass spectrometry observed peptides map non-uniquely to multiple variants shown in dark grey. Amino acids shown underlined indicate non-trypsin cleavage sites in the indicated variants. Cleavage is presumed to occur at these sites through chymotrypsin cleavage or additional mutations (not observed among the cloned variants) that place lysine or arginine residues. Figure 10A shows SEQ ID NOs: 1020-1061; Figure 10B shows SEQ ID NOs: 1062-1113, and Figure 10c shows SEQ ID NOs: 1114-1138; all in order of appearance, respectively. [Figure 10B-1] Figures 10A-C show antibody sequences from one expanded neutralizing clone in (A) patient (Pt) 1, (B) Pt3, and (C) Pt8, respectively. Peptides identified by mass spectrometry are shown in color. Variants marked with an asterisk are uniquely identified by one or more mass spectrometry observed peptides (shown in light grey). The remaining mass spectrometry observed peptides map non-uniquely to multiple variants shown in dark grey. Amino acids shown underlined indicate non-trypsin cleavage sites in the indicated variants. Cleavage is presumed to occur at these sites through chymotrypsin cleavage or additional mutations (not observed among the cloned variants) that place lysine or arginine residues. Figure 10A shows SEQ ID NOs: 1020-1061; Figure 10B shows SEQ ID NOs: 1062-1113, and Figure 10c shows SEQ ID NOs: 1114-1138; all in order of appearance, respectively. [Figure 10B-2]Figures 10A-C show antibody sequences from one expanded neutralizing clone in (A) patient (Pt) 1, (B) Pt3, and (C) Pt8, respectively. Peptides identified by mass spectrometry are shown in color. Variants marked with an asterisk are uniquely identified by one or more mass spectrometry observed peptides (shown in light grey). The remaining mass spectrometry observed peptides map non-uniquely to multiple variants shown in dark grey. Amino acids shown underlined indicate non-trypsin cleavage sites in the indicated variants. Cleavage is presumed to occur at these sites through chymotrypsin cleavage or additional mutations (not observed among the cloned variants) that place lysine or arginine residues. Figure 10A shows SEQ ID NOs: 1020-1061; Figure 10B shows SEQ ID NOs: 1062-1113, and Figure 10c shows SEQ ID NOs: 1114-1138; all in order of appearance, respectively. [Figure 10C-1] Figures 10A-C show antibody sequences from one expanded neutralizing clone in (A) patient (Pt) 1, (B) Pt3, and (C) Pt8, respectively. Peptides identified by mass spectrometry are shown in color. Variants marked with an asterisk are uniquely identified by one or more mass spectrometry observed peptides (shown in light grey). The remaining mass spectrometry observed peptides map non-uniquely to multiple variants shown in dark grey. Amino acids shown underlined indicate non-trypsin cleavage sites in the indicated variants. Cleavage is presumed to occur at these sites through chymotrypsin cleavage or additional mutations (not observed among the cloned variants) that place lysine or arginine residues. Figure 10A shows SEQ ID NOs: 1020-1061; Figure 10B shows SEQ ID NOs: 1062-1113, and Figure 10c shows SEQ ID NOs: 1114-1138; all in order of appearance, respectively. [Figure 10C-2]Figures 10A-C show antibody sequences from one expanded neutralizing clone in (A) patient (Pt) 1, (B) Pt3, and (C) Pt8, respectively. Peptides identified by mass spectrometry are shown in color. Variants marked with an asterisk are uniquely identified by one or more mass spectrometry observed peptides (shown in light grey). The remaining mass spectrometry observed peptides map non-uniquely to multiple variants shown in dark grey. Amino acids shown underlined indicate non-trypsin cleavage sites in the indicated variants. Cleavage is presumed to occur at these sites through chymotrypsin cleavage or additional mutations (not observed among the cloned variants) that place lysine or arginine residues. Figure 10A shows SEQ ID NOs: 1020-1061; Figure 10B shows SEQ ID NOs: 1062-1113, and Figure 10c shows SEQ ID NOs: 1114-1138; all in order of appearance, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Detailed Description of the Invention 1.HIV neutralizing antibody In one aspect of the invention, there is provided a broadly neutralizing antibody against HIV. In one aspect, the invention provides an isolated HIV antibody comprising a heavy chain comprising the following consensus amino acid sequence: QXXLXQSGGXVKKPGXSVXVSCXASGYXXFXXYXIHWXRQAPGXGXXWVGXIXPRXGXXXXAXXFQGRLSLTRDXXXXXXTXXXFMDLXGLRXDDTAVYFCARXXXXXXXXXXXXXXXXXXDX (SEQ ID NO: 1), where X represents any amino acid or the absence of an amino acid.

[0036] In another aspect, the invention provides an isolated HIV antibody comprising a light chain comprising the following consensus amino acid sequence: EIXLTQSPXSLSXSXGEXXTISCXXXQXXXXXXXLXWYQQRXGXAPRLLIXXXSXXXXGVPXRFSGXXXGXXYXLXISXLXXDDXAXYFCXXYEXXXXXXX (SEQ ID NO: 2), where X represents any amino acid or the absence of an amino acid.

[0037] In another aspect, the invention provides an isolated HIV antibody comprising a heavy chain comprising the consensus sequence of SEQ ID NO: 1, and a light chain comprising the consensus sequence of SEQ ID NO: 2. In a further aspect, the invention provides an isolated HIV antibody comprising a heavy chain sequence of SEQ ID NO: 1, and a light chain sequence of SEQ ID NO: 2, or a light chain sequence at least 70%, or at least 80%, or at least 85%, or at least 85% similar to the amino acid sequence of VRC01, provided that the antibody does not have the amino acid sequence of VRC01. The present invention provides isolated HIV antibodies comprising one or both of the sequences having 90%, or at least 95%, or at least 98%, or at least 99% identity to the HIV antibody of the present invention, wherein the specific percentages are determined as set forth below.

[0038] In another aspect, the invention provides an isolated antibody. The invention provides an isolated anti-HIV antibody comprising a heavy chain comprising a highly conserved heavy chain amino acid sequence, and a light chain comprising a highly conserved light chain amino acid sequence. A highly conserved heavy chain amino acid sequence is defined herein as having at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% amino acid identity with the sequence of SEQ ID NO:1. A highly conserved light chain amino acid sequence is defined herein as having at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% amino acid identity with the sequence of SEQ ID NO:2. The percentages are determined as set forth below.

[0039] In another aspect, the invention provides an isolated HIV antibody comprising a heavy chain that comprises a highly conserved amino acid sequence of the heavy chain and a light chain that comprises a highly conserved amino acid sequence of the light chain, provided that the antibody does not have the amino acid sequence of VRC01.

[0040] In another aspect, the invention provides an isolated HIV antibody comprising one or both of a heavy chain sequence of SEQ ID NO:1 and a light chain sequence of SEQ ID NO:2, wherein the antibody has an IC of less than 1.0 μg / ml. 50 Neutralizes HIV virus ZM53M.PB12 at concentrations less than 1.0 μg / ml 50 Neutralizes HIV virus R1166.c1 at concentrations below IC of 30 μg / ml 50 In another aspect, the invention provides an isolated HIV antibody comprising one or both of a heavy chain sequence of SEQ ID NO:1 and a light chain sequence of SEQ ID NO:2, wherein the antibody has an IC of 30 μg / ml or less. 50 The VRC01-resistant HIV virus was neutralized here at an IC of 50 μg / ml. 50 The HIV virus is defined as a virus that is resistant to neutralization by VRC01 at a specific antibody level.

[0041] In another aspect, the invention provides an isolated HIV antibody selected from the group consisting of 3BNC117, 3BNC60, 12A12, 12A21, NIH45-46, bANC131, (8ANC131), 8ANC134, IB2530, INC9, and 8ANC196.

[0042] In another aspect, the invention provides an isolated HIV antibody comprising heavy chain CDR1, CDR2 and CDR3 regions, and light chain CDR1, CDR2 and CDR3 regions, which comprise the amino acid sequences of the matching regions of an HIV antibody selected from the group consisting of 3BNC117, 3BNC60, 12A12, 12A21, NIH45-46, bANC131, (8ANC131), 8ANC134, IB2530, INC9 and 8ANC196.

[0043] In another aspect, the invention provides an isolated HIV antibody comprising a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:5-438.

[0044] In another aspect, the invention provides an isolated HIV antibody comprising a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 439-583.

[0045] In another aspect, the invention provides a method for the preparation of a nucleic acid sequence comprising the amino acid sequence shown in Table A or Table B. An isolated HIV antibody is provided, comprising a heavy chain and a light chain.

[0046] In another aspect, the invention provides an isolated HIV antibody comprising an insert sequence comprising the amino acid sequence: ASWDFDF (SEQ ID NO: 3). In a further aspect, the invention provides an isolated HIV antibody, in which the insert sequence SEQ ID NO: 3, which corresponds to the FR3 of the heavy chain beginning at amino acid 74 of 3BNC117 and 3BNC60 as shown in Figure 5A, is used in place of the corresponding region of an HIV antibody of the invention (as determined by sequence alignment). For example, SEQ ID NO: 3 may be inserted after the 7th amino acid of FR3 of the heavy chain.

[0047] In another aspect, the invention provides an isolated HIV antibody comprising an insert sequence comprising the amino acid sequence: TARDY (SEQ ID NO: 4). In a further aspect, the invention provides an isolated HIV antibody, in which an insert sequence corresponding to the CDR3 of the heavy chain beginning at amino acid 103 of NIH45-46, SEQ ID NO: 4, as shown in Figure 5A, is substituted for the corresponding region of an HIV antibody of the invention (as determined by sequence alignment). For example, SEQ ID NO: 4 may be inserted after the 4th amino acid of the CDR3 of the heavy chain.

[0048] In another aspect, the invention provides an isolated HIV antibody, in which an insert sequence, SEQ ID NO:3, corresponding to the FR3 of the heavy chain starting at amino acid 74 of 3BNC117 and 3BNC60, as shown in Figure 5A, is substituted for the corresponding region (as determined by sequence alignment) of an HIV antibody of the invention, and an insert sequence, SEQ ID NO:4, corresponding to the CDR3 of the heavy chain starting at amino acid 103 of NIH45-46, as shown in Figure 5A, is substituted for the corresponding region (as determined by sequence alignment) of an HIV antibody of the invention. For example, SEQ ID NO:3 can be inserted after the 7th amino acid of FR3 of the heavy chain, and SEQ ID NO:4 can be inserted after the 4th amino acid of CDR3 of the heavy chain.

[0049] In a further aspect, the invention provides a method for improving the neutralizing potency and breadth of an isolated HIV antibody, including the production of an isolated HIV antibody, in which an insert sequence corresponding to the FR3 of the heavy chain starting at amino acid 74 of 3BNC117 and 3BNC60, as shown in FIG. 5A, SEQ ID NO: 3, is used in place of the corresponding region of an inventive HIV antibody (as determined by sequence alignment), and / or an insert sequence corresponding to the CDR3 of the heavy chain starting at amino acid 103 of NIH45-46, as shown in FIG. 5A, SEQ ID NO: 4, is used in place of the corresponding region of an inventive HIV antibody (as determined by sequence alignment). For example, SEQ ID NO: 3 can be inserted after the 7th amino acid of the FR3 of the heavy chain, and / or SEQ ID NO: 4 can be inserted after the 4th amino acid of the CDR3 of the heavy chain. Those skilled in the art can modify the amino acid sequence of an antibody utilizing recombinant and / or synthetic chemistry techniques for the production of peptides or antibodies. One of skill in the art can also use HIV neutralization assays, as provided below, to identify improved HIV antibodies with better neutralizing potency and breadth.

[0050] In another aspect, the invention provides an improved isolated HIV antibody comprising at least one of the inserts of SEQ ID NO: 3 and SEQ ID NO: 4, wherein the improved isolated HIV antibody has a greater potency and breadth of HIV neutralization than an isolated HIV antibody that does not have the inserts of SEQ ID NO: 3 and SEQ ID NO: 4. One skilled in the art can identify improved HIV antibodies with better potency and breadth of HIV neutralization using HIV neutralization assays, as provided below.

[0051] One skilled in the art can modify the amino acid sequence of an antibody utilizing recombinant and / or synthetic chemical techniques for the production of peptides or antibodies.

[0052] In another aspect, the invention provides a method for making an isolated HIV antibody comprising the heavy chain consensus sequence of SEQ ID NO:1, and the light chain sequence of SEQ ID NO:2.

[0053] In a further aspect, the invention provides a method for producing an isolated HIV antibody comprising either or both of the heavy chain consensus sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 2, or a sequence having at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identity thereto, provided that the antibody does not have the amino acid sequence of VRC01. The percentages are determined as set forth below.

[0054] In another aspect, the invention provides a method for detecting an isolated HIV antibody, the method comprising: a biological sample containing immunoglobulin from a mammalian subject, isolating an HIV antibody from the sample, determining the amino acid sequence of the HIV antibody, and identifying the presence of a heavy chain sequence of SEQ ID NO: 1 and a light chain sequence of SEQ ID NO: 2. In a further aspect, the invention provides a method for selecting an isolated HIV antibody, the method comprising determining the presence of one or both of the heavy chain consensus sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 2, or sequences having at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identity thereto, provided that the antibody does not have the amino acid sequence of VRC01. The identifying percentages are determined as set forth below. Biological Sample The biological sample may be blood, serum, saliva, urine, sputum, a cell swab sample, or a tissue biopsy. The amino acid sequence may be determined by known methods including, for example, PCR and mass spectrometry.

[0055] The term "antibody" (Ab) as used herein includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific and polyreactive antibodies), and antibody fragments. Thus, within the scope of this specification, the term "antibody" (Ab) also includes, but is not limited to, any specific binding member, immunoglobulin class and / or isotype (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM), and biologically relevant fragments or specific binding members thereof, including, but not limited to, Fab, F(ab')2, Fv, and scFv (single chain or related entities). As is well known in the art, an antibody is a glycoprotein that contains at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antibody binding protein thereof. The heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH1, CH2, and CH3). The light chain consists of a light chain variable region (VL) and a light chain constant region (CL). Both heavy and light chain variable regions contain framework regions (FWR) and complementarity determining regions (CDR). The four FWR regions are relatively conserved, while the CDR regions (CDR1, CDR2 and CDR3) represent highly variable regions and are arranged from the NH2-terminus to the COOH-terminus as follows: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4. The heavy and light chain variable regions contain the binding domains that interact with the antibody, while the constant regions, depending on the isotype, may mediate the binding of the immunoglobulin to host tissues or factors.

[0056] The definition of antibody as used herein also includes chimeric antibodies, humanized antibodies, and recombinant antibodies, human antibodies made from transgenic non-human animals, as well as antibodies selected from libraries using enrichment techniques available to one of skill in the art.

[0057] The word "variable" refers to the fact that certain segments of the variable (V) domains differ in sequence among antibodies. This refers to the fact that the V domain mediates antigen binding and determines the specificity of a particular antibody for a particular antigen. However, mutations are not uniformly distributed throughout the 110-amino acid span of the variable region. Instead, V regions contain relatively invariant stretches of 15-30 amino acids called framework regions (FRs) separated by shorter regions of extreme variability called hypervariable regions of 9-12 amino acids each. Natural heavy and light chain variable regions each consist of four FRs, adopting a beta-sheet configuration, and connected to three hypervariable regions, which form loops that connect to, and in some cases are part of, the beta-sheet structure. The hypervariable regions in each chain are held together in a contiguous fashion by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of the antibody. (See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0058] As used herein, hypervariable region refers to the amino acid residues of an antibody which are responsible for antigen binding. Hypervariable regions generally contain amino acid residues from the complementarity determining regions (CDRs).

[0059] The term monoclonal antibody as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, in which the individual antibodies comprising the population are identical, except for naturally occurring mutations which may be present in minor amounts, etc. Polyclonal antibody refers to a preparation containing different antibodies directed against different determinants (epitopes).

[0060] As used herein, monoclonal antibodies include "chimeric" antibodies in which a portion of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains are identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and also include fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). The described invention provides antigen-binding sequences in the variable regions that are derived from human antibodies. Thus, chimeric antibodies of primary interest herein include antibodies that have one or more human antigen-binding sequences (e.g., CDRs) and contain one or more derived from a non-human antibody (e.g., FR or C-region sequences). In addition, chimeric antibodies included herein are those that contain antigen-binding sequences of human variable regions that contain one antibody class and subclass and other sequences (e.g., FR or C-region sequences) from another antibody class or subclass.

[0061] A "humanized antibody" is generally considered to be a human antibody having one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are generally taken from an "import" variable region. Humanization can be performed according to the method of Winter et al. (see, e.g., Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)) by substituting the corresponding sequences of a human antibody with the imported variable region sequences. Such humanized antibodies are thus chimeric antibodies (US Pat. No. 4,816,567), in which substantially less intact human variable regions have been introduced into it from a non-human species. was replaced by the corresponding sequence.

[0062] Antibody fragments include portions of an intact antibody, such as the antigen-binding or variable regions of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, bispecific antibodies, linear antibodies (see, e.g., US Pat. No. 5,641,870; Zapata et al., Protein Eng. 8(10): 1057-1062

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

[0063] "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This fragment contains a dimer of one heavy and one light chain variable domain in tight, non-covalent association. From these folds, the two domains contribute amino acid residues for antigen binding and give rise to six hypervariable loops (three loops from each of the H and L chains) that present the antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv that contains only the three CDRs specific for an antigen) has the ability to recognize and bind the antigen, albeit with a lower affinity than the entire binding site.

[0064] "Single-chain Fv" (sFv, scFv) is an antibody fragment comprising the VH and VL antibody domains linked together into a single peptide chain. The sFv polypeptide further comprises a polypeptide peptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. For a review of sFv, see, e.g., Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra.

[0065] The term bispecific antibody refers to small antibody fragments prepared by constructing sFv fragments with a short linker (5-10 residues) between the VH and VL domains to achieve inter-chain, rather than intra-chain, binding of the V domains, thereby resulting in a bivalent fragment (i.e., a fragment with two antigen-binding sites). With dual specificity, bispecific antibodies are heterodimers of two crossover sFv fragments in which the VH and VL domains of the two antibodies are present on different peptide chains. For example, bispecific antibodies are described more fully in, e.g., EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0066] Domain antibodies (dAbs), which can be generated in a fully human form, are the smallest known antibody antigen-binding fragments, measuring approximately 11-15 kDa. dAbs are the robust variable regions of immunoglobulin heavy and light chains (VH and VL, respectively). They are highly expressed in microbial cell cultures, exhibit favorable biophysical properties (e.g., but not limited to, solubility and temperature stability), and are amenable to selection and affinity maturation by in vitro selection systems (e.g., phage display). dAbs are bioactive as monomers, and due to their small size and inherent stability, can be formatted into larger molecules to generate drugs with long serum half-lives or other pharmacological activity. Examples of this technology are found, for example, in WO9425591 for antibodies derived from camelid heavy chain Ig, as well as in WO9425592 for the isolation of single domain full-length human antibodies from phage libraries. No. 6,399,496, filed on Oct. 23, 2003.

[0067] Fv and sFv are the only types that have intact combining portions that lack the constant regions. Thus, they are suitable for in vivo use to reduce non-specific binding. sFv fusion proteins can be constructed to produce fusion of an effector protein at either the amino or carboxy terminus of an sFv. See, e.g., Antibody Engineering, ed. Borrebaeck, supra. Antibody fragments are linear antibodies. Such linear antibody fragments are described, for example, in US Pat. No. 5,641,870. The antibodies may be monospecific or bispecific, such as

[0068] In some embodiments, the antibodies of the invention are bispecific or multispecific. A bispecific antibody is an antibody that has binding specificities for at least two different epitopes. A typical bispecific antibody can bind to two different epitopes of an antigen. Other such antibodies can combine the first antigen-binding site with a binding site for a second antigen. Alternatively, the anti-HIV arm can be combined with an arm that binds to a target molecule on white blood cells, such as a T cell receptor molecule (e.g., CD3), or an Fc receptor for IgG (Fc gamma R), such as Fc gamma RI (CD64), Fc gamma RII (CD32), and Fc gamma RIII (CD16), to focus and localize cellular defense mechanisms to infected cells. Bispecific antibodies can also be used to localize cytotoxic drugs to infected cells. Bispecific antibodies can be made as full length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0069] For example, WO 96 / 16673 discloses a bispecific anti-ErbB2 / anti-Fc gamma RIII antibody. US Pat. No. 5,837,234 discloses a bispecific anti-ErbB2 / anti-Fc gamma RI antibody. For example, a bispecific anti-ErbB2 / Fc alpha antibody is reported in WO 98 / 02463. US Pat. No. 5,821,337 discloses a bispecific anti-ErbB2 / anti-CD3 The authors disclose antibodies that are capable of binding to HIV antibodies. See also, for example, Mouquet et al., Polyreactivity Increases The Apparent Affinity Of Anti-HIV Antibodies By Heteroligation. NATURE. 467, 591-5 (2010). Methods for producing bispecific antibodies can be known. Previous methods for producing full-length bispecific antibodies are based on co-expressing two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (see, for example, Millstein et al., Nature, 305:537-539 (1983)). Similar production methods are described, for example, in WO 93 / 08829, Traunecker et al., EMBO J., 10:3655-3659 (1991) and Mouquet et al., Polyreactivity Increases The Apparent Affinity Of Anti-HIV Antibodies By Heteroligation. NATURE. 467, 591-5 (2010). Increases The Apparent Affinity Of Anti-HIV Antibodies By Heteroligation. NATURE. 467, 591-5 (2010)).

[0070] Alternatively, antibody variable regions with the desired binding specificities (antibody-antigen binding sites) are fused to immunoglobulin constant domain sequences. The fusions are of the constant domain of an Ig heavy chain, including at least part of the hinge, CH2 and CH3 regions. According to some embodiments, the constant region of the first heavy chain (CH1), which contains the site necessary for light chain binding, is present in at least one of the fusions. DNA encoding the immunoglobulin heavy chain fusions and, optionally, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host cell. This allows greater flexibility in adjusting the mutual ratio of the three polypeptide fragments in embodiments where unequal ratios of the three polypeptide chains used in the construction provide optimal yields of the desired bispecific antibody. However, it is possible to insert sequences encoding two or all three polypeptide chains into one vector when expression of at least two polypeptide chains in balanced ratios results in high yields or when the ratios do not significantly affect the yield of the desired chain combination.

[0071] Techniques for producing bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be produced using chemical linkage. For example, Brennan et al., Science, 229: 81 (1985) report the use of proteolytic cleavage of intact antibodies to generate F(ab')2 fragments. The Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

[0072] Other modifications of proteins are contemplated herein. For example, antibodies may be modified with a variety of nonproteinaceous polymers (e.g., polyethylene glycol and polypropylene glycol). The antibodies can also be coupled to one of the following polymers: polyoxyalkylene, polypropylene glycol, polyoxyalkylene or copolymers. The antibodies can also be coupled to one of the following polymers: polyoxyalkylene, polypropylene glycol, polyoxyalkylene or copolymers. The antibodies can also be coupled to one of the following polymers: polyoxyalkylene, polypropylene glycol, polyoxyalkylene or copolymers. The antibodies can also be coupled to one of the following polymers: polyoxyalkylene, polypropylene glycol, polyoxyalkylene or copolymers. The antibodies can also be coupled to one of the following polymers: polyoxyalkylene, polyoxyalkylene or polyoxyalkylene copolymers. The compound can be encapsulated in microcapsules made by the methods described herein, such as by the techniques described in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).

[0073] Typically, the antibodies of the invention are produced recombinantly using vectors and methods available in the art. Human antibodies are produced by in vitro activated B cells (see, e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275). Molecular Gene General methods in genetic engineering and genetic engineering useful in this invention are described in the current edition of Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), gene expression techniques (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), enzyme engineering (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), and the like. "A Guide to Protein Purification" in Basic Methods (MP Deutschcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA); Animal Cell Culture: A Manual of Basic Techniques, Second Edition, (RI Freshney. 1987. Liss, Inc. New York, NY); See Protocols for Gene Transfer and Expression, pp. 109-128, ed. EJ Murray, The Humana Press Inc., Clifton, NJ. Reagents for genetic manipulation, cloning vectors, Vectors and kits are available from commercial vendors (eg, Bio-Rad, Stratagene, Invitrogen, Clontech, and Sigma-Aldrich).

[0074] Human antibodies can also be produced in transgenic animals (e.g., mice) capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice has been described, resulting in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge. For example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno., 7:33 (1993); US Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all of GenPharm); See US Pat. No. 5,545,807; and WO 97 / 17852. Such animals can be genetically engineered for the production of human antibodies comprising the polypeptides of the described invention.

[0075] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were obtained via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from E. coli. In this manner, large amounts of these fragments can be readily produced. Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (see, e.g., Carter et al., Bio / Technology 10:163-167 (1992)). Other approaches have been developed to produce antibody fragments using recombinant host cells. According to the present invention, F(ab')2 fragments can be directly isolated from recombinant host cell cultures. Fab and F(ab')2 fragments with increased half-life in vivo that contain salvage receptor binding epitope residues are disclosed in US Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.

[0076] Other techniques known to those skilled in the art for the selection of antibody fragments from libraries using enrichment techniques include, but are not limited to, phage display, ribosome display (Hanes and Pluckthun, 1997, Proc. Nat. Acad. Sci. 94: 4937-4942), bacterial display, and the like. Prey (Georgiou, et al., 1997, Nature Biotechnology 15: 29-34) and / or yeast display (Kieke, et al., 1997, Protein Engineering 10: 1303-1310) can be used to replace the previously mentioned techniques for selecting single chain antibodies. Single chain antibodies can be selected from libraries of single chain antibodies generated directly using filamentous phage technology. Phage display technology is known in the art (see, e.g., U.S. Patent Nos. 5,565,332; 5,733,743; 5,871,907; 5,872,215; 5,885,793; 5,962,255; 6,140,471; 6,225,447; 6,291650; 6,492,160; 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081 and other U.S. family members, as well as the priority claim of GB9206318 filed May 24, 1992). to Cambridge Antibody Technology (CAT), as disclosed in the applications on which the application is based. see also Vaughn, et al. 1996, Nature Biotechnology 14: 309-314. (See references ). Single chain antibodies can be designed and assembled using available recombinant gene techniques such as DNA amplification techniques (eg, PCR) or by using the respective hybridoma cDNA as a template.

[0077] Modified antibodies are also included within the scope of the invention. Modifications of the sequences detailed in this application are included within the scope of the invention. Furthermore, modifications of antibody sequences with improved affinity can be obtained using methods known in the art and are also included within the scope of the invention. For example, amino acid substitutions can be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of nucleic acid sequences can be used to improve the efficiency of translation in expression systems for the production of antibodies.

[0078] The sequences of such variant antibodies share 70% or more (i.e., 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) sequence identity with the sequences detailed in this application. Such sequence identity is calculated with respect to the full length of such referenced sequences (i.e., the sequences detailed in the application). As referred to herein, the percentage of identity is determined using BLAST version 2.1.3 using the default parameters specified by NCBI (the National Center for Biotechnology Information; http: / / www.ncbi.nlm.nih.gov / ) [Blosum 62 matrix; gap open penalty=11 and gap extension penalty=1]. For example, in accordance with the present invention, peptide sequences are provided that include at least about 5, 10, 15, 20, 30, 40, 50, 75, 100, 150 or more contiguous peptides of one or more of the sequences disclosed herein, as well as all intermediate lengths thereof. As used herein, the term "intermediate length" is meant to refer to any length between the recited values ​​(e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.).

[0079] The present invention provides antibodies that have broad range detoxifying activity in serum, either alone or in combination with other antibodies, such as, but not limited to, VRC01 and PG9.

[0080] According to another embodiment, the invention provides methods for the manufacture and administration of HIV antibody compositions suitable for administration to a human or non-human primate patient infected with or at risk of HIV infection in an amount and according to a schedule sufficient to induce a protective immune response against HIV or a reduction in HIV virus in humans.

[0081] In another aspect, the invention provides a vaccine comprising at least one antibody of the invention and a pharmacologically acceptable carrier. In one aspect, the vaccine is a vaccine comprising at least one antibody described herein and a pharmacologically acceptable carrier. The vaccine can include multiple antibodies having the characteristics described herein in any combination, and can further include antibodies known in the art that neutralize HIV.

[0082] It should be understood that the composition can be a single or combination of antibodies disclosed herein, either the same or different, to treat the progression of various subtypes of HIV infection after vaccination, either prophylactically or therapeutically. Such combinations can be selected according to the desired immunity. When the antibody is administered to an animal or human, it can be combined with one or more pharmacologically acceptable carriers, excipients or adjuvants known to those skilled in the art. The composition can further include broadly neutralizing antibodies known in the art, including, but not limited to, VRC01, PG9 and b12.

[0083] Furthermore, particularly with regard to determining effective levels in patients for the treatment of HIV, suitable animal models are available and are widely implemented to evaluate the in vivo efficacy against HIV of various gene therapy protocols (Sarver et al. (1993b), supra). These models include mice, monkeys and cats. Even though these animals are not naturally susceptible to HIV disease, chimeric mouse models (e.g., SCID, bg / nu / xid, NOD / SCID, SCID-hu, immunocompetent SCID-hu, myeloablated BALB / c) reconstituted with human peripheral blood mononuclear cells (PBMCs), lymph nodes, fetal liver / thymus or other tissues can be infected with lentiviral vectors or HIV and used as HIV pathogenesis models. Similarly, simian immunodeficiency virus (SIV) / monkey models can be used, as can feline immunodeficiency virus (FIV) / cat models. When used therapeutically to treat AIDS, the pharmaceutical composition can contain other pharmaceuticals along with the vector of the invention. In their traditional form (i.e., as an antiviral drug to treat HIV infection), Examples of HIV agents include non-nucleoside reverse transcriptase inhibitors, protease inhibitors, entry or fusion inhibitors and integrase inhibitors.

[0084] In another aspect, the invention provides pharmaceutical compositions based on antibodies, including isolated HIV antibodies, or affinity-improved versions thereof, in an effective amount, providing prophylactic or therapeutic treatment options for reducing infection with the HIV virus. The antibody-based pharmaceutical compositions of the invention can be prepared by a number of methods known in the art (see, for example, McGoff and Scher, 2000, Solution Formulation of Proteins / Peptides: In McNally, EJ, ed. Protein Formulation and Delivery. New York, NY: Marcel Dekker; pp. 139-158; Akers and Defilippis, 2000, Peptides and Proteins as Parenteral Solutions In: Pharmaceutical Formulation Development of Peptides and Proteins. Philadelphia, PA: Talyor and Francis; pp. 145-177; Akers, et al., 2002, Pharm. Biotechnol. 14:47-127), which promotes maximum stability during storage within acceptable temperature ranges, A pharmacologically acceptable composition suitable for administration to a patient in a formulation that retains both biological activity comprises an effective amount of the antibody. The pharmaceutical composition may also contain a pharmacologically acceptable diluent, a pharmacologically acceptable carrier, or a pharmacologically acceptable excipient, depending on the formulation required. Alternatively, such excipients are typically used to prepare pharmaceutical compositions for administration to animals or humans. The diluent is selected so that it does not affect the biological activity in combination. Examples of such diluents are distilled water, phosphate buffered saline, Ringer's solution, dextrose solution, and Hank's aqueous solution. The amount of excipient useful in a pharmaceutical composition or formulation of this invention is an amount useful for distributing the antibody evenly throughout the composition so that it can be evenly dispersed when it is to be delivered to a subject as required. It is also preferred that the amount of excipient used in the pharmaceutical composition be sufficient to distribute the antibody evenly throughout the composition so that it can be evenly dispersed when it is to be delivered to a subject as required. In fact, it may be useful to dilute the antibody to a concentration that provides the desired beneficial palliative or curative results while minimizing any adverse side effects that may arise from higher concentrations. It may also have a preservative effect. Thus, for antibodies with high physiological activity, more of the excipient is used. On the other hand, for any active ingredient that exhibits low physiological activity, a smaller amount of excipient is used.

[0085] The antibody, antibody composition, or vaccine composition described above comprises one or a combination of the antibodies described herein and can be administered for the prophylactic and therapeutic treatment of HIV viral infection. can.

[0086] The invention relates to isolated polypeptides comprising the light and heavy chain amino acid sequences shown in Table A, Table B and Figure 10A-C; the heavy and light chain consensus sequences of SEQ ID NOs: 1 and 2; and the insert sequences, SEQ ID NOs: 3 and 4.

[0087] In other related embodiments, the invention provides polypeptide variants encoding the amino acid sequences of HIV antibodies listed in Tables A, B and Figures 10A-C; consensus sequences for the heavy and light chains of SEQ ID NOs: 1 and 2; and insert sequences, SEQ ID NOs: 3 and 4. These polypeptide variants have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% or more sequence identity compared to the polypeptide sequences of the invention, where sequence identity is determined by the methods described herein (e.g., BLAST analysis using standard parameters). Those skilled in the art will understand that these values ​​may be appropriately adjusted to determine the corresponding identity of the encoded proteins by taking into account amino acid similarity, etc.

[0088] The word "polypeptide" is used in its conventional sense, i.e., as a sequence of amino acids. A polypeptide is not limited to a particular length of the product. Peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such words may be used interchangeably herein, unless specifically indicated otherwise. This word also includes post-expression modifications of a polypeptide (e.g., glycosylation, acetylation, phosphorylation, etc.), as well as other naturally occurring and non-naturally occurring modifications known in the art. A polypeptide can be any protein or subsequence thereof. A particular polypeptide of interest in the context of this invention is an amino acid subsequence that includes a CDR, a VH, and a VL and is capable of binding an antigen or an HIV-infected cell.

[0089] A polypeptide "variant," as the word is used herein, is a polypeptide that generally differs from the polypeptides disclosed herein, particularly by one or more substitutions, deletions, additions, or insertions. Such variants can be naturally occurring or synthetically produced, for example, by modifying one or more of the above-described polypeptide sequences according to the invention using any of a number of techniques described herein and / or known in the art, and evaluating one or more biological activities of the polypeptide.

[0090] For example, certain amino acids can be substituted for other amino acids in a protein structure without appreciable loss of its ability to bind to other polypeptides (e.g., antigens) or cells. Since it is the binding ability and properties of a protein that determine its biological functional activity, certain amino acid sequence substitutions can be made in the protein sequence, and correspondingly, in the underlying DNA coding sequence, to obtain a protein with favorable properties. Various changes can be made without appreciable loss of the peptide sequences of the disclosed compositions or their biological utility or activity. It is thus believed that the peptide is not produced by the corresponding DNA sequence encoding said peptide.

[0091] In many instances, a polypeptide variant will contain one or more conservative substitutions, which are substitutions of an amino acid with another amino acid having similar properties, such that those skilled in the art of peptide chemistry would expect the secondary structure and hydropathic properties of a polypeptide to remain largely unchanged.

[0092] Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents (e.g., their hydrophobicity, hydrophilicity, charge, size, etc.). Exemplary substitutions taking into account variability in the aforementioned characteristics are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0093] "Homology" or "sequence identity" refers to the percentage of residues in a polynucleotide or polypeptide sequence variant that match the non-variant sequence after aligning and gapping, as appropriate, the sequences to achieve the maximum percent homology. In certain embodiments, polynucleotide and polypeptide variants have at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98% or at least about 99% polynucleotide or polypeptide homology with the polynucleotides or polypeptides described herein.

[0094] Such variant polypeptide sequences share 70% (i.e., 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) sequence identity with the sequences detailed in this application. In further embodiments, the described invention provides polypeptide fragments of various lengths comprising contiguous stretches of the amino acid sequences disclosed herein. For example, peptide sequences are provided in the present invention that comprise one or more contiguous peptides of the sequences disclosed herein of at least about 5, 10, 15, 20, 30, 40, 50, 75, 100, 150 or more lengths, as well as all intermediate lengths therebetween.

[0095] The invention also includes nucleotide sequences encoding part or all of the light and heavy chains of the antibodies of the described invention, and fragments thereof. Due to redundancy in genetic information, variants of these sequences exist that encode the same amino acid sequences.

[0096] The invention includes isolated nucleic acid sequences encoding polypeptides for the heavy and light chains of HIV antibodies displayed in Tables A, B and Figures 10A-C; consensus sequences for the heavy and light chains of SEQ ID NOs: 1 and 2; and insert sequences, SEQ ID NOs: 3 and 4.

[0097] In other related embodiments, the described invention provides polynucleotide variants encoding the heavy and light chain peptide sequences of HIV antibodies displayed in Tables A, B and Figures 10A-C; the consensus sequences for the heavy and light chains of SEQ ID NOs: 1 and 2; and the insert sequences, SEQ ID NOs: 3 and 4. These polypeptide variants have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity compared to the polynucleotide sequences of the invention as determined using the methods described herein (e.g., BLAST analysis with standard parameters). One of skill in the art will understand that these values ​​may be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.

[0098] The words "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to single- or double-stranded RNA, DNA, or mixed polymers. Polynucleotides may include expressed genomic sequences, extragenomic and plasmid sequences, and smaller engineered gene segments, or may be suitable for expressing a polypeptide.

[0099] An "isolated nucleic acid" is a nucleic acid that is separated from other genomic DNA sequences, as well as proteins or complexes such as ribosomes and polymerases, and necessarily has a naturally occurring sequence. The term includes nucleic acid sequences that have been removed from their naturally occurring environment, including recombinant or cloned DNA isolates, and chemically synthesized analogs or biologically synthesized analogs by heterologous systems. Substantially purified nucleic acids include isolated forms of nucleic acids. Thus, this refers to nucleic acids as originally isolated, and does not exclude genes or sequences that are later added to the isolated nucleic acid by the hand of man.

[0100] The term "variant" of a polynucleotide as used herein refers to a polynucleotide that generally differs from the polynucleotides disclosed herein, particularly by one or more substitutions, deletions, additions, and / or insertions. Such variants can occur naturally or can be produced synthetically. For example, this can be achieved by modifying one or more of the polynucleotide sequences of the invention and evaluating one or more biological activities of the polypeptide, as described herein, and / or by using a number of techniques that are well known in the art.

[0101] Modifications can be made in the structure of the polynucleotides of the invention as described and functional molecules can be obtained that encode variant or derivative polypeptides with desirable characteristics. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent or improved variant or portion of the polypeptide of the invention, one skilled in the art will generally alter one or more of the codons in the encoded DNA sequence.

[0102] Polynucleotide variants generally contain one or more substitutions, deletions, additions and / or insertions such that the immunogenic binding properties of the polypeptides encoded by the variant polynucleotides are not substantially diminished, particularly with respect to the polypeptides encoded by the polynucleotide sequences described herein.

[0103] In further embodiments, the described invention provides polynucleotide fragments that include various lengths of contiguous stretches of sequences identical or complementary to one or more of the sequences disclosed herein, for example, at least about 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, or 1000 or more contiguous nucleotides of one or more of the sequences disclosed herein, as well as all intermediate lengths therebetween, and may further include any length between the recited values, such as: 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; and 200-500; including all integers between 500-1000.

[0104] In another embodiment of the invention, the polynucleotide composition is capable of hybridizing to the polynucleotide sequences provided herein, or fragments thereof, or complementary sequences thereof, under moderately high stringency conditions. Hybridization techniques are well known in molecular biology. For illustrative purposes, moderately stringent conditions suitable for testing hybridization of the polynucleotides of the invention with other polynucleotides include the following: Prewash in 5xSSC, 0.5% SDS, 1.0mM EDT. A (pH 8.0); hybridization in 5x SSC at 50-60°C overnight; followed by two washes in 2x, 0.5x, and 0.2x SSC containing 0.1% SDS at 65°C for 20 minutes each. One of skill in the art will appreciate that the stringency of hybridization can be readily manipulated by altering the salt content of the hybridization solution and / or the temperature at which hybridization is carried out. For example, in other embodiments, suitable high stringency conditions include those described above, except that the temperature of hybridization is increased, e.g., to 60-65°C or 65-70°C.

[0105] In some embodiments, the polypeptides encoded by the polynucleotide variants or fragments have the same binding specificity (i.e., bind specifically or preferentially to the same epitope or HIV strain) as the polypeptides encoded by the native polynucleotides. The nucleotides, polynucleotide variants, fragments and hybridizing sequences are intended to be polypeptides having a level of binding activity for the specific polypeptide sequences set forth herein that is at least about 50%, at least about 70%, and at least about 90% of that of the specific polypeptide sequences set forth herein. Code the code.

[0106] Regardless of the length of the coding sequence itself, the polynucleotides described in the invention, or fragments thereof, can be combined with other DNA sequences (e.g., promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc.) such that their total length can vary considerably. Nucleic acid fragments of almost any length can be used. For example, illustrative polynucleotide segments having total lengths of about 10,000, about 5,000, about 3,000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs in length, etc. (including all intermediate lengths) are included in many implementations of the invention.

[0107] In some embodiments, the polynucleotide sequences provided herein are used as probes or primers for nucleic acid hybridization (e.g., as PCR primers). The ability of these nucleic acid probes to specifically hybridize to a sequence of interest allows them to be used to detect the presence of complementary sequences in a given sample. However, other uses are also contemplated by the described invention, such as the use of sequence information for the creation of mutant primers, or primers used to create other genetic constructs. As such, nucleic acid segments of the described invention that contain sequence regions of at least approximately 15 nucleotide long continuous sequences that are similar to or complementary to the 15 nucleotide long continuous sequences disclosed herein are particularly useful. Longer continuous identical or complementary sequences, such as approximately 20, 30, 40, 50, 100, 200, 500, 1000 of them (including all intermediate lengths), including the full length sequences and all lengths in between, are also used in some embodiments.

[0108] Polynucleotide molecules having sequence regions that include contiguous nucleotide ranges of 10-14, 15-20, 30, 50, or 100-200 nucleotides (including intermediate lengths as well) that are identical or complementary to the polynucleotide sequences disclosed herein are specifically designed as hybridization probes, e.g., for Southern and Northern blotting, and / or as primers, e.g., for PCR. The total size of the fragments, as well as the size of the complementary range, will ultimately depend on the use or application of the particular nucleic acid segment. Commonly used fragments in hybridization embodiments are used. Here, the length of the contiguous complementary region can vary, such as between about 15 and about 100 nucleotides, but longer contiguous complementary ranges are used depending on the length of the complementary sequence to detect the desired sequence.

[0109] The use of hybridization probes approximately 15-25 nucleotides in length allows the formation of duplex molecules that are stable and selective. Molecules having contiguous complementary sequences ranging from 12 bases in length or more can be utilized to increase the stability and selectivity of the hybrids and thereby improve the quality and degree of specificity of the resulting particular hybridized molecules. Where required, nucleic acid molecules having complementary stretches of 15 to 25 contiguous nucleotides or longer can be utilized.

[0110] Hybridization probes may be selected from any portion of any of the sequences disclosed herein. All that is necessary is to identify any contiguous portion of the sequences described herein, or sequences, from about 15-25 nucleotides up to and including the full length sequence, that one wishes to use as a probe or primer. For example, one may wish to use primers from towards the ends of the full length sequence.

[0111] Furthermore, vectors, such as expression vectors, that contain a nucleic acid sequence according to the invention are included within the scope of the invention. Cells transformed with such vectors are also included within the scope of the invention.

[0112] The invention also provides vectors and host cells containing the nucleic acids of the invention, as well as recombinant techniques for the production of the polypeptides of the invention. Vectors of the invention include those capable of replication in any type of cell or organism (e.g., plasmids, phages, cosmids, and minichromosomes). In some embodiments, a vector containing a polynucleotide of the invention described is a vector suitable for propagation or replication of the polynucleotide, or a vector suitable for expressing a polypeptide of the invention described. Such vectors are known in the art and are commercially available.

[0113] "Vector" includes shuttle vectors and expression vectors. Generally, plasmid constructs also contain an origin of replication (e.g., ColE1 origin of replication) and a selection marker (e.g., ampicillin or tetracycline) for replication and selection of the plasmid in bacteria, respectively. An "expression vector" refers to a vector that contains the necessary control sequences or regulatory elements for the expression of an antibody, including an antibody fragment, of the invention in bacterial or eukaryotic cells.

[0114] As used herein, a "cell" can be any cell, including, but not limited to, a cell of a eukaryotic, multicellular species (e.g., as opposed to a unicellular yeast cell), such as a mammalian cell or a human cell. A cell can exist as a single cell or can be part of a larger collection of cells. Such a "larger collection of cells" can include, for example, a cell culture (mixed or pure), a tissue (e.g., endothelial, epithelial, mucosal, or other tissue), an organ (e.g., lung, liver, muscle, and other organs), an organ system (e.g., circulatory system, respiratory system, gastrointestinal system, urinary system, nervous system, integumentary system, or other organ system), or an organism (e.g., a bird, mammal, or other of that species).

[0115] Inventive polynucleotides are synthesized in whole or in combined portions and inserted into a vector using routine molecular and cell biology techniques, including, for example, subcloning the polynucleotide into a linearized vector using appropriate restriction sites and restriction enzymes. The described inventive polynucleotides are amplified by the polymerase chain reaction using oligonucleotide primers complementary to each strand of the polynucleotide. These primers also contain restriction enzyme cleavage sites to facilitate subcloning into a vector. Components of a replicable vector generally include, but are not limited to: a signal sequence, an origin of replication, and one or more Marker or selectable gene.

[0116] To express a polypeptide of the invention, a nucleotide sequence encoding the polypeptide or a functional equivalent can be inserted into an appropriate expression vector (i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence). Methods well known to those skilled in the art can be used to generate expression vectors containing a sequence encoding the polypeptide of interest and appropriate transcriptional and translational control elements. These methods can be used in These techniques include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination techniques, as described, for example, in Sambrook, J., et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY, and Ausubel, FM et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York. NY.

[0117] The invention provides kits useful for carrying out diagnostic or prognostic assays using the antibodies, polypeptides and nucleic acids of the invention. The kits of the invention include suitable containers containing the HIV antibodies, polypeptides or nucleic acids of the invention, in labeled or unlabeled form. In addition, when the antibodies, polypeptides or nucleic acids are provided in a labeled form suitable for indirect binding experiments, the kits further include reagents for carrying out the appropriate indirect assay. For example, depending on the nature of the label, the kits may include one or more suitable containers containing an enzyme substrate or a derivatizing agent. Control samples and / or instructions may also be included. The invention also provides kits for detecting the presence of HIV antibodies or nucleotide sequences of HIV antibodies of the invention in biological samples by PCR or mass spectrometry.

[0118] As used herein, a "label" refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody to produce a "labeled" antibody. Labels can also be conjugated to the polypeptides and / or nucleic acid sequences disclosed herein. The label can be detectable alone (e.g., radioisotope labels or fluorescent labels) or, in the case of enzymatic labels, can cause chemical alteration of a substrate compound or composition that is detectable. The antibodies and polypeptides of the described invention can be modified to include epitope tags or labels for use, for example, in purification or diagnostic applications. Suitable detection means include the use of labels such as radionucleotides, enzymes, coenzymes, fluorescers, chemiluminescers, chromogens, enzyme substrates or cofactors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes, and the like.

[0119] According to another alternative embodiment, the invention provides a method of diagnosis, which generally involves contacting a biological sample obtained from a patient (e.g., blood, serum, saliva, urine, sputum, a cell swab sample, or a tissue biopsy) with an HIV antibody and determining whether the antibody preferentially binds to the sample compared to a control sample or a predicted cutoff value, thereby indicating the presence of the HIV virus.

[0120] According to another alternative embodiment, the invention provides a method for detecting the presence of an HIV antibody of the invention in a biological sample from a patient. The detection method generally involves obtaining a biological sample from the patient (e.g., blood, serum, saliva, urine, sputum, a cell swab sample or a tissue biopsy) and isolating an HIV antibody or a fragment thereof, a nucleic acid encoding the HIV antibody, and analyzing the presence of the HIV antibody in the biological sample. The invention also provides a method for detecting the nucleotide sequence of an HIV antibody in a cell. The nucleotide sequence of the HIV antibody may be detected using the primers disclosed herein. The presence of the HIV antibody in the biological sample from the patient may be detected using known recombinant techniques and / or mass spectrometry. The amount of the ion exchange reaction may be determined by use of a meter.

[0121] In another embodiment, the invention provides a method for detecting HIV antibodies comprising a heavy chain comprising a highly conserved consensus sequence and a light chain comprising a highly conserved consensus sequence in a biological sample. The method includes obtaining a biological sample comprising immunoglobulins from a mammalian subject, isolating HIV antibodies from the sample, and identifying the highly conserved consensus sequences of the heavy and light chains. The biological sample may be blood, serum, saliva, urine, sputum, a cell swab sample, or a tissue biopsy. The amino acid sequence may be determined by methods well known in the art, including, for example, PCR and mass spectrometry.

[0122] The word "assessing" includes any form of measurement and includes making a determination as to whether an element is present or not. The words "determining," "measuring," "assessing," "evaluating," and "analyzing" are used interchangeably and include quantitative and qualitative determinations. Evaluating may be relative or absolute. "Assessing the presence of" includes determining the amount of something present and / or determining whether it is present or not. As used herein, the words "determining," "measuring," "evaluating," and "analyzing" are used interchangeably and include both quantitative and qualitative determinations.

[0123] II. Methods of Reducing Viral Replication Additionally, methods are provided for reducing HIV viral titer, viral replication, viral growth, or an increase in the amount of HIV viral proteins in a subject. According to another aspect, the method includes administering to the subject an amount of an HIV antibody effective to reduce in the subject an HIV viral titer, viral replication, or an increase in the amount of HIV proteins of one or more HIV strains or isolates.

[0124] According to another embodiment, the invention provides a method for reducing viral replication or transmission of HIV to further host cells or tissues comprising contacting a mammalian cell with an antibody, or portion thereof, that binds to an antigenic epitope on gp120.

[0125] III. Treatment Methods According to another embodiment, the invention provides a method of treating a mammal infected with a virus (such as HIV), comprising administering to the mammal a pharmaceutical composition comprising an HIV antibody disclosed herein. According to one embodiment, the method of treating a mammal infected with HIV comprises administering to the mammal a pharmaceutical composition comprising an antibody, or fragment thereof, according to the invention. The composition according to the invention can comprise one or more antibodies with the disclosed characteristics (e.g., a plurality or pool of antibodies). It can also comprise other HIV neutralizing antibodies known in the art, such as, but not limited to, VRC01, PG9 and b12.

[0126] Passive immunization has proven to be an effective and safe strategy for the prevention and treatment of viral diseases (see, e.g., Keller et al., Clin. Microbiol. Rev. 13:602-14 (2000); Casadevall, Nat. Biotechnol. 20:114 (2002); Shibata et al., Nat. Med. 5:204-10 (1999); and Igarashi et al., Nat. Med. 5:211-16 (1999), each of which is incorporated herein by reference). Passive immunization using human monoclonal antibodies provides a first aid method for the prevention and treatment of HIV emergencies.

[0127] A subject at risk for an HIV-related disease or disorder includes a patient who has been in contact with an infected person or has been exposed to HIV in any way. Administration of a prophylactic agent may be prior to the onset of symptoms characteristic of an HIV-related disease or disorder, such that the disease or disorder is prevented or, alternatively, delayed in its progression. It could happen.

[0128] For in vivo treatment of human and non-human patients, the patient is administered or provided with a pharmaceutical formulation comprising an HIV antibody according to the invention. When used for in vivo treatment, the antibody according to the invention is administered to the patient in a therapeutically effective amount (i.e., an amount that eliminates or reduces the viral burden in the patient). The antibody is administered to the patient by intravenous injection as a bolus dose or by continuous infusion over a period of time, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes according to known techniques. The antibody can be administered parenterally, where possible, to the target cell site or intravenously. In some embodiments, the antibody is administered intravenously or subcutaneously. The therapeutic composition according to the invention can be administered to the patient or subject systemically, parenterally, or locally. The above parameters for assessing the success of treatment and improvement in the disease are readily measurable by routine procedures known to the physician.

[0129] For parenteral administration, the antibody may be formulated in a unit dose injectable form (solution, suspension, emulsion) in association with a pharmacologically acceptable, parenteral vehicle. Examples of such vehicles include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles include, but are not limited to, fixed oils and ethyl oleate. Liposomes may be used as carriers. The vehicle may contain small amounts of additives, such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. The antibody may be formulated in such vehicles at a concentration of approximately 1 mg / ml to 10 mg / ml.

[0130] The dosage and administration schedule will depend on various factors (e.g., the nature of the infection, such as its therapeutic index, the patient, and the patient's medical history) as determined by the physician. In general, a therapeutically effective amount of the antibody is administered to the patient. In some embodiments, the amount of antibody administered ranges from about 0.1 mg / kg to about 50 mg / kg of the patient's body weight. Depending on the type and severity of the infection, the amount of antibody per body weight is about 0.1 mg / kg to about 50 mg / kg (e.g., about 0.1-15 mg / kg / dose), and this range is a candidate for an initial dosage for administration to the patient, for example, either by one or more divided doses, or by continuous infusion. The progress of this treatment is readily monitored by conventional methods and assays and based on criteria known to the physician or skilled artisan. The above parameters for evaluating the success of the treatment and improvement in the disease are readily measurable by routine procedures known to the physician.

[0131] Other optimal therapeutic regimes may be combined with the administration of the HIV antibody of the present invention. The combined administration includes simultaneous administration, in either order, using separate or single pharmaceutical formulations, and sequential administration, where it is preferred that there is a time period during which both (or all) active agents exert their biological activity. Such combined treatments may result in therapeutic synergy. The above parameters for assessing the success of treatment and improvement in the disease are readily measurable by routine procedures known to physicians.

[0132] The words "treating" or "treatment" or "palliative" are used interchangeably and refer to therapeutic treatment and prophylactic or preventative methods; where the objective is to prevent or slow (reduce) the progression of the targeted pathological condition or disorder. Populations in need of treatment include populations already suffering from the disorder as well as those susceptible to having the disorder or in which the disorder is to be prevented. A subject or mammal is successfully treated for an infection if, after receiving a therapeutic amount of an antibody according to the methods of the invention, the patient exhibits an observable and / or measurable reduction or absence of one or more of the following: a reduction in the number of infected cells or an increase in the number of infected cells. absence; reduction in the percentage of total infected cells; and / or to some extent elimination of one or more symptoms specifically associated with the infection; reduction in morbidity and mortality, and improvement in quality of life issues. The above parameters for assessing treatment success and improvement in the disease are readily measurable by routine procedures known to the physician.

[0133] The phrase "therapeutically effective amount" refers to an amount of an antibody or drug effective to treat a disease or disorder in a subject or mammal.

[0134] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration, and may be in any order.

[0135] As used herein, a "carrier" includes a pharmacologically acceptable carrier, excipient, or stabilizer that is nontoxic to cells or mammals exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants, including but not limited to ascorbic acid; low molecular weight (less than approximately 10 residues) polypeptides; proteins (e.g., serum albumin, gelatin, or immunoglobulins); hydrophilic polymers such as polyvinylpyrrolidone; amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates, including but not limited to glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols (e.g., mannitol or sorbitol); salt-forming counterions such as sodium; and / or nonionic surfactants such as polyoxyethylene sorbitan monolaurate (e.g., TWEEN); polyethylene glycols (PEGs) and poloxamers (e.g., PLURONICS).

[0136] Where a range of values ​​is given, each intervening value between the upper and lower limit of that range (rounded to the nearest tenth of the unit of the lower limit unless the context clearly states otherwise), and any other stated or intervening value in that stated range, is included in the invention. The upper and lower limits of these smaller ranges (which may be independently included in the smaller ranges) are also included in the invention, subject to any specifically excluded end value in the stated range. When the stated range includes one or both of the end values, ranges excluding either both of those included end values ​​are also included in the invention.

[0137] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this invention, the preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference in their entirety.

[0138] As used herein and in the appended claims, the singular forms "a," "and," and "the" refer to plural unless specifically stated otherwise.

[0139] The publications disclosed herein are provided solely for their disclosure prior to the filing date of the present invention. Nothing in this specification should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the actual publication dates, which may need to be independently confirmed, may be different from the publication dates provided.

[0140] Each application and patent cited herein is hereby incorporated by reference or patent law. The patent literature or non-patient literature (patient or non-patient literature), as well as the patent literature cited in each application or patent (including each issued patent pending prosecution; including application cited literature), and the PCT and foreign applications or patents corresponding to and / or claiming priority from each of these applications or patents, and the documents cited or references in each application cited literature are expressly incorporated herein by reference. More generally, in this specification, a document or reference is cited in a reference list before the claims; or in the text itself; and each of these documents or references ("references cited herein") is expressly incorporated herein by reference, including any manufacturer's specifications, instructions, etc. of the references cited herein.

[0141] The following non-limiting examples serve to further illustrate this invention. EXAMPLES

[0142] Example 1 Materials, methods and equipment Human samples were collected after signed informed consent according to Institutional Review Board (IRB) reviewed protocols by all participating institutions. Patient 1 was selected from a group of long-term nonprogressors followed at the Aaron Diamod Aids Research Center (New York). Patients 3 and 8 were selected from a group of long-term nonprogressors followed at the Ragon Institute, Boston. Patients 1, 3, and 8 were selected on the basis of their broadly neutralizing serological activity against a standard panel of HIV isolates. Patient 12 was selected from the International Aids Vaccine Initiative protocol G group on the basis of broadly neutralizing serological activity.

[0143] Staining, single cell sorting, and antibody cloning Staining and single-cell sorting of 2CC-Core and gp140-specific Ig+ memory B cells was performed (JF Scheid et al., Nature 458, 636 (Apr 2, 2009)). Briefly, CD19+ B cells were enriched from peripheral blood mononuclear cells using anti-human CD19 magnetic MACS beads (Miltenyi Biotec) and subsequently stained with biotinylated 2CC-Core (B. Dey et al., PLoS Pathog 5, e1000445 (May, 2009)) or YU2-gp140 trimers (R. Diskin, PM Marcovecchio, PJ Bjorkman, Nat Struct Mol Biol 17, 608 (May, 2010)), stained with anti-human CD20 and anti-human IgG antibodies (Becton Dickinson) and then detected with streptavidin-conjugated phycoerythrin (PE, Becton Dickinson). Cells were sorted using a FACSAria III cell sorter (Becton Dickinson) to remove cell doublets, and then incubated with 4 μl of 10 mM DTT per well. 8 U RNAsin™ (Promega), 0.4 U 5'-3' Pri me RNAse Inhibitor TM The wells were placed in a 96-well PCR plate (Denville) containing cold 0.5x phosphate buffered saline (Eppendorf). The plate was sealed with Microseal™ 'F' film (BioRad). The tubes were sealed, quickly frozen on dry ice, and stored at -80°C.

[0144] cDNA synthesis and Ig amplification were performed with the following modifications (H. Wardemann et al., Science 301, 1374 (Sep 5, 2003)): Instead of using the original primer sets, the first and second immunoglobulin-specific PCRs were performed using the primers listed in Table 1 in a semi-nested approach. Cloning of the heavy and light chain PCR products into their respective expression vectors was performed and the 100% identity of the original PCR products and the cloned expression plasmids was confirmed by sequencing prior to expression of the antibodies in HEK293 cells.

[0145] ELISA. High-binding 96-well ELISA plate (High-binding 96-well E LISA plates (Costar) contain 100 ng per well in PBS. Plates were coated overnight with purified antibodies (gp140, gp120, gp41, gp120core and 2CC-core) (B. Dey et al., PLoS Pathog 5, e1000445 (May, 2009)) and mutant proteins (gp120 D368R, gp120 I420R). After washing, plates were blocked for 2 h with 2% BSA, 1 μM EDTA, 0.05% Tween-PBS (blocking buffer). They were then incubated with IgG antibodies diluted to 4 μg / ml and several serial 1:4 dilutions in PBS. Plates were then stained with goat HRP-conjugated anti-mouse IgG (Jackson Immun Incubate with oResearch (0.8 μg / ml in blocking buffer) for 1 hour. and 100 μl of HRP chromogen substrate (ABTS sol The optical density was measured at 405 nm (OD ) using an ELISA microplate reader (Molecular Devices). 405nm ) was measured. The background value was subtracted, which was measured by incubating the coated wells with PBS alone. The IgG antibodies were verified to be polyreactive (H. Mouquet et al., Nature 467, 591 (Sep 30, 2010)) and were found to be polyreactive in four or more Polyreactive antibodies were considered to be polyreactive when they recognized at least two structurally distinct antigens; ssDNA, dsDNA, insulin, and LPS. The threshold for reactivity was determined by using the control antibodies mGO53 (negative), eiJB40 (low positive), and ED38 (high positive).

[0146] Neutralization assay: A neutralization screen was performed (DC Montefiori, Curr Protoc Immunol Chapter 12, Unit 12 11 (Jan, 2005)). Briefly, neutralization was measured as a reduction in luciferase reporter gene expression after a single-round infection in Tzm-bl. To exclude non-specific antiviral activity in the antibody samples, MuLV (murine leukemia virus) was used as a negative control.

[0147] Clone specific identification of bone marrow plasma cells. Bone marrow plasma cell ) were stained with anti-human CD138 and anti-CD19 antibodies (Becton Dickinson) after Ficoll purification of mononuclear cells from bone marrow aspirates using Ficoll-Paque (GE Healthcare). CD138+, CD19+ human plasma cells were bulk sorted on a FACSAriaIII cell sorter (Becton Dickinson) and RNA isolation was performed on 100,000 cells using Trizol LS reagent (Invitrogen) according to the manufacturer's instructions. RNA was reverse transcribed with Superscript III reverse transcriptase (Invitrogen) according to the manufacturer's instructions. cDNA was then generated using the following: Immunoglobulin-specific PCR was performed with the following modifications: 1 μl of cDNA was amplified using the first forward leader sequence primer and reverse constant region primer shown in Table 1, followed by two nested immunoglobulin heavy chain clone specific PCRs using clone-specific forward and reverse primers designed based on the sequences from the single-cell analysis. The PCR products were gel purified and then cloned into the TOPO TA vector according to the manufacturer's instructions. Colonies were screened by PCR using clone-specific primers and sequences.

[0148] Surface Plasmon Resonance. All experiments were performed with a Biacore T100 (Biacore) in HBS-EP+ running buffer (Biacore) at 25°C as previously described (Mouquet 2010). YU-2 gp140 and 2CC-core proteins were immobilized at 12.5 μg / mL on a CM5 chip (Biacore) by amine coupling at pH 4.5 resulting in an immobilization level of 100 RUs. For kinetic analysis of gp140 and 2CC-core derivatized chips, IgG was injected at 700 nM and four serial 1:2 dilutions in HBS-EP+ running buffer (Biacore) at a flow rate of 40 μl / min through the flow cell with 3 min of association and 5 min of dissociation. The sensor surface was resuspended between each experiment with a 30 second injection of 10 mM glycine-HCl pH 2.5 at a flow rate of 50 μL / min. Off rate ((k d (s -1 ))), on rate (ka ( M -1 s -1 ) and the binding constant (KD (M) or KA (M -1) were calculated after subtracting background values ​​(binding to control flow cell and HBS-EP + running buffer signal) using Biacore T100 evaluation software using kinetic analysis and a 1:1 binding model. The sensorgrams shown in Figures 2 and 8 are derived from Biacore data processing using Scrubber2 software (Center for Biomolecular Interaction Analysis, University of Utah).

[0149] CD4i site induction. 293T cells were transfected with Fugene at a ratio of 1:2. TM 6 (Roche) to transfect gp160 in the pMX-IRE-GFP construct (Pietzsch et al. 2010). BAL.26 Δc also is gp160 YU.2 After 46 hours, 293T cells were transfected with P The cells were washed with PBS and detached with trypsin-free cell dissociation buffer (Gibco) and then sonicated for 10 min in FACS buffer (1xPBS, 2% FBS, 2mM EDTA). 7 The cells were resuspended at a concentration of 1000 cells / ml. sCD4 (Progenics Pharmaceutica ls, Inc.) and mAbs were added to gp160-expressing 293T cells in a 1:4 dilution series starting at a final concentration of 40 μg / ml. mGO is a negative control antibody that does not bind to gp160Δc (H. Mouquet et al., Nature 467, 591 (Sep 30, After 15 min of incubation on ice, cells were detached and probed with Alexa64. 7. Labeled CD4-derived cytoplasmic antibody (3-67; (JF Scheid et al., Nature 458, 636 (Apr 2, 2009)), or Alexa647-labeled control mAb (i.e., PG16; LM Walker et al., Science 326, 285 (Oct 9, 2009) or gp160 YU.2 2G12 and gp160 for BAL.26 2G12) on ice. Staining was performed for 25 min. Antibody labeling was performed using the Alexa Fluor™ 647 Microscale Protein Labeling Kit (Invitrogen). Cells were analyzed on an LSRFortessa cell analyzer (BD Bioscience).

[0150] Crystallization. 3BNC60 IgG was expressed by transient expression in HEK293-6E cells and purified by papain cleavage (R. Diskin, PM Marcovecchio, PJ Bjorkman, Nat Struct Mol Biol 17, 608 (May, 2010). Crystallization screens were performed using the MRC Crystallography. On a plate (Jena Bioscience), Mosquito TM (TTP LabTech) crystallization robot was used to perform the crystallization of nL sitting drops (nL sitting drops). The assay was carried out at 20 °C by vapor diffusion in 400 nL of 100 ng drops. To generate the droplets, 3BNC60 Fab at a concentration of 9.5 mg / ml was combined with the reservoir solution in a 1:1 ratio. The first crystallization hit was PEGRx HT TM (Hampton Research) crystallization screens were used and further optimized manually. Crystals suitable for data collection were prepared in 11.7% polyethylene glycol 20000, 0.1M sodium acetate, p in the monoclinic space group with two Fabs in the asymmetric unit. Crystals were grown in H5.0, 100 mM potassium / sodium tartrate, 20 mM lithium sulfate, 10 mM N-cyclohexyl-2-aminoethanesulfonic acid (CHES) pH 9.5 for several weeks. Crystals were immersed in receiver solution supplemented with 15% glycerol for 2 hours, then immersed in receiver solution supplemented with 30% glycerol and flash cooled in liquid nitrogen. Diffraction data were collected at 100K by the Stanford Synchrotron Radiation Lightsource (SSRL) beam-line 12-2 using a Pilatus 6M detector. Data were indexed, integrated, and scaled using XDS (W. Kabsch, Acta Crystallogr D Biol Crystallogr 66, 125 (Feb, 2010)) (Table 8). Molecular replacement was performed using the V H and CH1 domain, and V of anti-gp120 b13 antibody (PDB code 3IDX). L and C L Model binding and refinement to the analysis of 2.65A was performed iteratively using Phenix P. Emsley, B. Lohkamp, ​​WG Scott, K. Cowtan, Acta Crystallogr D Biol Crystallogr 66, 486 (Apr, 2010) and Coot (P. Emsley, B. Lohkamp, ​​WG Scott, K. Cowtan, Acta Crystallogr D Biol Crystallogr 66, 486 (Apr, 2010). The structure was optimized using the Maximum-likelihood target function and non-crystallographic symmetry constraints It was refined using the mmetry restraint. The final model is (R work =20.7%;R free= 25.7%) contains 6478 protein atoms, 146 water molecules, and 28 sugar atoms (Table 8). 91.9%, 7.6%, and 0.5% of the residues are in the favorable, allowed, and disallowed regions of the Ramachandran plot, respectively. Structural analysis and visualization were performed using PyMol (The PyMOL Molecular Graphics System, Version 1.3, Schrodinger, LLC). The 3BNC60 structure has 6478 protein atoms, 146 water molecules, and 28 sugar atoms (Table 8). 91.9%, 7.6%, and 0.5% of the residues are in the favorable, allowed, and disallowed regions of the Ramachandran plot, respectively. Residues 3-205 of the ribosome (including the first N-acetylglucosamine in the N-linked carbohydrate attached to Asn72) and residues 2-217 of the heavy chain. H Residues 133-140 in one domain were lesioned.

[0151] Mass spectrometry. Protein G Sepharose (GE Heal) was used according to the manufacturer's instructions. IgG was purified from serum using a ELISA kit (Thcare). IgG was then digested with immobilized papain (Pierce) and the Fab-Fc fragment mixture was incubated with saturating amounts of biotinylated 2CC-Core protein. Streptavidin-conjugated Dynabeads (Invitrogen) were added after 15 min incubation at room temperature and washed ten times with phosphate-buffered saline (Gibco). Bound Fab fragments were eluted with lithium dodecyl sulfate buffer (Invitrogen) at 95°C and sample purity was confirmed by SDS-polyacrylamide gel electrophoresis, silver or Coomassie staining, and subsequent mass spectrometry analysis.

[0152] The isolated Fab fragments were reduced with dithiothreitol, alkylated using iodoacetamide, and separated by 1D gel electrophoresis on 4-12% NuPAGE Novex Bis-Tris gels (Invitrogen) and stained with Coomassie Blue (Thermo Fisher). The Fab fragments were excised from the gel and digested using 200 ng of trypsin. The resulting peptides were isolated using reversed-phase resin (PORS 20 R2, Applied Biosystem) and eluted with 40% trypsin in 0.5% acetic acid. An aliquot of acetonitrile and a second aliquot of 80% acetonitrile in 0.5% acetic acid were used for extraction. The acetonitrile was removed using a speedvac (Thermo Fisher Scientific) and an aliquot of the remaining solution was pressure loaded onto a PicoFrit column self-packed with an integrated emitter tip (360 μm OD, 50 μm ID, 10 μm tip) and packed with 6 cm of reversed-phase C18 material (ReproSi from Dr. Maisch GmbH). l-Pur C18-AQ, 3 μm beads) and a homemade microelectrochemical LTQ Orbitrap with spray source TM XL mass analyzer or LTQ Orbitrap Velos TM The system was interfaced to an Agilent 1200 series HPLC system (Agilent) with either a mass spectrometer (Thermo Fisher Scientific). Peptides were extracted into the mass spectrometer with the following gradient: 0–5% B for 5 min, 40% B for 125 min, 60% B for 150 min, and 100% B for 165 min (A = 0.1 M acetic acid, B = 70% acetonitrile in 0.1 M acetic acid, flow rate 90 nM). Both instruments were operated in data-dependent mode, and for both mass spectrometers, the target values ​​were set to 5e5 ions and a resolution of 60,000 (at 400 m / z). LTQ Orbitrap TMFor analysis on the XL, a full scan was followed by eight MS / MS scans on the eight most abundant ions from that full scan. Peptides (charge state >1 only) were isolated with a 2 Da window, dissociated via a 1e4 ion target window, CAD (normalized collision energy = 35, activation Q = 0.25, activation time = 30 ms), and masses were analyzed on the LTQ. LTQ Orbitrap TM For analysis on Velos. Complete Skip After the scan, 10 MS / MS scans were performed at a resolution of 75000 on the 10 most abundant ions from the immediate preceding full scan. Peptides (only charge states >2) were isolated with a 3Da window, a target window of 2e5 ions, dissociated via HCD (normalized collision energy = 40, activation time = 0.100 ms), and mass analyzed in the Orbitrap. For both instruments, ions selected from MS / MS were placed in the exclusion list for 30 seconds. The resulting MS / MS spectra were searched against the Human IPI or an in-house patient specific IgG database using Xtandem, and peptides were automatically compared to tryptic peptides in the Human IPI and our in-house patient specific IgG database. Peptides that matched the patient's specific IgG were manually confirmed.

[0153] Multiple sequence alignments. All multiple sequence alignments were performed using CLUSTALW2 with default parameters (weight matrix: GONNET for proteins and UIB for DNA, gap open = 10, gap extension 0.1 ). Shading of the alignment was done using the TeXshade package.

[0154] Consensus of alignments. Consensus sequences for multiple alignments were generated based on identity and similarity (>=70%) between residues. Amino acids were split along similarity as follows: FYW, ILVM, RK, DE, GA, ST and NQ. Germline tree. Relationships between sequences were generated using the Neighbor-Joining method. A bootstrapped consensus tree inferred from 1000 replicates was taken to show relatedness. Branches that coincided with splits reproduced in 50% of the bootstrap replicates were collapsed. Of the related sequences that came together in the bootstrap test (1000 replicates), the percentage of replicates in the tree is shown in the next branch. The tree was drawn with branch lengths in the same units as those of the evolutionary distances used to infer the tree. Evolutionary distances were calculated using the number of differences method. The mean amino acid difference was calculated as the number of amino acid differences per sequence. All ambiguous positions were removed for each sequence pair. Evolutionary analysis was performed with MEGA5.

[0155] R / S ratio calculations. DNA sequences were aligned with protein sequences for replacement / substitution calculations. All gap positions were removed from the analysis. R / S ratio analysis was performed using a Perl script.

[0156] Example 2 To determine whether HIV antibody cloning is limited due to somatic mutations, a new set of primers was designed to avoid this potential problem (Table 1). The new primer set was validated by sorting B cells that bound to HIV-gp120 that lacks the V1-3 loop and contains a stable disulfide bond (2CC-core). In contrast to the resurfaced bait used to clone VRC01, the 2CC-core bait also contains the CD4-derived coreceptor binding site (CD4i ) is permitted to be captured.

[0157] In a side-by-side comparison, the new primer set increased the recovery of IgH chains when compared to the original primer set (Figure 4(a)). Antibodies obtained with the new primer set were more mutated (mean 35.6 vs. 19.8 p=<0.0001 and maximum 85 vs. 50 for IgH) and contained clones not found with the original primer set. To determine whether the new primers would rescue VRC01-like antibodies from YU2 gp140-sorted cells, we tested cDNA samples from individuals that had already been thoroughly screened with the original primer set that did not produce any VRC01-related clones. In 80 wells, we found three antibodies that matched VRC01 mutants as determined by IgH and IgL sequences (Figures 5A and B). VRC01-like antibodies were captured by gp140 trimers, and primers specifically designed to clone hypermutated antibodies were found to capture large fragments of anti-HIV antibodies from memory B cells of patients with high titers of broadly neutralizing antibodies.

[0158] Of four unrelated HIV-infected individuals, including two Caucasians, one Hispanic, and one African, who showed high titers of broadly neutralizing antibodies when tested with the 2CC-core bait, their previously cloned antibodies could not explain the serological activity in two of them (Table 2 and Fig. 6A and B). 576 antibodies, representing 201 different unique diversified clones, were identified in a total of 1.5 × 10 5 IgG + Obtained from a starting population of memory B cells.

[0159] Example 3 HIV antibody binding specificity The sizes of the antibody clones captured by the 2CC-core bait varied widely from 2-76 diversified members (Table 3). To determine whether the antibodies captured by the 2CC-core bound to the HIV spike, ELISA was performed using YU2 gp120 with representative members of each expanded clone. All antibodies tested bound to gp120 (Table 3).

[0160] The site of antibody binding on the HIV spike was mapped using mutant proteins that interfere with the CD4bs (gp120(D368R)) or CD4-induced coreceptor binding site (CD4i, gp120(I420R)). VRC01 was sensitive to the D368R mutant as reported in X. Wu et al., Science 329, 856 (Aug 13, 2010). Because of its close proximity to the CD4i site, it is classified as a CD4bs antibody. However, it also showed some sensitivity to the I420R variant due to its close proximity to the CD4i site. The VRC01 variant, NIH45-46, and antibodies 3BNC60, 8ANC131, and 12A12 showed ELISA patterns similar to VRC01 (these clone members were not neutralized based on their neutralizing activity). The other clones, including 1B2530 and 8ANC195, were equally sensitive to both mutants and could be accurately classified based on ELISA alone. There wasn't.

[0161] To determine whether the antibodies were polyreactive, ELISAs were performed on purified ssDNA, dsDNA, insulin, and LPS. 63% of the anti-2CC-core antibodies tested were polyreactive. The majority of antibodies captured by 2CC-bait recognized either the CD4bs or CD4i sites on gp120, and many were polyreactive. It was also sexual.

[0162] Example 4 somatic cell hypertransformation Somatic hypermutation is necessary for the development of high affinity antigen binding and in some cases contributes to the polyreactivity of anti-HIV antibodies. To validate the hypermutated 2CC-core specific antibodies, four representative antibodies were backmutated to the corresponding germline. Backmutation resulted in a complete loss of antigen binding and lack of polyreactivity in all four clones validated.

[0163] Example 5 HIV neutralization HIV neutralization activity was measured using a standardized in vitro assay using an initial panel of eight viruses, including three tier 1 clade A, B, and C, and five tier 2 clade B Env pseudovirus variants (MS Seaman et al., J Virol 84, 1439 (Feb, 2010)). Neutralizing activity of antibodies was measured by ELISA. Antibody neutralization activity was compared to VRC01 and purified serum IgG from donors (Fig. 1A, Table 4 or Fig. 6). Antibodies that showed high levels of neutralizing activity were further validated on a panel of 15 additional tier 2 clade A, B, C, D, G, AG and AE Env pseudovirus variants (Fig. 1B, Table 5), including five viruses resistant to VRC01 (Fig. 1B, Table 5).

[0164] All 90 of the antibodies tested showed some neutralizing activity, and six clones contained antibody variants that displayed high levels of potency and breadth (Fig. 1A, B, and C, and Tables 4 and 5). These clones were the most abundant among those captured by the 2CC-bait in each of the four patient studies (Table 3). The most potent novel antibody, 3BNC117, belonging to a 76-member clone, had a mean IC in combination with a group of 14 tier 2 viruses of 0.5 μg / ml, compared to 1.8 μg / ml with VRC01. 80 showed.

[0165] Only 4 of the 20 viruses tested were more sensitive to VRC01 than 3BNC117, whereas 14 were more sensitive to 3BNC117, including DU172.17, which was completely VRC01 resistant and 3BNC117 sensitive (Fig. 1B and C). NIH45-46, a novel VRC01 variant, was more competent than VRC01 in 15 of the 20 viruses tested, but less competent than 3BNC117 (Fig. 1B and C, and Figs. 4 and 5).

[0166] There is a great deal of variation in the potency and breadth of neutralization among the five most potent neutralizing antibody clone members. For example, in the first panel, a mutant of 3BNC117, 3BNC156, neutralized only two viruses, and at much higher concentrations than 3BNC117 (Fig. 1A and Table 4), whereas another mutant, 3BNC55, neutralized only two viruses, with an average IC of 4 μg / ml. 50 It showed activity against six viruses, which was intermediate between the two above. Finally, The antibodies were highly mutated. The average number of mutations for the top 10 antibodies was V H 72 in, and V LThe mutated residues were 45 at 100–1500 ng / mL, which correlated with potency and breadth. Reversion of the mutated residues to germline resulted in a complete loss of neutralizing activity for all antibodies tested at that residue.

[0167] Example 6 Identification of diagnostic peptides The above cloning method captured antibodies produced by antigen binding to memory B cells, however, circulating antibodies are not produced by these cells, but instead originate from plasma cells in the bone marrow. However, the cognate antigen cannot be used as bait to capture plasma cells, since they do not express surface IgA (Radbruch et al., Nat Rev Immunol 6, 741 (Oct, 2006)). Furthermore, the plasma cells in the bone marrow and the circulating memory cells are not produced by these cells, but instead originate from plasma cells in the bone marrow. The relationship of memory B cells to IgV expression has not been precisely defined. To determine whether antibodies cloned from memory B cells are also found in the bone marrow plasma cell compartment, CD138-expressing plasma cells were purified from combined bone marrow samples from two of the four individuals studied, and IgV expression was determined for the more potent antibodies cloned from memory B cells in these individuals. H PCR was used to amplify the gene. The clone-specific primers for RU01 are as follows: CTGCAACCGGTGTACATTCTCAAGTGCAACTGGTGC (FWRD) (SEQ ID NO: 584), CT GCAACCGGTGTACATTCTCAGGTCCATTTGTCACAG (FWRD), (SEQ ID NO: 585) TGCGAAGTCGACGCTGACGAGACAGTGACCTGC (REV) (SEQ ID NO: 586), TGCGAAGTCGACGCTGAAGAGACAATAATTTG (REV) (SEQ ID NO: 587), TGCGAAGTCGACGCTGACGAGACAATAACT (REV) (SEQ ID NO: 588), and RU10 for: CTGCAACCGGTGTACATTTTCAGGGGCACTTGGTG (FWRD) (SEQ ID NO: 589), TGCGAAGTCGACGCTGAGGTGACGATGACCGTG (REV) (SEQ ID NO: 590). Members of the selected clone and numerous additional variants were quickly identified in both patients.

[0168] To confirm that these antibodies were also found in serum, IgG purified from the same two individuals and additional sera was adsorbed onto 2CC-core baits and mass spectrometry was performed on the eluted IgG (Fig. 1D, Fig. 7 and Fig. 10A-C). Diagnostic peptides were found for highly active antibody variants in all cases (Fig. 7, Fig. 10A-C). Broad and potent anti-HIV antibodies cloned from memory B cells were also found to be found in the bone marrow plasma cell compartment and in the circulating IgG of patients with high serum titers of broadly neutralizing antibodies.

[0169] Example 7 HIV antibody binding properties To determine whether antibody affinity for gp120 correlated with neutralizing activity, specular plasmon resonance (SPR) was used to compare binding of highly active antibodies, analogs of selected clones, and germline revertant precursors (Fig. 2A and B, Fig. 8, and Table 6).

[0170] The top neutralizing antibodies were ranked by affinity (K A ) is 10 on the YU2 gp140 trimer. 7 -10 12 and 10 on 2CC-core 7 -10 11 (M -1 ) (Tables 2A and B, and Table 6). Consistent with their potency and breadth of neutralization, 3BNC66, 3BNC156, and 3BNC55 showed lower affinity for the YU2 gp140 trimer than 3BNC117, but surprisingly, affinity for the 2CC-core did not correlate with neutralizing activity ( (Figure 1, Figure 8, Table 4 and Table 6). No binding by SPR was detected for any of the germline revertant antibodies tested (Figure 2B, Table 6). It was found that anti-HIV antibodies captured by the YU2 2CC-core tended to show higher affinity for the gp140 trimer than the 2CC-core.

[0171] When VRC01 binds to the HIV spike, it undergoes major conformational changes that mimic CD4 binding and expose the CD4i site. In contrast, B12 and most other known anti-CD4bs antibodies do not.

[0172] To determine whether this was a shared feature of highly active antibodies, HIV-BAL.26Δc or -YU2 gp160Δc was expressed on the surface of HEK293 cells. The antibodies were then expressed and CD4i antibody binding was measured in the presence or absence of CD4 or anti-CD4bs antibodies (Figure 2C). With one exception, all of the highly active antibodies tested were anti-CD4i antibodies that bound to HIV-BAL.26Δc or -YU2 gp160Δc or both. It was similar to CD4 and VRC01 in terms of promoting antibodies.

[0173] The only highly active antibody that does not share this feature, 8ANC195, is a typical antibody in that it is equally sensitive to the D368R and I420R mutants. It differed from the CD4bs antibodies (Table 3). In addition, its neutralization pattern differed from other highly active antibodies: it did not neutralize any tier viruses and showed strong activity against a clade B virus, H086.8, which was resistant to all other antibodies tested, including 3BNC117, VRC01, and b12.

[0174] Example 8 HIV antibody sequence identity To determine whether the highly active anti-CD4bs antibodies shared common sequence features, the 10 best antibodies: 2 variants from 5 independent antibodies derived from 5 different patients were aligned (Figure 3). H Comparison of regions shows that 68 IgV H The IgV nucleotide sequence was associated with a highly conserved consensus sequence covering the nucleotide residues (Figure 3A). H The consensus includes six VRC01-gp120 contact residues, Arg59 CD4 and Asp368 gp120 The VRC01-Arg 71 contact residues mimic the key interaction of VRC01 (Figure 3A). In addition, six contact residues are included in the consensus, which is consistent with the closely related germline IgV H Gene (V H 1-2 and V H 1-46) were conserved.

[0175] Codons encoding consensus residues were highly somatically mutated in the 10 selected antibodies, yet the amino acid sequences were conserved (Figure 9). Substitution rates for silent mutations in consensus residues ranged from 0.7-1.7, whereas they ranged from 3.5-22 in non-consensus residues, indicating that conservation of consensus is strongly selected for (Table 7). In contrast to the heavy chain, the light chain of VRC01 resulted in only 8 of 32 contacts with gp120. Consistent with a more limited role, comparison of the light chain sequences of the same antibodies revealed that 53 IgVs containing 3VRC01-gp120 contact residues were conserved. LA less extensive consensus covering residues became evident (Figure 3B). Finally, like the heavy chains, the light chains arose from a limited set of germline genes: two from IgK1D-33, two from IgK3-11, and one from IgL1-47 (Figure 3). Antibody 8ANC195 differed from the others in several important aspects, did not completely follow the consensus, and was not ascribed to related heavy and light chains (Figures 3A and B). The important sequence overlap between the ti-CD4bs antibodies (HAADs) It was confirmed that there was convergence.

[0176] Example 9 Crystal structure of 3BNC60 Fab To determine whether the structure of the antibody in different patients is also conserved, 3BNC60 The crystal structure of Fab was analyzed at 2.65 A resolution. The structure was compared with VRC01, which forms the antigen-binding site. H and V L We characterized the four domains of canonical Fab and the complementarity determining regions (CDRs), VH, CH1, VL, and CL, in the asymmetric unit of 3BNC60. The two Fabs in the asymmetric unit of 3BNC60 were nearly identical; however, the conformation of residues 74-78 in the loop connecting the D and E chains differed slightly due to the different chemical environment formed by crystal lattice contacts.

[0177] Overlapped images of the VH domains from 3BNC60 and VRC01 in the VRC01- gp120 co-crystal structure (T. Zhou et al., Science 329, 811 (Aug 13, 2010)) show major differences restricted to residues 58-65 (3BNC60 numbering) in CDR2 and a 1.3A ( The root mean square deviation (rmsd) of the 111 Cα atoms was calculated. Superimposing the structures showed conservation of the recognition interface in gp120. For example, Arg72 3BNC60 Arg71 VRC01 It adopts a similar morphology as Arg59 CD4 and Asp368 gp120 In addition, it mimics the important salt bridge formed between Trp47 3BNC60 , Trp47 VRC01 Gln64 adopts a similar conformation to residues that contact gp120, and contains a complex network of aromatic and aliphatic residue interactions that stabilize the conformation of CDRH3 and CDRL3. VRC01 Gln65, which matches 3BNC60 is in a residue segment (residues 58-65) that differs from the structure from VRC01. The conformation of this region of 3BNC60, which is involved in lattice contacts in the crystal, is likely altered upon binding to gp120 by clashing with the CD4-binding loop on gp120.

[0178] 3BNC60 and VRC01 V L The overlapping of the domains is 0.9A (95 Cα original) The rmsd of the α-terminal region (calculated for each gene) was calculated for each gene, and some of the gp120-contacting residues were structurally conserved; Tyr91 3BNC60 and Glu91a 3BNC60 is Tyr91 VRC01 and Glu96 VRC01 , which engages loop D of gp120 through a polar interaction. Overall, these structural comparisons suggested that 3BNC60 binds gp120 with the same configuration as observed for VRC01 binding.

[0179] Example 10 HIV antibody consensus sequence The aforementioned experiments demonstrated that VRC01 and the HIV spike are closely related to IgV, including eight of the contact residues. H and IgVL We determined a class of agonistic anti-CD4bs antibodies, HAADs, that share a consensus sequence (Fig. 3A and B). Selected for their high levels of serological anti-HIV activity in five different donors, these antibodies were classified as two closely related IgVs that fit the HAAD consensus. H and three IgV L Derived exclusively from germline genes; V H 1-2 and V H 1-46 differ by only seven amino acids and are not part of the consensus (Figure 3A). Despite extensive somatic hypermutation, the consensus residues were retained in their germline forms.

[0180] The only exception to the consensus, 8ANC195, differs in several ways that suggest it may have a unique method of antigen recognition: the critical Arg59 CD4 and Asp368 gp120 lack of an Arg in the heavy chain similar to the contact site of IgG; a unique neutralization pattern; and an inability to facilitate anti-CD4i antibody binding. It is one of two different highly active antibodies that occur in patients with rheumatoid arthritis.

[0181] [Table 1]

[0182] [Table 2]

[0183] [Table 3]

[0184] [Table 4]

[0185]

Table 5

[0186]

Table 6

[0187]

Table 7

[0188]

Table 8

[0189]

Table 9

[0190]

Table 10

[0191]

Table 11

[0192]

Table 12

[0193]

Table 13

[0194]

Table 14

[0195]

Table 15

[0196]

Table 16

[0197]

Table 17

[0198]

Table 18

[0199]

Table 19

[0200]

Table 20

[0201]

Table 21

[0202]

Table 22

[0203]

Table 23

[0204]

Table 24

[0205]

Table 25

[0206]

Table 26

[0207]

Table 27

[0208]

Table 28

[0209]

Table 29

[0210]

Table 30

[0211]

Table 31

[0212]

Table 32

[0213]

Table 33

[0214]

Table 34

[0215]

Table 35

[0216]

Table 36

[0217]

Table 37

[0218]

Table 38

[0219]

Table 39

[0220]

Table 40

[0221]

Table 41

[0222]

Table 42

[0223]

Table 43

[0224]

Table 44

[0225]

Table 45

[0226]

Table 46

[0227]

Table 47

[0228]

Table 48

[0229]

Table 49

[0230]

Table 50

[0231]

Table 51

[0232]

Table 52

[0233]

Table 53

[0234]

Table 54

[0235]

Table 55

[0236]

Table 56

[0237]

Table 57

[0238]

Table 58

[0239]

Table 59

[0240]

Table 60

[0241]

Table 61

[0242]

Table 62

[0243]

Table 63

[0244]

Table 64

[0245]

Table 65

[0246]

Table 66

[0247]

Table 67

[0248]

Table 68

[0249]

Table 69

[0250]

Table 70

[0251]

Table 71

[0252]

Table 72

[0253]

Table 73

[0254]

Table 74

[0255]

Table 75

[0256]

Table 76

[0257]

Table 77

[0258]

Table 78

[0259]

Table 79

[0260]

Table 80

[0261]

Table 81

[0262]

Table 82

[0263]

Table 83

[0264]

Table 84

[0265]

Table 85

[0266]

Table 86

[0267]

Table 87

[0268]

Table 88

[0269]

Table 89

[0270]

Table 90

[0271]

Table 91

[0272]

Table 92

[0273]

Table 93

[0274]

Table 94

[0275]

Table 95

[0276]

Table 96

[0277]

Table 97

[0278]

Table 98

[0279]

Table 99

[0280]

Table 100

[0281]

Table 101

[0282]

Table 102

[0283]

Table 103

[0284]

Table 104

[0285]

Table 105

[0286]

Table 106

[0287]

Table 107

[0288]

Table 108

[0289]

Table 109

[0290]

Table 110

[0291]

Table 111

[0292]

Table 112

[0293]

Table 113

[0294]

Table 114

[0295]

Table 115

Claims

1. 1. An isolated anti-HIV antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the sequence of SEQ ID NO:892, and a light chain variable region comprising the sequence of SEQ ID NO:

906.

2. The isolated anti-HIV antibody or antigen-binding fragment thereof of claim 1 , wherein the isolated anti-HIV antibody is a recombinant antibody or a human antibody.

3. The isolated anti-HIV antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the isolated anti-HIV antibody is a recombinant antibody.

4. The isolated anti-HIV antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antigen-binding fragment is selected from the group consisting of a Fab fragment, an F(ab')2 fragment, an Fv fragment and a single-chain Fv fragment.

5. A composition comprising the isolated anti-HIV antibody or antigen-binding fragment thereof of any one of claims 1 to 4.

6. A nucleic acid molecule encoding the isolated anti-HIV antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

7. A vector comprising the nucleic acid of claim 6.

8. A cultured cell comprising the vector of claim 7.

9. A pharmaceutical composition comprising at least one of the anti-HIV antibodies or antigen-binding fragments thereof according to any one of claims 1 to 4, and a pharmacologically acceptable carrier.

10. The pharmaceutical composition according to claim 9, for use in the prevention or treatment of HIV infection or an HIV-related disease.

11. The pharmaceutical composition of claim 10 further comprising a second therapeutic agent.

12. The pharmaceutical composition of claim 11 , wherein the second therapeutic agent is an antiviral agent.

13. 13. The pharmaceutical composition of claim 12, wherein the antiviral agent is selected from the group consisting of non-nucleoside reverse transcriptase inhibitors, protease inhibitors, entry or fusion inhibitors and integrase inhibitors.

14. The pharmaceutical composition of claim 11 , wherein the second therapeutic agent comprises a second anti-HIV antibody or antigen-binding fragment thereof.

15. The pharmaceutical composition according to any one of claims 9 to 14, wherein the anti-HIV antibody or antigen-binding fragment thereof is a recombinant antibody or antigen-binding fragment thereof.

16. A method for producing the anti-HIV antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising the steps of: Culturing a cell comprising a vector comprising nucleic acid encoding the heavy and light chains of the anti-HIV antibody or antigen-binding fragment thereof under conditions in which the nucleic acid is expressed; and isolating the anti-HIV antibody or antigen-binding fragment thereof; A manufacturing method comprising:

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