Anti-human immunodeficiency virus-1 antibody and method of use thereof

Abstract

The problem that this invention aims to solve is to provide an anti-HIV-1 antibody with improved binding ability to the HIV-1 p24 protein compared to similar commercially available reagents. [Solution] The present invention relates to an anti-HIV-1 antibody comprising L-CDR1, L-CDR2, and L-CDR3, wherein L-CDR1 is selected from the group consisting of sequences different from SEQ ID NOs. 15, SEQ ID NOs. 18, SEQ ID NOs. 21, and SEQ ID NOs. 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 sequences different from SEQ ID NOs. 16, SEQ ID NOs. 19, SEQ ID NOs. 22, and SEQ ID NOs. 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 sequences different from SEQ ID NOs. 17, SEQ ID NOs. 20, SEQ ID NOs. 23, and SEQ ID NOs. 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 Art

[0002] Human immunodeficiency virus 1 (HIV-1) is a retrovirus that infects 37.9 million people worldwide and kills approximately 1 million people each year, particularly in vulnerable populations that cannot access diagnosis and treatment (Soliman, M. et al., "Mechanisms of HIV Control", Current HIV / AIDS Reports 2017, vol. 14(3); pp. 101-109). HIV-1 is the main cause of acquired immunodeficiency syndrome (AIDS) and is an incurable disease that is transmitted from HIV-1-infected individuals through sexual contact or exposure to contaminated products of blood or blood origin. The virus targets the immune system by disrupting and impairing the function of immune cells. Infected individuals become immunodeficient and are more susceptible to opportunistic infections and certain types of cancer (source: WHO website - https: / / www.who.int / news-room / fact-sheets / detail / hiv-aids). Currently, only 46% of people living with HIV-1 know their infection status. Therefore, the detection of HIV-1 during acute infection is a major 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 (acute phase) of an individual's infection, as this is likely to prevent secondary infections and allow for 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. Here, p24 is considered an important biomarker for early HIV-1 detection because it is the most abundant structural protein of the HIV-1 viral envelope and is secreted at high levels in the serum during the early stages of infection. p24 is a polymerized capsid protein that acts as a major structural component of the HIV-1 envelope around the viral RNA molecule. p24 is a 24-25 kDa protein derived from a Gag polyprotein precursor that can be detected before seroconversion, similar to HIV-1 RNA (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 narrow the diagnostic gap from 4 weeks to 2 weeks after exposure (Gray, ER 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 TS., "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 possess high binding ability and excellent manufacturing characteristics.

[0006] Accordingly, 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. 101-109. [Non-Patent Document 2] Source: 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 edition, Public Health Service, NIH, Washington DC. [Non-Patent Document 10] Chothia et al., Nature 342: pp. 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: pp. 1156-1166, 2008. [Non-Patent Document 14] Retter et al., Nucleo Acids Res., 33 (Database issue): pp. D671-D674 (2005) [Non-Patent Document 15] Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: pp. 2264-2268. [Non-Patent Document 16] Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: pp. 5873-5877. [Non-Patent Document 17] Altschul et al., 1990, J. Mol. Biol. 215: pp. 403-410. [Non-Patent Document 18] Altschul et al., 1997, Nucleic Acids Res. 25: pp. 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 [Overview of the project] [Means for solving the problem]

[0008] Where this specification refers to the sequence of CDR X, or a sequence that differs from CDR X due to one or two substitutions, deletions, or additions, it should be understood that such substitutions, deletions, or additions may occur at any amino acid within the range of amino acids defined by CDR X. This specification individualizes each specific amino acid within the range of amino acids defined by CDR X as suitable for such substitutions, deletions, or additions.

[0009] As a non-limiting example, the L-CDR1 of antibody #A can be defined to include 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 refined by subsequent correction.

[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 that 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-CDRB is selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, and sequences that 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 that differ from any of SEQ ID NO: 17, 20, or 23 by one or two substitutions, deletions, or additions.

[0011] In other embodiments, 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 that 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 that 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 that 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 the anti-HIV-1 antibody of the present invention comprises a sequence having about 90% homology with 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 the anti-HIV-1 antibody of the present invention comprises a sequence having about 90% homology with 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 includes sequence number 24, or a sequence different from sequence number 24 by one or two substitutions, deletions, or additions; the amino acid sequence of H-CDR2 includes sequence number 25, or a sequence different from sequence number 25 by one or two substitutions, deletions, or additions; and the amino acid sequence of H-CDR3 includes sequence number 26, or a sequence different from sequence number 26 by one or two substitutions, deletions, or additions. In other embodiments, the amino acid sequence of H-CDR1 includes sequence number 27, or a sequence different from sequence number 27 by one or two substitutions, deletions, or additions; the amino acid sequence of H-CDR2 includes sequence number 28, or a sequence different from sequence number 28 by one or two substitutions, deletions, or additions; and the amino acid sequence of H-CDR3 includes sequence number 29, or a sequence different from sequence number 29 by one or two substitutions, deletions, or additions. In other embodiments, the amino acid sequence of H-CDR1 includes sequence number 30, or a sequence different from sequence number 30 by one or two substitutions, deletions, or additions; the amino acid sequence of H-CDR2 includes sequence number 31, or a sequence different from sequence number 31 by one or two substitutions, deletions, or additions; and the amino acid sequence of H-CDR3 includes sequence number 32, or a sequence different from sequence number 32 by one or two substitutions, deletions, or additions.

[0016] In some embodiments, the amino acid sequence of L-CDR1 includes sequence number 15, or a sequence different from sequence number 15 by one or two substitutions, deletions, or additions; the amino acid sequence of L-CDR2 includes sequence number 16, or a sequence different from sequence number 16 by one or two substitutions, deletions, or additions; the amino acid sequence of L-CDR3 includes sequence number 17, or a sequence different from sequence number 17 by one or two substitutions, deletions, or additions; the amino acid sequence of H-CDR1 includes sequence number 24, or a sequence different from sequence number 24 by one or two substitutions, deletions, or additions; the amino acid sequence of H-CDR2 includes sequence number 25, or a sequence different from sequence number 25 by one or two substitutions, deletions, or additions; and the amino acid sequence of H-CDR3 includes sequence number 26, or a sequence different from sequence number 26 by one or two substitutions, deletions, or additions.

[0017] In some embodiments, the amino acid sequence of L-CDR1 includes sequence number 18, or a sequence different from sequence number 18 by one or two substitutions, deletions, or additions; the amino acid sequence of L-CDR2 includes sequence number 19, or a sequence different from sequence number 19 by one or two substitutions, deletions, or additions; the amino acid sequence of L-CDR3 includes sequence number 20, or a sequence different from sequence number 20 by one or two substitutions, deletions, or additions; the amino acid sequence of H-CDR1 includes sequence number 27, or a sequence different from sequence number 27 by one or two substitutions, deletions, or additions; the amino acid sequence of H-CDR2 includes sequence number 28, or a sequence different from sequence number 28 by one or two substitutions, deletions, or additions; and the amino acid sequence of H-CDR3 includes sequence number 29, or a sequence different from sequence number 29 by one or two substitutions, deletions, or additions.

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

[0019] In some embodiments, the light chain of the anti-HIV-1 antibody of the present 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 the anti-HIV-1 antibody of the present 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 antibody of the present invention specifically binds to an epitope of the HIV-1 p24 protein containing the amino acid sequence of SEQ ID NO: 33.

[0022] In some embodiments, the amino acid sequence of L-CDR1 of the anti-HIV-1 antibody of the present invention includes SEQ ID NO: 21, or a sequence different from SEQ ID NO: 21 by one or two substitutions, deletions, or additions; the amino acid sequence of L-CDR2 includes SEQ ID NO: 22, or a sequence different from SEQ ID NO: 22 by one or two substitutions, deletions, or additions; and the amino acid sequence of L-CDR3 includes SEQ ID NO: 23, or a sequence different 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 the anti-HIV-1 antibody of the present invention includes SEQ ID NO: 30, or a sequence different from SEQ ID NO: 30 by one or two substitutions, deletions, or additions; the amino acid sequence of H-CDR2 includes SEQ ID NO: 31, or a sequence different from SEQ ID NO: 31 by one or two substitutions, deletions, or additions; and the amino acid sequence of H-CDR3 includes SEQ ID NO: 32, or a sequence different 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 variable fragments (Fv), single-chain Fv (scFv), bispecific antibodies (sc(Fv)2), single-chain antibodies, single-domain antibodies, Fab fragments, F(ab')2 fragments, Fab' fragments, disulfide-bonded Fv (dsFv), chemically conjugated Fv (ccFv), diabodies, anti-idiotype (anti-Id) antibodies, affibodies, nanobodies, and unibodies.

[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 antibody of the present invention.

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

[0030] The present invention also discloses compositions comprising an anti-HIV-1 antibody and a solid support as described herein, wherein the anti-HIV-1 antibody is covalently or non-covalently bound to the solid support. In some embodiments, the solid support includes particles, beads, membranes, surfaces, polypeptide chips, microtiter plates, or the solid phase of a chromatography column.

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

[0032] [Figure 1] The monomer percentage of antibody #A determined by SE-UPLC analysis, and SDS-PAGE of a single clone of antibody #A (lanes 1, 2, and 3 represent subclones run under reducing and non-reducing conditions, respectively). [Figure 2] The monomer percentage of antibody #B determined by SE-UPLC analysis, and SDS-PAGE of antibody #B single clone (lanes 1, 2, and 3 represent subclones run under reducing and non-reducing conditions, respectively). [Figure 3] The monomer percentage of antibody #D determined by SE-UPLC analysis, and SDS-PAGE of a single clone of antibody #D (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, PDB structure code 2XKN was used in the homology query, and for antibodies B# and #D, codes 5OPY and 1F3D were used, respectively. [Figure 5] Sensorgrams of saturated antibody #A and competing antibodies #B and #D. Antibodies #B and #D add signals to #A, indicating that these antibodies do not compete for binding within the same epitope region. [Figure 6] Sensorgrams of saturated antibody #B and competing antibodies #A and #D. Antibodies #A and #D add signals to #B, indicating that these antibodies do not compete for binding within the same epitope region. [Figure 7] Sensorgrams of saturated antibody #D and competing antibodies #A and #B. Antibodies #A and #B add signals to #D, indicating that these antibodies do not compete for binding within the same epitope region. [Figure 8] Sensorgrams of the association of antibodies #A, #B, and #D to HIV-1 p24 in the absence of competing antibodies. Each antibody achieves its complete binding signal (experimental control). [Figure 9] The binding kinetics of antibodies #A, #B, and #D, as well as the commercially available mAb #1, to the HIV-1 p24 antigen, calculated by biolayer interferometry (BLI). Sensorgrams were performed at gradient concentrations from 0.1 to 33 nM, and a 1:1 binding model was fitted to calculate ka (association rate constant), kd (dissociation rate constant), and KD (equilibrium dissociation constant). [Figure 10] Conjugation of antibodies #A, #B, and #D, as well as commercially available mAb #2, to the HIV-1 p24 capsid protein by indirect ELISA. The titration curves for each antibody begin at a concentration of 2 μg / mL and are then diluted 1:10 (left). Signal-versus-noise data at an antibody concentration of 200 ng / mL is shown on the right, indicating that commercially available mAb #2 performs poorly compared to antibodies #A, #B, and #D. [Modes for carrying out the invention]

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

[0034] As used in this specification and the attached claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise.

[0035] Throughout this specification, unless the context otherwise requires, it will be understood that the word “comprise,” or variations such as “comprises,” or “comprising,” mean the inclusion of the element or integer, or group of elements or integers, mentioned, 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 those skilled in the art in which this invention pertains. Exemplary methods and materials are described below, but similar or equivalent methods and materials may also be used and will be apparent to those skilled in the art. All publications and other references referenced herein are incorporated by reference as a whole. In case of any conflict, this specification, including definitions, shall prevail. Materials, methods, and examples are illustrative and not intended to limit the scope of the invention.

[0037] Each embodiment in this specification may be applied to any other embodiment, with modifications as necessary, unless otherwise explicitly stated.

[0038] The following terms should be understood to have the following meanings unless otherwise indicated.

[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, "nucleotide" refers to a nucleic acid subunit consisting of a phosphate group, a pentagonal sugar, and a nitrogen-containing base. The pentagonal sugar found in RNA is ribose. In DNA, the pentagonal sugar is 2'-deoxyribose. The term also includes analogues of such subunits.

[0041] As used herein, the term “polynucleotide” refers to a polymer 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), in addition to DNA or RNA analogs containing unnatural nucleotide analogs, unnatural nucleoside bonds, or both. Nucleic acids can have any topological structure. For example, nucleic acids may be single-stranded, double-stranded, triple-stranded, quadruple-stranded, partially double-stranded, branched, hairpin-shaped, cyclic, or padlock structures.

[0042] As used herein, the term "protein" refers to a large biomolecule or macromolecule consisting of one or more chains of amino acid residues. Many proteins are enzymes that catalyze biochemical reactions and are essential for metabolism. Proteins also have structural or mechanical functions, such as actin and myosin in muscle, and proteins in the cytoskeleton that form scaffold systems that maintain the shape of cells. Other proteins are important in cell signaling, immune responses, cell adhesion, and the cell cycle. However, proteins may be entirely artificial or recombinant, that is, they may not exist naturally in biological systems.

[0043] As used herein, the term "polypeptide" refers to both naturally occurring and non-naturally occurring proteins, as well as their fragments, variants, derivatives, and analogues. Polypeptides may be monomers or polymers. Polypeptides may contain several distinct domains (peptides), each of which has one or more distinct activities.

[0044] As used herein, the term “recombinant” means a biomolecule, such as a gene or protein, that (1) is isolated from its naturally occurring environment, (2) whose gene is not related to all or part of a naturally occurring polynucleotide, (3) is operably linked to a polynucleotide that is not naturally linked, or (4) does not exist in nature. The term “recombinant” can be used in relation to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs synthesized in vivo by heterologous systems, as well as proteins and / or mRNA 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 within naturally occurring proteins. 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, without stop codons between them, and are typically translated from mRNA as part of the fusion protein.

[0046] When referring to proteins as described in (3), the terms “fusion protein” and “recombinant” may be used interchangeably herein.

[0047] As used herein, the terms “antibody” and “immunoglobulin” have the same meaning and are used interchangeably in this invention. As used herein, the term “antibody” refers to an immunoglobulin molecule and a molecule comprising an immunoactive portion of an immunoglobulin molecule, i.e., an antigen-binding site that specifically binds to an antigen. Thus, the term antibody encompasses not only the entire antibody molecule 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 disulfide bonds. 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 different sequence domains. The light chain contains two domains: a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively called CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine the binding recognition and specificity to the antigen. The constant domains of the light chain (CL) and the heavy chain (CH) give rise to important biological properties such as antibody chain association, secretion, transplacental migration, complement binding, and binding to the Fc receptor (FcR). The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin, consisting of one variable region of the light chain and one variable region of the heavy chain. Antibody specificity lies in the structural complementarity between the antibody-binding site and the antigenic determinant. The antibody-binding site is composed primarily of residues derived from the hypervariable or complementarity-determining region (CDR). Occasionally, residues derived from the non-hypervariable or framework region (FR) affect the overall domain structure and, therefore, the binding site. The complementarity-determining region or CDR refers to the amino acid sequence that collectively defines the binding affinity and specificity of the native Fv region of the native immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs, which are named L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3, respectively. Therefore, the antigen-binding site typically contains six CDRs, including sets of CDRs from the heavy chain V region and the light chain V region, respectively. The framework region (FR) refers to the amino acid sequence inserted between CDRs.

[0049] The CDR can be identified according to the definitions of Kabat, Chothia, accumulation by both Kabat and Chothia, AbM, contact, IMGT unique numbering, and / or structural definition, or any method of CDR determination known in the art. The antibody CDR can be identified as a hypervariable region, as first defined by Kabat et al. (see, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, NIH, Washington DC). The location of the CDR can also be identified as a loop structure of the structure first described by Chothia et al. (see, e.g., Chothia et al., Nature 342: pp. 877-883, 1989). Other approaches to CDR identification include "AbM definition" (a hybrid of Kabat and Chothia, guided by Oxford Molecular's AbM antibody modeling software (now Accelrys0)), "contact definition" of CDRs based on observed antigen contact (as shown in MacCallum et al., J. Mol. Biol., 262:732-745, 1996), or "IMGT unique numbering" (relying on the high structural conservation of the variable region (see Lefranc, M.-P. Nucl. Acids Res., 33, D593-D597, 2005)). In another approach, referred to herein as "conceptual definition" of CDRs, the location of a CDR can be identified as a residue that contributes to the enthalpy of antigen binding. See, for example, Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008. Furthermore, other CDR boundary definitions, while not strictly following one of the approaches described above, still overlap with at least a portion of the Kabat CDR. However, given predictions or experimental findings that specific residues or groups of residues, or even the entire CDR, do not significantly affect antigen binding, these definitions may be shortened or extended.As used herein, CDR refers to a CDR defined by any approach known in the art, including combinations of approaches. The methods used herein may utilize a CDR defined according to any of these approaches. For any given embodiment including multiple CDRs, the CDR 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 available on the "Abysis" website at www.bioinf.org.uk / abs (maintained by AC Martin of the 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): pp. D671-D674 (2005), and can be accessed through them. Preferably, sequences are analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT, and the Protein Data Bank (PDB) along with structural data from the PDB. Unless otherwise indicated, all CDRs presented herein are 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 terms “monoclonal antibody” or “mAb” refer to an antibody composition having a homogeneous population of antibodies that bind to the same epitope. The term is not limited in terms of the species or source of the antibody, nor is it intended to be limited by the method by which it is produced. Accordingly, the term includes antibodies obtained from mouse hybridomas, as well as human monoclonal antibodies obtained using humans rather than mouse hybridomas. The term also includes antibodies obtained by other methods for producing monoclonal antibodies known in the art, such as the establishment of eukaryotic cell lines by transient or stable transfection.

[0053] As used herein, the term “recombinant antibody” means an antibody expressed from a cell or cell line transfected with one or more expression vectors containing an antibody coding sequence, the coding sequence being unrelated to the cell or natively. Recombinant antibodies or fragments thereof are prepared, expressed, created or isolated by any recombinant means, as is well known to those skilled in the art.

[0054] In some embodiments, the “recombinant antibody” may also be a “monoclonal antibody” if it originates from a homogeneous population of antibodies that bind to the same epitope.

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

[0056] As used herein, the term “antigen-binding fragment (Fab)” refers to an antibody fragment comprising one constant domain and one variable domain in both the heavy and light chains. The variable domain contains an antigen-binding site. Generally, an antibody comprises a fragment crystallizable region (Fc) and two antigen-binding fragments (Fab). The Fab fragment can be separated from the Fc region to yield 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 chemical substances. For example, when an isolated antibody is expressed by cells of a different species, a human antibody expressed in mouse cells, for instance, is substantially free of other proteins of a different species. Proteins can be made substantially free of naturally occurring components (or components associated with the cell expression system used to produce the antibody) by isolating them using protein purification techniques well known in the art.

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

[0059] As used herein, the term “epitope” refers to a portion of an antigen to which an antibody specifically binds. Therefore, the term “epitope” includes any protein determinant that has the ability to specifically bind to an immunoglobulin or T cell receptor.

[0060] A polypeptide is "immunologically reactive" with an antibody if it binds to the antibody by antibody recognition of a specific 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 known polypeptides containing the epitope to which the antibody is directed as a competitor. 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 concentrated from a blood sample, and cultured cells (e.g., B cells derived from a subject). Samples may also include biopsy or tissue samples, including nerve tissue. In yet another embodiment, a sample may include whole cells and / or cell solubilization solutions.

[0062] The sample may be processed to physically or mechanically destroy the structure of the tissue or cells, thereby releasing intracellular components into a solution, which may further contain enzymes, buffers, salts, surfactants, etc., used to prepare biological samples for analysis using standard methods. The sample may also include processed samples, such as those obtained by filtering or passing the sample through a filter, after centrifugation, or by adhesion to a medium, matrix, or support.

[0063] The terms “patient” or “individual” are used interchangeably herein and refer to the mammalian subject being diagnosed or treated, with human patients preferred. In some cases, the methods of the present invention are used in laboratory animals, in veterinary applications, and, non-limitingly, in the development of animal models of diseases, including rodents such as mice, rats, and hamsters; and primates.

[0064] The term "vector" refers to a nucleic acid that can be used to introduce another nucleic acid, to which it is bound, 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-deficient 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 within the host cell into which they are introduced (e.g., bacterial vectors containing bacterial origins of replication and episomatic mammalian vectors). Other vectors (e.g., non-episomatic mammalian vectors) are integrated into the host cell's genome upon introduction into the host cell and thereby replicate together with the host genome.

[0065] An "expression vector" is a type of vector that can direct the expression of selected polynucleotides. An "expressing cell" is a cell that contains an expression vector.

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

[0067] As used herein, the terms “diagnostic” or “diagnosed” mean identifying the presence or nature of a pathological condition or individuals susceptible to the disease. Diagnostic methods differ in their sensitivity and specificity. The “sensitivity” of a diagnostic assay is the percentage of affected individuals who test positive (the “percentage of true positives”). Individuals not detected by the assay are “false negatives.” Individuals who are not affected and test negative 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 individuals who test positive but do not have the disease. While no particular diagnostic method can provide a definitive diagnosis of a condition, it is sufficient if it provides a positive indication that is helpful in diagnosis.

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

[0069] This invention relates to novel antibodies specific for the detection of the human immunodeficiency virus 1 (HIV-1) p24 protein. These antibodies recognize novel, non-cross-reactive epitopes of the HIV-1 p24 protein and exhibit higher affinity and sensitivity compared to other commercially available products. Accordingly, the antibodies described herein can be used as diagnostic reagents, standards, or positive controls in immunoassays for early detection of HIV-1. They can be used for the detection of any of the three major groups of HIV-1 (group M (main strain), group N (novel strain), and group O (unclassified)).

[0070] The present invention also relates to compositions and kits comprising the anti-HIV-1 antibody 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” refer to two or more nucleic acid or polypeptide sequences that, when compared and aligned for the greatest match, are identical or have a specific percentage of identical nucleotides or amino acid residues. To determine percent homology / identity, sequences are aligned for the purpose of best comparison (for example, gaps may be introduced in the first amino acid or nucleic acid sequence for best alignment with the second amino acid or nucleic acid sequence). Then, amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then those molecules are identical at that position. Percent identity between two sequences is a function of the number of identical positions shared by those 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 of the same length after gaps are introduced within them, if necessary (e.g., excluding additional sequences that extend beyond the sequences to be compared). For sequence comparisons between two sequences, the "corresponding" CDR refers to the CDR at the same location in both sequences (e.g., CDR-H1 of each sequence).

[0072] The determination of percent identity, percent similarity, or percent similarity between two sequences can be achieved using mathematical algorithms. A preferred non-restrictive example of a mathematical algorithm used to compare two sequences is the algorithm from Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as described in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such algorithms are incorporated into the NBLAST and XBLAST programs in Altschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searching can be performed using the NBLAST program, score=100, word length=12, to obtain nucleotide sequences homologous to the nucleic acid encoding the target protein. BLAST protein search can be performed using the XBLAST program, score=50, and word length=3 to obtain amino acid sequences homologous to the target protein. For comparison purposes, gapped BLAST can be used to obtain gapped alignment, as described by Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. When using BLAST and gapped BLAST, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. Another preferred non-restrictive example of a mathematical algorithm used for sequence comparison is the algorithm by Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the 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 another embodiment 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 as defined in 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 variable fragments (Fv), single-chain Fv (scFv), bispecific antibodies (sc(Fv)2), single-chain antibodies, single-domain antibodies, Fab fragments, F(ab')2 fragments, Fab' fragments, disulfide-linked Fv (dsFv), chemically conjugated Fv (ccFv), diabodies, anti-idiotype (anti-Id) antibodies, affibodies, nanobodies, and unibodies.

[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 contain 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 may be any type known to those skilled in the art (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or 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 yet 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 aforementioned 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 the mouse IgG1 class or the mouse IgG2a class.

[0080] A. Light chain In some embodiments described herein, the anti-HIV-1 antibody comprises a light chain containing a complementation-determining region (CDR). The CDR corresponds to a sequence identified according to any CDR definition approach known to those skilled in the art. In some preferred embodiments, the CDR region corresponds to a sequence identified according to the Kabat numbering scheme. In other preferred embodiments, the CDR region may correspond to a sequence identified according to other numbering methods or a combination of Kabat and other numbering methods. For example, the CDR region may correspond to a sequence identified according to the Chothia numbering scheme.

[0081] In some embodiments described herein, the anti-HIV-1 antibody comprises a light chain including 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 sequence 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 present invention comprises the amino acid sequence of SEQ ID NO: 7, 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 with the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In some preferred embodiments, the light chain of the anti-HIV-1 antibody of the present invention comprises a sequence having about 90% homology with the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0083] In another embodiment described herein, the recombinant antibody comprises a light chain containing 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 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 present 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 antibody comprises a heavy chain containing a complementation-determining region (CDR). The CDR corresponds to a sequence identified according to any CDR definition approach known to those skilled in the art. In some preferred embodiments, the CDR region corresponds to a sequence identified according to the Kabat numbering scheme. In other preferred embodiments, the CDR region may correspond to a sequence identified according to other numbering methods or a combination of Kabat and other numbering methods. For example, the CDR region may correspond to a sequence identified according to the Chothia numbering scheme.

[0085] In some embodiments described herein, the anti-HIV-1 antibody comprises a heavy chain including 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 sequence 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 present invention comprises the amino acid sequence of SEQ ID NO: 10, 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 with the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some preferred embodiments, the heavy chain of the anti-HIV-1 antibody of the present invention comprises a sequence having about 90% homology with the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

[0087] In another embodiment described herein, the recombinant antibody comprises a heavy chain containing 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 with the amino acid sequence 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 present invention comprises a sequence having about 90% homology with the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0088] C. Exemplary anti-HIV-1 antibody In one embodiment described herein, the anti-HIV-1 antibody comprises a light chain containing 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 containing 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 containing 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 containing 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 containing 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 containing 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 antibody of the present invention may comprise any combination of the CDR regions of both the light chain and the heavy chain as described herein.

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

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

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

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

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

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

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

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

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

[0104] In one embodiment described herein, the anti-HIV-1 antibody comprises a light chain containing 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 containing 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 containing the amino acid sequence of SEQ ID NO: 7 and a heavy chain containing the amino acid sequence of SEQ ID NO: 10.

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

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

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

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

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

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

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

[0113] In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 9 and a heavy chain containing 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 with the amino acid sequence 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 with the amino acid sequence 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 containing 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 containing 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 containing the amino acid sequence of SEQ ID NO: 1 and a heavy chain containing the amino acid sequence of SEQ ID NO: 4.

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

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

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

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

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

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

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

[0124] In a preferred embodiment, the anti-HIV-1 antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 3 and a heavy chain containing 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 with the amino acid sequence 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 with the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0126] In some preferred embodiments, the anti-HIV-1 antibody of the present invention specifically binds to the HIV-1 p24 protein. In some embodiments, the anti-HIV-1 antibody of the present invention binds to an epitope of the HIV-1 p24 protein. In some preferred embodiments, the anti-HIV-1 antibody of the present invention binds to a linear epitope of the HIV-1 p24 protein. In some preferred embodiments, the anti-HIV-1 antibody of the present invention binds to a linear epitope comprising at least five 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 to the said sequence. In other embodiments, the amino acid sequence of the HIV-1 p24 protein is defined 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 containing 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 characterized by comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 33. The antibody binds to the epitope of the p24 protein, and comprises a light chain containing 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 NOs. 15, 18, and 21; the amino acid sequence of L-CDR2 is selected from the group consisting of SEQ ID NOs. 16, 19, and 22; and the amino acid sequence of L-CDR3 is selected from the group consisting of SEQ ID NOs. 17, 20, and 23. Furthermore, it comprises a heavy chain containing 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 NOs. 24, 27, and 30; the amino acid sequence of H-CDR2 is selected from the group consisting of SEQ ID NOs. 25, 28, and 31; and the amino acid sequence of H-CDR3 is selected from the group consisting of SEQ ID NOs. 26, 29, and 32.

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

[0130] In some embodiments, the anti-HIV-1 antibody of the present invention is conjugated to a solid support.

[0131] D. Affinity tags The anti-HIV-1 antibody according to the present invention may contain affinity tags. Affinity tags are useful for purification. Examples of 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 selection of affinity tags is not particularly limited. However, anti-HIV-1 antibodies may lack affinity tags if, for example, 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 example of an affinity tag is polyhistidine, which typically contains an amino acid sequence with between 4 and 10 consecutive histidines.

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

[0133] II. Nucleic acids, cloned cells, and expression cells The present invention also relates to a nucleic acid comprising a nucleotide sequence encoding the anti-HIV-1 antibody described herein. The nucleic acid may be an isolated nucleic acid. The nucleic acid may be DNA or RNA. The DNA comprising the nucleotide sequence encoding the anti-HIV-1 antibody described herein typically comprises a promoter operably linked to the nucleotide sequence. The promoter preferably has the ability to drive constitutive or inducible expression of the nucleotide sequence in the expression cells of interest. The nucleic acid may also comprise a selection marker useful for selecting cells containing the nucleic acid of interest. Useful selection 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 the anti-HIV-1 antibody described herein. Codons may be selected, for example, to fit the codon bias of the expression cells of interest (e.g., mammalian cells such as human cells) and / or for convenience during cloning. The DNA may comprise a replication origin (e.g., for plasmid replication in prokaryotic cells), for example, a plasmid.

[0134] In one embodiment described herein, the nucleic acid comprises a nucleotide sequence encoding the anti-HIV-1 antibody of the present invention, a promoter operably linked to the nucleotide sequence, and a selection 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 a more preferred embodiment, the nucleic acid of the light chain of the anti-HIV-1 antibody of the present 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 present 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 present 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 present 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 present 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 comprising nucleic acids containing a nucleotide sequence encoding an anti-HIV-1 antibody as described herein. The cells may be expression cells or cloned cells. The nucleic acids are typically cloned in Escherichia coli (E. coli), but other cloned cells may be used.

[0138] If the cell is an expressing cell, the nucleic acid may be the nucleic acid of a chromosome, that is, its nucleotide sequence is incorporated into the chromosome; however, in that case, the nucleic acid may exist in the expressing cell as, for example, extrachromosomal DNA or a vector, such as a plasmid, cosmid, or phage. The type of vector should not be considered restrictive.

[0139] In one embodiment described herein, the cells are typically expression cells. The nature of the expression cells is not particularly limiting. Mammalian expression cells may enable the convenient folding, post-translational modification, and / or secretion of recombinant antibodies or oligomeric recombinant antibodies, but other eukaryotic or prokaryotic cells may 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 vector incorporation, more preferably for plasmid DNA incorporation.

[0140] The anti-HIV-1 antibody of the present invention can be produced by a suitable transfection strategy for mammalian cells of nucleic acids containing a nucleotide sequence encoding the anti-HIV-1 antibody. Those skilled in the art are aware of various techniques (lipofection, electroporation, etc.) available for transfection of nucleic acids into select cell lines. Therefore, the selection of mammalian cell lines and transfection strategies should not be considered limiting. Cell lines may also be selected for plasmid DNA integration.

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

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

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

[0144] In other embodiments, the composition comprises the 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 specific binding, such as between an antibody and its antigen, between a ligand and its receptor, or between an enzyme and its substrate, exemplified by interactions between a streptavidin-binding protein and streptavidin, or between an antibody and its antigen.

[0145] In other embodiments, the composition comprises the anti-HIV-1 antibody of the present invention and a solid support, wherein the anti-HIV-1 antibody is directly or indirectly conjugated to the solid support. As used herein, the term “direct” conjugation refers to the direct conjugation of a molecule to a solid support, for example, a gold-thiol interaction that conjugates the cysteinethiol of the anti-HIV-1 antibody to a gold surface. As used herein, the term “indirect” conjugation includes the specific conjugation of the anti-HIV-1 antibody to another molecule that is directly conjugated to the solid support, for example, the anti-HIV-1 antibody may conjugate an antibody that is directly conjugated to the solid support, thereby indirectly conjugating the anti-HIV-1 antibody to the solid support. The term “indirect” conjugation 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 chains of molecules is specific or covalent, and (b) the terminal molecules of the daisy chain are directly conjugated to the solid support.

[0146] The solid support may include particles, beads, membranes, surfaces, polypeptide chips, microtiter plates, or the solid phase of a chromatography column. Preferably, the solid support may be latex beads.

[0147] The composition may comprise a plurality of beads or particles, each of which is 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 of which is covalently or noncovalently bound to at least one anti-HIV-1 antibody as described herein.

[0148] Various embodiments of the embodiments relate to a kit for detecting the presence of HIV-1 in a sample, the kit comprising at least one anti-HIV-1 antibody and a solid support or composition described herein. In some embodiments, 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 may be used, for example, in assays for detecting the presence of HIV-1 in a sample or for measuring the concentration of HIV-1 in a sample, but are not limited to such assays. The anti-HIV-1 antibodies, compositions, and kits of the present invention may 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 antibody of the present invention is used in methods and assays in which other RNA viruses are also detected. In other embodiments, the anti-HIV-1 antibody 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 a more preferred embodiment, the anti-HIV-1 antibody 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] In methods and assays for detecting HIV-1 in a sample, the use of several anti-HIV-1 antibodies described herein is also intended within the scope of the present invention.

[0152] The present invention will be described in more detail below with reference to exemplary embodiments, but this does not constitute a limitation of the present invention. [Examples]

[0153] (Example 1) Desirable anti-HIV antibody and stable cell line generation As disclosed herein, specific combinations of light and heavy chains of the present invention have yielded preferred antibodies:

[0154] [Table 1]

[0155] The variable and constant regions of each antibody were cloned into bicistronic vectors and expressed in Chinese hamster ovary (CHO) cells. The production characteristics of each antibody were evaluated based on their ability to generate clones of their respective stable cell lines, as well as the reproducible expression and purification of functional antibodies.

[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 create a stable cell line. After 24 hours, the transfected cells were counted and placed in selective medium for stable integration of the protein gene.

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

[0158] Production Pool: A 1-liter production process was performed using a stable pool to evaluate VCD, titer, and viability. Cells were scaled up to production medium in a 3-liter shaking flask (working volume 1 liter). The supernatant of the acclimatized medium collected from each stable pool production process was clarified by centrifugation, and proteins were purified by affinity purification using a Protein A column (Tables 2 to 4).

[0159] Cell line banking: 2.5 × 10⁶ cells per 1 mL 6The cells were grown to a maximum of 15 × 10¹⁴ cells. At the time of harvesting for cell banking, the viability exceeded 95%. Next, the cells were centrifuged, and the cell pellet was resuspended in CHO complete medium containing 7.5% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, D1435) per vial. 6 The cell count was expressed as cells / mL. A total of five vials were prepared for each pool and frozen for storage in liquid nitrogen.

[0160] [Table 2]

[0161] [Table 3]

[0162] [Table 4]

[0163] Production of stable antibodies Starting with banked best pooled cell lines for antibodies #A, #B, and #D, stable clones were obtained by single-cell cloning. The best clone for each antibody was selected based on expression levels and bioanalytical characterization of the purified material of antibodies #A, #B, and #D from the manufacturing process. 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 using the computational modeling software Bioiluminate (Schrodinger), version 3.5, via antibody homology. Briefly, the amino acid sequences of the VH and VL regions of antibodies #A, #B, and #D were loaded into Bioiluminate. The framework region and CDR were identified by searching the Protein Databank (PDB) for antibody structures and selecting a PDB template 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, PDB structure code 2XKN was used in the homology query, and for antibodies B# and #D, codes 5OPY and 1F3D were used, respectively.

[0165] Antibodies #A and #B were the results for the IgG1k isotype, while antibody #D was the result for the IgG2ak isotype.

[0166] [Table 5]

[0167] Analysis of the nucleotide sequences of the three antibodies, when queried against IgBLAST, an algorithm developed by the National Center for Biotechnology Information (NCBI) to facilitate the analysis of immunoglobulin variable domain sequences against the ImMunoGeneTics Database (IMGT), showed that all generated heavy chains (VH) and light chains (VL) possessed 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 cleavage of the peptide, the HIV-p24 sequence was extended at the C-terminus and N-terminus using a neutral GSGGSGG linker. The extended antigen sequence was translated into a 15-amino acid linear peptide with a 14-amino acid peptide-peptide duplication. The resulting HIV-p24 peptide microarray contained 232 different linear peptides (464 spots) printed in pairs, and was composed of additional HA (YPYDVPDYAG, 38 spots) and c-Myc (EQKLISEEDL, 38 spots) control peptides.

[0169] Washing buffer: PBS containing 0.05% Tween 20, pH 7.4; wash for 10 seconds three times after each incubation step.

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

[0171] Incubation buffer: Wash 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 at 4°C for 16 hours; shaking at 140 rpm.

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

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

[0175] Scanner: LI-COR Odyssey Imaging System; Scan offset 0.65mm, Resolution 21μm, Scan intensity 7 / 7 (Red=680nm / Green=800nm)

[0176] Pre-staining of the HIV-p24 peptide microarray was performed using 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. Subsequently, other HIV-p24 peptide microarray copies were incubated with monoclonal antibody D at concentrations of 1 μg / ml, 10 μg / ml, and 100 μg / ml in incubation buffer, stained with the secondary antibody and control antibody, and read at a 7 / 7 (red / green) scan intensity. Additional HA peptides forming the peptide microarray were then stained as an internal quality control to confirm assay quality and peptide microarray integrity.

[0177] Epitope mapping of mAb D to HIV-p24, followed by an epitope substitution scan, highlighted the conserved 7-amino acid core motif PIAPGQM (SEQ ID NO: 33).

[0178] (Example 4) Epitope Binning Examination Molecular docking and Western blot evaluation 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 was biotinylated (bt-p24) and loaded onto a streptavidin (SA) biosensor for 300 seconds. The loaded sensor was immersed in saturated antibody (100 μg / mL) for 600 seconds, followed by immersion in competitive antibody (25 μg / mL) for 300 seconds. The results showed that when antibody #A was bound to HIV-1 p24, antibodies #B and #D added an increase to the BLI signal response, indicating that antibodies #B and #D bind to different epitopes compared to antibody A. Similarly, when antibody #A or #D is used as a saturated antibody, the remaining antibodies do not compete for the same epitope (Figures 9-10).

[0179] Table 6 summarizes the epitope binning data for antibodies #A, #B, and #D. Briefly, the BLI signals of competing and saturated antibodies were normalized to the buffer. A threshold of 0.02 was set to determine antibody blocking or binding, allowing self-blocking pairs to be recognized on the matrix diagonal (gray indicates binding, bold indicates self-blocking). The PEARSON correlation coefficient for the first 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 different 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 using 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 biosensor chips (ForteBio) specifically coated with anti-mouse Fc. The concentration gradients used for each antibody ranged from 0.1 to 33 nM, and each dilution was prepared in phosphate buffer (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 dissociation rate to association rate (kd / ka). The tested antibodies were ranked as follows based on the calculated affinity constants: Antibody #B ≈ Antibody #D > Antibody #A > Commercial mAb #1. Due to the length of the observed dissociation curves, it was not possible to calculate the exact KD values ​​for antibodies #B and #D. However, the presented data indicates that the calculated KD values ​​for antibodies #A, #B, and #D are lower than those 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, indirect ELISA assays were performed. The titration curves for each antibody were calculated using a starting concentration of 2 μg / mL, with a ratio of 2 × 10⁻⁶. -2 The antibodies were generated by serial dilution at a 1:10 ratio until a low concentration of ng / mL was reached. The performance of each antibody was compared with that of a commercially available clone (commercial mAb #2) (Figure 10, left). The data shows that antibodies #A, #B, and #D bind with a higher signal-to-noise ratio (S / N) than the commercially available antibody, mainly at concentrations of 20–2000 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 derived from commercially available mAbs #1 and #2. The binding affinity and potency of each antibody were evaluated by BLI and indirect ELISA. In both experiments, HIV-1 antibodies #A, #B, and #D showed superior affinity and EC50 values ​​compared to the commercially available antibodies tested (see Figure 9 and Table 7 for kinetic analysis, and Figure 10 for ELISA data).

[0185] The experimental data presented herein demonstrate that the p24 structural protein of HIV-1 can be detected using the anti-HIV-1 antibody of the present invention. Compared to similar commercially available products, the antibody exhibits improved properties in terms of affinity, sensitivity, potency, expression, solubility, and manufacturability. Furthermore, their use in serological testing contributes to shortening the time frame between HIV-1 infection and diagnostic events, and thus can prevent secondary viral infection.

[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 containing complementarity-determining regions L-CDR1, L-CDR2, and L-CDR3, The amino acid sequence of L-CDR1 is selected from the group consisting of sequences different from SEQ ID NOs. 15, 18, 21, and any of SEQ ID NOs. 15, 18, or 21, by one or two substitutions, deletions, or additions. The amino acid sequence of L-CDR2 is selected from a group consisting of sequences different from SEQ ID NOs. 16, 19, 22, and SEQ ID NOs. 16, 19, or 22, by one or two substitutions, deletions, or additions. An anti-HIV-1 antibody in which the amino acid sequence of L-CDR3 is selected from the group consisting of sequences different from SEQ ID NOs. 17, SEQ ID NOs. 20, SEQ ID NOs. 23, and SEQ ID NOs. 17, 20, or 23 by one or two substitutions, deletions, or additions.

2. The antibody comprises a heavy chain containing complementarity-determining regions H-CDR1, H-CDR2, and H-CDR3. The amino acid sequence of H-CDR1 is selected from the group consisting of sequences different from SEQ ID NOs. 24, 27, 30, and any of SEQ ID NOs. 24, 27, or 30, with one or two substitutions, deletions, or additions. The amino acid sequence of H-CDR2 is selected from the group consisting of sequences different from SEQ ID NOs. 25, 28, 31, and any of SEQ ID NOs. 25, 28, or 31, by one or two substitutions, deletions, or additions. The anti-HIV-1 antibody according to claim 1, wherein the amino acid sequence of H-CDR3 is selected from the group consisting of sequences different from SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, and SEQ ID NO: 26, 29, or 32 by one or two substitutions, deletions, or additions.

3. The anti-HIV-1 antibody according to claim 1 or 2, wherein the light chain comprises a sequence having approximately 90% homology to the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO:

9.

4. The anti-HIV-1 antibody according to claim 3, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO:

9.

5. The anti-HIV-1 antibody according to any one of claims 1 to 4, wherein the heavy chain comprises a sequence having approximately 90% homology to the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:

12.

6. The anti-HIV-1 antibody according to claim 5, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:

12.

7. The amino acid sequence of L-CDR1 includes a sequence that differs from sequence number 15, or from sequence number 15 by one or two substitutions, deletions, or additions. The amino acid sequence of L-CDR2 includes a sequence that differs from SEQ ID NO: 16, or from SEQ ID NO: 16 by one or two substitutions, deletions, or additions. The anti-HIV-1 antibody according to any one of claims 1 to 6, wherein the amino acid sequence of L-CDR3 comprises a sequence different from SEQ ID NO: 17, or a sequence with one or two substitutions, deletions, or additions from SEQ ID NO:

17.

8. The amino acid sequence of L-CDR1 includes a sequence that differs from sequence number 18, or from sequence number 18 by one or two substitutions, deletions, or additions. The amino acid sequence of L-CDR2 includes a sequence that differs from sequence number 19, or from sequence number 19 by one or two substitutions, deletions, or additions. The anti-HIV-1 antibody according to any one of claims 1 to 6, wherein the amino acid sequence of L-CDR3 comprises a sequence different from SEQ ID NO: 20, or a sequence with one or two substitutions, deletions, or additions from SEQ ID NO:

20.

9. The amino acid sequence of L-CDR1 includes a sequence that differs from sequence number 21, or from sequence number 21 by one or two substitutions, deletions, or additions. The amino acid sequence of L-CDR2 includes a sequence that differs from SEQ ID NO: 22, or a sequence that differs from SEQ ID NO: 22 by one or two substitutions, deletions, or additions. The anti-HIV-1 antibody according to any one of claims 1 to 6, wherein the amino acid sequence of L-CDR3 comprises a sequence different from SEQ ID NO: 23, or a sequence with one or two substitutions, deletions, or additions from SEQ ID NO:

23.

10. The amino acid sequence of H-CDR1 includes a sequence that differs from sequence number 24, or a sequence that differs from sequence number 24 by one or two substitutions, deletions, or additions. The amino acid sequence of H-CDR2 includes a sequence that differs from SEQ ID NO: 25, or a sequence that differs from SEQ ID NO: 25 by one or two substitutions, deletions, or additions. The anti-HIV-1 antibody according to any one of claims 1 to 7, wherein the amino acid sequence of H-CDR3 comprises a sequence different from SEQ ID NO: 26, or a sequence with one or two substitutions, deletions, or additions from SEQ ID NO:

26.

11. The amino acid sequence of H-CDR1 includes a sequence that differs from sequence number 27, or from sequence number 27 by one or two substitutions, deletions, or additions. The amino acid sequence of H-CDR2 includes a sequence that differs from SEQ ID NO: 28, or a sequence that differs from SEQ ID NO: 28 by one or two substitutions, deletions, or additions. The anti-HIV-1 antibody according to any one of claims 1 to 6 or 8, wherein the amino acid sequence of H-CDR3 comprises a sequence different from SEQ ID NO: 29, or a sequence with one or two substitutions, deletions, or additions from SEQ ID NO:

29.

12. The amino acid sequence of H-CDR1 includes a sequence that differs from 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 includes a sequence that differs from SEQ ID NO: 31, or a sequence that differs from SEQ ID NO: 31 by one or two substitutions, deletions, or additions. The anti-HIV-1 antibody according to any one of claims 1 to 6 or 9, wherein the amino acid sequence of H-CDR3 comprises a sequence different from SEQ ID NO: 32, or a sequence with one or two substitutions, deletions, or additions from SEQ ID NO:

32.

13. The amino acid sequence of L-CDR1 includes a sequence that differs from sequence number 15, or from sequence number 15 by one or two substitutions, deletions, or additions. The amino acid sequence of L-CDR2 includes a sequence that differs from SEQ ID NO: 16, or from SEQ ID NO: 16 by one or two substitutions, deletions, or additions. The amino acid sequence of L-CDR3 includes a sequence that differs from SEQ ID NO: 17, or from SEQ ID NO: 17 by one or two substitutions, deletions, or additions. The amino acid sequence of H-CDR1 includes a sequence that differs from sequence number 24, or a sequence that differs from sequence number 24 by one or two substitutions, deletions, or additions. The amino acid sequence of H-CDR2 includes a sequence that differs from SEQ ID NO: 25, or a sequence that differs from SEQ ID NO: 25 by one or two substitutions, deletions, or additions. The anti-HIV-1 antibody according to any one of claims 1 to 6, wherein the amino acid sequence of H-CDR3 comprises a sequence different from SEQ ID NO: 26, or a sequence with one or two substitutions, deletions, or additions from SEQ ID NO:

26.

14. The amino acid sequence of L-CDR1 includes a sequence that differs from sequence number 18, or from sequence number 18 by one or two substitutions, deletions, or additions. The amino acid sequence of L-CDR2 includes a sequence that differs from sequence number 19, or from sequence number 19 by one or two substitutions, deletions, or additions. The amino acid sequence of L-CDR3 includes a sequence that differs from 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 includes a sequence that differs from sequence number 27, or from sequence number 27 by one or two substitutions, deletions, or additions. The amino acid sequence of H-CDR2 includes a sequence that differs from SEQ ID NO: 28, or a sequence that differs from SEQ ID NO: 28 by one or two substitutions, deletions, or additions. The anti-HIV-1 antibody according to any one of claims 1 to 6, wherein the amino acid sequence of H-CDR3 comprises a sequence different from SEQ ID NO: 29, or a sequence with one or two substitutions, deletions, or additions from SEQ ID NO:

29.

15. The amino acid sequence of L-CDR1 includes a sequence that differs from sequence number 21, or from sequence number 21 by one or two substitutions, deletions, or additions. The amino acid sequence of L-CDR2 includes a sequence that differs from 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 includes sequences different from sequence number 23, or sequences with one or two substitutions, deletions, or additions to sequence number 23. The amino acid sequence of H-CDR1 includes a sequence that differs from 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 includes a sequence that differs from SEQ ID NO: 31, or a sequence that differs from SEQ ID NO: 31 by one or two substitutions, deletions, or additions. The anti-HIV-1 antibody according to any one of claims 1 to 6, wherein the amino acid sequence of H-CDR3 comprises a sequence different from SEQ ID NO: 32, or a sequence with one or two substitutions, deletions, or additions from SEQ ID NO:

32.

16. The anti-HIV-1 antibody according to any one of claims 1 to 6, wherein 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.

17. The anti-HIV-1 antibody according to any one of claims 1 to 6, wherein 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.

18. The anti-HIV-1 antibody according to any one of claims 1 to 6, wherein 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.

19. An anti-HIV-1 antibody according to any one of claims 1 to 18, which is a monoclonal antibody or a recombinant antibody.

20. An anti-HIV-1 antibody according to any one of claims 1 to 19, which is an antibody fragment.

21. Antibody fragments include variable fragments (Fv), single-stranded Fv (scFv), and bispecific antibodies (sc(Fv)). 2 ), single-chain antibody, single-domain antibody, Fab fragment, F(ab') 2 An anti-HIV-1 antibody according to claim 20, selected from a 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.

22. An anti-HIV-1 antibody according to any one of claims 1 to 21, comprising a constant region of the mouse IgG1 class or the mouse IgG2a class.

23. An anti-HIV-1 antibody according to any one of claims 1 to 22, which is bound to a solid support.

24. A cell comprising the anti-HIV-1 antibody according to any one of claims 1 to 23.

25. A nucleic acid comprising a nucleotide sequence encoding an anti-HIV-1 antibody according to any one of claims 1 to 20, a promoter operably linked to the nucleotide sequence, and a selection marker.

26. A cell containing the nucleic acid described in claim 25.

27. A composition comprising an anti-HIV-1 antibody according to any one of claims 1 to 20, wherein the anti-HIV-1 antibody is covalently or noncovalently bound to the solid support, and a solid support.

28. The composition according to claim 27, wherein the solid support comprises particles, beads, a membrane, a surface, a polypeptide chip, a microtiter plate, or a solid phase of a chromatography column.

29. A kit for detecting the presence of HIV-1 in a sample, wherein the kit comprises at least one anti-HIV-1 antibody as described in any one of claims 1 to 23, and a solid support, wherein the at least one anti-HIV-1 antibody is covalently or noncovalently bound to the solid support.

30. An anti-HIV-1 antibody characterized by its specific binding to the epitope of the HIV-1 p24 protein containing the amino acid sequence of SEQ ID NO:

33.

31. The amino acid sequence of L-CDR1 includes a sequence that differs from sequence number 21, or from sequence number 21 by one or two substitutions, deletions, or additions. The amino acid sequence of L-CDR2 includes a sequence that differs from SEQ ID NO: 22, or a sequence that differs from SEQ ID NO: 22 by one or two substitutions, deletions, or additions. The anti-HIV-1 antibody according to claim 30, wherein the amino acid sequence of L-CDR3 comprises a sequence different from SEQ ID NO: 23, or a sequence with one or two substitutions, deletions, or additions from SEQ ID NO:

23.

32. The amino acid sequence of H-CDR1 includes a sequence that differs from 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 includes a sequence that differs from SEQ ID NO: 31, or a sequence that differs from SEQ ID NO: 31 by one or two substitutions, deletions, or additions. The anti-HIV-1 antibody according to any one of claims 30 to 31, wherein the amino acid sequence of H-CDR3 comprises a sequence different from SEQ ID NO: 32, or a sequence with one or two substitutions, deletions, or additions from SEQ ID NO: 32.

Images