Anti-human immunodeficiency virus-1 antibodies and methods of use thereof

Abstract

An anti-HIV-1 antibody comprising L-CDR1, L-CDR2 and L-CDR3, wherein L-CDR1 is selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, and sequences which differ from any of SEQ ID NO: 15, 18 or 21 by one or two substitutions, deletions or additions; the amino acid sequence of L-CDR2 is selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, and sequences which differ from any of SEQ ID NO: 16, 19 or 22 by one or two substitutions, deletions or additions; and the amino acid sequence of L-CDR3 is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, and sequences which differ from any of SEQ ID NO: 17, 20 or 23 by one or two substitutions, deletions or additions.

Description

[Technical Field]

[0001] This application relates to antibodies against human immunodeficiency virus-1 (anti-HIV-1) that specifically bind to the HIV-1 p24 protein. The invention also refers to methods and assays for detecting HIV-1 in a sample using said antibodies. [Background technology]

[0002] Human immunodeficiency virus 1 (HIV-1) is a retrovirus that infects 37.9 million people worldwide and causes approximately 1 million deaths annually, especially among vulnerable populations lacking access to diagnosis and treatment (Soliman, M. et al., "Mechanisms of HIV Control," Current HIV / AIDS Reports 2017, vol. 14(3); pp. 101-9). HIV-1 is the primary cause of acquired immunodeficiency syndrome (AIDS), an incurable disease transmitted by HIV-1-infected individuals through sexual contact or exposure to contaminated blood or blood-derived products. The virus targets the immune system by disrupting and impairing immune cell function. Infected individuals become immunocompromised and susceptible to other opportunistic infections, as well as certain cancers (Source: WHO website - https: / / www.who.int / news-room / fact-sheets / detail / hiv-aids). Currently, only 46% of people infected with HIV-1 are aware of their infection status. Therefore, detection of HIV-1 during acute infection is a significant public health concern (Stone, M. et al., "Comparison of detection limits of fourth- and fifth-generation combination HIV antigen-antibody, p24 antigen, and viral load assays on diverse HIV isolates." Journal of Clinical Microbiology 2018, vol. 56(8); pp. 1-12).

[0003] In this particular situation, the goal is to diagnose HIV-1 within the first few weeks after an individual is infected (the acute phase), as this is likely to prevent secondary infection and allow early access to treatment and care (Lewis J. et al., "Field accuracy of fourth-generation rapid diagnostic tests for acute HIV-1: a systematic review," AIDS 2015, vol. 29(18); pp. 2465-71). To achieve this goal in a timely manner, the use of early biomarkers for HIV-1 detection is key. The most commonly used biomarkers for diagnosing HIV-1 infection are antibodies against viral structural proteins. p24 is the most abundant structural protein of the HIV-1 viral envelope and is secreted at high levels into serum during the early stages of infection, making it an important biomarker for early HIV-1 detection. p24 is a polymerized capsid protein that serves as the main structural component of the HIV-1 envelope around the viral RNA molecule. p24 is a 24-25 kDa protein derived from the Gag polyprotein precursor that, like HIV-1 RNA, is detectable before seroconversion (Gray, ER et al., "p24 revisited: a landscape review of antigen detection for early HIV diagnosis." AIDS. 2018, vol. 32(15); pp. 2089-102).

[0004] Current guidelines from the Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) recommend the use of fourth-generation antibody-antigen assays as the preferred method for HIV-1 screening. These tests detect p24 antigen and anti-HIV-1 antibodies and have narrowed the diagnostic gap from 4 weeks to 2 weeks after exposure (Gray, E.R. et al., "p24 revisited: a landscape review of antigen detection for early HIV diagnosis." AIDS. 2018, vol. 32(15); pp. 2089-102; Codoner, F. et al., "Gag protease coevolution analyses define novel structural surfaces in the HIV-1 matrix and capsid involved in resistance to protease inhibitors." Scientific Reports 2017, vol. 7(3717); pp. 1-10; Alexander, T.S., "Human Immunodeficiency Virus Diagnostic Testing: 30 Years of Evolution." Clinical and Vaccine Immunology 2016, vol. 23(4); pp. 249-53; WHO, "World Health Organization Model List of Essential In Vitro Diagnostics.'', 1st edition, Geneva, 2018; Centers for Disease Control and Prevention, 2017, ``National HIV testing day and new testing recommendations.'', Morbidity and Mortality Weekly Report vol. 63(25); pp. 537-37).

[0005] However, some currently available antibodies have low sensitivity for early p24 detection, so there is still a need for anti-HIV-1 antibodies that specifically bind to the p24 antigen and have high binding capacity and excellent manufacturing characteristics.

[0006] Thus, the present invention provides anti-HIV-1 antibodies with improved binding ability to the HIV-1 p24 protein compared to similar commercially available reagents. These antibodies recognize novel, non-cross-reactive epitopes and can be used alone or as capture / detection partners in multiple HIV-1 immunoassays, such as immunodiagnostic or blood screening platforms. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Soliman, M. et al., "Mechanisms of HIV Control," Current HIV / AIDS Reports 2017, vol. 14(3); pp. 101–9 [Non-patent document 2] Source from WHO website - https: / / www.who.int / news-room / fact-sheets / detail / hiv-aids [Non-patent document 3] Stone, M. et al., "Comparison of detection limits of fourth- and fifth-generation combination HIV antigen-antibody, p24 antigen, and viral load assays on diverse HIV isolates." Journal of Clinical Microbiology 2018, vol. 56(8); pp. 1-12 [Non-patent document 4] Lewis J. et al., “Field accuracy of fourth-generation rapid diagnostic tests for acute HIV-1: a systematic review.” AIDS 2015, vol. 29(18); pp. 2465–71 [Non-patent document 5] Gray, ER et al., “p24 revisited: a landscape review of antigen detection for early HIV diagnosis.” AIDS. 2018, vol. 32(15); pp. 2089–102. [Non-patent document 6] Codoner, F. et al., “Gag protease coevolution analyzes define novel structural surfaces in the HIV-1 matrix and capsid involved in resistance to Protease Inhibitors.” Scientific Reports 2017, vol. 7(3717); pp. 1-10 [Non-Patent Document 7] Alexander TS., “Human Immunodeficiency Virus Diagnostic Testing: 30 Years of Evolution.” Clinical and Vaccine Immunology 2016, vol. 23(4); pp. 249-53 [Non-patent document 8] WHO, “World Health Organization Model List of Essential In Vitro Diagnostics.”, 1st edition, Geneva, 2018; Centers for Disease Control and Prevention, 2017, “National HIV testing day and new testing recommendations.”, Morbidity and Mortality Weekly Report vol. 63(25); pp. 537–37 [Non-Patent Document 9] Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington DC [Non-Patent Document 10] Chothia et al., Nature 342:877-883, 1989 [Non-Patent Document 11] MacCallum et al., J. Mol. Biol., 262:732-745, 1996 [Non-Patent Document 12] Lefranc, M.-P. Nucl. Acids Res., 33, pp. D593-D597, 2005 [Non-Patent Document 13] Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008 [Non-Patent Document 14] Retter et al., Nucl. Acids Res., 33 (Database issue): pp. D671-D674 (2005) [Non-Patent Document 15] Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268 [Non-Patent Document 16] Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877 [Non-Patent Document 17] Altschul et al., 1990, J. Mol. Biol. 215:403-410 [Non-Patent Document 18] Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402 [Non-Patent Document 19] Lefranc MP, Lefranc G, "IMGT(R) and 30 years of Immunoinformatics Insight in Antibody V and C Domain Structure and Function.", Jefferis R; Strohl WR, Kato K., Antibodies 2019, vol. 8(29); pp. 1-21 [Non-Patent Document 20] Liao-Chan S. et al., "Monoclonal Antibody Binding-site Diversity Assessment with a Cell-based Clustering Assay.", Journal of Immunological Methods 2014, vol.405; pp. 1-14 Summary of the Invention [Means for solving the problem]

[0008] Where the specification refers to a sequence of CDR X, or a sequence which differs from CDR X by one or two substitutions, deletions or additions, it is to be understood that such substitutions, deletions or additions can occur at any amino acid within the range of amino acids defined by CDR X. The specification individuates each particular amino acid within the range of amino acids defined by CDR X as being suitable for such substitution, deletion or addition.

[0009] As a non-limiting example, L-CDR1 of antibody #A can be defined as comprising the sequence of amino acids (1)-(11), RASQDISNYLH [shown schematically in SEQ ID NO: 15]. Each of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 is considered suitable for substitution, deletion, or addition unless otherwise specified or later refined by amendment.

[0010] In a first aspect, the present invention discloses an anti-HIV-1 antibody comprising a light chain comprising complementarity determining regions L-CDR1, L-CDR2 and L-CDR3, wherein the amino acid sequence of L-CDR1 is selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, and sequences which differ from any of SEQ ID NO: 15, 18 or 21 by one or two substitutions, deletions or additions; the amino acid sequence of L-CDR2 is selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, and sequences which differ from any of SEQ ID NO: 16, 19 or 22 by one or two substitutions, deletions or additions; and the amino acid sequence of L-CDR3 is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, and sequences which differ from any of SEQ ID NO: 17, 20 or 23 by one or two substitutions, deletions or additions.

[0011] In another embodiment, the anti-HIV-1 antibody of the present invention comprises a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2, and H-CDR3, wherein the amino acid sequence of H-CDR1 is selected from the group consisting of SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, and sequences which differ from any of SEQ ID NO:24, 27, or 30 by one or two substitutions, deletions, or additions; the amino acid sequence of H-CDR2 is selected from the group consisting of SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, and sequences which differ from any of SEQ ID NO:25, 28, or 31 by one or two substitutions, deletions, or additions; and the amino acid sequence of H-CDR3 is selected from the group consisting of SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, and sequences which differ from any of SEQ ID NO:26, 29, or 32 by one or two substitutions, deletions, or additions.

[0012] In some embodiments, the light chain of an anti-HIV-1 antibody of the invention comprises a sequence having about 90% homology to the amino acid sequence of SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9. In other embodiments, the light chain comprises the amino acid sequence of SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9.

[0013] In some embodiments, the heavy chain of an anti-HIV-1 antibody of the invention comprises a sequence having about 90% homology to the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In other embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

[0014] In some embodiments, the amino acid sequence of L-CDR1 comprises SEQ ID NO: 15 or a sequence that differs from SEQ ID NO: 15 by one or two substitutions, deletions, or additions, the amino acid sequence of L-CDR2 comprises SEQ ID NO: 16 or a sequence that differs from SEQ ID NO: 16 by one or two substitutions, deletions, or additions, and the amino acid sequence of L-CDR3 comprises SEQ ID NO: 17 or a sequence that differs from SEQ ID NO: 17 by one or two substitutions, deletions, or additions. In other embodiments, the amino acid sequence of L-CDR1 comprises SEQ ID NO: 18 or a sequence that differs from SEQ ID NO: 18 by one or two substitutions, deletions, or additions, the amino acid sequence of L-CDR2 comprises SEQ ID NO: 19 or a sequence that differs from SEQ ID NO: 19 by one or two substitutions, deletions, or additions, and the amino acid sequence of L-CDR3 comprises SEQ ID NO: 20 or a sequence that differs from SEQ ID NO: 20 by one or two substitutions, deletions, or additions. In other embodiments, the amino acid sequence of L-CDR1 comprises SEQ ID NO: 21 or a sequence that differs from SEQ ID NO: 21 by one or two substitutions, deletions, or additions; the amino acid sequence of L-CDR2 comprises SEQ ID NO: 22 or a sequence that differs from SEQ ID NO: 22 by one or two substitutions, deletions, or additions; and the amino acid sequence of L-CDR3 comprises SEQ ID NO: 23 or a sequence that differs from SEQ ID NO: 23 by one or two substitutions, deletions, or additions.

[0015] In some embodiments, the amino acid sequence of H-CDR1 comprises SEQ ID NO: 24 or a sequence that differs from SEQ ID NO: 24 by one or two substitutions, deletions, or additions, the amino acid sequence of H-CDR2 comprises SEQ ID NO: 25 or a sequence that differs from SEQ ID NO: 25 by one or two substitutions, deletions, or additions, and the amino acid sequence of H-CDR3 comprises SEQ ID NO: 26 or a sequence that differs from SEQ ID NO: 26 by one or two substitutions, deletions, or additions. In other embodiments, the amino acid sequence of H-CDR1 comprises SEQ ID NO: 27 or a sequence that differs from SEQ ID NO: 27 by one or two substitutions, deletions, or additions, the amino acid sequence of H-CDR2 comprises SEQ ID NO: 28 or a sequence that differs from SEQ ID NO: 28 by one or two substitutions, deletions, or additions, and the amino acid sequence of H-CDR3 comprises SEQ ID NO: 29 or a sequence that differs from SEQ ID NO: 29 by one or two substitutions, deletions, or additions. In other embodiments, the amino acid sequence of H-CDR1 comprises SEQ ID NO: 30 or a sequence that differs from SEQ ID NO: 30 by one or two substitutions, deletions, or additions; the amino acid sequence of H-CDR2 comprises SEQ ID NO: 31 or a sequence that differs from SEQ ID NO: 31 by one or two substitutions, deletions, or additions; and the amino acid sequence of H-CDR3 comprises SEQ ID NO: 32 or a sequence that differs from SEQ ID NO: 32 by one or two substitutions, deletions, or additions.

[0016] In some embodiments, the amino acid sequence of L-CDR1 comprises SEQ ID NO: 15 or a sequence that differs from SEQ ID NO: 15 by one or two substitutions, deletions, or additions; the amino acid sequence of L-CDR2 comprises SEQ ID NO: 16 or a sequence that differs from SEQ ID NO: 16 by one or two substitutions, deletions, or additions; the amino acid sequence of L-CDR3 comprises SEQ ID NO: 17 or a sequence that differs from SEQ ID NO: 17 by one or two substitutions, deletions, or additions; the amino acid sequence of H-CDR1 comprises SEQ ID NO: 24 or a sequence that differs from SEQ ID NO: 24 by one or two substitutions, deletions, or additions; the amino acid sequence of H-CDR2 comprises SEQ ID NO: 25 or a sequence that differs from SEQ ID NO: 25 by one or two substitutions, deletions, or additions; and the amino acid sequence of H-CDR3 comprises SEQ ID NO: 26 or a sequence that differs from SEQ ID NO: 26 by one or two substitutions, deletions, or additions.

[0017] In some embodiments, the amino acid sequence of L-CDR1 comprises SEQ ID NO: 18 or a sequence that differs from SEQ ID NO: 18 by one or two substitutions, deletions, or additions; the amino acid sequence of L-CDR2 comprises SEQ ID NO: 19 or a sequence that differs from SEQ ID NO: 19 by one or two substitutions, deletions, or additions; the amino acid sequence of L-CDR3 comprises SEQ ID NO: 20 or a sequence that differs from SEQ ID NO: 20 by one or two substitutions, deletions, or additions; the amino acid sequence of H-CDR1 comprises SEQ ID NO: 27 or a sequence that differs from SEQ ID NO: 27 by one or two substitutions, deletions, or additions; the amino acid sequence of H-CDR2 comprises SEQ ID NO: 28 or a sequence that differs from SEQ ID NO: 28 by one or two substitutions, deletions, or additions; and the amino acid sequence of H-CDR3 comprises SEQ ID NO: 29 or a sequence that differs from SEQ ID NO: 29 by one or two substitutions, deletions, or additions.

[0018] In some embodiments, the amino acid sequence of L-CDR1 comprises SEQ ID NO:21 or a sequence that differs from SEQ ID NO:21 by one or two substitutions, deletions, or additions; the amino acid sequence of L-CDR2 comprises SEQ ID NO:22 or a sequence that differs from SEQ ID NO:22 by one or two substitutions, deletions, or additions; the amino acid sequence of L-CDR3 comprises SEQ ID NO:23 or a sequence that differs from SEQ ID NO:23 by one or two substitutions, deletions, or additions; the amino acid sequence of H-CDR1 comprises SEQ ID NO:30 or a sequence that differs from SEQ ID NO:30 by one or two substitutions, deletions, or additions; the amino acid sequence of H-CDR2 comprises SEQ ID NO:31 or a sequence that differs from SEQ ID NO:31 by one or two substitutions, deletions, or additions; and the amino acid sequence of H-CDR3 comprises SEQ ID NO:32 or a sequence that differs from SEQ ID NO:32 by one or two substitutions, deletions, or additions.

[0019] In some embodiments, the light chain of an anti-HIV-1 antibody of the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0020] In some embodiments, the heavy chain of an anti-HIV-1 antibody of the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0021] In some embodiments, the anti-HIV-1 antibodies of the invention specifically bind to an epitope of the HIV-1 p24 protein comprising the amino acid sequence of SEQ ID NO:33.

[0022] In some embodiments, the amino acid sequence of L-CDR1 of an anti-HIV-1 antibody of the present invention comprises SEQ ID NO:21 or a sequence that differs from SEQ ID NO:21 by one or two substitutions, deletions, or additions; the amino acid sequence of L-CDR2 comprises SEQ ID NO:22 or a sequence that differs from SEQ ID NO:22 by one or two substitutions, deletions, or additions; and the amino acid sequence of L-CDR3 comprises SEQ ID NO:23 or a sequence that differs from SEQ ID NO:23 by one or two substitutions, deletions, or additions.

[0023] In some embodiments, the amino acid sequence of H-CDR1 of an anti-HIV-1 antibody of the invention comprises SEQ ID NO: 30 or a sequence that differs from SEQ ID NO: 30 by one or two substitutions, deletions, or additions; the amino acid sequence of H-CDR2 comprises SEQ ID NO: 31 or a sequence that differs from SEQ ID NO: 31 by one or two substitutions, deletions, or additions; and the amino acid sequence of H-CDR3 comprises SEQ ID NO: 32 or a sequence that differs from SEQ ID NO: 32 by one or two substitutions, deletions, or additions.

[0024] In some embodiments, the light chain of the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and the heavy chain of the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0025] In some embodiments, the anti-HIV-1 antibody of the present invention is a monoclonal antibody or a recombinant antibody. In other embodiments, the antibody is an antibody fragment. When the anti-HIV-1 antibody is an antibody fragment, it is selected from a variable fragment (Fv), a single-chain Fv (scFv), a bispecific antibody (sc(Fv)2), a single-chain antibody, a single-domain antibody, a Fab fragment, a F(ab')2 fragment, a Fab' fragment, a disulfide-linked Fv (dsFv), a chemically conjugated Fv (ccFv), a diabody, an anti-idiotype (anti-Id) antibody, an affibody, a nanobody, and a unibody.

[0026] In some embodiments, the anti-HIV-1 antibody comprises a constant region of the mouse IgG1 class or the mouse IgG2a class.

[0027] In some embodiments, the anti-HIV-1 antibody is bound to a solid support.

[0028] In some embodiments, the present invention discloses cells comprising the anti-HIV-1 antibodies of the present invention.

[0029] In another aspect, the present invention discloses a nucleic acid comprising a nucleotide sequence encoding an anti-HIV-1 antibody, a promoter operably linked to the nucleotide sequence, and a selectable marker. Cells comprising the nucleic acid are also disclosed herein.

[0030] The present invention also discloses compositions comprising an anti-HIV-1 antibody described herein and a solid support, wherein the anti-HIV-1 antibody is covalently or non-covalently bound to the solid support. In some embodiments, the solid support comprises a particle, bead, membrane, surface, polypeptide chip, microtiter plate, or solid phase of a chromatography column.

[0031] The present invention also discloses a kit for detecting the presence of HIV-1 in a sample, said kit comprising at least one anti-HIV-1 antibody according to the present invention and a solid support, wherein said at least one antibody is covalently or non-covalently bound to the solid support. [Brief explanation of the drawings]

[0032] [Figure 1] Monomer percentage by SE-UPLC analysis of antibody #A, and SDS-PAGE of antibody #A single clone (lanes 1, 2, and 3 represent subclones run under reducing and non-reducing conditions, respectively). [Figure 2] Monomer percentage by SE-UPLC analysis of antibody #B, and SDS-PAGE of antibody #B single clones (lanes 1, 2, and 3 represent subclones run under reducing and non-reducing conditions, respectively). [Figure 3] Monomer percentage by SE-UPLC analysis of antibody #D, and SDS-PAGE of antibody #D single clones (lanes 1, 2, and 3 represent subclones run under reducing and non-reducing conditions, respectively). [Figure 4] PDB predicted structures of antibodies #A, #B, and #D (4A, 4B, and 4D, respectively). For antibody #A, the PDB structure code 2XKN was used in the homology query, and for antibodies B# and #D, the codes 5OPY and 1F3D were used, respectively. [Figure 5] Sensorgram of saturated antibody #A and competing antibodies #B and #D. Antibodies #B and #D add signal to #A, indicating that these antibodies do not compete for binding within the same epitope region. [Figure 6] Sensorgram of saturated antibody #B and competing antibodies #A and #D. Antibodies #A and #D add signal to #B, indicating that these antibodies do not compete for binding within the same epitope region. [Figure 7] Sensorgram of saturated antibody #D and competing antibodies #A and #B. Antibodies #A and #B add signal to #D, indicating that these antibodies do not compete for binding within the same epitope region. [Figure 8] Sensorgram of the association of antibodies #A, #B, and #D to HIV-1 p24 in the absence of competing antibodies, with each antibody achieving its full binding signal (experimental control). [Figure 9] Binding kinetics of antibodies #A, #B, and #D and commercial mAb #1 to the antigen HIV-1 p24 calculated by biolayer interferometry (BLI). Sensorgrams were run over a concentration gradient from 0.1 to 33 nM and fitted with a 1:1 binding model to calculate the association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD). [Figure 10] Binding of antibodies #A, #B, and #D and commercial mAb #2 to HIV-1 p24 capsid protein by indirect ELISA. Titration curves for each antibody start at a concentration of 2 μg / mL and are then diluted 1:10 (left). Signal-to-noise data at an antibody concentration of 200 ng / mL are shown on the right, demonstrating the poor performance of commercial mAb #2 compared to antibodies #A, #B, and #D. DETAILED DESCRIPTION OF THE INVENTION

[0033] The following description is intended merely to illustrate various embodiments of the present disclosure. As such, the specific modifications discussed are not intended to be limiting. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the spirit or scope of the subject matter presented herein, and it is understood that such equivalent embodiments are to be included herein.

[0034] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0035] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer, or group of elements or integers, but not the exclusion of any other element or integer, or group of elements or integers.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below; however, methods and materials similar or equivalent to those described herein can also be used and will be apparent to those skilled in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0037] Each embodiment in this specification may be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.

[0038] The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0039] As used herein, the term "nucleic acid" refers to any material composed of DNA or RNA. Nucleic acids can be produced synthetically or by living cells.

[0040] As used herein, a "nucleotide" is a nucleic acid subunit consisting of a phosphate group, a five-carbon sugar, and a nitrogenous base. The five-carbon sugar found in RNA is ribose. In DNA, the five-carbon sugar is 2'-deoxyribose. The term also includes analogs of such subunits.

[0041] As used herein, the term "polynucleotide" refers to a polymeric chain of nucleotides. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-natural internucleoside linkages, or both. Nucleic acids can have any topological structure. By way of example, nucleic acids can be single-stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular, or padlock-shaped.

[0042] As used herein, the term "protein" refers to a large biological molecule or macromolecule composed of one or more chains of amino acid residues. Many proteins are enzymes that catalyze biochemical reactions and are crucial to metabolism. Proteins also have structural or mechanical functions, such as actin and myosin in muscles and proteins in the cytoskeleton, which form the scaffolding system that maintains the shape of cells. Other proteins are important in cell signaling, immune response, cell adhesion, and the cell cycle. However, proteins may also be entirely artificial or recombinant, i.e., not naturally occurring in living systems.

[0043] As used herein, the term "polypeptide" refers to both naturally occurring and non-naturally occurring proteins, as well as fragments, variants, derivatives, and analogs thereof. A polypeptide can be a monomer or a polymer. A polypeptide can contain several different domains (peptides), each of which has one or more distinct activities.

[0044] As used herein, the term "recombinant" refers to a biomolecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) whose gene is not associated with all or a portion of a polynucleotide found in nature, (3) is operably linked to a polynucleotide with which it is not naturally linked, or (4) is not found in nature. The term "recombinant" can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs synthesized in vivo by heterologous systems, as well as proteins and / or mRNAs encoded by such nucleic acids.

[0045] As used herein, the term "fusion protein" refers to a protein containing two or more amino acid sequences that do not coexist in a naturally occurring protein. A fusion protein may contain two or more amino acid sequences from the same or different organisms. The two or more amino acid sequences of a fusion protein are typically in-frame, do not contain a stop codon between them, and are typically translated from mRNA as part of the fusion protein.

[0046] When referring to a protein according to (3), the terms "fusion protein" and "recombinant" may be used interchangeably herein.

[0047] As used herein, the terms "antibody" or "immunoglobulin" have the same meaning and are used equivalently in the present invention. As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that specifically binds to an antigen. Thus, the term antibody encompasses not only whole antibody molecules, but also antibody fragments or derivatives.

[0048] In natural antibodies, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinct sequence domains. Light chains contain two domains: a variable domain (VL) and a constant domain (CL). Heavy chains contain four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine binding recognition and specificity to antigens. The constant region domains of the light chain (CL) and heavy chain (CH) confer important biological properties, such as antibody chain assembly, secretion, transplacental mobility, complement fixation, and Fc receptor (FcR) binding. An Fv fragment is the N-terminal portion of an immunoglobulin Fab fragment and consists of one light chain variable portion and one heavy chain variable portion. Antibody specificity resides in the structural complementarity between the antibody-binding site and an antigenic determinant. An antibody-binding site is composed of residues primarily from hypervariable or complementarity-determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FRs) contribute to the overall domain structure and, therefore, the binding site. Complementarity-determining regions or CDRs refer to amino acid sequences that collectively define the binding affinity and specificity of the native Fv region of a native immunoglobulin-binding site. Each immunoglobulin light and heavy chain has three CDRs, designated L-CDR1, L-CDR2, and L-CDR3, and H-CDR1, H-CDR2, and H-CDR3, respectively. Thus, an antigen-binding site typically contains six CDRs, including a set of CDRs from each of the heavy and light chain V regions. Framework region (FR) refers to the amino acid sequences interposed between the CDRs.

[0049] CDRs can be identified according to the definitions of Kabat, Chothia, both Kabat and Chothia, AbM, contact, IMGT unique numbering, and / or conformational definitions, or any method of CDR determination known in the art. Antibody CDRs can be identified as hypervariable regions as originally defined by Kabat et al. See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington, DC. CDR locations can also be identified as loop structures of the structure originally described by Chothia et al. (See, e.g., Chothia et al., Nature 342:877-883, 1989). Other approaches to CDR identification include the "AbM definition" (a compromise of Kabat and Chothia, derived using Oxford Molecular's AbM antibody modeling software (now Accelrys0)), the "contact definition" of CDRs based on observed antigen contacts (as set forth in MacCallum et al., J. Mol. Biol., 262:732-745, 1996), or "IMGT unique numbering," which relies on the high structural conservation of variable regions (see Lefranc, M.-P. Nucl. Acids Res., 33, D593-D597, 2005). In another approach, referred to herein as the "conformational definition" of CDRs, CDR positions can be identified as residues that generate enthalpic contributions to antigen binding. See, e.g., Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008). Still other CDR boundary definitions do not strictly follow one of the above approaches, but still overlap with at least a portion of the Kabat CDRs, although they may be shortened or extended in light of predictions or experimental findings that particular residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding.As used herein, CDR refers to CDRs defined by any approach known in the art, including a combination of approaches. The methods used herein may utilize CDRs defined according to any of these approaches. For any given embodiment that includes multiple CDRs, the CDRs may be defined according to any of Kabat numbering, Chothia numbering, extended numbering, AbM numbering, contact numbering, IMGT specific numbering, and / or conformational definitions, unless otherwise specified.

[0050] Exemplary databases of antibody sequences are described and can be accessed through the "Abysis" website at www.bioinf.org.uk / abs (maintained by A.C. Martin, Department of Biochemistry & Molecular Biology, University College London, London, England) and the VBASE2 website at www.vbase2.org as described in Retter et al., Nucl. Acids Res., 33 (Database issue): D671-D674 (2005). Preferably, sequences are analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT, and the Protein Data Bank (PDB) with structural data from the PDB. Unless otherwise indicated, all CDRs presented herein have been derived according to the scheme shown in accordance with the Abysis database website.

[0051] As used herein, the term "antibody" includes isolated antibodies, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies, recombinant antibodies, chimeric antibodies, and antibody fragments.

[0052] As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody composition having a homogeneous antibody population that binds to the same epitope. The term is not limited with respect to the species or source of the antibody, nor is it intended to be limited by the method by which it is produced. Thus, the term encompasses antibodies obtained from murine hybridomas, as well as human monoclonal antibodies obtained using human rather than murine hybridomas. The term also encompasses antibodies obtained by other methods for producing monoclonal antibodies known in the art, such as establishment of eukaryotic cell lines by transient or stable transfection.

[0053] As used herein, the term "recombinant antibody" refers to an antibody expressed from a cell or cell line transfected with one or more expression vectors containing antibody coding sequences, wherein the coding sequences are not naturally associated with the cell. Recombinant antibodies or fragments thereof may be prepared, expressed, produced, or isolated by any recombinant means, as known to those of skill in the art.

[0054] In some embodiments, a "recombinant antibody" can also be a "monoclonal antibody" if it is derived from a homogeneous population of antibodies that bind to the same epitope.

[0055] Thus, the term "antibody fragment," as used herein, includes, but is not limited to, variable fragments (Fv), single-chain Fvs (scFv), diabodies (sc(Fv)2), single-chain antibodies, single-domain antibodies, Fab fragments, F(ab')2 fragments, Fab' fragments, disulfide-linked Fvs (dsFv), chemically conjugated Fvs (ccFv), diabodies, and anti-idiotypic (anti-Id) antibodies, as well as functionally active, epitope-binding fragments of any of the above. In certain embodiments, antibodies also include affibodies, nanobodies, and unibodies. In certain embodiments, specific antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgAi, and IgA2) or subclass.

[0056] As used herein, the term "antigen-binding fragment (Fab)" refers to an antibody fragment containing one constant domain and one variable domain of each of the heavy and light chains. The variable domains contain the antigen-binding site. Generally, an antibody contains a fragment crystallizable region (Fc) and two antigen-binding fragments (Fab). The Fab fragment can be separated from the Fc region to generate two Fab fragments, which are also known as F(ab')2 fragments or dimeric fragment antigen binding.

[0057] The term "isolated" refers to a protein (e.g., an antibody) or nucleic acid that is substantially free of other cellular material and / or chemicals. For example, when an isolated antibody is expressed by cells from a different species, e.g., a human antibody expressed in a murine cell will be substantially free of other proteins from the different species. A protein may be rendered substantially free of components associated with it in nature (or components associated with the cellular expression system used to produce the antibody) by isolation using protein purification techniques well known in the art.

[0058] As used herein, the term "antigen" refers to a biological molecule that specifically binds to a respective antibody. Antibodies from a diverse repertoire bind specific antigenic structures through their variable region interactions.

[0059] As used herein, the term "epitope" refers to the portion of an antigen to which an antibody specifically binds. Thus, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor.

[0060] A polypeptide is "immunologically reactive" with an antibody if it binds to the antibody due to antibody recognition of a particular epitope contained within that polypeptide. Immunological reactivity can be determined by antibody binding, more specifically, by the kinetics of antibody binding, and / or by competition in binding using as a competitor a known polypeptide containing the epitope to which the antibody is directed. Techniques for determining whether a polypeptide is immunologically reactive with an antibody are known in the art.

[0061] As used herein, the term "sample" refers to any biological material obtained from a subject or patient. In one embodiment, a sample may include blood, ascites, CSF, saliva, or urine. In other embodiments, a sample may include whole blood, plasma, serum, B cells enriched from a blood sample, and cultured cells (e.g., B cells from a subject). Samples may also include biopsies or tissue samples, including neural tissue. In yet other embodiments, a sample may include whole cells and / or cell lysates.

[0062] The sample may be treated to physically or mechanically disrupt the structure of the tissue or cells, thereby releasing the intracellular components into solution, which may further include enzymes, buffers, salts, detergents, etc., used to prepare the biological sample for analysis using standard methods. Samples may also include processed samples, such as those obtained by applying or passing the sample through a filtration device, or after centrifugation, or by adherence to a medium, matrix, or support.

[0063] The terms "patient" or "individual" are used interchangeably herein and refer to a mammalian subject being diagnosed or treated, with human patients being preferred. Optionally, the methods of the present invention are used in laboratory animals, in veterinary applications, and in the development of animal models of disease, including, but not limited to, rodents, including mice, rats, and hamsters; and primates.

[0064] The term "vector" refers to a nucleic acid that can be used to introduce another nucleic acid linked to it into a cell. One type of vector is a "plasmid," which refers to a linear or circular double-stranded DNA molecule into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), in which additional DNA segments can be introduced into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors containing a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

[0065] An "expression vector" is a type of vector that is capable of directing the expression of a selected polynucleotide. An "expression cell" is a cell that contains an expression vector.

[0066] A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression (e.g., level, timing, or location of expression) of the nucleotide sequence. A "regulatory sequence" is a nucleic acid that affects the expression (e.g., level, timing, or location of expression) of an operably linked nucleic acid. A regulatory sequence can exert its effect, for example, directly on the nucleic acid being regulated or through the action of one or more other molecules (e.g., a polypeptide that binds to the regulatory sequence and / or nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements.

[0067] As used herein, the terms "diagnostic" or "diagnosed" refer to identifying the presence or nature of a pathological condition or a patient predisposed to a disease. Diagnostic methods vary in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (the "percent true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and test negative in the assay are called "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it is sufficient if the method provides a positive indication that aids in diagnosis.

[0068] As used herein, the term "binding affinity" refers to the strength of the interaction between an epitope of an antigen and the antigen-binding site of an antibody.

[0069] The present invention relates to novel antibodies specific for the detection of human immunodeficiency virus 1 (HIV-1) p24 protein. These antibodies recognize a novel, non-cross-reactive epitope on the HIV-1 p24 protein and exhibit a high degree of affinity and sensitivity compared to other commercially available products. Therefore, the antibodies described herein can be utilized as diagnostic reagents, standards, or positive controls in immunoassays for early HIV-1 detection. They can be used to detect any of the three major HIV-1 groups: group M (main lineage), group N (novel), and group O (unclassified).

[0070] The present invention also relates to compositions and kits comprising said anti-HIV-1 antibodies for detecting the presence of HIV-1 in a sample.

[0071] I. Anti-HIV-1 antibodies As used herein, the terms "homology," "similarity," or "identity," in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are identical or have a certain percentage of identical nucleotides or amino acid residues when compared and aligned for maximum correspondence. To determine percent homology / identity, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (e.g., overlapping positions) × 100). In some embodiments, the two sequences being compared are the same length (e.g., excluding additional sequences extending beyond the sequences being compared), after introducing gaps into the sequences, if necessary. For sequence comparison between two sequences, a "corresponding" CDR refers to a CDR in the same location in both sequences (e.g., CDR-H1 in each sequence).

[0072] Determining percent identity, percent similarity, or percent similarity between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm has been incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed using the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleic acid encoding a protein of interest. BLAST protein searches can be performed using the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein of interest. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. Another preferred, non-limiting example of a mathematical algorithm utilized for sequence comparison is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm has been incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0073] In one embodiment described herein, the recombinant antibody comprises a light chain and a heavy chain. In other embodiments described herein, the recombinant antibody comprises two light chains and two heavy chains. The light chain of the recombinant antibody of the present invention may comprise two domains: a variable domain (VL) and a constant domain (CL). The heavy chain of the recombinant antibody of the present invention may comprise four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH).

[0074] In some embodiments, the anti-HIV-1 antibody of the present invention is a monoclonal antibody. In other embodiments, the anti-HIV-1 antibody of the present invention is a recombinant antibody. In other embodiments, the anti-HIV-1 antibody is a recombinant monoclonal antibody according to the definition of the present invention. In other embodiments, the anti-HIV-1 antibody is an isolated antibody.

[0075] In some embodiments, the anti-HIV-1 antibody is an antibody fragment. In preferred embodiments, the antibody fragment is selected from a variable fragment (Fv), a single-chain Fv (scFv), a bispecific antibody (sc(Fv)2), a single-chain antibody, a single-domain antibody, a Fab fragment, a F(ab')2 fragment, a Fab' fragment, a disulfide-linked Fv (dsFv), a chemically conjugated Fv (ccFv), a diabody, an anti-idiotypic (anti-Id) antibody, an affibody, a nanobody, and a unibody.

[0076] In one embodiment described herein, the anti-HIV-1 antibody comprises an Fc region and two Fab fragments. In another embodiment described herein, the anti-HIV-1 antibody is an antigen-binding fragment and does not comprise an Fc region. In another embodiment described herein, the anti-HIV-1 antibody consists of one Fab fragment. In another embodiment described herein, the anti-HIV-1 antibody consists of two Fab fragments (F(ab)2).

[0077] In one embodiment described herein, the anti-HIV-1 antibody can be of any type known to those skilled in the art (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or of any class known to those skilled in the art (e.g., IgG1, IgG2, IgG3, IgG4, IgAi, and IgA2), or any known subclass.

[0078] In one embodiment described herein, the anti-HIV-1 antibody is of the IgG type. In a preferred embodiment, the anti-HIV-1 antibody is of the IgG1, IgG2, IgG3, or IgG4 class. In another preferred embodiment, the anti-HIV-1 antibody is of the IgG1 or IgG2 class. In another preferred embodiment, the anti-HIV-1 antibody is of the IgG2a class.

[0079] The species of the constant region of the antibody of the present invention may be human, mouse, rabbit, rat, hamster, guinea pig, goat, sheep, horse, chicken, or a chimera of any of the above species, but the species of the antibody of the present invention is not particularly limited. In some preferred embodiments, the anti-HIV antibody of the present invention comprises a constant region of mouse IgG1 class or mouse IgG2a class.

[0080] A. Light Chain In some embodiments described herein, the anti-HIV-1 antibodies comprise a light chain comprising complementarity-determining regions (CDRs). The CDRs correspond to sequences identified according to any CDR definition approach known to those of skill in the art. In some preferred embodiments, the CDR regions correspond to sequences identified according to the Kabat numbering scheme. In other preferred embodiments, the CDR regions may correspond to sequences identified according to other numbering methods or a combination of Kabat and other numbering methods. For example, the CDR regions may correspond to sequences identified according to the Chothia numbering scheme.

[0081] In some embodiments described herein, the anti-HIV-1 antibodies comprise a light chain comprising complementarity determining regions L-CDR1, L-CDR2, and L-CDR3, each of which comprises a sequence of at least five consecutive amino acids selected from the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. In some preferred embodiments, the sequence of L-CDR1 is selected from the group consisting of SEQ ID NO:15, SEQ ID NO:18, and SEQ ID NO:21. In some preferred embodiments, the sequence of L-CDR2 is selected from the group consisting of SEQ ID NO:16, SEQ ID NO:19, and SEQ ID NO:22. In some preferred embodiments, the sequence of L-CDR3 is selected from the group consisting of SEQ ID NO:17, SEQ ID NO:20, and SEQ ID NO:23. In other preferred embodiments, the sequence of L-CDR1 is selected from the group consisting of SEQ ID NO:15, SEQ ID NO:18, and SEQ ID NO:21, the sequence of L-CDR2 is selected from the group consisting of SEQ ID NO:16, SEQ ID NO:19, and SEQ ID NO:22, and the sequence of L-CDR3 is selected from the group consisting of SEQ ID NO:17, SEQ ID NO:20, and SEQ ID NO:23.

[0082] In another embodiment described herein, the variable region of the light chain of the anti-HIV-1 antibody of the invention comprises the amino acid sequence of SEQ ID NO:7, or SEQ ID NO:8, or SEQ ID NO:9. In yet another embodiment, the variable region of the light chain of the recombinant antibody may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence consisting of SEQ ID NO:7, or SEQ ID NO:8, or SEQ ID NO:9. In some preferred embodiments, the light chain of the anti-HIV-1 antibody of the invention comprises a sequence having about 90% homology to the amino acid sequence of SEQ ID NO:7, or SEQ ID NO:8, or SEQ ID NO:9.

[0083] In another embodiment described herein, the recombinant antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In other embodiments, the light chain of the recombinant antibody may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence consisting of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In some preferred embodiments, the light chain of the anti-HIV-1 antibody of the invention comprises a sequence having about 90% homology to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0084] B. Heavy Chain In some embodiments described herein, the anti-HIV-1 antibodies comprise a heavy chain comprising complementarity-determining regions (CDRs). The CDRs correspond to sequences identified according to any CDR definition approach known to those skilled in the art. In some preferred embodiments, the CDR regions correspond to sequences identified according to the Kabat numbering scheme. In other preferred embodiments, the CDR regions may correspond to sequences identified according to other numbering methods or a combination of Kabat and other numbering methods. For example, the CDR regions may correspond to sequences identified according to the Chothia numbering scheme.

[0085] In some embodiments described herein, the anti-HIV-1 antibodies comprise a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2, and H-CDR3, each of which comprises a sequence of at least five consecutive amino acids selected from the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some preferred embodiments, the sequence of H-CDR1 is selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 27, and SEQ ID NO: 30. In some preferred embodiments, the sequence of H-CDR2 is selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 28, and SEQ ID NO: 31. In some preferred embodiments, the sequence of H-CDR3 is selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 29, and SEQ ID NO: 32. In some preferred embodiments, the sequence of H-CDR1 is selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 27, and SEQ ID NO: 30, the sequence of H-CDR2 is selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 28, and SEQ ID NO: 31, and the sequence of H-CDR3 is selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 29, and SEQ ID NO: 32.

[0086] In another embodiment described herein, the variable region of the heavy chain of the anti-HIV-1 antibody of the invention comprises the amino acid sequence of SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12. In yet another embodiment, the variable region of the heavy chain of the recombinant antibody may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence of SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12. In some preferred embodiments, the heavy chain of the anti-HIV-1 antibody of the invention comprises a sequence having about 90% homology to the amino acid sequence of SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12.

[0087] In another embodiment described herein, the recombinant antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. In other embodiments, the heavy chain of the recombinant antibody may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence consisting of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. In some preferred embodiments, the heavy chain of the anti-HIV-1 antibody of the invention comprises a sequence having about 90% homology to the amino acid sequence of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0088] C. Exemplary Anti-HIV-1 Antibodies In one embodiment described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2 and L-CDR3, wherein the amino acid sequence of L-CDR1 is SEQ ID NO: 15, the amino acid sequence of L-CDR2 is SEQ ID NO: 16 and the amino acid sequence of L-CDR3 is SEQ ID NO: 17.

[0089] In other embodiments described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2, and L-CDR3, wherein the amino acid sequence of L-CDR1 is SEQ ID NO: 18, the amino acid sequence of L-CDR2 is SEQ ID NO: 19, and the amino acid sequence of L-CDR3 is SEQ ID NO: 20.

[0090] In other embodiments described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2, and L-CDR3, wherein the amino acid sequence of L-CDR1 is SEQ ID NO: 21, the amino acid sequence of L-CDR2 is SEQ ID NO: 22, and the amino acid sequence of L-CDR3 is SEQ ID NO: 23.

[0091] In one embodiment described herein, the anti-HIV-1 antibody comprises a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2 and H-CDR3, wherein the amino acid sequence of H-CDR1 is SEQ ID NO: 24, the amino acid sequence of H-CDR2 is SEQ ID NO: 25 and the amino acid sequence of H-CDR3 is SEQ ID NO: 26.

[0092] In other embodiments described herein, the anti-HIV-1 antibody comprises a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2 and H-CDR3, wherein the amino acid sequence of H-CDR1 is SEQ ID NO: 27, the amino acid sequence of H-CDR2 is SEQ ID NO: 28 and the amino acid sequence of H-CDR3 is SEQ ID NO: 29.

[0093] In other embodiments described herein, the anti-HIV-1 antibody comprises a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2 and H-CDR3, wherein the amino acid sequence of H-CDR1 is SEQ ID NO: 30, the amino acid sequence of H-CDR2 is SEQ ID NO: 31 and the amino acid sequence of H-CDR3 is SEQ ID NO: 32.

[0094] The anti-HIV-1 antibodies of the present invention may comprise any combination of the CDR regions of both the light and heavy chains described herein.

[0095] In a preferred embodiment described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2, and L-CDR3, wherein the amino acid sequence of L-CDR1 is SEQ ID NO: 15, the amino acid sequence of L-CDR2 is SEQ ID NO: 16, and the amino acid sequence of L-CDR3 is SEQ ID NO: 17, and a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2, and H-CDR3, wherein the amino acid sequence of H-CDR1 is SEQ ID NO: 24, the amino acid sequence of H-CDR2 is SEQ ID NO: 25, and the amino acid sequence of H-CDR3 is SEQ ID NO: 26.

[0096] In a preferred embodiment described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2, and L-CDR3, wherein the amino acid sequence of L-CDR1 is SEQ ID NO: 15, the amino acid sequence of L-CDR2 is SEQ ID NO: 16, and the amino acid sequence of L-CDR3 is SEQ ID NO: 17, and a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2, and H-CDR3, wherein the amino acid sequence of H-CDR1 is SEQ ID NO: 27, the amino acid sequence of H-CDR2 is SEQ ID NO: 28, and the amino acid sequence of H-CDR3 is SEQ ID NO: 29.

[0097] In a preferred embodiment described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2, and L-CDR3, wherein the amino acid sequence of L-CDR1 is SEQ ID NO: 15, the amino acid sequence of L-CDR2 is SEQ ID NO: 16, and the amino acid sequence of L-CDR3 is SEQ ID NO: 17, and a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2, and H-CDR3, wherein the amino acid sequence of H-CDR1 is SEQ ID NO: 30, the amino acid sequence of H-CDR2 is SEQ ID NO: 31, and the amino acid sequence of H-CDR3 is SEQ ID NO: 32.

[0098] In a preferred embodiment described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2, and L-CDR3, wherein the amino acid sequence of L-CDR1 is SEQ ID NO: 18, the amino acid sequence of L-CDR2 is SEQ ID NO: 19, and the amino acid sequence of L-CDR3 is SEQ ID NO: 20, and a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2, and H-CDR3, wherein the amino acid sequence of H-CDR1 is SEQ ID NO: 24, the amino acid sequence of H-CDR2 is SEQ ID NO: 25, and the amino acid sequence of H-CDR3 is SEQ ID NO: 26.

[0099] In a preferred embodiment described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2 and L-CDR3, wherein the amino acid sequence of L-CDR1 is SEQ ID NO: 18, the amino acid sequence of L-CDR2 is SEQ ID NO: 19 and the amino acid sequence of L-CDR3 is SEQ ID NO: 20, and a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2 and H-CDR3, wherein the amino acid sequence of H-CDR1 is SEQ ID NO: 27, the amino acid sequence of H-CDR2 is SEQ ID NO: 28 and the amino acid sequence of H-CDR3 is SEQ ID NO: 29.

[0100] In a preferred embodiment described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2, and L-CDR3, wherein the amino acid sequence of L-CDR1 is SEQ ID NO: 18, the amino acid sequence of L-CDR2 is SEQ ID NO: 19, and the amino acid sequence of L-CDR3 is SEQ ID NO: 20, and a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2, and H-CDR3, wherein the amino acid sequence of H-CDR1 is SEQ ID NO: 30, the amino acid sequence of H-CDR2 is SEQ ID NO: 31, and the amino acid sequence of H-CDR3 is SEQ ID NO: 32.

[0101] In a preferred embodiment described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2, and L-CDR3, wherein the amino acid sequence of L-CDR1 is SEQ ID NO: 21, the amino acid sequence of L-CDR2 is SEQ ID NO: 22, and the amino acid sequence of L-CDR3 is SEQ ID NO: 23, and a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2, and H-CDR3, wherein the amino acid sequence of H-CDR1 is SEQ ID NO: 24, the amino acid sequence of H-CDR2 is SEQ ID NO: 25, and the amino acid sequence of H-CDR3 is SEQ ID NO: 26.

[0102] In a preferred embodiment described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2, and L-CDR3, wherein the amino acid sequence of L-CDR1 is SEQ ID NO: 21, the amino acid sequence of L-CDR2 is SEQ ID NO: 22, and the amino acid sequence of L-CDR3 is SEQ ID NO: 23, and a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2, and H-CDR3, wherein the amino acid sequence of H-CDR1 is SEQ ID NO: 27, the amino acid sequence of H-CDR2 is SEQ ID NO: 28, and the amino acid sequence of H-CDR3 is SEQ ID NO: 29.

[0103] In a preferred embodiment described herein, the anti-HIV-1 antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2 and L-CDR3, wherein the amino acid sequence of L-CDR1 is SEQ ID NO: 21, the amino acid sequence of L-CDR2 is SEQ ID NO: 22 and the amino acid sequence of L-CDR3 is SEQ ID NO: 23, and a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2 and H-CDR3, wherein the amino acid sequence of H-CDR1 is SEQ ID NO: 30, the amino acid sequence of H-CDR2 is SEQ ID NO: 31 and the amino acid sequence of H-CDR3 is SEQ ID NO: 32.

[0104] In one embodiment described herein, the anti-HIV-1 antibody comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, and a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

[0105] In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:7 and a heavy chain comprising the amino acid sequence of SEQ ID NO:10.

[0106] In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:7 and a heavy chain comprising the amino acid sequence of SEQ ID NO:11.

[0107] In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:7 and a heavy chain comprising the amino acid sequence of SEQ ID NO:12.

[0108] In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:8 and a heavy chain comprising the amino acid sequence of SEQ ID NO:10.

[0109] In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:8 and a heavy chain comprising the amino acid sequence of SEQ ID NO:11.

[0110] In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:8 and a heavy chain comprising the amino acid sequence of SEQ ID NO:12.

[0111] In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:9 and a heavy chain comprising the amino acid sequence of SEQ ID NO:10.

[0112] In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:9 and a heavy chain comprising the amino acid sequence of SEQ ID NO:11.

[0113] In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:9 and a heavy chain comprising the amino acid sequence of SEQ ID NO:12.

[0114] In other preferred embodiments, the light chain of the anti-HIV-1 antibody may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence consisting of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, and the heavy chain of the anti-HIV-1 antibody may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence consisting of SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

[0115] In one embodiment described herein, the anti-HIV-1 antibody comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0116] In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain comprising the amino acid sequence of SEQ ID NO:4.

[0117] In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain comprising the amino acid sequence of SEQ ID NO:5.

[0118] In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain comprising the amino acid sequence of SEQ ID NO:6.

[0119] In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:2 and a heavy chain comprising the amino acid sequence of SEQ ID NO:4.

[0120] In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:2 and a heavy chain comprising the amino acid sequence of SEQ ID NO:5.

[0121] In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:2 and a heavy chain comprising the amino acid sequence of SEQ ID NO:6.

[0122] In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:3 and a heavy chain comprising the amino acid sequence of SEQ ID NO:4.

[0123] In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:3 and a heavy chain comprising the amino acid sequence of SEQ ID NO:5.

[0124] In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:3 and a heavy chain comprising the amino acid sequence of SEQ ID NO:6.

[0125] In other preferred embodiments, the light chain of the anti-HIV-1 antibody may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence consisting of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and the heavy chain of the anti-HIV-1 antibody may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence consisting of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0126] In some preferred embodiments, the anti-HIV-1 antibodies of the present invention specifically bind to HIV-1 p24 protein. In some embodiments, the anti-HIV-1 antibodies of the present invention bind to an epitope of the HIV-1 p24 protein. In some preferred embodiments, the anti-HIV-1 antibodies of the present invention bind to a linear epitope of the HIV-1 p24 protein. In some preferred embodiments, the anti-HIV-1 antibodies of the present invention bind to a linear epitope comprising at least 5 consecutive amino acids selected from the amino acid sequence of the HIV-1 p24 protein (SEQ ID NO: 35) or a sequence having at least 90% homology thereto. In other embodiments, the amino acid sequence of the HIV-1 p24 protein is set forth in SEQ ID NO: 36.

[0127] In another preferred embodiment, the anti-HIV-1 antibody of the present invention binds to an epitope of the HIV-1 p24 protein characterized by comprising the amino acid sequence of SEQ ID NO:33.

[0128] In a more preferred embodiment, the anti-HIV-1 antibody of the present invention is an HIV-1 antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 33. The antibody binds to an epitope of p24 protein, and the antibody comprises a light chain comprising complementarity determining regions L-CDR1, L-CDR2, and L-CDR3, wherein the amino acid sequence of L-CDR1 is selected from the group consisting of SEQ ID NO:15, SEQ ID NO:18, and SEQ ID NO:21, the amino acid sequence of L-CDR2 is selected from the group consisting of SEQ ID NO:16, SEQ ID NO:19, and SEQ ID NO:22, and the amino acid sequence of L-CDR3 is selected from the group consisting of SEQ ID NO:17, SEQ ID NO:20, and SEQ ID NO:23. The antibody further comprises a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2, and H-CDR3, wherein the amino acid sequence of H-CDR1 is selected from the group consisting of SEQ ID NO:24, SEQ ID NO:27, and SEQ ID NO:30, the amino acid sequence of H-CDR2 is selected from the group consisting of SEQ ID NO:25, SEQ ID NO:28, and SEQ ID NO:31, and the amino acid sequence of H-CDR3 is selected from the group consisting of SEQ ID NO:26, SEQ ID NO:29, and SEQ ID NO:32.

[0129] In some preferred embodiments, the anti-HIV-1 antibody of the present invention binds to an epitope of HIV-1 p24 protein comprising the amino acid sequence of SEQ ID NO: 33, and the antibody comprises a light chain comprising complementarity-determining regions L-CDR1, L-CDR2, and L-CDR3, wherein the amino acid sequence of L-CDR1 is SEQ ID NO: 18, the amino acid sequence of L-CDR2 is SEQ ID NO: 19, and the amino acid sequence of L-CDR3 is SEQ ID NO: 20, and further comprises a heavy chain comprising complementarity-determining regions H-CDR1, H-CDR2, and H-CDR3, wherein the amino acid sequence of H-CDR1 is SEQ ID NO: 27, the amino acid sequence of H-CDR2 is SEQ ID NO: 28, and the amino acid sequence of H-CDR3 is SEQ ID NO: 29.

[0130] In some embodiments, the anti-HIV-1 antibodies of the invention are bound to a solid support.

[0131] D. Affinity Tags The anti-HIV-1 antibody of the present invention may comprise an affinity tag. Affinity tags are useful for purification. Exemplary affinity tags include polyhistidine, glutathione S-transferase (GST), chitin-binding protein, maltose-binding protein (MBP), streptavidin-binding peptide (Strep tag), isopeptide bond formation, FLAG tag, V5 tag, Myc tag, HA tag, NE tag, AviTag, calmodulin tag, polyglutamic acid, S tag, SBP tag, Softag 1, Softag 3, TC tag, VSV tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, biotin carboxyl carrier protein, green fluorescent protein tag, HaloTag, Nus tag, and thioredoxin tag, but the choice of affinity tag is not particularly limited. However, the anti-HIV-1 antibody may lack an affinity tag, for example, if the affinity tag is removed after use or if the anti-HIV-1 antibody is purified using a strategy that does not require an affinity tag. An exemplary affinity tag is polyhistidine, which typically comprises an amino acid sequence containing between 4 and 10 consecutive histidines.

[0132] The anti-HIV-1 antibodies of the present invention may comprise an affinity tag and may be purified using said affinity tag. Several methods for purifying anti-HIV-1 antibodies are available in the state of the art and are well known to those skilled in the art. Exemplary methods of purification for anti-HIV-1 antibodies, with or without an affinity tag, are immobilized metal affinity chromatography (IMAC), protein A / G affinity, exchange chromatography (IEX or IEC), hydrophobic interaction chromatography (HIC), and / or the additional use of tag and affinity chromatography techniques beyond IMAC or protein A / G. The purification methods and tags employed should not be considered limiting.

[0133] II. Nucleic Acids, Cloning, and Expression Cells The present invention also relates to a nucleic acid comprising a nucleotide sequence encoding an anti-HIV-1 antibody described herein. The nucleic acid may be isolated. The nucleic acid may be DNA or RNA. DNA comprising a nucleotide sequence encoding an anti-HIV-1 antibody described herein typically comprises a promoter operably linked to the nucleotide sequence. The promoter is preferably capable of driving constitutive or inducible expression of the nucleotide sequence in a target expression cell. The nucleic acid may also comprise a selectable marker useful for selecting cells containing the target nucleic acid. Useful selectable markers are well known to those skilled in the art. The exact nucleotide sequence of the nucleic acid is not particularly limited, as long as the nucleotide sequence encodes an anti-HIV-1 antibody described herein. Codons may be selected, for example, to match the codon bias of the target expression cell (e.g., a mammalian cell such as a human cell) and / or for convenience during cloning. The DNA may comprise an origin of replication (e.g., for replication of a plasmid in a prokaryotic cell).

[0134] In one embodiment described herein, the nucleic acid comprises a nucleotide sequence encoding an anti-HIV-1 antibody of the invention, a promoter operably linked to the nucleotide sequence, and a selectable marker.

[0135] In some preferred embodiments, the nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42. In more preferred embodiments, the nucleic acid of the light chain of the anti-HIV-1 antibody of the invention comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 39, and SEQ ID NO: 41, and the nucleic acid of the heavy chain of the anti-HIV-1 antibody of the invention comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 38, SEQ ID NO: 40, and SEQ ID NO: 42.

[0136] In some embodiments, the light and heavy chain nucleic acids of the anti-HIV-1 antibody of the invention comprise the nucleotide sequence of SEQ ID NO: 37 and the nucleotide sequence of SEQ ID NO: 38, respectively. In other embodiments, the light and heavy chain nucleic acids of the anti-HIV-1 antibody of the invention comprise the nucleotide sequence of SEQ ID NO: 39 and the nucleotide sequence of SEQ ID NO: 40, respectively. In other embodiments, the light and heavy chain nucleic acids of the anti-HIV-1 antibody of the invention comprise the nucleotide sequence of SEQ ID NO: 41 and the nucleotide sequence of SEQ ID NO: 42, respectively.

[0137] Various aspects of the present invention also relate to cells containing nucleic acids comprising nucleotide sequences encoding anti-HIV-1 antibodies as described herein. The cells can be expression cells or cloning cells. Nucleic acids are typically cloned in E. coli, although other cloning cells may be used.

[0138] If the cell is an expression cell, the nucleic acid may be a chromosomal nucleic acid, i.e., where the nucleotide sequence is integrated into the chromosome, although the nucleic acid may then be present in the expression cell, for example, as extrachromosomal DNA or as a vector, e.g., a plasmid, cosmid, phage, etc. The type of vector should not be considered limiting.

[0139] In one embodiment described herein, the cell is typically an expression cell. The nature of the expression cell is not particularly limiting. Mammalian expression cells may allow for convenient folding, post-translational modification, and / or secretion of the recombinant antibody or oligomeric recombinant antibody, although other eukaryotic or prokaryotic cells may also be used as expression cells. Exemplary expression cells include CHO cell lines such as TunaCHO or ExpiCHO, Expi293, BHK, NS0, Sp2 / 0, COS, C127, HEK, HT-1080, PER.C6, HeLa, and Jurkat cells. Cells may also be selected for integration of vectors, more preferably for integration of plasmid DNA.

[0140] The anti-HIV-1 antibodies of the present invention can be produced by appropriate transfection strategies of a nucleic acid containing a nucleotide sequence encoding the anti-HIV-1 antibody into mammalian cells. Those skilled in the art are aware of the various techniques (lipofection, electroporation, etc.) available for transfecting a nucleic acid into a cell line of choice. Therefore, the choice of mammalian cell line and transfection strategy should not be considered limiting. The cell line can further be selected for integration of the plasmid DNA.

[0141] In one preferred embodiment described herein, the cells comprise an anti-HIV-1 antibody of the invention.

[0142] III. Compositions and Kits Various aspects of the present invention relate to compositions comprising anti-HIV-1 antibodies as described herein.

[0143] In one embodiment described herein, a composition comprises an anti-HIV-1 antibody of the invention and a solid support.

[0144] In another embodiment, a composition comprises an anti-HIV-1 antibody of the present invention and a solid support, wherein the anti-HIV-1 antibody is covalently or non-covalently bound to the solid support. As used herein, the term "non-covalently bound" refers to a specific bond, such as between an antibody and its antigen, a ligand and its receptor, or an enzyme and its substrate, exemplified by the interaction between a streptavidin-binding protein and streptavidin or between an antibody and its antigen.

[0145] In another embodiment, a composition comprises an anti-HIV-1 antibody of the present invention and a solid support, wherein the anti-HIV-1 antibody is directly or indirectly bound to the solid support. As used herein, the term "direct" binding refers to direct conjugation of a molecule to the solid support, e.g., a gold-thiol interaction that binds a cysteine ​​thiol of the anti-HIV-1 antibody to a gold surface. As used herein, the term "indirect" binding includes specific binding of an anti-HIV-1 antibody to another molecule that is directly bound to the solid support, e.g., an anti-HIV-1 antibody may bind an antibody that is directly bound to the solid support, thereby indirectly binding the anti-HIV-1 antibody to the solid support. The term "indirect" binding is independent of the number of molecules between the anti-HIV-1 antibody and the solid support, as long as (a) each interaction between the daisy chain of molecules is a specific or covalent interaction, and (b) the terminal molecule of the daisy chain is directly bound to the solid support.

[0146] The solid support may comprise a particle, bead, membrane, surface, polypeptide chip, microtiter plate, or the solid phase of a chromatography column. Preferably, the solid support may be a latex bead.

[0147] The composition may comprise a plurality of beads or particles, each bead or particle of said plurality of beads or particles being directly or indirectly bound to at least one anti-HIV-1 antibody as described herein.The composition may comprise a plurality of beads or particles, each bead or particle of said plurality of beads or particles being covalently or non-covalently bound to at least one anti-HIV-1 antibody as described herein.

[0148] Various aspects of the embodiments relate to kits for detecting the presence of HIV-1 in a sample, the kits comprising at least one anti-HIV-1 antibody and a solid support or composition described herein, in some embodiments, the at least one antibody is covalently or non-covalently bound to the solid support.

[0149] The anti-HIV-1 antibodies, compositions, and kits described herein can be for use in assays, for example, but not limited to, for detecting the presence of HIV-1 in a sample or for measuring the concentration of HIV-1 in a sample. The anti-HIV-1 antibodies, compositions, and kits of the present invention can also be used for the detection of HIV-1 alone or in combination with other antibodies for the detection of other pathogens, such as in multiplex assays and methods.

[0150] In some preferred embodiments, the anti-HIV-1 antibodies of the present invention are used in methods and assays in which other RNA viruses are also detected. In other embodiments, the anti-HIV-1 antibodies and other anti-HIV-2 antibodies are used in methods and assays for the simultaneous detection of HIV-1 and HIV-2 in a sample. In more preferred embodiments, the anti-HIV-1 antibodies and other anti-HIV-2 antibodies are used in methods and assays for the specific detection of HIV-1 p24 protein and HIV-2 p26 protein in a sample.

[0151] It is also contemplated within the scope of the present invention to use multiple anti-HIV-1 antibodies described herein in methods and assays for detecting HIV-1 in a sample.

[0152] The present invention will now be described in more detail with reference to illustrative examples, which do not constitute a limitation of the present invention. [Example]

[0153] Example 1 Generation of preferred anti-HIV antibodies and stable cell lines As disclosed herein, specific combinations of light and heavy chains of the present invention have resulted in preferred antibodies:

[0154] [Table 1]

[0155] The variable and constant regions of each antibody were cloned into a bicistronic vector and expressed in Chinese hamster ovary (CHO) cells. The manufacturing characteristics of each antibody were assessed based on their ability to generate respective stable cell line clones and reproducible expression and purification of functional antibody.

[0156] Pool Development Transfection: Expression of three constructs containing the nucleotide sequences of antibodies #A, #B, and #D (SEQ ID NOs: 37-42) was generated in a bicistronic expression vector containing the heavy and light chains of each antibody. To express the antibodies, 200 μg of DNA was electroporated into CHO cells to generate stable cell lines. After 24 hours, transfected cells were counted and placed under selective medium for stable integration of the protein gene.

[0157] Pool generation: Transfected cells were diluted to 0.5 x 10 6 Cells were seeded at a cell density of 1000 cells / mL in selective medium in 250 mL shaker flasks with a working volume of 50 mL and incubated at 37°C and 5% CO2. During the selection process, cells were spun down and resuspended in fresh selective medium every 2-3 days until the pool regained its growth rate and viability. Cell cultures were monitored for growth via viable cell density (VCD), percent viability, and titer.

[0158] Production Pools: A 1-liter production run was performed on stable pools to assess VCD, titer, and viability. Cells were scaled up to production medium in 3 L shake flasks (1 L working volume). Conditioned medium supernatants from each stable pool production run were clarified by centrifugation, and proteins were purified by affinity purification using a Protein A column (Tables 2-4).

[0159] Cell line banking: 2.5 x 10 cells per mL 6The cells were expanded to 15 × 10 cells per vial. At the time of harvest for cell banking, viability was greater than 95%. Cells were then centrifuged, and the cell pellet was resuspended in CHO complete medium containing 7.5% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, D1435) and diluted to 15 × 10 cells per vial. 6 The cell count was expressed as cells / mL. A total of five vials of each pool were prepared and cryopreserved for storage in liquid nitrogen.

[0160] [Table 2]

[0161] [Table 3]

[0162] [Table 4]

[0163] Stable antibody generation Starting from the best banked pooled cell lines of antibodies #A, #B, and #D, stable clones were obtained by single-cell cloning. The best clones for each antibody were selected based on expression levels and bioanalytical characterization of purified material from the manufacturing process for antibodies #A, #B, and #D. Bioanalytical characterization included SE-UPLC and SDS-PAGE (Figures 1-3).

[0164] Example 2 Antibody modeling and evaluation Three-dimensional structural models of antibodies #A, #B, and #D were constructed by antibody homology using the computational modeling software Bioiluminate (Schrodinger, Inc., version 3.5). Briefly, the amino acid sequences of the VH and VL regions of antibodies #A, #B, and #D were loaded into Bioiluminate. Framework regions and CDRs were identified by searching antibody structures in the Protein Data Bank (PDB) and selecting PDB templates based on high sequence similarity and structural compatibility (Table 5). The predicted CDR sequences for each antibody of the present invention are shown in Table 5, and the PDB predicted structures of antibodies #A, #B, and #D are shown in Figures 4A, 4B, and 4D, respectively. For antibody #A, the PDB structure code 2XKN was used in the homology query, and for antibodies B# and #D, the codes 5OPY and 1F3D were used, respectively.

[0165] Antibodies #A and #B were found to be of the IgG1k isotype, and antibody #D was found to be of the IgG2ak isotype.

[0166] [Table 5]

[0167] Analysis of the nucleotide sequences of the three antibodies, queried against IgBLAST, an algorithm developed by the National Center for Biotechnology Information (NCBI) that facilitates the analysis of immunoglobulin variable domain sequences against the ImMunoGeneTics Database (IMGT) database, shows that all generated heavy (VH) and light (VL) chains have unique complementarity-determining regions (CDRs) (Lefranc MP, Lefranc G, "IMGT® and 30 years of immunoinformatics insight in antibody V and C domain structure and function." Jefferis R; Strohl WR, Kato K., Antibodies 2019, vol. 8(29); pp. 1-21).

[0168] Example 3 Epitope mapping of mAb D To avoid cleaved peptides, the HIV-p24 sequence was extended at the C- and N-termini with neutral GSGSGSG linkers. The extended antigen sequence was translated into 15-amino acid linear peptides with 14-amino acid peptide-peptide overlaps. The resulting HIV-p24 peptide microarray contained 232 different linear peptides (464 spots) printed in duplicate, framed by additional HA (YPYDVPDYAG, 38 spots) and c-Myc (EQKLISEEDL, 38 spots) control peptides.

[0169] Wash buffer: PBS containing 0.05% Tween 20, pH 7.4; three 10-second washes after each incubation step

[0170] Blocking buffer: Rockland Blocking Buffer MB-070 (30 min before first assay)

[0171] Incubation buffer: Washing buffer containing 10% blocking buffer

[0172] Assay conditions: antibody concentrations of 1 μg / ml, 10 μg / ml, and 100 μg / ml in incubation buffer; incubation for 16 hours at 4°C; shaking at 140 rpm

[0173] Secondary antibody: Goat anti-mouse IgG (H+L) DyLight680 (0.2 μg / ml); staining for 45 min in incubation buffer at RT

[0174] Control antibody: Mouse monoclonal anti-HA (12CA5) DyLight800 (0.5 μg / ml); stained for 45 min in incubation buffer at RT

[0175] Scanner: LI-COR Odyssey Imaging System; scan offset 0.65 mm, resolution 21 μm, scan intensity 7 / 7 (red = 680 nm / green = 800 nm)

[0176] Prestaining of the HIV-p24 peptide microarray was performed with a secondary goat anti-mouse IgG (H+L) DyLight680 antibody in incubation buffer to investigate background interactions with antigen-derived peptides that may interfere with the primary assay. Subsequent incubation of other HIV-p24 peptide microarray copies with monoclonal antibody D at concentrations of 1 μg / ml, 10 μg / ml, and 100 μg / ml in incubation buffer followed by staining with the secondary and control antibodies was performed at a scan intensity of 7 / 7 (red / green). An additional HA peptide forming the peptide microarray was then stained as an internal quality control to verify the quality of the assay and the integrity of the peptide microarray.

[0177] Epitope mapping to HIV-p24 for mAb D and subsequent epitope substitution scanning highlighted a conserved seven amino acid core motif, PIAPGQM (SEQ ID NO: 33).

[0178] Example 4 Epitope binning study Molecular docking and Western blot analysis of antibodies #A, #B, and #D suggested that these antibodies recognize linear epitopes in regions 1, 4, and 7 of the HIV-1 p24 protein, respectively. To further confirm these observations, a tandem epitope binning assay was performed using biolayer interferometry (BLI). A yeast-derived version of the HIV-1 p24 antigen (bt-p24) was biotinylated and loaded onto a streptavidin (SA) biosensor for 300 seconds. The loaded sensor was immersed in a saturating antibody (100 μg / mL) for 600 seconds, followed by a competing antibody (25 μg / mL) for 300 seconds. The results showed that when antibody #A bound to HIV-1 p24, antibodies #B and #D added an increase in the BLI signal response, indicating that antibodies #B and #D bind to different epitopes compared to antibody #A. Similarly, when antibody #A or #D was used as a saturating antibody, the remaining antibodies did not show competition for the same epitope (Figures 9 to 10).

[0179] Table 6 summarizes the epitope binning data for antibodies #A, #B, and #D. Briefly, the BLI signals of competing and saturating antibodies were normalized to buffer. The threshold for determining antibody blocking or binding was set at 0.02 to allow for the recognition of self-blocking pairs on the diagonal of the matrix (gray indicates binding, bold indicates self-blocking). The PEARSON correlation coefficient for the primary antibody #A was calculated using the PEARSON function in Microsoft Excel (Liao-Chan S. et al., "Monoclonal Antibody Binding-Site Diversity Assessment with a Cell-Based Clustering Assay." Journal of Immunological Methods 2014, vol. 405; pp. 1-14). Three distinct bins were identified for antibodies #A, #B, and #D. No antibody blocking was observed.

[0180] [Table 6]

[0181] Example 5 Affinity evaluation of anti-HIV-1 antibodies #A, #B, and #D To investigate the interaction between antibodies #A, #B, and #D and the HIV-1 p24 antigen in more detail, affinity analysis was performed by BLI. Antibodies #A, #B, and #D were compared with a commercially available monoclonal antibody (commercial mAb #1). Antibodies #A, #B, and #D, as well as commercial mAb #1, were captured using a biosensor chip (ForteBio) specifically coated with anti-mouse Fc. A concentration gradient ranging from 0.1 to 33 nM was used for each antibody, and each dilution was prepared in phosphate buffered saline (PBS) containing 0.01% (w / v) bovine serum albumin (BSA) and 0.02% (v / v) surfactant Tween-20. The recorded sensorgrams were fitted using a 1:1 binding model, and the equilibrium constant, KD, was calculated from the ratio of the dissociation rate to the association rate (kd / k). The tested antibodies were ranked based on their calculated affinity constants as follows: Antibody #B ≈ Antibody #D > Antibody #A > Commercial mAb #1. While the exact KD values ​​for antibodies #B and #D could not be calculated due to the long observed dissociation curves, the data presented indicate that the calculated KD values ​​for antibodies #A, #B, and #D are lower than that observed for commercial mAb #1 (Table 7). This data supports the observation that antibodies #A, #B, and #D exhibit higher affinity for HIV-1 p24 than commercial mAb #1.

[0182] [Table 7]

[0183] Example 6 Binding ability of anti-HIV-1 antibodies #A, #B, and #D To further evaluate the binding of antibodies #A, #B, and #D to the HIV-1 p24 antigen, an indirect ELISA assay was performed. A titration curve for each antibody was performed using a starting concentration of 2 μg / mL and a titration of 2 × 10 -2 The antibodies were generated by serial 1:10 dilutions until low concentrations of ng / mL were reached. The performance of each antibody was compared to a commercially available clone (commercial mAb #2) (Figure 10, left). The data show that antibodies #A, #B, and #D bind with higher signal-to-noise ratios (S / N) than the commercial antibody, primarily at concentrations between 20 and 2000 ng / mL, and also exhibit lower EC50 values ​​compared to commercial mAb #2 (Figure 10, right).

[0184] conclusion Functional assays were performed comparing anti-HIV-1 antibodies #A, #B, and #D with commercially available anti-HIV-1 p24 antibodies from commercial mAbs #1 and #2. The binding affinity and potency of each antibody were assessed by BLI and indirect ELISA. In both experiments, HIV-1 antibodies #A, #B, and #D exhibited superior affinity and superior EC50 values ​​compared to the commercially available antibodies tested (Figure 9 and Table 7 for kinetic analysis, Figure 10 for ELISA data).

[0185] The experimental data presented here demonstrate that the anti-HIV-1 antibodies of the present invention can be used to detect the p24 structural protein of HIV-1. The antibodies exhibit improved properties in terms of affinity, sensitivity, potency, expression, solubility, and manufacturability when compared to similar commercially available products, and their use in serological tests can contribute to shortening the time frame between HIV-1 infection and the diagnostic event, thus preventing secondary viral transmission.

[0186] [Table 8A]

[0187] [Table 8B]

[0188] Table 8C

[0189]

Table 8D

[0190]

Table 8E

[0191] Table 8F

[0192]

Table 8G

[0193]

Table 8H

Claims

1. An anti-HIV-1 antibody comprising a light chain comprising complementarity determining regions L-CDR1, L-CDR2 and L-CDR3 and a heavy chain comprising complementarity determining regions H-CDR1, H-CDR2 and H-CDR3, The amino acid sequence of L-CDR1 is SEQ ID NO: 15, the amino acid sequence of L-CDR2 is SEQ ID NO: 16, and the amino acid sequence of L-CDR3 is SEQ ID NO: 17; and The amino acid sequence of H-CDR1 is SEQ ID NO: 24, the amino acid sequence of H-CDR2 is SEQ ID NO: 25, and the amino acid sequence of H-CDR3 is SEQ ID NO:

26. Anti-HIV-1 antibody.

2. The anti-HIV-1 antibody of claim 1, wherein the light chain comprises a sequence having approximately 90% homology with the amino acid sequence of SEQ ID NO:

7.

3. The anti-HIV-1 antibody of claim 2, wherein the light chain comprises the amino acid sequence of SEQ ID NO:

7.

4. The anti-HIV-1 antibody according to any one of claims 1 to 3, wherein the heavy chain comprises a sequence having approximately 90% homology with the amino acid sequence of SEQ ID NO:

10.

5. The anti-HIV-1 antibody of claim 4, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:

10.

6. The anti-HIV-1 antibody according to any one of claims 1 to 5, wherein the light chain of the antibody comprises the amino acid sequence of SEQ ID NO:

1.

7. The anti-HIV-1 antibody according to any one of claims 1 to 6, wherein the heavy chain of the antibody comprises the amino acid sequence of SEQ ID NO:

4.

8. The anti-HIV-1 antibody according to any one of claims 1 to 6, wherein the light chain of the antibody comprises the amino acid sequence of SEQ ID NO: 1 and the heavy chain of the antibody comprises the amino acid sequence of SEQ ID NO:

4.

9. The anti-HIV-1 antibody according to any one of claims 1 to 8, which is a monoclonal antibody or a recombinant antibody.

10. The anti-HIV-1 antibody according to any one of claims 1 to 9, which is an antibody fragment.

11. The antibody fragment may be a variable fragment (Fv), a single-chain Fv (scFv), a bispecific antibody (sc(Fv) 2 ), single chain antibody, Fab fragment, F(ab') 2 The anti-HIV-1 antibody of claim 10, which is selected from a Fab' fragment, a disulfide-linked Fv (dsFv), a chemically conjugated Fv (ccFv), a diabody, an anti-idiotypic (anti-Id) antibody, and a unibody.

12. The anti-HIV-1 antibody according to any one of claims 1 to 11, comprising a constant region of mouse IgG1 class or mouse IgG2a class.

13. The anti-HIV-1 antibody of any one of claims 1 to 12, which is bound to a solid support.

14. A cell comprising the anti-HIV-1 antibody of any one of claims 1 to 13.

15. A nucleic acid comprising a nucleotide sequence encoding the anti-HIV-1 antibody of any one of claims 1 to 10, a promoter operably linked to the nucleotide sequence, and a selection marker.

16. A cell comprising the nucleic acid of claim 15.

17. A composition comprising the anti-HIV-1 antibody of any one of claims 1 to 10 and a solid support, wherein the anti-HIV-1 antibody is covalently or non-covalently bound to the solid support.

18. 18. The composition of claim 17, wherein the solid support comprises a particle, bead, membrane, surface, polypeptide chip, microtiter plate, or the solid phase of a chromatography column.

19. 14. A kit for detecting the presence of HIV-1 in a sample, said kit comprising at least one anti-HIV-1 antibody according to any one of claims 1 to 13 and a solid support, wherein said at least one anti-HIV-1 antibody is covalently or non-covalently bound to the solid support.

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