Antibodies that neutralize human immunodeficiency virus, and methods for using them.

Abstract

This invention provides a wide range of neutralizing antibodies targeting the epitopes of human immunodeficiency virus (HVM) or HIV. Furthermore, it provides compositions containing antibodies for use in prevention, and methods for the diagnosis and treatment of HIV infection. [Solution] An isolated anti-HIV antibody or its antigen-binding fragment is provided, comprising a heavy chain variable region containing a specific sequence and a light chain variable region containing a specific sequence. The antibody is a recombinant antibody or a human antibody.

Description

Technical Field

[0001] Cross - reference to Related Applications This application is a related application of U.S. Provisional Patent Application No. 61 / 486,960, filed on May 17, 2011, and claims the priority based on this provisional application under 35 U.S.C. § 119.

[0002] (Statement Regarding Federally Sponsored Research) The research leading to this invention has been supported in part by National Institutes of Health grant number P01 AI08677 - 01. Accordingly, the U.S. government may have certain rights in this invention.

[0003] Field of the Invention This invention relates to antibodies that bind to epitopes of the human immunodeficiency virus (HIV). This invention further relates to methods of making such antibodies and to their use in broadly neutralizing antibodies that bind to the HIV gp120 envelope protein for the prevention and treatment of HIV infection.

Background Art

[0004] Background of the Invention HIV causes acquired immunodeficiency syndrome (AIDS). The immune response to HIV infection in long - term non - progressors suggests that specific viral immunity can limit infection and disease manifestations. Among individuals infected with HIV, some show broadly neutralizing IgG antibodies in their sera. Despite their potential importance in the design of effective vaccines, little is known about the specificity and activity of these antibodies, and no correlation with protective immunity has been found. In animal models, passive transfer of neutralizing antibodies can contribute to protection against viral challenge. Neutralizing antibodies can develop in individuals infected with HIV, but the detailed composition of the serological response has not yet been fully elucidated.

[0005] Several immunological abnormalities are described in AIDS. These include, but are not limited to, abnormalities in B cell function, abnormal antibody responses, defective monocyte function, impaired cytokine production, suppressed natural killer and cytotoxic cell function, impaired lymphocyte ability to recognize and react to soluble antigens, and reduced T4 helper / trigger lymphocyte counts.

[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 virus particles (violins) are covered by a membrane or envelope derived from the surface membrane of the host cell. This membrane contains a group of envelope glycoproteins (gp160) that are fixed to the membrane bilayer in their C-terminal region. Each glycoprotein contains two parts: an N-terminal portion and a C-terminal portion. The N-terminal portion, called gp120 due to its relative molecular weight of approximately 120 kD, protrudes 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 peptide bonds, which are particularly susceptible to proteolytic cleavage. European Patent Application Publication No. 0 335 635, McCune et al., and the citations contained herein are incorporated herein by reference in their entirety.

[0007] Various approaches to AIDS vaccines have been proposed, including, but are not limited to, inactivated and attenuated viral vaccines, subunit vaccines from virus-infected cells, recombinantly produced viral antigens, vaccines based on synthetic peptides, anti-idiotype vaccines, and vaccines based on viral carriers. Further approaches to therapeutic and prophylactic treatment of HIV include producing highly competent, broad-spectrum monoclonal neutralizing antibodies. Several studies have reported the cloning and production of monoclonal antibodies by various techniques to target the CD4 binding site as well as other parts of the virion spike, and to detoxify HIV. These techniques typically include autofusion or phage display techniques. Typically, in the production of HIV neutralizing antibodies using phage display techniques, random combinations of heavy and light chains are fused, and random pairs are selected. Studies have reported a limited number of monoclonal antibodies (e.g., phage display antibody b12) that are broadly competent and broadly detoxifying (meaning antibodies that detoxify multiple strains of HIV in serum) against HIV. Monoclonal antibody b12 is a broad-spectrum neutralizing antibody that has been reported to prevent HIV infection in macaques. Other broad-spectrum neutralizing antibodies include 2G12, which atypically possesses a structure not found in other antibodies with three binding sites.

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

[0009] Several studies have reported that certain patients develop antibodies against HIV that neutralize a wide range of pathogens. These studies have shown that antibodies can provide protection against initial HIV infection and can regulate viral load during infection in passive transmission experiments in non-human primates. See, for example, 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. [Overview of the Initiative]

[0010] Summary of the Invention This invention provides, in one embodiment, a broad-spectrum neutralizing antibody against HIV. In one embodiment, this invention provides an isolated HIV antibody comprising a heavy chain containing the following consensus amino acid sequence: QXXLXQSGGXVKKPGXSVXVSCXASGYXXFXXYXIHWXRQAPGXGXXWVGXIXPRXGXXXXAXXFQGRLSLTRDXXXXXXTXXXFMDLXGLRXDDTAVYFCARXXXXXXXXXXXXXXXXXXDX (Sequence ID 1). In the formula, X represents any amino acid or indicates the absence of an amino acid.

[0011] In another embodiment, the invention provides an isolated HIV antibody comprising a light chain having the following consensus amino acid sequence: EIXLTQSPXSLSXSXGEXXTISCXXXQXXXXXXXLXWYQQRXGXAPRLLIXXXSXXXXGVPXRFSGXXXGXXYXLXISXLXXDDXAXYFCXXYEXXXXXXX (Sequence ID 2). In the formula, X represents any amino acid or indicates the absence of an amino acid.

[0012] In another embodiment, the invention provides an isolated HIV antibody comprising a heavy chain containing a highly conserved consensus sequence and a light chain containing a highly conserved consensus sequence. The invention further provides a method for producing an isolated HIV antibody comprising a heavy chain containing a highly conserved consensus sequence and a light chain containing a highly conserved consensus sequence.

[0013] In another embodiment, the present invention provides an isolated HIV antibody comprising a heavy chain containing the consensus sequence of SEQ ID NO: 1 and a light chain containing the consensus sequence of SEQ ID NO: 2. In a further embodiment, the present invention provides an isolated HIV antibody comprising either or both of the heavy chain containing the consensus sequence of SEQ ID NO: 1 and the light chain containing the sequence of SEQ ID NO: 2, or comprising 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.

[0014] In another embodiment, the present invention provides an isolated HIV antibody comprising one or both of a heavy chain containing the consensus sequence of SEQ ID NO: 1 and a light chain containing the consensus sequence of SEQ ID NO: 2, wherein the antibody has an IC50 concentration of less than 1.0 μg / ml. 50 Neutralizes HIV virus ZM53M.PB12 at concentrations of less than 1.0 μg / ml. 50 Neutralizes HIV virus R1166.c1 at concentrations below 30 μg / ml. 50 Neutralize DU172.17 by concentration. In another embodiment, the present invention provides an isolated HIV antibody comprising one or both of a heavy chain containing the consensus sequence of SEQ ID NO: 1 and a light chain containing the consensus acid sequence of SEQ ID NO: 2, wherein the antibody has an IC50 concentration of 30 μg / ml or less. 50 It neutralizes VRC01-resistant HIV viruses at a certain concentration.

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

[0016] In another embodiment, the present invention provides an isolated HIV antibody comprising heavy chain CDR1, CDR2 and CDR3 regions and light chain CDR1, CDR2 and CDR3 regions, comprising amino acid sequences of a matching region 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 embodiment, the present invention provides an isolated HIV antibody comprising a heavy chain containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 438.

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

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

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

[0021] In another embodiment, the invention provides an isolated HIV antibody comprising the insertion sequences of SEQ ID NOs: 3 and SEQ ID NOs: 4.

[0022] In another embodiment, the present invention provides a method for improving the ability and breadth of an isolated HIV antibody to neutralize HIV, comprising inserting at least one of the insertion sequences of SEQ ID NO: 3 and SEQ ID NO: 4.

[0023] In another embodiment, the present invention provides a composition comprising isolated HIV according to the invention.

[0024] In another embodiment, the present invention provides a pharmaceutical composition comprising an antibody according to the invention and a pharmacologically acceptable carrier.

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

[0026] In another embodiment, the present invention provides a vector comprising a nucleic acid molecule encoding an isolated HIV antibody, and cells comprising such a vector.

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

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

[0029] Another embodiment of this invention provides a method for reducing viral replication or the spread of infection to additional host cells or tissues, comprising contacting mammalian cells with at least one of the antibodies according to this invention. In another aspect, this 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 according to this invention.

[0030] In another embodiment, the invention provides a method for producing and administering an HIV antibody preparation suitable for administration to mammalian subjects infected with or at risk of HIV in an amount sufficient to induce a protective immune response to HIV or a reduction of the HIV virus in the mammalian subjects, and according to a sufficient schedule. In another embodiment, the invention provides a method for detecting an HIV antibody comprising a heavy chain containing a highly conserved consensus sequence and a light chain containing a highly conserved consensus sequence.

[0031] In another embodiment, the present invention provides isolated antibodies according to the present invention for use in the treatment of HIV.

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

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

[0034] [Figure 1A-B]Figures 1A-D show the neutralizing activity IC50 of HIV antibodies. (A) Limited panel. The top line shows the donor number, clone, or antibody (Table 4). The virus is shown on the left. The color indicates the concentration in IC50. Red indicates ≤0.1 μg / ml, orange indicates 0.1-1 μg / ml, orange indicates 0.1-1 μg / ml, yellow indicates 1-10 μg / ml, green indicates ≥10 μg / ml, and white indicates that no neutralization was shown in any concentration test. (B) Expanded panel. (C) Neutralization summary graph comparing VRCO1, NIH45-46, and 3BNC117. Line length and circle size are inversely proportional to IC50. The colors indicate the viral clade (strain). Red represents A, blue represents B, green represents C, reddish-purple represents D, black represents AE, and gold represents AG. (D) The heavy chain sequences of 3BNC60 (SEQ ID NO: 893), 1B2530, and 8ANC134, which have coverage by peptides discovered by mass spectrometry, are shown in light gray. Red dots indicate differences from their respective germline sequences. [Figure 1C-D] Figures 1A-D show the neutralizing activity IC50 of HIV antibodies. (A) Limited panel. The top line shows the donor number, clone, or antibody (Table 4). The virus is shown on the left. The color indicates the concentration in IC50. Red indicates ≤0.1 μg / ml, orange indicates 0.1-1 μg / ml, orange indicates 0.1-1 μg / ml, yellow indicates 1-10 μg / ml, green indicates ≥10 μg / ml, and white indicates that no neutralization was shown in any concentration test. (B) Expanded panel. (C) Neutralization summary graph comparing VRCO1, NIH45-46, and 3BNC117. Line length and circle size are inversely proportional to IC50. The colors indicate the viral clade (strain). Red represents A, blue represents B, green represents C, reddish-purple represents D, black represents AE, and gold represents AG. (D) The heavy chain sequences of 3BNC60 (SEQ ID NO: 893), 1B2530, and 8ANC134, which have coverage by peptides discovered by mass spectrometry, are shown in light gray. Red dots indicate differences from their respective germline sequences. [Figure 2]Figures 2A-C show the binding characteristics of HIV antibodies. (A) Representative surface plasmon resonance (SPR) sensograms for binding of 12A12, 12A21, and 12A-germ cell lineage (GL) reverted antibodies to YU2-gpl40 and 2CC-core. (B) Shows KA for representative antibodies. (C) The graph shows the mean fluorescence intensity of anti-CD4i antibodies bound to Bal.26 expressed in 293T cells, with incubation with indicator antibodies. The table shows whether the antibody induces accessibility to the CD4i site. [Figure 3A-1] Figures 3A and 3B show the consensus sequence and amino acid sequence of the HIV antibody. (A) Alignment of amino acids related to the framework (FR) and CDR regions for the consensus, germ cell gene, 10 selected antibodies, and 8ANC195 (SEQ ID NOs. 1 and 890-902, in order of appearance). Residues are numbered according to the structure of 3BNC60. (B) For the light chain (SEQ ID NOs. 2 and 903-916, in order of appearance), as in (A). (C, D, and E) Crystal structures of the antigen-binding fragment of 3BNC60. [Figure 3A-2] Figures 3A and 3B show the consensus sequence and amino acid sequence of the HIV antibody. (A) Alignment of amino acids related to the framework (FR) and CDR regions for the consensus, germ cell gene, 10 selected antibodies, and 8ANC195 (SEQ ID NOs. 1 and 890-902, in order of appearance). Residues are numbered according to the structure of 3BNC60. (B) For the light chain (SEQ ID NOs. 2 and 903-916, in order of appearance), as in (A). (C, D, and E) Crystal structures of the antigen-binding fragment of 3BNC60. [Figure 3B]Figures 3A and 3B show the consensus sequence and amino acid sequence of the HIV antibody. (A) Alignment of amino acids related to the framework (FR) and CDR regions for the consensus, germ cell gene, 10 selected antibodies, and 8ANC195 (SEQ ID NOs. 1 and 890-902, in order of appearance). Residues are numbered according to the structure of 3BNC60. (B) For the light chain (SEQ ID NOs. 2 and 903-916, in order of appearance), as in (A). (C, D, and E) Crystal structures of the antigen-binding fragment of 3BNC60. [Figure 4A-1] 4A and B demonstrate the repair of highly mutated immunoglobulin heavy chains using specific primers. (A) Comparison of new and old primer sets side by side. Red boxes indicate successful amplification of the IgVH gene. 4A shows sequence numbers 917-979 (in order of appearance). (B) HIV antibody binding to the 2CC-core from Pt 8. Clonal families are shown by differently expanded slices. Two highly mutated clones that were not amplified with the old primer set are shown in striped pie slices. [Figure 4A-2] 4A and B demonstrate the repair of highly mutated immunoglobulin heavy chains using specific primers. (A) Comparison of new and old primer sets side by side. Red boxes indicate successful amplification of the IgVH gene. 4A shows sequence numbers 917-979 (in order of appearance). (B) HIV antibody binding to the 2CC-core from Pt 8. Clonal families are shown by differently expanded slices. Two highly mutated clones that were not amplified with the old primer set are shown in striped pie slices. [Figure 4B]4A and B demonstrate the repair of highly mutated immunoglobulin heavy chains using specific primers. (A) Comparison of new and old primer sets side by side. Red boxes indicate successful amplification of the IgVH gene. 4A shows sequence numbers 917-979 (in order of appearance). (B) HIV antibody binding to the 2CC-core from Pt 8. Clonal families are shown by differently expanded slices. Two highly mutated clones that were not amplified with the old primer set are shown in striped pie slices. [Figure 5A] Figure 5 shows the heavy chain (A) (sequence numbers 980-984, in order of appearance) and light chain (B) (sequence numbers 985-989, in order of appearance) of IgV from the new VRC01 clone member. [Figure 5B] Figure 5 shows the heavy chain (A) (sequence numbers 980-984, in order of appearance) and light chain (B) (sequence numbers 985-989, in order of appearance) of IgV from the new VRC01 clone member. [Figure 6A] Figure 6 shows the patient serum neutralizing activity. (A) The table summarizes the neutralizing activity of purified serum IgG against a panel of Tier 2 viruses in Tzm-bl analysis. Dark red boxes indicate IC50 values ​​lower than 10 μg / ml, orange boxes indicate values ​​between 10 and 100 μg / ml, and yellow boxes indicate values ​​higher than 100 μg / ml. (B) The dot plot summarizes the subject IC50 values ​​higher than 4 in A. [Figure 6B] Figure 6 shows the patient serum neutralizing activity. (A) The table summarizes the neutralizing activity of purified serum IgG against a panel of Tier 2 viruses in Tzm-bl analysis. Dark red boxes indicate IC50 values ​​lower than 10 μg / ml, orange boxes indicate values ​​between 10 and 100 μg / ml, and yellow boxes indicate values ​​higher than 100 μg / ml. (B) The dot plot summarizes the subject IC50 values ​​higher than 4 in A. [Figure 7A-B]Figure 7 shows the detection of antibodies by mass spectrometry. Collisions activated the separation MS / MS spectra recorded on the double-charged peptides, HSDYCDFDVWGSGSQVIVSSASTK (SEQ ID NO: 888) (A) from 3BNC153HC and 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) were labeled in the spectra. Water loss from the fragment ions was indicated by *. The ions corresponding to water loss from the original ions were labeled in the spectra. Observed backbone cleavage was indicated in the sequences using (a symbol with the left-right inverted ") for b-type ions and (a symbol with the left-right inverted ") for y-type ions. [Figure 8A] Figures 8A and 8B show the affinity of HIV antibodies. (A) Antibodies bound to gp140 and 2CC-core were measured by surface plasmon resonance (SPR). Overtime, SPR sensograms for the bound antibodies of selected 3BNC antibody clones are shown. (B) The bar graph shows the binding affinity (KA) for the antigens of gp140 and 2CC-core for the selected IgG antibodies shown in A. [Figure 8B] Figures 8A and 8B show the affinity of HIV antibodies. (A) Antibodies bound to gp140 and 2CC-core were measured by surface plasmon resonance (SPR). Overtime, SPR sensograms for the bound antibodies of selected 3BNC antibody clones are shown. (B) The bar graph shows the binding affinity (KA) for the antigens of gp140 and 2CC-core for the selected IgG antibodies shown in A. [Figure 9A-1]Figures 9A-C illustrate somatic hypermutation analysis of selected HIV antibodies for the gene sequences of (A) the heavy chain immunoglobulin, (B) the light chain kappa, and (C) the light chain lambda. The sequences are aligned with their respective germline nucleotide sequences. Somatic mutations are shown in red, and gray boxes indicate substitution mutations. Germline amino acid sequences are shown above the nucleotide alignments with * indicating consensus residues. Figure 9A-1 shows sequence numbers 991, 990, and 992-997; Figure 9A-2 shows sequence numbers 991, 990, and 992-997 (continued) and sequence numbers 999, 998, and 1000-1003; Figure 9A-3 shows sequence numbers 999, 998, and 1000-1003 (continued); and Figure 9B-1 shows sequence numbers 1005, 1004, and 1006-1003. Figure 9B-2 shows sequence numbers 1005, 1004, and 1006-1009 (continued), as well as sequence numbers 1011, 1010, and 1012-1015; Figure 9B-3 shows sequence numbers 1011, 1010, and 1012-1015 (continued); Figure 9C shows sequence numbers 1017, 1016, and 1018-1019; all are in order of appearance. [Figure 9A-2]Figures 9A-C illustrate somatic hypermutation analysis of selected HIV antibodies for the gene sequences of (A) the heavy chain immunoglobulin, (B) the light chain kappa, and (C) the light chain lambda. The sequences are aligned with their respective germline nucleotide sequences. Somatic mutations are shown in red, and gray boxes indicate substitution mutations. Germline amino acid sequences are shown above the nucleotide alignments with * indicating consensus residues. Figure 9A-1 shows sequence numbers 991, 990, and 992-997; Figure 9A-2 shows sequence numbers 991, 990, and 992-997 (continued) and sequence numbers 999, 998, and 1000-1003; Figure 9A-3 shows sequence numbers 999, 998, and 1000-1003 (continued); and Figure 9B-1 shows sequence numbers 1005, 1004, and 1006-1003. Figure 9B-2 shows sequence numbers 1005, 1004, and 1006-1009 (continued), as well as sequence numbers 1011, 1010, and 1012-1015; Figure 9B-3 shows sequence numbers 1011, 1010, and 1012-1015 (continued); Figure 9C shows sequence numbers 1017, 1016, and 1018-1019; all are in order of appearance. [Figure 9A-3]Figures 9A-C illustrate somatic hypermutation analysis of selected HIV antibodies for the gene sequences of (A) the heavy chain immunoglobulin, (B) the light chain kappa, and (C) the light chain lambda. The sequences are aligned with their respective germline nucleotide sequences. Somatic mutations are shown in red, and gray boxes indicate substitution mutations. Germline amino acid sequences are shown above the nucleotide alignments with * indicating consensus residues. Figure 9A-1 shows sequence numbers 991, 990, and 992-997; Figure 9A-2 shows sequence numbers 991, 990, and 992-997 (continued) and sequence numbers 999, 998, and 1000-1003; Figure 9A-3 shows sequence numbers 999, 998, and 1000-1003 (continued); and Figure 9B-1 shows sequence numbers 1005, 1004, and 1006-1003. Figure 9B-2 shows sequence numbers 1005, 1004, and 1006-1009 (continued), as well as sequence numbers 1011, 1010, and 1012-1015; Figure 9B-3 shows sequence numbers 1011, 1010, and 1012-1015 (continued); Figure 9C shows sequence numbers 1017, 1016, and 1018-1019; all are in order of appearance. [Figure 9B-1]Figures 9A-C illustrate somatic hypermutation analysis of selected HIV antibodies for the gene sequences of (A) the heavy chain immunoglobulin, (B) the light chain kappa, and (C) the light chain lambda. The sequences are aligned with their respective germline nucleotide sequences. Somatic mutations are shown in red, and gray boxes indicate substitution mutations. Germline amino acid sequences are shown above the nucleotide alignments with * indicating consensus residues. Figure 9A-1 shows sequence numbers 991, 990, and 992-997; Figure 9A-2 shows sequence numbers 991, 990, and 992-997 (continued) and sequence numbers 999, 998, and 1000-1003; Figure 9A-3 shows sequence numbers 999, 998, and 1000-1003 (continued); and Figure 9B-1 shows sequence numbers 1005, 1004, and 1006-1003. Figure 9B-2 shows sequence numbers 1005, 1004, and 1006-1009 (continued), as well as sequence numbers 1011, 1010, and 1012-1015; Figure 9B-3 shows sequence numbers 1011, 1010, and 1012-1015 (continued); Figure 9C shows sequence numbers 1017, 1016, and 1018-1019; all are in order of appearance. [Figure 9B-2]Figures 9A-C illustrate somatic hypermutation analysis of selected HIV antibodies for the gene sequences of (A) the heavy chain immunoglobulin, (B) the light chain kappa, and (C) the light chain lambda. The sequences are aligned with their respective germline nucleotide sequences. Somatic mutations are shown in red, and gray boxes indicate substitution mutations. Germline amino acid sequences are shown above the nucleotide alignments with * indicating consensus residues. Figure 9A-1 shows sequence numbers 991, 990, and 992-997; Figure 9A-2 shows sequence numbers 991, 990, and 992-997 (continued) and sequence numbers 999, 998, and 1000-1003; Figure 9A-3 shows sequence numbers 999, 998, and 1000-1003 (continued); and Figure 9B-1 shows sequence numbers 1005, 1004, and 1006-1003. Figure 9B-2 shows sequence numbers 1005, 1004, and 1006-1009 (continued), as well as sequence numbers 1011, 1010, and 1012-1015; Figure 9B-3 shows sequence numbers 1011, 1010, and 1012-1015 (continued); Figure 9C shows sequence numbers 1017, 1016, and 1018-1019; all are in order of appearance. [Figure 9B-3]Figures 9A-C illustrate somatic hypermutation analysis of selected HIV antibodies for the gene sequences of (A) the heavy chain immunoglobulin, (B) the light chain kappa, and (C) the light chain lambda. The sequences are aligned with their respective germline nucleotide sequences. Somatic mutations are shown in red, and gray boxes indicate substitution mutations. Germline amino acid sequences are shown above the nucleotide alignments with * indicating consensus residues. Figure 9A-1 shows sequence numbers 991, 990, and 992-997; Figure 9A-2 shows sequence numbers 991, 990, and 992-997 (continued) and sequence numbers 999, 998, and 1000-1003; Figure 9A-3 shows sequence numbers 999, 998, and 1000-1003 (continued); and Figure 9B-1 shows sequence numbers 1005, 1004, and 1006-1003. Figure 9B-2 shows sequence numbers 1005, 1004, and 1006-1009 (continued), as well as sequence numbers 1011, 1010, and 1012-1015; Figure 9B-3 shows sequence numbers 1011, 1010, and 1012-1015 (continued); Figure 9C shows sequence numbers 1017, 1016, and 1018-1019; all are in order of appearance. [Figure 9C]Figures 9A-C illustrate somatic hypermutation analysis of selected HIV antibodies for the gene sequences of (A) the heavy chain immunoglobulin, (B) the light chain kappa, and (C) the light chain lambda. The sequences are aligned with their respective germline nucleotide sequences. Somatic mutations are shown in red, and gray boxes indicate substitution mutations. Germline amino acid sequences are shown above the nucleotide alignments with * indicating consensus residues. Figure 9A-1 shows sequence numbers 991, 990, and 992-997; Figure 9A-2 shows sequence numbers 991, 990, and 992-997 (continued) and sequence numbers 999, 998, and 1000-1003; Figure 9A-3 shows sequence numbers 999, 998, and 1000-1003 (continued); and Figure 9B-1 shows sequence numbers 1005, 1004, and 1006-1003. Figure 9B-2 shows sequence numbers 1005, 1004, and 1006-1009 (continued), as well as sequence numbers 1011, 1010, and 1012-1015; Figure 9B-3 shows sequence numbers 1011, 1010, and 1012-1015 (continued); Figure 9C shows sequence numbers 1017, 1016, and 1018-1019; all are in order of appearance. [Figure 10A-1] Figures 10A–C show the sequence of antibodies from a single enlarged neutralization clone in (A) Patient (Pt)1, (B) Pt3, and (C) Pt8, respectively. Peptides identified by mass spectrometry are shown in color. Mutants marked with an asterisk are uniquely identified by peptides observed by one or more mass spectrometers (shown in light gray). The remaining mass spectrometers non-uniquely observed peptide maps for multiple mutants shown in dark gray. Underlined amino acids indicate non-trypsin cleavage sites in the indicated mutants. Cleavage is presumed to occur through chymotrypsin cleavage or additional mutations (not observed between cloned mutants) that place lysine or arginine residues at these sites. Figure 10A shows sequence numbers 1020–1061; Figure 10B shows sequence numbers 1062–1113; and Figure 10c shows sequence numbers 1114–1138; all in chronological order of appearance. [Figure 10A-2]Figures 10A–C show the sequence of antibodies from a single enlarged neutralization clone in (A) Patient (Pt)1, (B) Pt3, and (C) Pt8, respectively. Peptides identified by mass spectrometry are shown in color. Mutants marked with an asterisk are uniquely identified by peptides observed by one or more mass spectrometers (shown in light gray). The remaining mass spectrometers non-uniquely observed peptide maps for multiple mutants shown in dark gray. Underlined amino acids indicate non-trypsin cleavage sites in the indicated mutants. Cleavage is presumed to occur through chymotrypsin cleavage or additional mutations (not observed between cloned mutants) that place lysine or arginine residues at these sites. Figure 10A shows sequence numbers 1020–1061; Figure 10B shows sequence numbers 1062–1113; and Figure 10c shows sequence numbers 1114–1138; all in chronological order of appearance. [Figure 10B-1] Figures 10A–C show the sequence of antibodies from a single enlarged neutralization clone in (A) Patient (Pt)1, (B) Pt3, and (C) Pt8, respectively. Peptides identified by mass spectrometry are shown in color. Mutants marked with an asterisk are uniquely identified by peptides observed by one or more mass spectrometers (shown in light gray). The remaining mass spectrometers non-uniquely observed peptide maps for multiple mutants shown in dark gray. Underlined amino acids indicate non-trypsin cleavage sites in the indicated mutants. Cleavage is presumed to occur through chymotrypsin cleavage or additional mutations (not observed between cloned mutants) that place lysine or arginine residues at these sites. Figure 10A shows sequence numbers 1020–1061; Figure 10B shows sequence numbers 1062–1113; and Figure 10c shows sequence numbers 1114–1138; all in chronological order of appearance. [Figure 10B-2]Figures 10A–C show the sequence of antibodies from a single enlarged neutralization clone in (A) Patient (Pt)1, (B) Pt3, and (C) Pt8, respectively. Peptides identified by mass spectrometry are shown in color. Mutants marked with an asterisk are uniquely identified by peptides observed by one or more mass spectrometers (shown in light gray). The remaining mass spectrometers non-uniquely observed peptide maps for multiple mutants shown in dark gray. Underlined amino acids indicate non-trypsin cleavage sites in the indicated mutants. Cleavage is presumed to occur through chymotrypsin cleavage or additional mutations (not observed between cloned mutants) that place lysine or arginine residues at these sites. Figure 10A shows sequence numbers 1020–1061; Figure 10B shows sequence numbers 1062–1113; and Figure 10c shows sequence numbers 1114–1138; all in chronological order of appearance. [Figure 10C-1] Figures 10A–C show the sequence of antibodies from a single enlarged neutralization clone in (A) Patient (Pt)1, (B) Pt3, and (C) Pt8, respectively. Peptides identified by mass spectrometry are shown in color. Mutants marked with an asterisk are uniquely identified by peptides observed by one or more mass spectrometers (shown in light gray). The remaining mass spectrometers non-uniquely observed peptide maps for multiple mutants shown in dark gray. Underlined amino acids indicate non-trypsin cleavage sites in the indicated mutants. Cleavage is presumed to occur through chymotrypsin cleavage or additional mutations (not observed between cloned mutants) that place lysine or arginine residues at these sites. Figure 10A shows sequence numbers 1020–1061; Figure 10B shows sequence numbers 1062–1113; and Figure 10c shows sequence numbers 1114–1138; all in chronological order of appearance. [Figure 10C-2]Figures 10A–C show the sequence of antibodies from a single enlarged neutralization clone in (A) Patient (Pt)1, (B) Pt3, and (C) Pt8, respectively. Peptides identified by mass spectrometry are shown in color. Mutants marked with an asterisk are uniquely identified by peptides observed by one or more mass spectrometers (shown in light gray). The remaining mass spectrometers non-uniquely observed peptide maps for multiple mutants shown in dark gray. Underlined amino acids indicate non-trypsin cleavage sites in the indicated mutants. Cleavage is presumed to occur through chymotrypsin cleavage or additional mutations (not observed between cloned mutants) that place lysine or arginine residues at these sites. Figure 10A shows sequence numbers 1020–1061; Figure 10B shows sequence numbers 1062–1113; and Figure 10c shows sequence numbers 1114–1138; all in chronological order of appearance. [Modes for carrying out the invention]

[0035] Detailed description of the invention 1.HIV neutralizing antibodies In one embodiment of this invention, a broad-spectrum neutralizing antibody against HIV is provided. In one embodiment, this invention provides an isolated HIV antibody comprising a heavy chain having the following consensus amino acid sequence: QXXLXQSGGXVKKPGXSVXVSCXASGYXXFXXYXIHWXRQAPGXGXXWVGXIXPRXGXXXXAXXFQGRLSLTRDXXXXXXTXXXFMDLXGLRXDDTAVYFCARXXXXXXXXXXXXXXXXXXDX (Sequence ID 1). In the formula, X represents any amino acid or indicates the absence of an amino acid.

[0036] In another embodiment, the invention provides an isolated HIV antibody comprising a light chain having the following consensus amino acid sequence: EIXLTQSPXSLSXSXGEXXTISCXXXQXXXXXXXLXWYQQRXGXAPRLLIXXXSXXXXGVPXRFSGXXXGXXYXLXISXLXXDDXAXYFCXXYEXXXXXXX (Sequence ID 2), where X represents any amino acid or indicates the absence of an amino acid.

[0037] In another embodiment, the invention provides an isolated HIV antibody comprising a heavy chain containing the consensus sequence of SEQ ID NO: 1 and a light chain containing the consensus sequence of SEQ ID NO: 2. In a further embodiment, the invention provides an isolated HIV antibody comprising one or both of the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 2, or, on the condition that the antibody does not have the amino acid sequence of VRC01, 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. The percentages were determined as shown below.

[0038] This invention provides, in other forms, isolated antibodies. It provides isolated HIV antibodies comprising a heavy chain containing a highly conserved heavy chain amino acid sequence and a light chain containing a highly conserved light chain amino acid sequence. The highly conserved heavy chain amino acid sequence is hereby identified as amino acids 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% homology with the sequence of SEQ ID NO: 1. The highly conserved light chain amino acid sequence is hereby identified as amino acids 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% homology with the sequence of SEQ ID NO: 2. The percentage determinations were made as shown below.

[0039] In another embodiment, the present invention provides an isolated HIV antibody comprising heavy chains containing highly conserved amino acid sequences of the heavy chains and light chains containing highly conserved amino acid sequences of the light chains, provided that the antibody does not have the amino acid sequence of VRC01.

[0040] In another embodiment, the present invention provides an isolated HIV antibody comprising one or both of the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 2, wherein the antibody neutralizes HIV virus ZM53M.PB12 at an IC 50 concentration of less than 1.0 μg / ml, or neutralizes HIV virus R1166.c1 at an IC 50 concentration of less than 1.0 μg / ml, or neutralizes DU172.17 at an IC 50 concentration of less than 30 μg / ml. In another embodiment, the present invention provides an isolated HIV antibody comprising one or both of the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 2, wherein the antibody neutralizes VRC01-resistant HIV virus at an IC 50 concentration of 30 μg / ml or less. The VRC01-resistant HIV virus is herein defined as an HIV virus that exhibits resistance to neutralization by VRC01 at an IC 50 value of 50 μg / ml.

[0041] In another embodiment, the present 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 embodiment, the present invention provides an isolated HIV antibody comprising heavy chain CDR1, CDR2 and CDR3 regions and light chain CDR1, CDR2 and CDR3 regions, comprising amino acid sequences of a matching region 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 embodiment, the present invention provides an isolated HIV antibody comprising a heavy chain containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 438.

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

[0045] In another embodiment, the present invention provides an isolated HIV antibody comprising a heavy chain and a light chain having amino acid sequences shown in Table A or Table B.

[0046] In another embodiment, the invention provides an isolated HIV antibody comprising an insertion sequence having the amino acid sequence:ASWDFDF (SEQ ID NO: 3). In a further embodiment, the invention provides an isolated HIV antibody in which, as shown in Figure 5A, an insertion sequence matching the FR3 of the heavy chain beginning at the 74th amino acid of 3BNC117 and 3BNC60, SEQ ID NO: 3, is used in place of the corresponding region of the 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 the FR3 of the heavy chain.

[0047] In another embodiment, the invention provides an isolated HIV antibody comprising an insertion sequence containing the amino acid sequence:TARDY(SEQ ID NO: 4). In a further embodiment, the invention provides an isolated HIV antibody in which, as shown in Figure 5A, an insertion sequence matching the CDR3 of the heavy chain beginning at the 103rd amino acid of NIH45-46, SEQ ID NO: 4, is used in place of the corresponding region of the 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 embodiment, the invention provides an isolated HIV antibody in which an insertion sequence, SEQ ID NO: 3, matching the FR3 of the heavy chain starting at the 74th amino acid of 3BNC117 and 3BNC60, is used in place of the corresponding region of the HIV antibody of the invention (as determined by sequence alignment), as shown in Figure 5A, and an insertion sequence, SEQ ID NO: 4, matching the CDR3 of the heavy chain starting at the 103rd amino acid of NIH45-46, as shown in Figure 5A, is used in place of the corresponding region of the 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 the FR3 of the heavy chain, and SEQ ID NO: 4 may be inserted after the 4th amino acid of the CDR3 of the heavy chain.

[0049] In a further embodiment, the invention provides a method for improving the neutralizing ability and breadth of an isolated HIV antibody, including the production of an isolated HIV antibody. Here, as shown in Figure 5A, an insertion sequence matching the FR3 of the heavy chain beginning at the 74th amino acid of 3BNC117 and 3BNC60, SEQ ID NO: 3, is used in place of the corresponding region of the HIV antibody of the invention (as determined by sequence alignment), and / or an insertion sequence matching the CDR3 of the heavy chain beginning at the 103rd amino acid of NIH45-46, SEQ ID NO: 4, is used in place of the corresponding region of the HIV antibody of the invention (as determined by sequence alignment), as shown in Figure 5A. For example, SEQ ID NO: 3 may be inserted after the 7th amino acid of the FR3 of the heavy chain, and / or SEQ ID NO: 4 may 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 by utilizing recombinant and / or synthetic chemistry techniques for the production of peptides or antibodies. Furthermore, those skilled in the art can use HIV neutralization analysis to identify improved HIV antibodies with better neutralization ability and breadth, as shown below.

[0050] In another embodiment, the invention provides an improved isolated HIV antibody comprising at least one of the insertion sequences of SEQ ID NO: 3 and SEQ ID NO: 4, wherein the improved isolated HIV antibody has superior HIV neutralizing ability and broadness compared to an isolated HIV antibody without the insertion sequences of SEQ ID NO: 3 and SEQ ID NO: 4. Those skilled in the art can identify the improved HIV antibody with better HIV neutralizing ability and broadness using HIV neutralization analysis, as shown below.

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

[0052] In another embodiment, the present invention provides a method for producing 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 embodiment, the present 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, on the condition that the antibody does not have the amino acid sequence of VRC01, 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. The percentages were determined as shown below.

[0054] In another embodiment, the invention provides a method for detecting isolated HIV antibodies, the method comprising a biological sample containing immunoglobulin from a mammalian subject, isolation of HIV antibodies from the sample, determination of the amino sequence of the HIV antibodies, and identification of the presence of the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 2. In a further embodiment, the invention provides a method for selecting isolated HIV antibodies, 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, given that the antibody does not have the amino acid sequence of VRC01, 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 to it. The percentage determinations were determined as shown below. Biological sample A biological sample may be blood, serum, saliva, urine, sputum, cell swab sample, or tissue biopsy. The amino acid sequence may be determined by known methods, for example, PCR and mass spectrometry.

[0055] The term “antibody” (Ab) here includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies and polyreactive antibodies), and antibody fragments. Thus, the term “antibody” (Ab) within the scope of this specification also includes any specific binding member, class and / or isotype of immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM), but is not limited to the following. And biologically related fragments, or members that specifically bind to them, include Fab, F(ab')2, Fv, and scFv (single-chain or related entities), but is not limited to the following. As is well known in this art, an antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interlinked by disulfide bonds, or an antibody that binds to a protein. The heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH1, CH2, and CH3). The light chain consists of a light chain variable region (VL) and a light chain constant region (CL). Both the heavy and light chain variable regions contain a framework region (FWR) and a complementarity-determining region (CDR). The four FWR regions are relatively conserved, while the CDR regions (CDR1, CDR2, and CDR3) represent the highly variable regions and are located 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 binding domains that synergize with antibodies, while, depending on the isotype, the constant region can mediate the binding of immunoglobulins to host tissues or factors.

[0056] Furthermore, the definition of antibody used herein includes chimeric antibodies, humanized antibodies, recombinant antibodies, and human antibodies produced from transgenic non-human animals, as well as antibodies selected from libraries using enrichment techniques available to those skilled in the art.

[0057] The term "variable" refers to the fact that a segment of the variable (V) domain differs from the sequence of other antibodies. The V domain mediates antigen binding and determines the specificity of a particular antibody for a particular antigen. However, the mutations are not uniformly distributed across the entire 110-amino acid span of the variable region. Instead, the V region contains relatively invariant ranges called framework regions (FRs) of 15-30 amino acids, separated by shorter, highly variable regions called high-frequency variable regions of 9-12 amino acids each. The variable regions of the natural heavy and light chains each consist of four FRs, employing a beta-sheet configuration and bound to three high-frequency variable regions, forming loops that bind to, or in some cases form part of, the beta-sheet structure. The high-frequency variable regions in each difference are held together in an adjacent state by the FRs and, together with the other chains, contribute to the formation of the antibody's antigen-binding site. (See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0058] The high-frequency variable region used here refers to the amino acid residues of the antibody that cause antigen binding. The high-frequency variable region generally includes amino acid residues from the complementarity-determining region (CDR).

[0059] The term "monoclonal antibody" used here essentially refers to an antibody obtained from a population of identical antibodies. For example, except for naturally occurring mutations that may exist in small quantities, individual antibodies within a population are identical. Polyclonal antibodies refer to a preparation containing different antibodies against different determinants (epitopes).

[0060] Here, monoclonal antibodies include “chimeric” antibodies in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence of an antibody belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence of an antibody belonging to another antibody class or subclass, and also include fragments of such antibodies insofar as they exhibit the desired biological activity (see, e.g., US Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). The described invention provides an antigen-binding sequence derived from a human antibody in the variable region. Thus, the chimeric antibody of primary interest here includes an antibody having one or more human antigen-binding sequences (e.g., CDRs) and one or more derived from a non-human antibody (e.g., FR or C region sequences). In addition, the chimeric antibodies included herein are those that contain antigen-binding sequences in human variable regions that include classes and subclasses of one antibody, as well as other sequences (e.g., FR or C region sequences) derived from classes or subclasses of other antibodies.

[0061] A "humanized antibody" is generally considered to be a human antibody that has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often called "imported" residues, and "imported" residues are generally taken from "imported" variable regions. Humanization can be carried out by substituting the corresponding sequence of a human antibody with an imported high-variability region sequence, 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)). Thus, such a humanized antibody is a chimeric antibody (US Pat. No. 4,816,567), where substantially less intact human variable regions are replaced by corresponding sequences from a non-human species.

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

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

[0063] "Fv" is a minimal antibody fragment containing a complete antigen-recognition site and an antigen-binding site. This fragment contains a dimer of variable regions of one heavy chain and one light chain in a tight, non-covalent relationship. From these foldings, the two domains derive six high-frequency variable loops (three from the H chain and three from the L chain) that contribute amino acid residues for antigen binding and offer the antigen that specifically binds to the antibody. However, even a single variable region (or half of Fv containing only the three specific CDRs for the antigen) has the ability to recognize and bind to an antigen, despite having lower affinity than the overall binding site.

[0064] A "single-chain Fv" (sFv, scFv) is an antibody fragment containing VH and VL antibody domains linked to a single peptide chain. The sFv polypeptide further contains a polypeptide peptide linker between the VH and VL domains, enabling the sFv to form a desirable structure for antigen binding. For a review of sFv, see, for example, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995 (see below).

[0065] The term "bispecific antibody" refers to a small antibody fragment prepared by constructing an sFv fragment having a short linker (5-10 residues) between the VH and VL domains, thereby achieving inter-chain binding of the V domain rather than intra-chain binding, and thereby producing a bivalent fragment (i.e., a fragment with two antigen-binding sites). Having dual specificity, a bispecific antibody is a heterodimer of two crossover sFv fragments in the VH and VL domains of two antibodies present on different peptide chains. For example, bispecific antibodies are fully described 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 produced in a fully human form, are the smallest known antibody antigen-binding fragments, approximately 11–15 kDa. dAbs are robust variable regions (VH and VL, respectively) of the heavy and light chains of immunoglobulins. They are highly expressed in microbial cell cultures, exhibit desirable biophysical properties (e.g., solubility and temperature stability, though not limited to these), and are also favorable for selection and affinity maturation by in vitro selection systems (e.g., phage display). dAbs are physiologically active as monomers, and their small size and inherent stability allow them to be formatted into larger molecules for producing drugs with long serum half-lives or other pharmacological activities. Examples of this technique are described, for example, in WO9425591 for antibodies derived from camelid heavy chain Ig, and also in US20030130496 for the isolation of full-length single-domain human antibodies from phage libraries.

[0067] Fv and sFv are the only types that have intact fusion regions lacking a constant region. Thus, they are well-suited for reducing nonspecific binding in in vivo use. sFv fusion proteins can be constructed to produce fusion of effector proteins at the amino or carboxyl terminus of sFv. See, e.g., Antibody Engineering, ed. Borrebaeck, supra. Antibody fragments are also linear antibodies, e.g., described in US Pat. No. 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

[0068] In some forms, the antibody according to the invention is bispecific or multispecific. A bispecific antibody is an antibody that has binding specificity for at least two different epitopes. A typical bispecific antibody can bind to two different epitopes of one antigen. Other such antibodies can bind to the first antigen-binding site for a second antigen. Alternatively, an anti-HIV arm can bind to a target molecule on leukocytes, such as a T cell receptor molecule (e.g., CD3), or to an Fc receptor for IgG (Fc gamma R), such as Fc gamma RI (CD64), Fc gamma RII (CD32), and Fc gamma RIII (CD16), in order to concentrate and localize cellular defense mechanisms on infected cells. Bispecific antibodies can also be used to localize cytotoxic drugs to infected cells. Bispecific antibodies can be produced 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, and 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 WO98 / 02463. US Pat. No. 5,821,337 discloses a bispecific anti-ErbB2 / anti-CD3 antibody. See also, for example, Mouquet et al., Polyreactivity Increases The Apparent Affinity Of Anti-HIV Antibodies By Heteroligation. NATURE. 467, 591-5 (2010). Bispecific antibodies can be prepared using known methods. Conventional methods for preparing full-length bispecific antibodies are based on co-expression of heavy-light chain pairs of two immunoglobulins. Here, the two strands have different specificities (see, for example, Millstein et al., Nature, 305:537-539 (1983)). Similar production is disclosed, for example, in WO 93 / 08829, Traunecker et al., EMBO J., 10:3655-3659 (1991) and Mouquet et al., PolyreactivityIncreases The Apparent Affinity Of Anti-HIV Antibodies By Heteroligation. NATURE. 467, 591-5 (2010).

[0070] Alternatively, an antibody variable region with the desired binding specificity (antibody-antigen binding site) is fused to the constant domain sequence of the immunoglobulin. The fusion includes the constant domain of the Ig heavy chain, at least the hinge portion, and the CH2 and CH3 regions. According to some specific examples, the constant region of the first heavy chain (CH1), which includes the site necessary for light chain binding, is present in at least one of the fusions. The immunoglobulin heavy chain fusion and, if necessary, the DNA encoding the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into suitable host cells. While an unbalanced ratio of the three polypeptide chains used in construction provides the optimal yield of the desired bispecific antibody, this gives greater flexibility in adjusting the relative ratios of the three polypeptide fragments in the embodiments. However, when the expression of at least two polypeptide chains in balanced ratios yields high yields, or when the ratio does not significantly affect the yield of the desired chain combination, it is possible to insert sequences encoding two or all three polypeptide chains into a single vector.

[0071] Techniques for producing bispecific antibodies from antibody fragments are described in the literature. For example, bispecific antibodies can be produced using chemical bonding. For instance, Brennan et al., Science, 229: 81 (1985), describe how intact antibodies are proteolytically cleaved to produce F(ab')2 fragments. These fragments are reduced in the presence of a dithiol complexing agent, sodium arsenite, to stabilize neighboring dithiols and prevent intermolecular disulfide formation. The produced Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to a Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'-TNB derivative to form a bispecific antibody. The produced bispecific antibodies can be used as chemicals for the selective immobilization of enzymes.

[0072] Other modifications of proteins are considered here. For example, antibodies can be conjugated to one of various non-protein polymers (e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol). Antibodies can also be encapsulated in microcapsules produced by coacervation techniques in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions, or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively). Such techniques are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).

[0073] Typically, the antibodies of this invention are recombinantly produced using vectors and methods available as known techniques. Human antibodies are produced in vitro by activated B cells (see, e.g., US Pat. Nos. 5,567,610 and 5,229,275). Common methods in molecular genetics and genetic engineering useful for this invention are described in the current edition of Molecular Cloning. See 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), “Guide to Protein Purification” in Enzymological Methods (MP Deutshcer, 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), and Gene Transfer and Expression Protocols pp. 109-128, ed. EJ Murray, The Humana Press Inc., Clifton, NJ). Reagents, cloning vectors, and kits for genetic engineering are available from commercial vendors (e.g., BioRad, Stratagene, Invitrogen, CloneTech, and Sigma-Aldrich).

[0074] Human antibodies can also be produced in transgenic animals (e.g., mice) that lack endogenous immunoglobulin production but are capable of producing a complete repertoire of human antibodies. For example, homozygous deletion of the heavy chain linkage (JH) region of antibody genes in chimeric and germline mutant mice has been shown to completely suppress endogenous antibody production. By transferring an array of human germline immunoglobulin genes to such germline mutant mice, human antibodies can be produced in response to antigen challenge. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno., 7:33 (1993); US Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all of GenPharm); US Pat. No. 5,545,807; and WO 97 / 17852. Such animals can be genetically designed for the production of human antibodies containing 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 directly obtained by recombinant host cells. Fab, Fv, and ScFv antibody fragments are all expressed in and secreted from E. coli. Thus, it is possible to easily produce large quantities of these fragments. Fab'-SH fragments can be directly recovered from E. coli and can be chemically bound to form F(ab')2 fragments (see, e.g., Carter et al., Bio / Technology 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be directly monoleaded from cultures of recombinant host cells. Fab and F(ab')2 fragments with increased half-lives in vivo, containing salvage receptors that bind to epitope residues, are described in US Pat. No. 5,869,046. Other techniques for the production of antibody fragments are apparent to those skilled in the art.

[0076] Other techniques known to those skilled in the art for selecting 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 (Georgiou, et al., 1997, Nature Biotechnology 15: 29-34), and / or yeast display (Kieke, et al., 1997, Protein Engineering 10: 1303-1310), which may replace the previously described techniques for selecting single-chain antibodies. Single-chain antibodies are selected from a library of single-chain antibodies produced by directly utilizing filamentous phage techniques. Phage display technology is known in the art (see, for example, the technology by Cambridge Antibody Technology (CAT) disclosed in US 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; 6,291,650; 6,492,160; 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081 and other US family members, as well as the technology disclosed in the application based on the priority application GB9206318 filed on May 24, 1992; and further, Vaughn, et al. (See Nature Biotechnology 14: 309-314, 1996). Single-chain antibodies can be designed and assembled using available recombination techniques such as DNA amplification techniques (e.g., PCR) or by using their respective hybridoma cDNA as a template.

[0077] Modified antibodies are also included within the scope of the invention. The sequence modifications detailed in this application are included within the scope of the invention. Furthermore, antibody sequence modifications 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, optimization of the codons in nucleic acid sequences can be used to improve translation efficiency in expression systems for antibody production.

[0078] The sequences of such deformed antibodies share 70% or more (i.e., 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) of 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., sequences detailed in this application). As noted herein, the percentage of identity is determined using BLAST version 2.1.3 with default parameters identified 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, according to the present invention, peptide sequences are provided that include at least about 5, 10, 15, 20, 30, 40, 50, 75, 100, 150 or more adjacent peptides of one or more sequences disclosed herein, as well as sequences of intermediate lengths between all of these. As used herein, the term “intermediate length” means any length between the cited 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] This invention provides antibodies that have broad detoxification activity in serum, either on their own or in combination with other antibodies, such as VRC01 and PG9, but not limited to the following.

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

[0081] In another embodiment, the invention provides a vaccine comprising at least one antibody according to the invention and a pharmacologically acceptable carrier. In one embodiment, the vaccine is a vaccine comprising at least one antibody described herein and a pharmacologically acceptable carrier. The vaccine may contain a number of antibodies having the characteristics described herein in any combination, and may further contain antibodies that detoxify HIV as known in the art.

[0082] It should be understood that the compositions may be the same or different single antibodies or combinations of antibodies disclosed herein, for the purpose of treating the progression of various subtypes of HIV infection after vaccination, either prophylactically or therapeutically. Such combinations may be selected based on the desired immunization. When administering antibodies to animals or humans, it is possible to combine them with one or more pharmacologically acceptable carriers, excipients, or adjuvants known to those skilled in the art. The compositions may further include, but are not limited to, broadly neutralizing antibodies known in the art, including VRC01, PG9, and b12.

[0083] Furthermore, with regard to measuring effective levels in patients for the treatment of HIV, suitable animal models are available and widely used to evaluate the in vivo efficacy of various gene therapy protocols against HIV (Sarver et al. (1993b), supra). These models include mice, monkeys, and cats. Even if these animals are not naturally susceptible to HIV disease, chimeric mouse models (e.g., SCID, bg / nu / xid, NOD / SCID, SCID-hu, immunocompetent SCID-hu, bone marrow excision BALB / c) reconstituted with human peripheral blood mononuclear cells (PBMCs), lymph nodes, fetal liver / thymus, or other tissues can be infected with lentiviral vectors or HIV and can be 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 for the treatment of AIDS, the pharmaceutical composition may include other pharmaceuticals along with the vector relating to the invention. These other medications can be used in the conventional way (i.e., as antiviral drugs to treat HIV infection). Examples of HIV drugs include non-nucleoside reverse transcriptase inhibitors, protease inhibitors, entry inhibitors or fusion inhibitors, and integrase inhibitors.

[0084] In another embodiment, the invention provides a pharmaceutical composition based on an antibody or an improved version thereof containing an effective amount of isolated HIV antibody, thereby providing a choice of prophylactic or therapeutic treatments for reducing HIV virus infection. The antibody-based pharmaceutical compositions of this invention can be prepared by many 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: Taylor and Francis; pp. 145-177; Akers, et al., 2002, Pharm. Biotechnol. 14:47-127). A pharmacokinetically acceptable composition suitable for administration to a patient contains an effective amount of antibody in a formulation that promotes maximum stability during storage within an acceptable temperature range while retaining both biological activity and bioactivity. The pharmaceutical composition may also contain a pharmacokinetically acceptable diluent, a pharmacokinetically acceptable carrier, or a pharmacokinetically acceptable excipient, depending on the required formulation. Alternatively, such excipients are commonly used to prepare pharmaceutical compositions for administration to animals or humans. The diluent is selected so as not to affect bioactivity in combination. Examples of such diluents include distilled water, phosphate buffer solutions, Ringer's solution, glucose solution, and Hank's aqueous solution. The amount of excipient useful in the pharmaceutical composition, or the formulation of this invention, is the amount useful for uniformly distributing the antibody throughout the composition so that it can be uniformly dispersed when it is to be delivered to the subject as needed.This can help dilute the antibody to a concentration that provides a desirable, beneficial mitigating or curative effect, while minimizing any adverse side effects that may occur from excessively high concentrations. It may also have a preservation effect. Thus, more excipients are used for antibodies with high physiological activity. On the other hand, smaller amounts of excipients are used for any active ingredient exhibiting low physiological activity.

[0085] The antibodies, antibody compositions, or vaccine compositions described herein, comprising one or a combination of the antibodies described herein, may be administered for prophylactic and therapeutic treatment of HIV virus infection.

[0086] This invention relates to an isolated polypeptide comprising the light and heavy chain amino acid sequences shown below: Table A, Table B and Figures 10A-C; consensus sequences of the heavy and light chains of SEQ ID NOs: 1 and 2; and insert sequences, SEQ ID NOs: 3 and 4.

[0087] In other related embodiments, the invention provides polypeptide variants encoding 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 insertion 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 greater sequence identity compared to the polypeptide sequences of the present invention, where sequence identity is determined by the method described herein (e.g., BLAST analysis using standard parameters). Those skilled in the art will understand that these values ​​can be appropriately adjusted to determine the corresponding identity of the encoded protein by considering amino acid similarity, etc.

[0088] The term "polypeptide" is used in its conventional sense, that is, as a sequence of amino acids. Polypeptides are not limited to a specific length of product. Peptides, oligopeptides, and proteins are included in the definition of polypeptide, and such terms can be used interchangeably here unless otherwise specified. This term also includes post-expression modifications of polypeptides (e.g., glycosylation, acetylation, phosphorylation, etc.), as well as other modifications known in the art, both naturally occurring and unnaturally occurring. Polypeptides can be any protein or its subsequences. The particular polypeptide of interest in the background of this invention is the amino acid subsequence comprising CDR, VH, and VL, which can bind antigens or HIV-infected cells.

[0089] As the term is used here, a polypeptide "mutant" is a polypeptide that is generally different from the polypeptide described herein, particularly by one or more substitutions, deletions, additions or insertions. Such mutants can occur spontaneously or be produced synthetically. For example, this can be done by modifying one or more of the polypeptide sequences described herein and / or known techniques, and by evaluating one or more physiological activities of the polypeptide.

[0090] For example, certain amino acids can be substituted for other amino acids in a protein structure without apparent loss of ability to bind to other polypeptides (e.g., antigens) or cells. Since the binding ability and properties of a protein determine its biological functional activity, substitutions of certain amino acid sequences can be made in the protein sequence and, accordingly, in the coding sequence of the underlying DNA, thereby yielding a protein with desirable properties. It is conceivable that various changes can be made in the peptide sequences of the disclosed compositions, or in the corresponding DNA sequences encoding such peptides without apparent loss of their biological utility or activity.

[0091] In many cases, polypeptide variants contain one or more conserved substitutions. A “conservative substitution” is the substitution of an amino acid with another amino acid that has similar properties, so that those skilled in peptide chemistry consider the properties of the secondary structure and the hydrophobicity and hydrophilicity indicators of the polypeptide to be largely invariant.

[0092] Amino acid substitutions are generally based on the relative similarities of amino acid side-chain substituents (e.g., their hydrophobicity, hydrophilicity, charge, size, etc.). Typical substitutions that take into account the diversity due to the aforementioned properties are well known to those skilled in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0093] "Homologousity" or "sequence identity" refers, where necessary, to the percentage of residues in a polynucleotide or polypeptide sequence variant that matches the non-mutant sequence after aligning the sequences and creating gaps to achieve the greatest possible percentage of homology. In certain embodiments, polynucleotide and polypeptide variants have at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 90%, at least approximately 95%, at least approximately 98%, or at least approximately 99% polynucleotide or polypeptide homology with the polynucleotide or polypeptide described herein.

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

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

[0096] This invention comprises isolated nucleic acid sequences encoding polypeptides for the heavy and light chains of an HIV antibody, as shown in Tables A and B and Figures 10A-C; consensus sequences for the heavy and light chains, 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 heavy and light chain peptide sequences of an HIV antibody, as shown in Tables A and 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 sequence identity of 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 higher, compared to the polynucleotide sequences of this invention, as determined using the methods described herein (e.g., BLAST analysis with standard parameters). Those skilled in the art will understand that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by considering codon degeneracy, amino acid similarity, reading frame positioning, etc.

[0098] The terms “nucleic acid” and “polynucleotide” are used here interchangeably to refer to single-stranded or double-stranded RNA, DNA, or mixed polymers. Polynucleotides may include expressed genomic sequences, extra-genomic and plasmid sequences, and smaller, manipulated gene segments, or they may be suitable for expressing polypeptides.

[0099] "Isolated nucleic acids" are nucleic acids separated from other genomic DNA sequences, as well as proteins or complexes such as ribosomes and polymerases, and necessarily possess a native sequence. This term includes nucleic acid sequences removed from their naturally occurring environment, including the isolation of recombinant or cloned DNA, and analogs synthesized chemically or biologically by non-homologous systems. Sufficiently purified nucleic acids include the isolated form of nucleic acids. Therefore, this refers to nucleic acids and does not exclude originally isolated nucleic acids, as well as genes or sequences added to nucleic acids that have been artificially isolated later.

[0100] The term "variant" as used herein refers to a polynucleotide that is generally different from the polynucleotide revealed herein, particularly by one or more substitutions, deletions, additions, and / or insertions. Such variants can occur spontaneously or be produced synthetically. For example, this is possible by modifying one or more of the polynucleotide sequences relating to the invention, as described herein, and by evaluating one or more of the physiological activities of the polypeptide and / or by using some well-known techniques.

[0101] Modifications can be made in the structure of the polynucleotide according to the described invention, and functional molecules encoding mutant or derived polypeptides with desirable characteristics can be obtained. If it is preferable to alter the amino acid sequence of the polypeptide to make an equivalent of the polypeptide according to this invention, or a further improved mutant or part thereof, those skilled in the art can modify one or more codons of the commonly encoded DNA sequence.

[0102] Generally, polynucleotide variants include one or more substitutions, deletions, additions, and / or insertions, so that the immunogenicity binding properties of polypeptides encoded by mutant polynucleotides are not substantially reduced, particularly in relation to polypeptides encoded by the polynucleotide sequences described herein.

[0103] In further embodiments, the described invention provides polynucleotide fragments comprising a variety of lengths within a range of adjacent sequences identical to or complementary to one or more sequences disclosed herein. For example, comprising at least approximately 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, or 1000 or more adjacent nucleotides in one or more sequences disclosed herein, as well as all intermediate lengths between them, and further comprising any length between the following cited values: 16, 17, 18, 19 and others; 21, 22, 23 and others; 30, 31, 32 and others; 50, 51, 52, 53 and others; 100, 101, 102, 103 and others; 150, 151, 152, 153 and others; and all integers between 200-500 and 500-1000.

[0104] In another embodiment of the invention, a polynucleotide composition can be hybridized to a polynucleotide sequence, or a fragment thereof, or a complementary sequence thereof, provided herein, under moderately high stringency conditions. Hybridization techniques are well known in molecular biology. For convenience of explanation, moderately stringency conditions suitable for testing the hybridization of the polynucleotide of this invention with other polynucleotides include: pre-washing with a solution of 5x SSC, 0.5% SDS, and 1.0 mM EDTA (pH 8.0); hybridization with 5x SSC at 50-60°C overnight; then washing twice with 2x, 0.5x, and 0.2x SSC containing 0.1% SDS, respectively, at 65°C for 20 minutes. Those skilled in the art will understand that the stringency of hybridization is readily manipulable by changing the salt content of the hybridization solution and / or the temperature at which hybridization takes place. For example, in other embodiments, suitable high stringency conditions include those described above, excluding increasing the hybridization temperature to, for example, 60-65°C or 65-70°C.

[0105] In some embodiments, polypeptides encoded by polynucleotide variants or fragments, as well as polypeptides encoded by natural polynucleotides, have the same binding specificity (i.e., they bind specifically or preferentially to the same epitope or HIV strain) as the polypeptides. In some embodiments, the described polynucleotides, polynucleotide variants, fragments, and hybridizing sequences encode polypeptides having levels of binding activity of at least approximately 50%, at least approximately 70%, and at least approximately 90% of the specific polypeptide sequences shown herein.

[0106] Regardless of the length of the coding sequence itself, the polynucleotides or fragments thereof described in the invention can be combined with other DNA sequences (e.g., promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc.) so that their total length can vary considerably. Nucleic acid fragments of almost any length can be used. For example, exemplary polynucleotide segments with total lengths of approximately 10,000, 5,000, 3,000, 2,000, 1,000, 500, 200, 100, and 50 base pairs (including all intermediate lengths) are included in many embodiments of this 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 target sequences allows them to be used to detect the presence of complementary sequences in a given sample. However, the described invention also includes other uses, such as the use of sequence information for the creation of mutant primers, or primers used to create other gene structures. In this sense, nucleic acid segments of the described invention that include sequence regions of long consecutive sequences of at least approximately 15 nucleotides, similar to or complementary to the long consecutive sequences of 15 nucleotides disclosed herein, are particularly useful. Longer consecutive identical or complementary sequences, such as full-length sequences and those of approximately 20, 30, 40, 50, 100, 200, 500, and 1000 (including all intermediate lengths), are also used in some embodiments.

[0108] Polynucleotide molecules having sequence regions containing a range of consecutive nucleotides of 10-14, 15-20, 30, 50, or 100-200 nucleotides (including intermediate lengths) that are identical or complementary to the polynucleotide sequences disclosed herein are specifically designed, for example, as probes for hybridization for Southern and Northern blotting, and / or as primers for PCR. The total size of the fragment, as well as the size of the complementary range, ultimately depends on the use or application of the particular nucleic acid segment. Fragments commonly used in hybridization embodiments are employed. Here, the length of the consecutive complementary region may vary, such as between approximately 15 and approximately 100 nucleotides, but longer consecutive complementary ranges are used depending on the length of the complementary sequence to detect the desired sequence.

[0109] The use of hybridization probes with a length of approximately 15–25 nucleotides enables the formation of stable and selective double-stranded molecules. Molecules with consecutive complementary sequences in the range of 12 bases or more can be utilized to increase the stability and selectivity of hybrids and thereby improve the degree of quality and specificity of the particular complex molecules obtained. Where required, nucleic acid molecules with complementary ranges of genes of 15 to 25 consecutive nucleotides, or longer nucleotides, can be utilized.

[0110] A hybridization probe can be selected from any portion of any sequence disclosed herein. All that is required is to identify the sequence described herein, or any contiguous portion of the sequence, from approximately 15–25 nucleotides to the full-length sequence, and the approximately 15–25 nucleotides containing the full-length sequence, which you wish to use as a probe or primer. For example, you might wish to use a primer from the end of the full-length sequence.

[0111] Furthermore, vectors such as expression vectors containing nucleic acid sequences 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] This invention, like the recombinant technology for the production of the polypeptide of the invention, provides a vector containing the nucleic acid of the invention and a host cell. The vector of the invention includes those that can replicate in any type of cell or organism (e.g., plasmids, phages, cosmids and minichromosomes). In some embodiments, the vector containing the polynucleotide of the invention described is a vector suitable for the proliferation or replication of the polynucleotide, or a vector suitable for expressing the polypeptide of the invention described. Such vectors are known in the art and are commercially available.

[0113] "Vectors" include shuttle vectors and expression vectors. Generally, plasmid constructs also include an origin of replication (e.g., ColE1 origin of replication) and a selection marker (e.g., ampicillin or tetracycline resistance) for plasmid replication and selection in bacteria, respectively. "Expression vector" refers to a vector containing necessary regulatory sequences or modulatory elements for the expression of an antibody containing the antibody fragment according to the invention in bacterial or eukaryotic cells.

[0114] As used here, “cell” can be any cell. This includes, but is not limited to, cells of eukaryotes, multicellular species (as opposed to, for example, unicellular yeast cells), such as mammalian or human cells. A cell may exist individually or as part of a larger collection of cells. Such “larger collections of cells” may include, for example, cell cultures (mixed or pure), tissues (e.g., endothelial, epithelial, mucous membranes or other tissues), organs (e.g., lungs, liver, muscles and other organs), organ systems (e.g., circulatory, respiratory, gastrointestinal, urinary, nervous, cutaneous systems or other organ systems), or organisms (e.g., birds, mammals or others of the same species).

[0115] The polynucleotides according to the invention are synthesized whole or in part and inserted into a vector using routine molecular and cell biology techniques (e.g., including subcloning the polynucleotides into a linearized vector using appropriate restriction sites and restriction enzymes). The polynucleotides according to the described invention are amplified by 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 the vector. The components of a replicable vector generally include, but are not limited to, a signal sequence, a replication origin, and one or more markers or selectable genes.

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

[0117] This invention provides a kit useful for performing diagnostic or prognostic analysis using the antibodies, polypeptides, and nucleic acids according to this invention. The kit according to this invention comprises a suitable container containing the HIV antibody, polypeptide, or nucleic acid according to the invention, in a labeled or unlabeled form. In addition, when the antibody, polypeptide, or nucleic acid is supplied in a labeled form suitable for indirect binding experiments, the kit further comprises reagents for performing appropriate indirect analysis. For example, depending on the nature of the label, the kit may comprise one or more suitable containers containing enzyme substrates or derivatizers. Control samples and / or instructions may also be included. This invention also provides a kit for detecting the presence of the HIV antibody or nucleotide sequence of the HIV antibody according to this invention in a biological sample by PCR or mass spectrometry.

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

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

[0120] According to another embodiment, the present invention provides a method for detecting the presence of the HIV antibody of the present invention in a biological sample from a patient. The detection method generally includes obtaining a biological sample from a patient (e.g., blood, serum, saliva, urine, sputum, cell swab sample or tissue biopsy), isolating the HIV antibody or a fragment thereof, the nucleic acid encoding the HIV antibody, and analyzing the presence of the HIV antibody in the biological sample. The present invention also provides a method for detecting the nucleotide sequence of the HIV antibody in cells. The nucleotide sequence of the HIV antibody can be detected using primers disclosed herein. The presence of the HIV antibody in a biological sample from a patient can be determined by utilizing known recombinant techniques and / or by using a mass spectrometer.

[0121] In another embodiment, the present invention provides a method for detecting HIV antibodies comprising a heavy chain containing a highly conserved consensus sequence and a light chain containing a highly conserved consensus sequence in a biological sample. The method comprises obtaining a biological sample containing 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, cell swab sample, or tissue biopsy. The amino acid sequence can 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, including determining whether an element exists or not. The words "determining," "measure," "assess," "evaluate," and "analyze" are used interchangeably and include both quantitative and qualitative decisions. Assessing can be correlational or absolute. "Assessing the existence of ~" includes determining the amount of something's existence and / or determining whether it exists or not. As used here, the words "determining," "measure," "evaluate," and "analyze" are used interchangeably and include both quantitative and qualitative decisions.

[0123] II. Methods to reduce virus replication Furthermore, methods are provided to reduce increases in HIV virus titer, viral replication, viral proliferation, or the amount of HIV viral protein in a subject. In another aspect, the method involves administering to a subject an amount of HIV antibody effective in reducing increases in HIV virus titer, viral replication, or the amount of HIV protein of one or more HIV strains or isolates in the subject.

[0124] According to another embodiment, the present invention provides a method for reducing the nucleic acid of HIV infection in further host cells or tissues, comprising contacting a mammalian animal cell bound to an antigen epitope on gp120 with an antibody, the portion thereof.

[0125] III. Treatment Methods According to another embodiment, the present invention provides a method for treating a mammal infected with a virus (e.g., HIV), the method comprising administering to the mammal a pharmaceutical composition comprising the HIV antibody disclosed herein. According to one embodiment, the method for treating an HIV-infected mammal comprises administering to the mammal a pharmaceutical composition comprising the antibody according to the present invention, or a fragment thereof. The composition according to the invention may contain one or more antibodies having the disclosed characteristics (e.g., a number or pool of antibodies). For example, it may also contain other HIV neutralizing antibodies well known in the art, such as VRC01, PG9, and b12, but is not limited to the following.

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

[0127] Subjects at risk of HIV-related illness or disability include individuals who have been in contact with an infected person or have been exposed to HIV in any way. Administration of prophylactic medication may occur before the onset of symptoms specific to HIV-related illness or disability, so as to prevent the illness or disability or, instead, delay its progression.

[0128] For the in vivo treatment of human and non-human patients, patients are administered or provided with a pharmaceutical formulation containing the 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 patient's viral burden). The antibody is administered to the patient by intramuscular, intraperitoneal, intraracerobrospinal, subcutaneous, intra-articular, intra-articular bursa, intramedullary, oral, topical, or inhalation routes, by intravenous injection as a bolus or by continuous intravenous infusion, according to known methods. The antibody may be administered parenterally, intravenously, or to target cell sites, where possible. In some embodiments, the antibody is administered intravenously or subcutaneously. The therapeutic composition according to the invention may be administered tissuely, parenterally, or topically to the patient or subject. The above parameters for evaluating the success and improvement of treatment in the disease are readily measurable by routine procedures known to physicians.

[0129] For parenteral administration, antibodies may be formulated in unit dose injectable forms (solutions, suspensions, emulsions) in association with pharmacologically acceptable parenteral excipients. Examples of such excipients include, but are not limited to, water, saline, Ringer's solution, glucose solution, and 5% human serum albumin. Non-aqueous excipients include, but are not limited to, non-volatile oils and ethyl oleates. Liposomes may be used as carriers. Excipients may contain small amounts of additives such as buffers and substances that enhance isotonicity and chemical stability, such as preservatives. Antibodies may be formulated in such excipients at concentrations of approximately 1 mg / ml to 10 mg / ml.

[0130] The dosage and administration plan depend on various factors determined by the physician (e.g., the nature of the infection, the patient, and the patient's medical history, such as the treatment index). Generally, a therapeutically effective amount of antibody is administered to the patient. In some embodiments, the amount of antibody administered ranges from approximately 0.1 mg / kg to approximately 50 mg / kg of the patient's body weight. Depending on the type and severity of the infection, the amount of antibody relative to body weight is approximately 0.1 mg / kg to approximately 50 mg / kg (e.g., approximately 0.1-15 mg / kg / dose), and this range is a candidate for the initial dosage for administration to the patient, either by one or more divided doses or by continuous intravenous infusion. Progress in this treatment is immediately monitored by conventional methods and analyses, based on criteria well known to physicians or those skilled in the art. The above parameters for evaluating the success and improvement of treatment in the disease are readily measurable by routine procedures known to physicians.

[0131] Other optimal treatment plans may be combined with the administration of the HIV antibody according to this invention. Combined administrations may be in either order, and include split prescriptions, simultaneous administration using individual drug prescriptions, and continuous administration. Here, it is preferable that there is a time cycle between the exertion of both (or all) activators. Such combined therapies may produce a synergistic therapeutic effect. The above parameters for evaluating the success and improvement of treatment in the disease are readily measurable by routine procedures known to physicians.

[0132] The words “to treat,” “treatment,” and “mitigation” are used interchangeably to refer to therapeutic treatment and methods of prevention or mitigation; here, the objective is to prevent or slow (reduce) the progression of a targeted pathological condition or disability. The group requiring treatment includes the group that is prone to the disability, as well as the group that already has the disability, as well as the group that is to be prevented from having the disability. A subject or mammal is successfully treated for an infection if, after receiving a therapeutic amount of antibodies according to the method of this invention, the patient shows one or more of the following observable and / or measurable reductions or absences: a reduction in the number of infected cells or absence of infected cells; a reduction in the percentage of the total number of infected cells; and / or the elimination of one or more signs specifically associated with the infection to some extent; a reduction in the pathological condition and mortality, and an improvement in quality of life. The above parameters for evaluating the success and improvement of treatment in a disease are readily measurable by routine treatments known to physicians.

[0133] The term "therapeutably effective dose" refers to the amount of antibody or drug that is effective in treating a disease or disorder in a subject or mammal.

[0134] Administration "in combination" with one or more additional therapeutic agents includes simultaneous and sequential administration, and may be in any order.

[0135] The term "carrier" as used herein includes pharmacologically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals to which they are exposed at the dosage and concentration used. Often, physiologically acceptable carriers are aqueous pH-buffered solutions. Examples of physiologically acceptable carriers include, but are not limited to, buffers such as phosphates, citrates and other organic acids; antioxidants, but not limited to ascorbic acid; low molecular weight (less than approximately 10 residues) polypeptides; proteins (e.g., serum albumin, gelatin, or immunoglobulin); hydrophilic polymers such as polyvinylpyrrolidone; amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates, but not limited to glucose, mannose, or dextrin; 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 glycol (PEG) and poloxamers (e.g., PLURONICS).

[0136] Given a range of values, each intermediate value between the upper and lower limits of that range (rounded to the nearest tenth of the unit of the lower limit unless explicitly stated otherwise in the context), and any other explicitly stated or intermediate values ​​within that range, are included in the present invention. The upper and lower limits of these smaller ranges (which may be independently contained within smaller ranges) are also included in the present invention, depending on the terminal values ​​specifically excluded from the range. If the range includes one or both of the terminal values, the range excluding either of these included terminal values ​​is also included in the present invention.

[0137] Unless otherwise specified, all technical and chemical terms used herein have meanings that will be understood as common to those with ordinary skill in the art to which this invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, but preferred methods and materials are described herein. All publications mentioned herein are incorporated herein by reference in their entirety.

[0138] Here, as used in the attached claims, the singular forms “one,” “and,” and “it” also refer to the plural unless otherwise specified.

[0139] The publications disclosed herein were provided independently for their disclosure prior to the filing date of this invention. This invention is not to be interpreted in any way that it does not have prior rights to such publications due to the effect of prior inventions. Furthermore, the actual publication date, which may need to be independently verified, may differ from the provided publication date.

[0140] Each application and patent cited herein, as well as each document or reference, or patent or non-patient document (patient or non-patient document), cited in each application or patent (including each issued patent in the application process; including application references), and corresponding to and / or claiming priority from each PCT and foreign application or patent, and references in the cited documents or each application reference are hereby expressed by reference. More generally herein, documents or references are cited, and the reference lists preceding the claims; or the text itself; and each of these documents or references ("Hereinafter Cited References"), and each of the documents or references of the Hereinafter Cited References (including any manufacturer's specifications, instructions, etc.) are hereby expressed by reference.

[0141] The following non-limiting embodiments may further illustrate this invention. [Examples]

[0142] Example 1 Materials, methods, and equipment use Human samples were collected after signed informed consent, following protocols checked by the Institutional Review Board (IRB) at all participating institutions. Patient 1 was selected from a group of long-term asymptomatic individuals followed at the Aaron Diamod Aids Research Center (New York). Patients 3 and 8 were selected from a group of long-term progression-free individuals followed at the Ragon Institute in Boston. Patients 1, 3, and 8 were selected based on their broad-spectrum neutralizing serological activity against a standard panel of HIV isolates. Patient 12 was selected from the International Aids Vaccine Initiative Protocol G group based on broad-spectrum 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 were performed (JF Scheid et al., Nature 458, 636 (Apr 2, 2009)). In short, CD19+ B cells were enriched from peripheral blood mononuclear cells using anti-human CD19 magnetic MACS beads (Miltenyi Biotec), and subsequently stained with anti-human CD20 and anti-human IgG antibodies (Becton Dickinson), as well as biotinylated 2CC-Core (B. Dey et al., PLoS Pathog 5, e1000445 (May, 2009)) or YU2-gp140 trimer (R. Diskin, PM Marcovecchio, PJ Bjorkman, Nat StructMol Biol 17, 608 (May, 2010)), and then detected by streptavidin-conjugated phycoerythrin (PE, Beckton Dickinson). Single cells were, Cells were sorted using a FACSAria III cell sorter (Becton Dickinson), cell doublets were removed, and 4 μl of 10 mM DTT, 8 U RNAsin (Promega), and 0.4 U 5'-3' Prime RNAse Inhibitor were added per well. TM The samples were placed in a 96-well PCR plate (Denville) containing a cold 0.5× phosphate buffer solution including (Eppendorf). The plate was sealed with Microseal (trademark) 'F' film (BioRad), rapidly 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 set, the first and second immunoglobulin-specific PCRs were performed using the primers listed in Table 1 in the seminested approach. Cloning of the heavy and light chain PCR products into their respective expression vectors was performed, and the identity of 100% of the original PCR products and the cloned expression plasmids was confirmed by sequencing prior to antibody expression in HEK293 cells.

[0145] ELISA. High-binding 96-well ELISA plates (Costar) were coated overnight with 100 ng of generated antibodies (gp140, gp120, gp41, gp120core, and 2CC-core) per well in PBS (B. Dey et al., PLoS Pathog 5, e1000445 (May, 2009)) and mutant proteins (gp120 D368R, gp120 I420R). After washing, the plates were blocked for 2 hours in 2% BSA, 1 μM EDTA, and 0.05% Tween-PBS (blocking buffer). Subsequently, the plates were incubated with IgG antibodies diluted to 4 μg / ml and several sequential 1:4 dilutions in PBS. Subsequently, the plates were developed by incubation for 1 hour with goat HRP-complexed anti-mouse IgG (Jackson ImmunoResearch) (0.8 μg / ml in blocking buffer) and by adding 100 μl of HRP dye substrate (ABTS solution, Invitrogen). Optical density was measured using an ELISA microplate reader (Molecular Devices) at 405 nm (OD 405nmThe values ​​were measured using ) and background values ​​were subtracted by incubation in PBS only in coated wells. IgG antibodies were validated as polyreactive (H. Mouquet et al., Nature 467, 591 (Sep 30, 2010)) and were considered polyreactive when they recognized at least two structurally different antigens from four: ssDNA, dsDNA, insulin, and LPS. The threshold for reactivity was determined by using control antibodies mGO53 (negative), eiJB40 (low positive), and ED38 (high positive).

[0146] Neutralization analysis: Neutralization screening was performed (DC Montefiori, Curr Protoc Immunol Chapter 12, Unit 12 11 (Jan, 2005)). Briefly, neutralization was detected as a decrease in luciferase reporter gene expression after single-round infection in Tzm-bl. MuLV (mouse leukemia virus) was used as a negative control to rule out nonspecific antiviral activity in antibody samples.

[0147] Clone-specific identification of bone marrow plasma cells. Bone marrow plasma cells were extracted from bone marrow fluid using Ficoll-Paque (GE Healthcare), and after Ficoll purification of mononuclear cells, they were stained with anti-human CD138 and anti-CD19 antibodies (Becton Dickinson). CD138+, CD19+ human plasma cells were bulk sorted using a FACSAria III 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 using Superscript III reverse transcriptase (Invitrogen) according to the manufacturer's instructions. Subsequently, the cDNA underwent immunoglobulin-specific PCR with the following modifications: 1 μl of cDNA was amplified twice by nested immunoglobulin heavy chain clone-specific PCR using the first forward reader sequence primer and reverse constant region primer shown in Table 1, followed by clone-specific forward and reverse primers designed based on the sequences obtained from single-cell analysis. After gel purification, the PCR products were incorporated into the TOPO TA vector according to the manufacturer's instructions. Colonies were selected by PCR using clone-specific primers and sequences.

[0148] Surface plasmon resonance. All experiments were performed using a Biacore T100 (Biacore) in HBS-EP + running buffer (Biacore) at 25°C, as previously described (Mouquet2010). YU-2 gp140 and 2CC-core proteins were immobilized on CM5 tips (Biacore) at 12.5 μg / mL with an amine coupling at pH 4.5, resulting in an immobilization level of 100 RUs. For kinetic analysis of the tips derivatized with gp140 and 2CC-core, IgG was injected through a flow cell at a flow rate of 40 μl / min in HBS-EP + running buffer (Biacore) at 700 nM and four sequential 1:2 dilutions, with 3 minutes of binding and 5 minutes of dissociation. The sensor surface was regenerated between each experiment by injecting 10 mM glycine-HCl pH 2.5 at a flow rate of 50 μL / min for 30 seconds. Off-rate ((k d (s -1 ))), ON rate (ka ( M -1 s -1 ) and coupling constant (KD (M) or KA (M) -1 The sensorograms were calculated after subtracting background values ​​(binding to the control flow cell and HBS-EP+ running buffer signals) using Biacore T100 evaluation software with kinetic analysis and a 1:1 binding model. The sensorograms 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 have plasmid:Fugene in a 1:2 ratio. TM Using 6 (Roche), gp160 in the pMX-IRE-GFP construct (Pietzsch et al. 2010) BAL.26 Δc or gp160 YU.2Transfection was performed with Δc. After 46 hours, 293T cells were washed with PBS and detached with trypsin-free cell dissociation buffer (Gibco), then 10°F in FACS buffer (1x PBS, 2% FBS, 2mM EDTA). 7 The cells were resuspended at a concentration of cell / ml. sCD4 (Progenics Pharmaceuticals, 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, 2010)). After 15 minutes of incubation on ice, the cells were isolated and treated with either an Alexa647-labeled CD4-derivative site mAb (3-67; (JF Scheid et al., Nature 458, 636 (Apr 2, 2009)) or an 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 Cells were stained on ice for 25 minutes using 2G12. Antibody labeling was performed using the Alexa Fluor® 647 microscale protein labeling kit (Invitrogen). Cells were analyzed on an LSR Fortessa cell analyzer (BD Bioscience).

[0150] Crystallization. 3BNC60 IgG was expressed transiently in HEK293-6E cells and prepared by papain cleavage (R. Diskin, PM Marcovecchio, PJ Bjorkman, Nat Struct Mol Biol 17, 608 (May, 2010)). The crystallization screen was prepared on an MRC crystallization plate (Jena Bioscience) using Mosquito TMCrystallization was performed at 20°C by vapor diffusion in nL sitting drops using a crystallization robot (TTP LabTech). We combined 3BNC60 Fab at a concentration of 9.5 mg / ml with a reservoir solution in a 1:1 ratio to prepare 400 nL droplets. The first hit for crystallization was PEGRx HT. TM Crystals were obtained using a crystallization screen (Hampton Research). Further manual optimization was performed. Crystals suitable for data acquisition were grown for several weeks in two Fabs in an asymmetric unit and in a monoclinic space group in 11.7% polyethylene glycol 20000, 0.1M sodium acetate pH 5.0, 100 mM potassium / sodium tartrate, 20 mM lithium sulfate, and 10 mM N-cyclohexyl-2-aminoethanesulfonic acid (CHES) pH 9.5. The crystals were immersed in a receiver solution supplemented with 15% glycerol for 2 hours, then immersed in a receiver solution supplemented with 30% glycerol, and instantaneously cooled in liquid nitrogen. Diffraction data were collected at 100 K using a Pilatus 6M detector by the Stanford synchrotron radiation source (SSRL) beamline 12-2. The data were indexed, aggregated, and scaled using XDS (W. Kabsch, Acta Crystallogr D Biol Crystallogr 66, 125 (Feb, 2010)) (Table 8). Molecular substitutions were performed as in the search model, from the antitumor antibody CTM01 (PDB code 1AD9) V H and CH1 domain, and V of anti-gp120 b13 antibody (PDB code 3IDX) L and C LThe analysis was performed using a feather with domains. Model coupling and purification of 2.65A for analysis were repeated 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 refined using the maximum-likelihood target function and non-crystallographic symmetry restraint. The final model was (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 preferred, permitted, and unpermitted Ramachandran plot regions, respectively. Structural analysis or visualization was performed using PyMol (The PyMOL Molecular Graphics System, Version 1.3, Schrodinger, LLC). The 3BNC60 structure consists of 3-205 residues for the light chain (including the first N-acetylglucosamine in the N-linked carbohydrate bound to Asn72) and 2-217 residues for the heavy chain. Terminal residues and C H There was a defect in residues 133-140 of one domain.

[0151] Mass spectrometry. IgG was purified from serum using Protein G Sepharose (GE Healthcare) according to the manufacturer's instructions. The IgG was then digested with immobilized papain (Pierce), and the Fab-Fc fragment mixture was incubated with a saturated amount of biotinylated 2CC-Core protein. Streptavidin-conjugated DynaBeads (Invitrogen) were added after incubation at room temperature for 15 minutes, and the mixture was washed 10 times with phosphate-buffered salt solution (Gibco). The conjugated Fab fragments were eluted with lithium dodecyl sulfate buffer (Invitrogen) at 95°C, and sample purity was confirmed by SDS-polyacrylamide gel electrophoresis, silver staining, or Coomassi staining, followed by mass spectrometry.

[0152] The isolated Fab fragments were reduced with dithiothreitol, alkylated with iodoacetamide, separated by 1D gel electrophoresis on 4-12% NuPAGE Novex bis-tris gel (Invitrogen), and stained with Coomassie blue (Thermo Fisher). The Fab fragments were scraped from the gel and digested with 200 ng of trypsin. The resulting peptides were isolated using reverse-phase resin (PORS 20 R2, Applied Biosystem) and extracted using aliquots of 40% acetonitrile in 0.5% acetic acid and a second aliquot of 80% acetonitrile in 0.5% acetic acid. Acetonitrile is removed using a Speedvac (Thermo Fisher Scientific), and aliquots of the remaining solution pressure are loaded onto a self-packed PicoFrit (trademark) column using integrated emitter tips (360 μm OD, 50 μm ID, 10 μm tips). A 6 cm reverse-phase C18 material (ReproSil-Pur C18-AQ, 3 μm beads from Dr. Maisch GmbH) is then packed, and an LTQ Orbitrap is used with a homemade micro-electrospray source. TM XL Mass Spectrometer or LTQ Orbitrap Velos TMOne of the mass spectrometers (Thermo Fisher Scientific) was interfaced with an Agilent 1200 series HPLC system (Agilent). Peptides were extracted into the mass spectrometer with the following gradient: 0–5% B for 5 minutes, 40% B for 125 minutes, 60% B for 150 minutes, and 100% B for 165 minutes (A = 0.1 M acetic acid, B = 70% acetonitrile in 0.1 M acetic acid, flow rate 90 nL / min). 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 TM For analysis on XL, eight MS / MS scans were performed on the eight most abundant ions from the full scan after the full scan. Peptides (charge state > 1 only) were isolated in a 2Da window, dissociated via CAD in a 1e4 ion target window (normalized collision energy = 35, activation Q = 0.25, activation time = 30 ms), and their mass was analyzed in LTQ. LTQ Orbitrap TMFor analysis on Velos, a 10 MS / MS scan was performed immediately after a full scan, with a resolution of 75,000 on the 10 most abundant ions from the aforementioned full scan. Peptides (charge state > 2 only) were isolated in a 3Da window, with a target ion window of 2e5, dissociated via HCD (normalized collision energy = 40, activation time = 0.100 ms), and their mass was analyzed by Orbitrap. For both instruments, ions selected from MS / MS were set to an exclusion list for 30 seconds. The resulting MS / MS spectra were searched using Xtandem against Human IPI or an in-house patient-specific IgG database, and peptides were automatically compared to trypsin peptides in Human IPI and our in-house patient-specific IgG database. Peptides matching patient-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). Alignment shading was performed using the TeXshade package.

[0154] Alignment consensus. Consensus sequences for multiple alignments were created based on residue-to-residue identity and similarity (>=70%). Amino acids were divided according to similarity as follows: FYW, ILVM, RK, DE, GA, ST, and NQ. Germline phylogenetic tree. Relationships between sequences were created using the Neighbor-Joining method. A bootstrap-style consensus phylogenetic tree inferred from 1000 replications was taken to show relevance. Branches corresponding to divisions reproduced in 50% of bootstrap replications collapse. Of the related sequences gathered in the bootstrap test (1000 replications), the percentage of replication in the phylogenetic tree is shown in the following branches. The phylogenetic tree was drawn with branch lengths in the same units as those of the evolutionary distance used to infer the phylogenetic tree. The evolutionary distance is calculated using the number of differences method and is a unit of the number of amino acid differences per sequence. All ambiguous positions were removed for each sequence pair. Evolutionary analysis was performed using MEGA5.

[0155] R / S ratio calculation. DNA sequences were overlaid 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 novel set of primers was designed to avoid potential problems (Table 1). The novel primer set was validated by sorting B cells bound to HIV-gp120 that lacked the V1-3 loop and contained a stable disulfide bond (2CC-core). In contrast to the resurfaced bait used to clone VRC01, the 2CC-core bait also allows antibody capture against the CD4-derivative co-receptor binding site (CD4i).

[0157] In the side-by-side comparison, the new primer set increased IgH chain recovery compared to the initial primer set (Figure 4(a)). The antibodies obtained with the new primer set were more mutated (mean 35.6 vs. 19.8 p=<0.0001 and max. 85 vs. 50 for IgH) and included clones not found in the original primer set. To determine whether the new primers recapture VRC01-like antibodies from cells sorted with YU2 gp140, cDNA samples from individuals that had already been thoroughly examined were validated with the original primer set, which does not produce any VRC01-related clones. In 80 wells, three antibodies consistent with VRC01 variants were found, as determined by the IgH and IgL sequences (Figures 5A and B). We found that VRC01-like antibodies are captured by the gp140 trimer, and primers specifically designed to clone highly mutant antibodies capture large fragments of anti-HIV antibodies from memory B cells of patients with high titers of broad-spectrum neutralizing antibodies.

[0158] Of four unrelated HIV-infected individuals, including two Caucasians, one Hispanic, and one African, who showed high titers of broad-spectrum neutralizing antibodies when examined using 2CC-core bait, the aforementioned clonal antibodies could not explain the serological activity of two of them (Table 2 and Figures 6A and B). 576 antibodies representing 201 distinct and unique diversified clones were found to have titers of 1.5 × 10⁶. 5 IgG + Obtained from the initial group of memory B cells.

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

[0160] The antibody binding sites on the HIV spike were mapped using mutant proteins that interfere with either the CD4bs (gp120(D368R)) or the CD4-derivative co-receptor binding site (CD4i, gp120(I420R)). As reported by X. Wu et al., Science 329, 856 (Aug 13, 2010), VRC01 is classified as a CD4bs antibody because it is sensitive to the D368R mutant. However, it also showed some sensitivity to the I420R mutant due to its proximity to the CD4i site. The VRC01 mutants NIH45-46 and the antibodies 3BNC60, 8ANC131, and 12A12 showed similar ELISA patterns to VRC01 (these clone members were selected based on neutralizing activity, Table 3). Other clones, including 1B2530 and 8ANC195, were equally sensitive to both variants and could not be accurately classified based solely on ELISA.

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

[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 hypermutant 2CC-core-specific antibodies, four representative antibodies were reversed into their corresponding germlines. The reverse mutations resulted in complete loss of antigen binding and lack of polyreactivity in all four clones validated.

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

[0164] All 90 antibodies tested showed some degree of neutralization, and six clones contained antibody variants that exhibited high levels of potency and breadth (Figures 1A, B, and C, and Tables 4 and 5). These clones were the most abundant among those captured by 2CC-bait in each of the four patient studies (Table 3). The most effective novel antibody, 3BNC117, belonging to a 76-member clone, showed an average IC50 in combination with 14 groups of tier 2 viruses at 0.5 μg / ml compared to 1.8 μg / ml at VRC01. 80 This was shown.

[0165] Of the 20 viruses tested, only 4 were more sensitive to VRC01 than to 3BNC117. However, 14 were completely VRC01 resistant, including DU172.17 which is susceptible to 3BNC117, and more sensitive to 3BNC117 (Figures 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 (Figures 1B and C, and Figures 4 and 5).

[0166] There is considerable variation in neutralization potency and breadth among the five most capable neutralizing antibody clones. For example, in the first panel, 3BNC156, a variant of 3BNC117, neutralized only two viruses, and at much higher concentrations than 3BNC117 (Figure 1A and Table 4), while another variant, 3BNC55, had an average IC50 of 4 μg / ml. 50 It showed activity against six viruses, falling between the two above. Ultimately, the antibody with the highest activity had undergone a high frequency of mutations. The average number of mutations for the top 10 antibodies was V H 72, and V L The value was 45, which was related to potency and breadth. Revert mutations of the mutated residue into the germline resulted in a complete loss of neutralizing activity for all antibodies validated for the residue.

[0167] Example 6 Identification of diagnostic peptides The aforementioned cloning method captured antibodies produced by antigens that bind to memory B cells; however, circulating antibodies are not produced by these cells, but instead originate from plasma cells in the bone marrow. However, since the congener antigens do not express surface IgA, they cannot be used as bait to capture plasma cells (Radbruch et al., Nat Rev Immunol 6, 741 (Oct, 2006)). Furthermore, the relationship between plasma cells in the bone marrow and circulating memory B cells is not 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 bone marrow samples combined from two of the four individuals studied, and IgV was used to obtain more potent antibodies cloned from memory B cells in these individuals. H The following clone-specific primers were used for gene amplification via PCR. For RU01: CTGCAACCGGTGTACATTCTCAAGTGCAACTGGTGC (FWRD) (SEQ ID NO: 584), CTGCAACCGGTGTACATTCTCAGGTCCATTTGTCACAG (FWRD), (SEQ ID NO: 585), TGCGAAGTCGACGCTGACGAGACAGTGACCTGC (REV) (SEQ ID NO: 586), TGCGAAGTCGACGCTGAAGAGACAATAATTTG (REV) (SEQ ID NO: 587), TGCGAAGTCGACGCTGACGAGACAATAACT (REV) (SEQ ID NO: 588), and for RU10: CTGCAACCGGTGTACATTTTCAGGGGCACTTGGTG (FWRD) (SEQ ID NO: 589), TGCGAAGTCGACGCTGAGGTGACGATGACCGTG (REV) (SEQ ID NO: 590). Members of the selected clones and numerous additional variants were promptly identified in both patients.

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

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

[0170] The top neutralizing antibody has affinity (K) on the 2CC-core. A ) is 10 on the YU2 gp140 trimer 7 -10 12 and 10 on 2CC-core 7 -10 11 (M -1 The range of affinities was shown (Tables 2A and B, and Table 6). In harmony with their neutralizing potency and breadth, 3BNC66, 3BNC156, and 3BNC55 showed lower affinity on the YU2 gp140 trimer than 3BNC117, but surprisingly, affinity to the 2CC-core was not associated with neutralizing activity (Figures 1, 8, Tables 4 and 6). SPR binding was not detected for any germline reverse mutation antibodies tested (Figure 2B, Table 6). Anti-HIV antibodies captured by the YU2 2CC-core tended to show higher affinity to the gp140 trimer than to the 2CC-core.

[0171] When VRC01 binds to the HIV spike, it mimics CD4 binding and results in a significant structural change that exposes the CD4i site. In contrast, B12 and most other known anti-CD4bs antibodies do not.

[0172] To determine if this is a shared characteristic of highly active antibodies, HIV-BAL.26Δc or -YU2 gp160Δc were expressed on the surface of HEK293 cells, and CD4i antibody binding was measured in the presence or absence of CD4 or anti-CD4bs antibodies (Figure 2C). One exception was that all of the highly active antibodies validated were similar to CD4 and VRC01 in that they promoted anti-CD4i antibodies to bind to HIV-BAL.26Δc or -YU2 gp160Δc or both.

[0173] Only 8ANC195, a highly active antibody that does not share this characteristic, differs from typical anti-CD4bs antibodies in that it is equally sensitive to the D368R and I420R variants (Table 3). In addition, it differs from other highly active antibodies in its neutralization pattern: it showed strong activity against clade B virus H086.8, which does not neutralize any tier virus and is resistant to all other validated antibodies, including 3BNC117, VRC01, and b12.

[0174] Example 8 Sequence identity of HIV antibodies To determine whether highly active anti-CD4bs antibodies shared common sequence features, 10 best antibodies were used: two variants from five independent antibodies derived from five different patients were aligned (Figure 3). IgV H The comparison of regions is based on 68 IgV H It was associated with a highly conserved consensus sequence covering the residue (Figure 3A). IgV H The consensus contains 6 VRC01-gp120 contact residues and Arg59 CD4 and Asp368 gp120It contains VRC01-Arg 71, which mimics the key interaction of (Figure 3A). Furthermore, it contains 6 contact residues, and consensus is that all antibodies in this class are closely related germline IgV. H Gene (V H 1-2 and V H It was preserved in both 1-46).

[0175] The codons encoding consensus residues underwent highly somatic mutations in 10 selected antibodies, yet the amino acid sequences remained conserved (Figure 9). The substitution rate for silent mutations in consensus residues ranged from 0.7 to 1.7, compared to 3.5 to 22 for non-consensus residues, indicating strong selection of consensus conservation (Table 7). In contrast to the heavy chain, only 8 of the 32 VRC01 light chain residues contacted gp120. Consistent with a more limited role, comparison of light chain sequences of the same antibodies revealed 53 IgV antibodies containing 3 VRC01-gp120 contact residues. L A less extensive consensus covering the residues was revealed (Figure 3B). Ultimately, like the heavy chain, the light chain was attributed to 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 key aspects, did not fully adhere to the consensus, and was not attributed to the associated heavy and light chains (Figures 3A and B). We confirmed that there is a convergence of key sequences among highly active agonistic anti-CD4bs antibodies (HAADs).

[0176] Example 9 Crystal structure of 3BNC60 Fab To determine whether the antibody structure is conserved in different patients, the crystal structure of 3BNC60Fab was analyzed at 2.65A resolution and compared with that of VRC01. The structure shows that the V forms the antigen-binding site. Hand V L We identified four domains within the standard Fab and complementarity-determining region (CDR): VH, CH1, VL, and CL. The two Fabs in the asymmetric unit of 3BNC60 were nearly identical; however, due to the different chemical environments formed by the contact of the crystal lattice, the morphology of residues 74-78 of the loop connecting D and E of the chain was slightly different.

[0177] Overlapping images of the VH domains from 3BNC60 and VRC01 in the VRC01-gp120 cocrystal structure (T. Zhou et al., Science 329, 811 (Aug 13, 2010)) showed significant differences limited to residues 58-65 of CDR2 (numbering of 3BNC60) and a mean squared deviation (rmsd) of 1.3A (calculated for 111 Cα atoms). The structural superposition demonstrated conservation of the recognition interface in gp120, e.g., Arg72 3BNC60 Arg71 VRC01 It adopts a similar form, which is usually Arg59 CD4 and Asp368 gp120 It mimics an important salt bridge formed between them. In addition, Trp47 3BNC60 Trp47 VRC01 It adopts a similar morphology to the residues in contact with gp120 and includes a complex network of interactions between aromatic and aliphatic residues that stabilize the morphology of CDRH3 and CDRL3. Gln64 VRC01 Gln65 matches 3BNC60 This is located in a residue segment (residues 58-65) that differs in structure from VRC01. In the crystal, the morphology of this region of 3BNC60, which is included in the lattice contact, appears to change to bind to gp120 upon collision with the CD4 binding loop on gp120.

[0178] 3BNC60 and VRC01 V LSuperimposing the domains yielded an rmsd of 0.9A (calculated for 95 Cα atoms), and showed that some of the gp120-contact residues are structurally conserved; Tyr91 3BNC60 and Glu91a 3BNC60 Tyr91 VRC01 and Glu96 VRC01 It adopted a similar morphology, and it engages in loop D of gp120 via a polar interaction. Overall, these structural comparisons suggest that 3BNC60 binds to gp120 with the same configuration as observed in the binding of VRC01.

[0179] Example 10 HIV antibody consensus sequence The aforementioned experiment involved the exchange of 8 contact residues between VRC01 and the HIV spike, including IgV H and IgV L We determined the class of HAADs (Hypoallergenic Anti-CD4bs Antibodies) that share a consensus sequence (Figure 3A and B). Selected for their high levels of serological anti-HIV activity in five different donors, these antibodies conform to the HAAD consensus of two closely related IgV H and three IgV L Derived solely from germline genes; V H 1-2 and V H Recipients 1-46 differ by only seven amino acids and are not part of the consensus (Figure 3A). Despite widespread somatic hypermutation, the consensus residues were retained in their germline forms.

[0180] The sole exception to the consensus, 8ANC195 differs from others in several ways, suggesting it may have its own unique method of antigen recognition: the important Arg59 CD4 and Asp368 gp120The absence of Arg in the heavy chain, similar to the contact site; a unique neutralization pattern; and inability to readily bind to anti-CD4i antibodies. This antibody is one of two different highly active antibodies occurring in a single patient.

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Claims

1. An isolated anti-HIV antibody or its antigen-binding fragment, comprising a heavy chain variable region containing the sequence of SEQ ID NO: 896 and a light chain variable region containing the sequence of SEQ ID NO:

910.

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

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

4. 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, as an isolated anti-HIV antibody or its antigen-binding fragment according to any one of claims 1 to 3.

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

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

7. A vector comprising the nucleic acid described in claim 6.

8. Cultured cells comprising the vector according to claim 7.

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

10. The pharmaceutical composition according to claim 9, used for the prevention or treatment of HIV infection or HIV-related diseases.

11. The pharmaceutical composition according to claim 10, further comprising a second therapeutic agent.

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

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

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

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

16. A method for producing an anti-HIV antibody or its antigen-binding fragment according to any one of claims 1 to 4, Culturing cells containing a vector comprising nucleic acids encoding the heavy and light chains of the anti-HIV antibody or its antigen-binding fragment, under conditions in which the nucleic acids are expressed, and Isolating the anti-HIV antibody or its antigen-binding fragment, A manufacturing method that includes this.

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