Broad spectrum neutralizing antibodies targeting V3 glycan sites on HIV ENV

Abstract

The present invention relates to monoclonal human antibodies or binding fragments thereof directed against the V3 glycan site of human immunodeficiency virus HIV-1, pharmaceutical compositions comprising such monoclonal human antibodies or binding fragments thereof, kits comprising such antibodies or binding fragments thereof, pharmaceutical compositions comprising such antibodies or binding fragments thereof, pharmaceutical compositions comprising such antibodies or binding fragments thereof, pharmaceutical compositions comprising such antibodies or binding fragments thereof, pharmaceutical compositions comprising such antibodies or binding fragments thereof, and pharmaceutical compositions comprising such antibodies or binding fragments thereof. And monoclonal antibodies or binding fragments thereof as well as pharmaceutical compositions and kits for use as medicaments and for the treatment or prevention of diseases caused by human immunodeficiency virus HIV-1.

Description

Technical Field

[0001] This invention relates to monoclonal human antibodies or their binding fragments targeting the V3 glycan site of human immunodeficiency virus HIV-1, pharmaceutical compositions comprising such monoclonal human antibodies or their binding fragments, kits comprising such antibodies or their binding fragments, and monoclonal antibodies or their binding fragments, pharmaceutical compositions, and kits used as pharmaceuticals and for the treatment or prevention of diseases caused by human immunodeficiency virus HIV-1. Background Technology

[0002] Despite the widespread use of effective antiretroviral therapy (ART), the fight against the global HIV-1 epidemic remains a public health priority. Highly effective broad-spectrum neutralizing anti-HIV-1 antibodies (bNAbs) represent a promising and powerful tool that opens unprecedented opportunities for alternative therapies and prevention strategies. In recent years, effective bNAbs have been isolated from HIV-1-infected donors, binding to various vulnerable epitopes on the HIV-1 envelope (Env) trimer. These epitopes include CD4 binding sites (CD4bs), V1 / V2 loops, V3 glycan sites, the proximal outer region of the membrane, fusion peptides, silencing surfaces, and the interface between the gp120 and gp41 Env subunits.

[0003] Among these epitopes, the V3 glycan site is a major site of viral vulnerability. This site acts as the binding site for the CCR5 co-receptor and mediates viral entry. The V3 glycan site is located at the base of the env V3 loop region and spans between two N-linked glycans at amino acid positions 301 and 332 in HIV-1. This region also includes the so-called “GDIR” amino acid motif (positions 324-327), which is the primary target of the V3 glycan site bNAb.

[0004] Members of the anti-HIV-1 bNAb class are not limited to specific heavy chain variable gene segments and typically exhibit a very long heavy chain complementarity-determining region (CDRH3) of 18 to 24 amino acids. The long CDRH3 enables V3 glycan site bNAbs to penetrate the glycan barrier of the env trimer and access the target amino acid residues below.

[0005] Importantly, the antiviral activity of all known V3 glycan site bNAbs isolated to date is highly dependent on the incorporation of the glycan at position 332 into the binding epitope. Therefore, removal of the glycan at position 332 impairs the neutralizing capacity and breadth of this previously described V3 glycan site bNAb. Representative members of this antibody class are 10-1074, BG18, BF520PGT121, PGT128, and PGT135.

[0006] In preclinical animal models, bNAb exhibited robust anti-infective protection even at low serum concentrations and has demonstrated favorable safety and pharmacokinetic characteristics. Furthermore, administration of bNAb to individuals infected with HIV-1 resulted in prolonged suppression of viremia and delayed viral rebound following analytical treatment interruption (ATI).

[0007] The most potent and broadest-spectrum neutralizing antibodies against HIV-1 target the CD4 binding site (CD4bs). CD4bs are particularly relevant because CD4 acts as the primary receptor for viral entry. However, bNAbs targeting other sites within the HIV-1 virus are also important because they can supplement the neutralization of certain viral strains by non-competitively inhibiting CD4bs bNAbs, and even improve neutralization of other viral strains due to their binding to different target sites.

[0008] Therefore, the identification and development of highly effective V3 glycan site bNAbs are highly relevant to anti-HIV-1 immunotherapy and prevention strategies. These bNAbs need to have enhanced neutralizing potency and breadth. Therefore, there remains an urgent need to isolate novel V3 glycan sites with antiviral activity exceeding that of known bNAbs targeting this epitope.

[0009] Therefore, the object of the present invention is to provide a novel human monoclonal antibody targeting HIV-1 that exhibits significant levels of breadth and potency against a wide range of viral strains and subtypes, and can be combined with known antibodies to further improve neutralization efficiency.

[0010] Furthermore, another object of the present invention is to provide a novel human monoclonal antibody against the V3 glycan site of HIV-1, which exhibits improved breadth and / or potency in neutralizing various viral strains and subtypes. Summary of the Invention

[0011] These objectives have been achieved through the various aspects of the invention as specified below.

[0012] According to a first aspect of the invention, an antibody or antigen-binding fragment thereof targeting the V3 glycan site of human immunodeficiency virus HIV-1 is provided, wherein the antibody or antigen-binding fragment thereof comprises a combination of a variable region heavy chain comprising heavy chains CDR1 to CDR3 and a variable region light chain comprising the amino acid sequences of light chains CDR1 to CDR3 of an antibody selected from the group consisting of: 007 (having the CDR-H1 amino acid sequence of SEQ ID No. 1, the CDR-H2 amino acid sequence of SEQ ID No. 2, the CDR-H3 amino acid sequence of SEQ ID No. 3, the CDR-L1 amino acid sequence of SEQ ID No. 4, the CDR-L2 amino acid sequence of SEQ ID No. 5, and the CDR-L3 amino acid sequence of SEQ ID No. 6), 03_A07 (having the CDR-H1 amino acid sequence of SEQ ID No. 7, the CDR-H2 amino acid sequence of SEQ ID No. 8, the CDR-H3 amino acid sequence of SEQ ID No. 9, the CDR-L1 amino acid sequence of SEQ ID No. 10, and the CDR-L3 amino acid sequence of SEQ ID No. 6), SEQ ID No. 7, the CDR-H2 amino acid sequence of SEQ ID No. 8, the CDR-H3 amino acid sequence of SEQ ID No. 9, and the CDR-L1 amino acid sequence of SEQ ID No. 10, and the CDR-L3 ... The following amino acids are listed: SEQ ID No. 11 (CDR-L2 amino acid sequence and SEQ ID No. 12 (CDR-L3 amino acid sequence)); 01_G04 (containing SEQ ID No. 13 (CDR-H1 amino acid sequence), SEQ ID No. 14 (CDR-H2 amino acid sequence), SEQ ID No. 15 (CDR-H3 amino acid sequence), SEQ ID No. 16 (CDR-L1 amino acid sequence), SEQ ID No. 17 (CDR-L2 amino acid sequence), and SEQ ID No. 18 (CDR-L3 amino acid sequence)); and 01_C10 (containing SEQ ID No. 19 (CDR-H1 amino acid sequence), SEQ ID No. 20 (CDR-H2 amino acid sequence), SEQ ID No. 21 (CDR-H3 amino acid sequence), SEQ ID No. 22 (CDR-L1 amino acid sequence), SEQ ID No. 23 (CDR-L2 amino acid sequence), and SEQ ID No. 24 (CDR-L3 amino acid sequence).

[0013] According to a preferred embodiment of the first aspect of the invention, the antibody or its antigen-binding fragment comprises a combination of a variable region heavy chain amino acid sequence and a variable region light chain amino acid sequence selected from the group consisting of: 007 (containing the amino acid sequences of SEQ ID No. 25 and SEQ ID No. 26, respectively), 03_A07 (containing the amino acid sequences of SEQ ID No. 27 and SEQ ID No. 28, respectively), 01_G04 (containing the amino acid sequences of SEQ ID No. 29 and SEQ ID No. 30, respectively), and 01_C10 (containing the amino acid sequences of SEQ ID No. 31 and SEQ ID No. 32, respectively).

[0014] According to a preferred embodiment of a first aspect of the invention, the antibody or its antigen-binding fragment comprises a combination of a variable region heavy chain amino acid sequence and a variable region light chain amino acid sequence selected from the group consisting of: 007 (composed of the amino acid sequences of SEQ ID No. 25 and SEQ ID No. 26, respectively), 03_A07 (composed of the amino acid sequences of SEQ ID No. 27 and SEQ ID No. 28, respectively), 01_G04 (composed of the amino acid sequences of SEQ ID No. 29 and SEQ ID No. 30, respectively), and 01_C10 (composed of the amino acid sequences of SEQ ID No. 31 and SEQ ID No. 32, respectively).

[0015] According to the first aspect of the present invention or a preferred embodiment of the foregoing embodiments, the amino acid sequence contained therein is an antibody selected from the group consisting of 007, 03_A07 and 01-G04, preferably an antibody selected from the group consisting of 007 and 03_A07, and more preferably antibody 007.

[0016] According to a second aspect of the invention, a pharmaceutical composition is provided comprising an antibody or an antigen-binding fragment thereof according to a first aspect of the invention, and at least one pharmaceutically acceptable excipient.

[0017] According to a preferred embodiment of the second aspect of the invention, the pharmaceutical composition is a therapeutic composition for use in human subjects.

[0018] According to a preferred embodiment of the second aspect of the invention, the pharmaceutical composition is a preventative composition for use in human subjects.

[0019] According to a further preferred embodiment of the second aspect of the invention, the pharmaceutical composition is a passive immunization composition for human subjects.

[0020] According to a preferred embodiment of the second aspect of the invention, the pharmaceutical composition is a vaccination composition for human subjects.

[0021] As used herein, "vaccination composition" means a pharmaceutical composition of the second aspect of the invention capable of providing active and / or passive immunity. "Active immunity" as used herein means inducing or enhancing an immune response in a subject to an antigen. "Passive immunity" as used herein and preferably means supplementing an antigen or pathogen immune response in a subject by providing antibodies and / or antigen-binding portions thereof that neutralize the antigen.

[0022] According to a second aspect of the invention or a preferred embodiment of the foregoing embodiments, the pharmaceutical composition further comprises at least one antibody targeting the CD4 binding site of HIV-1.

[0023] According to a third aspect of the present invention, a kit is provided comprising an antibody or antigen-binding fragment thereof according to a first aspect of the present invention, and a container.

[0024] According to a fourth aspect of the invention, antibodies or antigen-binding fragments thereof according to the first aspect of the invention, pharmaceutical compositions according to the second aspect of the invention, or kits according to the third aspect of the invention are provided for use as pharmaceuticals.

[0025] In a preferred embodiment of the fourth aspect of the invention, the use is in human subjects. In another preferred embodiment of the fourth aspect of the invention, the use is for imparting a preventive benefit to a subject and / or in the form of a composition capable of imparting a preventive benefit to a subject. In another preferred embodiment of the fourth aspect of the invention, the use is for imparting passive immunization to a subject and / or in the form of a composition capable of imparting passive immunization to a subject. In yet another embodiment of the fourth aspect of the invention, the use is for treating or preventing acquired immunodeficiency syndrome (AIDS).

[0026] According to a preferred embodiment of the fourth aspect, the antibody or antigen-binding fragment thereof according to the first aspect of the invention, or the pharmaceutical composition according to the second aspect of the invention, can be packaged into a single dosage form for parenteral administration. In a preferred embodiment, the antibody or antigen-binding fragment thereof or the pharmaceutical composition of the invention can be packaged into a single dosage form for intramuscular, intravenous, or subcutaneous administration.

[0027] According to a fifth aspect of the invention, an antibody or antigen-binding fragment thereof according to a first aspect of the invention, a pharmaceutical composition according to a second aspect of the invention, or a kit according to a third aspect of the invention are provided for use in the treatment or prevention of human immunodeficiency virus HIV-1 infection in mammalian subjects.

[0028] According to a sixth aspect of the invention, an antibody or antigen-binding fragment thereof according to a first aspect of the invention, a pharmaceutical composition according to a second aspect of the invention, or a kit according to a third aspect of the invention are provided for use in the treatment or prevention of disease caused by human immunodeficiency virus HIV-1 in mammalian subjects.

[0029] In a preferred embodiment of the fifth or sixth aspect of the invention, the use is performed on human subjects.

[0030] In another preferred embodiment of the fifth or sixth aspect of the invention, the use shall include intramuscular, subcutaneous or intravenous administration of an antibody or antigen-binding fragment thereof according to the first aspect of the invention, or a pharmaceutical composition according to the second aspect of the invention.

[0031] In a preferred embodiment of the fifth or sixth aspect of the invention, the use is for the treatment or prevention of acquired immunodeficiency syndrome (AIDS).

[0032] According to a seventh aspect of the invention, a nucleic acid is provided that encodes an antibody or an antigen-binding fragment thereof according to a first aspect of the invention. In one embodiment, the nucleic acid comprises DNA or RNA, such as mRNA. In one embodiment, the nucleic acid is codon-optimized for expression in a host cell.

[0033] According to an eighth aspect of the invention, an expression vector is provided that comprises a nucleic acid of the seventh aspect of the invention functionally associated with an expression control sequence. In one embodiment, the expression vector is a viral vector.

[0034] According to a ninth aspect of the invention, a host cell is provided comprising a nucleic acid according to a seventh aspect of the invention or an expression vector according to an eighth aspect of the invention. Examples of host cells that can be used include eukaryotic cells, such as yeast cells, mammalian cells, insect cells, and plant cells; and prokaryotic cells, including *Escherichia coli* (…). E. coli In one implementation, the mammalian cell is a human cell, such as a human B cell. The nucleic acid may be heterologous to the host cell.

[0035] According to a tenth aspect of the present invention, a method for preparing an antibody or antigen-binding fragment thereof of the first aspect of the present invention is provided, the method comprising culturing a host cell of the ninth aspect of the present invention under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof. Attached Figure Description

[0036] Figure 1 The neutralizing activity of the antibodies of the present invention against a group of 12 HIV-1 global reference pseudovirus strains with different envelope amino acid sequences (such as de Camp et al.) is shown when tested in a TZM-bl cell pseudovirus neutralization assay (see the Examples section below). J Virol. (As described in March 2014; 88(5): 2489–2507); All antibodies according to the invention described herein neutralized 100% (12 / 12) of the “global group” strains and showed high levels of potency; Neutralization data for reference bNAb PGT128, PGT121 and 10-1074 against the V3 glycan site were also retrieved from the antibody neutralization database CATNAP (Yoon et al., Nucleic Acids Research, Vol. 43, No. W1, July 1, 2015, pp. W213–W219, https: / / doi.org / 10.1093 / nar / gkv404).

[0037] Figure 2 The neutralizing activity (IC50) of antibody 007 of the present invention against multiple clades of 119 pseudoviruses with different HIV-1 envelope amino acid sequences was demonstrated when tested in a TZM-bl cell pseudovirus neutralization assay. 50 (as cited in whole by Schoofs et al., Immunity, June 18, 2019; 50(6):1513-1529.e9 and further described below); data for antibody 007 are shown compared to the most effective and extensive V3 glycan site bNAb (10-1074, BG18, PGT121, PGT128, PGT130 and PGT135) in the prior art; for presentation in this figure, 114 pseudovirus strains from 119 polyphyletic clades were selected, for which neutralization data for reference antibodies have been published; neutralization data for reference bNAb were retrieved from the antibody neutralization database CATNAP (Yoon et al., 2015, ibid.).

[0038] Figure 3 The invention's antibody 007 demonstrated neutralizing activity (IC50) against multiple clades of pseudoviruses with different HIV-1 envelope amino acid sequences, compared to prior art anti-HIV-1 antibodies, when tested in a TZM-bl cell pseudovirus neutralization assay.50 and breadth) (e.g. Figure 2 As shown, and as Schoofs et al., Immunity, June 18, 2019; 50(6):1513-1529.e9 and further described below); for the purposes of this figure, 114 pseudovirus strains from 119 polyphyletic clades were selected, of which neutralization data for reference antibodies have been published; Figure 3 The neutralizing activity of antibody 007 was presented compared with that of the most potent and broadest anti-HIV-1 bNAb targeting the V3 glycan site in existing technologies; neutralization data for bNAb were retrieved from the antibody neutralization database CATNAP (Yoon et al., 2015, ibid.).

[0039] Figure 4 The neutralizing activity (IC50) of the antibody of the present invention against a group of 12 pseudoviruses with different HIV-1 envelope amino acid sequences was demonstrated when tested in a TZM-bl cell pseudovirus neutralization assay. 50 ); It is known that the pseudoviruses in this group exhibit excellent resistance to the V3 glycan site bNAb; the viral strains in this group are part of the 119 multi-clade and the global pseudovirus group (e.g., Schoofs et al., Immunity, June 18, 2019; 50(6):1513-1529.e9; de Camp et al., J Virol. March 2014; 88(5): 2489–2507); data for antibodies 007, 01_C10, 01_G04 and 03_A07 are shown compared with the most effective and extensive V3 glycan site bNAb (10-1074, BG18, PGT121, PGT128, PGT130 and PGT135) in the prior art; neutralization data for bNAb were retrieved from the antibody neutralization database CATNAP (Yoon et al., 2015, ibid.).

[0040] Figure 5 The binding activity of the antibodies of the present invention (007, 01_C10, 01_G04, and 03_A07) against the BG505.SOSIP.664 HIV-1 envelope trimer is shown in the presence of reference bNAbs from known epitope classes (described in Schoofs et al., Immunity, June 18, 2019; 50(6):1513-1529.e9 and Schommers et al., Cell, February 6, 2020; 180(3):471-489.e22). The binding activity of all studied bNAbs was competitively inhibited only by V3 glycan bNAb BG18 and / or 10-1074, indicating that the V3 glycan site represents the binding epitope of the analyzed bNAb. Detailed Implementation

[0041] The inventors dedicated themselves to solving the problems of this invention and successfully discovered a novel and useful human monoclonal antibody against HIV-1, particularly against the V3 glycan site of HIV-1, overcoming the shortcomings and deficiencies of known antibodies.

[0042] In this paper, the inventors describe novel V3 glycan site antibodies that surpass the potency and breadth of known V3 glycan site bNAbs, such as antibodies 10-1074, BG18, PGT121, PGT128, PGT130, and PGT135. These properties open up unprecedented and new possibilities for antibody-based therapies and the prevention of HIV-1-related diseases.

[0043] Therefore, in a first aspect, the present invention provides an antibody or antigen-binding fragment thereof targeting the V3 glycan site of human immunodeficiency virus HIV-1, wherein the antibody or antigen-binding fragment thereof comprises a combination of a variable region heavy chain comprising heavy chains CDR1 to CDR3 and a variable region light chain comprising the amino acid sequences of light chains CDR1 to CDR3 of an antibody selected from the group consisting of: 007 (having the CDR-H1 amino acid sequence of SEQ ID No. 1, the CDR-H2 amino acid sequence of SEQ ID No. 2, the CDR-H3 amino acid sequence of SEQ ID No. 3, the CDR-L1 amino acid sequence of SEQ ID No. 4, the CDR-L2 amino acid sequence of SEQ ID No. 5, and the CDR-L3 amino acid sequence of SEQ ID No. 6), 03_A07 (having the CDR-H1 amino acid sequence of SEQ ID No. 7, the CDR-H2 amino acid sequence of SEQ ID No. 8, the CDR-H3 amino acid sequence of SEQ ID No. 9, the CDR-L1 amino acid sequence of SEQ ID No. 10, and the CDR-L3 amino acid sequence of SEQ ID No. 6), SEQ ID No. 7, the CDR-H2 amino acid sequence of SEQ ID No. 8, the CDR-H3 amino acid sequence of SEQ ID No. 9, and the CDR-L1 amino acid sequence of SEQ ID No. 10, SEQ ID No. 10, and the CDR-L3 ... The following amino acids were identified: SEQ ID No. 11 (CDR-L2 amino acid sequence and SEQ ID No. 12 CDR-L3 amino acid sequence), 01_G04 (with SEQ ID No. 13 CDR-H1 amino acid sequence, SEQ ID No. 14 CDR-H2 amino acid sequence, SEQ ID No. 15 CDR-H3 amino acid sequence, SEQ ID No. 16 CDR-L1 amino acid sequence, SEQ ID No. 17 CDR-L2 amino acid sequence and SEQ ID No. 18 CDR-L3 amino acid sequence), and 01_C10 (with SEQ ID No. 19 CDR-H1 amino acid sequence, SEQ ID No. 20 CDR-H2 amino acid sequence, SEQ ID No. 21 CDR-H3 amino acid sequence, SEQ ID No. 22 CDR-L1 amino acid sequence, SEQ ID No. 23 CDR-L2 amino acid sequence and SEQ ID No. 24 CDR-L3 amino acid sequence).

[0044] In the context of this invention, the antibodies generated and described herein may be used and claimed as complete monoclonal human antibodies or as any functional or binding fragment thereof. Preferably, the monoclonal human antibody or any type of functional or antigen-binding fragment thereof should contain at least the complementarity-determining regions (CDRs) 1 to 3 of the heavy chain and the CDRs 1 to 3 of the light chain of the human monoclonal antibody.

[0045] The CDR region of the antibody sequence described herein is preferably defined according to the IMGT numbering scheme, which is an adaptation of the Chothia numbering scheme (ImMunoGeneTics information system). ® ;Riechmann et al., NAR 27: 209-212 (1999); http: / / www.imgt.org).

[0046] According to the present invention, the CDR sequences of the light chain and heavy chain variable regions of the antibodies and their antigen-binding fragments described herein are as follows:

[0047] According to the present invention, the light chain and heavy chain variable region sequences of the preferred antibodies and their antigen-binding fragments having internal names 007, 03_A07, 01_G04 and 01_C10 described herein are as follows:

[0048] According to one embodiment of the present invention, the antibody or its antigen-binding fragment comprises the heavy chain variable region amino acid sequence of antibody 007 (SEQ ID No. 25). According to one embodiment of the present invention, the antibody or its antigen-binding fragment comprises the light chain variable region amino acid sequence of antibody 007 (SEQ ID No. 26). According to one embodiment of the present invention, the antibody or its antigen-binding fragment comprises the heavy chain variable region amino acid sequence of antibody 03_A07 (SEQ ID No. 27). According to one embodiment of the present invention, the antibody or its antigen-binding fragment comprises the light chain variable region amino acid sequence of antibody 03_A07 (SEQ ID No. 28). According to one embodiment of the present invention, the antibody or its antigen-binding fragment comprises the heavy chain variable region amino acid sequence of antibody 01_G04 (SEQ ID No. 29). According to one embodiment of the present invention, the antibody or its antigen-binding fragment comprises the light chain variable region amino acid sequence of antibody 01_G04 (SEQ ID No. 30). According to one embodiment of the present invention, the antibody or its antigen-binding fragment comprises the heavy chain variable region amino acid sequence of antibody 01_C10 (SEQ ID No. 31). According to an embodiment of the present invention, the antibody or its antigen-binding fragment comprises the amino acid sequence of the light chain variable region of antibody O1_C10 (SEQ ID No. 32).

[0049] According to a preferred embodiment of the present invention, the antibody or antigen-binding fragment comprises the heavy chain variable region amino acid sequence of SEQ ID No. 25 and the light chain variable region amino acid sequence of SEQ ID No. 26, or the antibody or antigen-binding fragment comprises the heavy chain variable region amino acid sequence of SEQ ID No. 27 and the light chain variable region amino acid sequence of SEQ ID No. 28, or the antibody or antigen-binding fragment comprises the heavy chain variable region amino acid sequence of SEQ ID No. 29 and the light chain variable region amino acid sequence of SEQ ID No. 30, or the antibody or antigen-binding fragment comprises the heavy chain variable region amino acid sequence of SEQ ID No. 31 and the light chain variable region amino acid sequence of SEQ ID No. 32.

[0050] According to one specific embodiment of the present invention, the antibody is composed of two heavy chains each containing the amino acid sequence of SEQ ID No. 25 and two light chains each containing the amino acid sequence of SEQ ID No. 26; or the antibody is composed of two heavy chains each containing the amino acid sequence of SEQ ID No. 27 and two light chains each containing the amino acid sequence of SEQ ID No. 28; or the antibody is composed of two heavy chains each containing the amino acid sequence of SEQ ID No. 29 and two light chains each containing the amino acid sequence of SEQ ID No. 30; or the antibody is composed of two heavy chains each containing the amino acid sequence of SEQ ID No. 31 and two light chains each containing the amino acid sequence of SEQ ID No. 32.

[0051] According to another specific embodiment of the present invention, the antibody is composed of two heavy chains, each consisting of an amino acid sequence of SEQ ID No. 25 and two light chains, each consisting of an amino acid sequence of SEQ ID No. 26; or the antibody is composed of two heavy chains, each consisting of an amino acid sequence of SEQ ID No. 27 and two light chains, each consisting of an amino acid sequence of SEQ ID No. 28; or the antibody is composed of two heavy chains, each consisting of an amino acid sequence of SEQ ID No. 29 and two light chains, each consisting of an amino acid sequence of SEQ ID No. 30; or the antibody is composed of two heavy chains, each consisting of an amino acid sequence of SEQ ID No. 31 and two light chains, each consisting of an amino acid sequence of SEQ ID No. 32.

[0052] According to a preferred embodiment of the present invention, the antibody used as the source of the sequence contained in the antibody or its antigen-binding fragment according to the present invention is selected from the group consisting of 007, 03_A07 and 01_G04, more preferably from an antibody consisting of the group consisting of 007 and 03_A07, and particularly preferably antibody 007.

[0053] In one embodiment of the present invention, the antibody used as the source of the sequence contained in the antibody of the present invention is 007. In another embodiment of the present invention, the antibody used as the source of the sequence contained in the antibody of the present invention is 03_A07. In one embodiment of the present invention, the antibody used as the source of the sequence contained in the antibody of the present invention is 01_G04. In another embodiment of the present invention, the antibody used as the source of the sequence contained in the antibody of the present invention is 01_C10.

[0054] In a preferred embodiment, the antibody is a monoclonal antibody or fragment thereof that retains the ability to bind specifically and neutralize infectious pathogens. In a preferred embodiment, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. For example, the antibody may be an antibody containing the Fc domain of any human IgG isotype (e.g., IgG1, IgG2, IgG3, or IgG4).

[0055] According to the present invention, the human IgG1 Fc domain (CH2-CH3), κ and λ light chain constant domains (CL), human IgG1 CH1 domain, and human IgG1 hinge domain of the antibody and its antigen-binding fragment described herein are as follows:

[0056] In one embodiment, the antibody or its antigen-binding fragment further comprises an Fc domain. In a preferred embodiment, the Fc domain is a human IgG1 Fc domain (CH2-CH3). In a preferred embodiment, the human IgG1 Fc domain (CH2-CH3) comprises the amino acid sequences of SEQ ID NO:33 and SEQ ID NO:34. In a preferred embodiment, the human IgG1 Fc domain (CH2-CH3) comprises the amino acid sequences of SEQ ID NO:33 and SEQ ID NO:35. In another embodiment, the human IgG1 Fc domain (CH2-CH3) consists of the amino acid sequences of SEQ ID NO:33 and SEQ ID NO:34. In yet another embodiment, the human IgG1 Fc domain (CH2-CH3) consists of the amino acid sequences of SEQ ID NO:33 and SEQ ID NO:35.

[0057] It should be understood that, for example, production in mammalian cell lines may remove one or more C-terminal lysine residues from the antibody heavy chain (see, for example, Liu et al., mAbs 6(5):1145-1154 (2014)). Therefore, the antibody or antigen-binding fragment of this disclosure may comprise a heavy chain, CH1-hinge-CH2-CH3, hinge-CH2-CH3, CH2-CH3, or CH3 polypeptide, wherein a C-terminal lysine residue may or may not be present; in other words, embodiments in which the C-terminal residue of the heavy chain, CH1-hinge-CH2-CH3, hinge-CH2-CH3, CH2-CH3, or CH3 polypeptide is not a lysine, and embodiments in which a lysine residue is a C-terminal residue.

[0058] In one embodiment, the antibody or its antigen-binding fragment further comprises a light chain constant domain (CL), preferably a human CL domain. In a preferred embodiment, the light chain constant domain is a human κ light chain constant domain. In a preferred embodiment, the light chain constant domain is a human λ light chain constant domain. In a preferred embodiment, the human κ light chain constant domain comprises the amino acid sequence of SEQ ID NO:36. In another preferred embodiment, the human κ light chain constant domain consists of the amino acid sequence of SEQ ID NO:36. In a preferred embodiment, the human λ light chain constant domain comprises the amino acid sequence of SEQ ID NO:37. In another preferred embodiment, the human λ light chain constant domain consists of the amino acid sequence of SEQ ID NO:37.

[0059] In one embodiment, the antibody or its antigen-binding fragment further comprises a hinge domain, preferably an IgG1 hinge domain, more preferably a human IgG1 hinge domain. In a preferred embodiment, the human IgG1 hinge domain comprises the amino acid sequence of SEQ ID No. 38. In another preferred embodiment, the human IgG1 hinge domain consists of the amino acid sequence of SEQ ID No. 38.

[0060] In one embodiment, the antibody or its antigen-binding fragment further comprises a heavy chain constant domain 1 (CH1), preferably a human CH1 domain. In a preferred embodiment, the human IgG1 CH1 domain comprises the amino acid sequence of SEQ ID No. 39. In another preferred embodiment, the human IgG1 CH1 domain consists of the amino acid sequence of SEQ ID No. 39. In one embodiment, the antibody or its antigen-binding fragment further comprises an Fc domain, a CH1 domain, a CL1 domain, a hinge domain, or any combination thereof. The Fc domain, CH1 domain, CL1 domain, hinge domain, or any combination thereof may be a human IgG isotype, preferably the same isotype.

[0061] Optionally, the antigen-binding fragment consists of or includes the following: Fab, Fab', Fab'-SH, F(ab)2, Fv, biantibody, single-chain antibody fragment, or multispecific antibody containing multiple different antibody fragments.

[0062] In this invention, an antibody or binding fragment targeting the V3 glycan site of HIV-1 means an antibody that binds to the V3 glycan site region within the gp120 envelope glycoprotein of HIV-1 with an affinity at least 10-fold, more preferably at least 50-fold, and particularly preferably at least 100-fold increased compared to unrelated epitopes, proteins, or protein regions. Generally, the term V3 glycan site refers herein to the V3 glycan site region within the gp120 envelope glycoprotein of HIV-1.

[0063] Generally, monoclonal human antibodies or their binding fragments, as described herein, also include antibody amino acid sequences that are at least 80% identical to the sequences defined above, provided they still target the V3 glycan site of human immunodeficiency virus HIV-1. This means sequences that include minor mutations in the antibody amino acid sequence that do not interfere with structural folding and antibody affinity for the V3 glycan site.

[0064] Typically and preferably, such mutations that do not substantially interfere with the structure and function of the antibody are located outside the antibody's CDR; more preferably, such mutations are located outside the antibody's CDR and outside the frame region. Therefore, it is preferable that, if present, the antibody amino acid sequence, which is at least 80% identical to the defined sequence, does not carry mutations in the CDR, and preferably does not carry mutations in the antibody's CDR or frame region.

[0065] Methods for determining the ability of antibodies with a certain degree of sequence identity to bind to the V3 glycan site of human immunodeficiency virus HIV-1 are known.

[0066] According to the present invention, the percentage of identity between two sequences is determined using a mathematical algorithm developed by Karlin and Altschul (Proc. Natl. Acad. Sci. USA (1993) 90: 5873-5877). This algorithm is based on the BLASTN and BLASTP procedures of Altschul et al. (J. Mol. Biol. (1990) 215: 403-410). BLAST nucleotide search is performed using the BLASTN procedure. To obtain vacancy alignments for comparison purposes, Gapped BLAST is used as described by Altschul et al. (Nucleic Acids Res. (1997) 25: 3389-3402). When using the BLAST and GappedBLAST procedures, the default parameters of the respective procedures are used.

[0067] According to a preferred embodiment of the invention, the antibody amino acid sequence, which forms part of the invention, consists of or comprises a nucleic acid sequence encoding a polypeptide having at least 85% identity, more preferably at least 90% identity, or even more preferably at least 95% identity with any of the sequences defined above and disclosed herein. According to a preferred embodiment, mutations in the amino acid sequence that result in less than 100% sequence identity are located outside the CDRs disclosed herein, and more preferably, such mutations are located outside the CDRs and frame regions of the antibody sequences disclosed herein.

[0068] In this invention, the neutralizing activity of the disclosed antibodies against a broad selection of pseudoviruses is tested. The TZM-bl cell pseudovirus neutralization assay, used to study the breadth and strength of neutralization, is a highly standardized assay commonly used in the field and in the technical field of this invention to analyze the neutralizing potency of antibodies against various HIV-1 strains.

[0069] In short, the antibody and viral strain are incubated together before the addition of TZM-bl target cells. These cells express Tat-regulated luciferase, which exhibits gene expression activity upon successful infection, resulting in a detectable luminescent signal in the presence of luciferin after cell lysis. The neutralizing antibody prevents infection, thereby preventing luminescence. The potency of the neutralizing antibody is determined by the antibody concentration required to reduce viral infectivity to a specific level.

[0070] Exemplary and standardized methods for establishing and adopting this determination are described in Sarzotti-Kelsoe et al., which are incorporated herein by reference in their entirety. J Immunol MethodsThe invention is disclosed and described in detail in July 2014; 409: 131–146. doi:10.1016 / j.jim.2013.11.022. Preferably, the neutralizing activity is determined using the methods described in the “Examples” section below. This specification, alone and in combination with common prior art, enables those skilled in the art to establish and determine conclusive readings for the TZM-bl cell pseudovirus neutralization assay as used herein.

[0071] Neutralizing efficacy, as defined herein, preferably considers only those viral variants that can be positively identified as being neutralized by the corresponding antibody. Based on the selection of positive neutralizing variants, the geometric mean IC50 of the neutralizing strains is then determined. 50 .

[0072] According to a preferred embodiment of the first aspect of the invention, the antibody or its antigen-binding fragment comprises a combination of the variable region heavy chain amino acid sequence and the variable region light chain amino acid sequence of antibody 007. In one preferred embodiment of the combination, the variable region heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID No. 7, and the variable region light chain amino acid sequence comprises the amino acid sequence of SEQ ID No. 8. In another preferred embodiment of the combination, the variable region heavy chain amino acid sequence consists of the amino acid sequence of SEQ ID No. 7, and the variable region light chain amino acid sequence consists of the amino acid sequence of SEQ ID No. 8.

[0073] Preferably, the antibody or antigen-binding fragment as described herein is mammalian, more preferably human. More preferably, the antibody or antigen-binding fragment as described herein is monoclonal and can be used and administered in a monoclonal manner, i.e., not simultaneously or overlappingly administered to a patient with another monoclonal antibody. Alternatively, preferably, the antibody or antigen-binding fragment of the present invention can be administered in combination with one or more different monoclonal antibodies, for example, simultaneously or overlappingly, such that all said antibodies deliver their effects simultaneously.

[0074] In the description of this application, antibody names may be used. It should be noted that an antibody comprises and is composed of heavy and light chains, and this also forms part of this specification. If an antibody is referred to by its name or SEQ ID No., it should be understood that these references are interchangeable.

[0075] In a second aspect, the invention also relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the first aspect of the invention, and at least one pharmaceutically acceptable excipient. In a preferred embodiment, the pharmaceutical composition according to the second aspect of the invention is a therapeutic composition for human subjects. In a preferred embodiment, the pharmaceutical composition according to the second aspect of the invention is a preventative composition for human subjects. In another preferred embodiment, the pharmaceutical composition according to the second aspect of the invention is a passive immunization composition for human subjects. In yet another preferred embodiment, the pharmaceutical composition according to the second aspect of the invention is a vaccination composition for human subjects.

[0076] In a preferred embodiment of the second aspect of the invention, the pharmaceutical composition further comprises at least one antibody targeting the CD4 binding site of HIV-1. The pharmaceutical composition according to this embodiment has the advantage of comprising at least two antibodies that bind to different epitopes of HIV-1, thus preventing competitive binding and consequently leading to an additive neutralizing effect.

[0077] Therefore, the combination of the unprecedentedly strong neutralizing effect of the V3 glycan site antibody according to the invention with the strong and broad neutralizing bNAb targeting the CD4 binding site is expected to exhibit particularly high and broad neutralizing effect against HIV-1 in vitro and in vivo.

[0078] In a third aspect, the present invention also covers a kit comprising an antibody or antigen-binding fragment thereof according to the first aspect of the present invention, and a container.

[0079] In a fourth aspect of the invention, an antibody or antigen-binding fragment thereof according to the first aspect of the invention, a pharmaceutical composition according to the second aspect, and a kit according to the third aspect are used as pharmaceuticals.

[0080] In a fifth aspect of the invention, an antibody or antigen-binding fragment thereof according to the first aspect of the invention, a pharmaceutical composition according to the second aspect, and a kit according to the third aspect of the invention are used for the treatment or prevention of human immunodeficiency virus (HIV-1) infection in mammalian subjects.

[0081] According to a sixth aspect of the invention, an antibody or antigen-binding fragment thereof according to a first aspect of the invention, a pharmaceutical composition according to a second aspect, and a kit according to a third aspect of the invention are used for the treatment or prevention of disease caused by human immunodeficiency virus HIV-1 in mammalian subjects.

[0082] In a preferred embodiment of the fifth or sixth aspect of the invention, the use is in human subjects. In a preferred embodiment of the fifth or sixth aspect of the invention, the use is in the form of a therapeutic composition. In a preferred embodiment of the fifth or sixth aspect of the invention, the use is in the form of a passive immunization composition. In another preferred embodiment of the fifth or sixth aspect of the invention, the use is in the form of a preventative composition. In another preferred embodiment of the fifth or sixth aspect of the invention, the use is for vaccination and / or in the form of a vaccination composition. In yet another preferred embodiment of the fifth or sixth aspect of the invention, the use is for the treatment or prevention of acquired immunodeficiency syndrome (AIDS).

[0083] In another aspect, the invention also relates to a method for treating a patient suffering from a disease caused by human immunodeficiency virus HIV-1 in a human subject, preferably for use in the treatment or prevention of acquired immunodeficiency syndrome (AIDS) in a human subject, wherein an effective amount of an antibody or antigen-binding fragment thereof according to the invention or a pharmaceutical composition of the invention is administered to the patient.

[0084] In another aspect, the present invention also relates to the use of antibodies or antigen-binding fragments thereof according to the invention, or pharmaceutical compositions according to the invention, in the manufacture of medicaments for treating human subjects with diseases caused by human immunodeficiency virus HIV-1, preferably for treating or preventing acquired immunodeficiency syndrome (AIDS) in human subjects.

[0085] According to a seventh aspect of the invention, a nucleic acid is provided that encodes an antibody or an antigen-binding fragment thereof according to a first aspect of the invention. In one embodiment, the nucleic acid comprises DNA or RNA, such as mRNA. In one embodiment, the nucleic acid is codon-optimized for expression in a host cell.

[0086] According to an eighth aspect of the invention, an expression vector is provided that comprises a nucleic acid of the seventh aspect of the invention functionally associated with an expression control sequence. In one embodiment, the expression vector is a viral vector.

[0087] According to a ninth aspect of the invention, a host cell is provided comprising a nucleic acid according to a seventh aspect of the invention or an expression vector according to an eighth aspect of the invention. Examples of host cells that can be used include eukaryotic cells, such as yeast cells, mammalian cells, insect cells, and plant cells; and prokaryotic cells, including *Escherichia coli*. In one embodiment, the mammalian cell is a human cell, such as a human B cell. The nucleic acid may be heterologous to the host cell, meaning that it is not natural for the subject to which the host cell is obtained or derived. In one embodiment, the nucleic acid or expression vector is altered or genetically engineered, for example by conjugation, transformation, transfection, electroporation, etc., to be added to the host cell.

[0088] According to a tenth aspect of the present invention, a method for preparing an antibody or antigen-binding fragment thereof of the first aspect of the present invention is provided, the method comprising culturing a host cell of the ninth aspect of the present invention under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof.

[0089] All embodiments of the invention described herein are considered to be possible in any combination unless those skilled in the art consider such combination to have no technical significance.

[0090] Example A) Experimental methods Isolation of monoclonal antibody sequences Blood and leukocyte-removed samples were obtained under protocols approved by the Institutional Review Board of the University of Cologne (Protocols 13-364 and 16-054), and written informed consent was provided by the participants. Peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation and stored at -150°C in 90% FBS and 10% DMSO. B cells were isolated from PBMCs by magnetic cell separation and labeled on ice for 30 minutes with anti-human CD19-AF700, anti-human IgG-APC, DAPI (BD), and HIV-1 Env bait protein.

[0091] The HIV-1 Env bait protein is BG505. SOSIP.664- GFP (Sliepen K et al., Biomolecules, 23 October 2015; 5(4):2919-34) or biotinylated (EZ-Link Sulfo NHS Biotinylated Kit, ThermoFisher) YU2 gp140(Yang et al., J Virol. 2000, 74(12):5716-25. doi: 10.1128 / jvi.74.12.5716-5725.2000), which uses streptavidin-PE labeling. Env reactive CD19 was sorted as previously described. + IgG + DAPI - Single cells (Gieselmann et al., Nat Protoc. 2021 Jul; 16(7):3639-3671. doi:10.1038 / s41596-021-00554-w.). Sorted cells were incubated at 65°C for 1 min with random hexamer primers, NP-40, and RNase-free H2O. cDNA was then generated using SuperScript IV in the presence of RT buffer, dNTPs, DTT, H2O, RNasin, and RNaseOUT. The antibody sequences used for single-cell analysis were subjected to semi-nested PCR using Taq polymerase and previously described primers CG_RT (Ozawa et al., Biotechniques. April 2006; 40(4):469-70, 472,474 passim. doi: 10.2144 / 000112123, first PCR), IgG_internal_RT (Tiller et al., J Immunol Methods. January 2008; 329(1-2):112-24. doi: 10.1016 / j.jim.2007.09.017.Epub October 31, 2007, second PCR) and a mixture of OPT5 / oPR primers (Kreer et al., J Immunol Methods. May 2020; 480:112752. doi: Amplification was performed using two PCR methods (10.1016 / j.jim.2020.112752).

[0092] Antibody sequence analysis Sequences of second PCR products with an average Phred score ≥28 and a minimum length of 240 nucleotides were annotated using IgBLAST (Ye et al., 2013), and trimmed from the frame region (FWR) 1 of the variable region to the end of the J gene. Base calls with a Phred score <16 were masked, and sequences with >15 masked nucleotides, frameshifts, or stop codons were excluded from further analysis. To analyze the potential for clones, all productive heavy chain sequences were grouped by the same V gene, and their CDRH3 pairwise Levenshtein distances were determined. Individual sequences that shared the same V gene and had a minimum CDRH3 identity of 75% were grouped as clones.

[0093] After 10 rounds of random sequence input, the results producing the fewest unassigned (non-clonal) sequences were selected for further analysis. All clones were manually revalidated by researchers to identify shared mutations. Sequences initially assigned to different clones but sharing the same VDJ gene and amino acid and / or silent nucleotide mutations were subsequently grouped into subclones. Nucleotide sequence identity with the phylogenetic line was calculated using IgBLAST.

[0094] Monoclonal antibody preparation To clone single-cell-derived antibodies, the first PCR product from a single-cell PCR was used as a template and amplified using Q5 high-fidelity polymerase and specific forward and reverse primers that resembled the corresponding nucleotide sequences of the V and J regions (Gieselmann et al., 2021, ibid.) and had expression vector overhangs for subsequent sequence-independent and ligation-independent cloning (SLIC).

[0095] PCR products were cloned into human antibody expression vectors (IgG1, κ, or λ chains) using the SLIC assembly method, as previously described (von Boehmer et al., Nat Protoc. Oct 2016; 11(10):1908-1923. doi: 10.1038 / nprot.2016.102). Antibodies were generated in HEK293-6E cells by transfection with polyethyleneimine. After 5–7 days, the antibodies were purified from the supernatant after incubation with Protein G and subsequent elution from the column with 0.1 M glycine (pH=3.0). The antibodies were neutralized in buffer, exchanged for PBS, and filtered for sterilization before being stored at 4°C.

[0096] Competition ELISA As previously described, a competitive ELISA was performed (in Schoofs et al., Immunity, June 18, 2019; 50(6):1513-1529.e9 and Schommers et al., Cell, February 6, 2020; 180(3):471-489.e22). The antibodies of this invention were biotinylated using the EZ-Link Sulfo-NHS-Biotin kit (Thermo Fisher Scientific) according to the manufacturer's protocol. Buffer exchange with PBS was performed by centrifugation using an Amicon 10 kDA centrifuge filter membrane (Millipore). High-binding ELISA plates (Greiner Bio-One) were coated overnight at 4°C with BG505.SOSIP.664 to coat the trimer protein or with anti-6x His-tagged antibody (Abcam 9108) at a concentration of 2 μg / ml. The plates were then blocked for 1 hour at 37°C with 3% BSA (Sigma Aldrich) in PBS. After blocking, wells initially coated with anti-6x His-tagged antibody were incubated with BG505SOSIP.664-His at a concentration of 2 μg / ml in PBS for 1 hour at room temperature. Competitive antibodies with known epitope specificity were used at an initial concentration of 32 μg / ml in PBS and serially diluted 1:3. After incubation with the competitive antibody for 1 hour at room temperature, biotinylated antibody of interest was added at a concentration of 0.5 μg / ml in PBS containing 3% BSA, and the plates were incubated at room temperature for 1 hour. Subsequently, the plates were incubated with peroxidase-streptoavidin (Jackson ImmunoResearch) diluted 1:5,000 in PBS containing 1% BSA and 0.05% Tween-20. Between each step, the plates were washed with PBS containing 0.05% Tween-20 (Carl Roth). The plates were developed using ABTS solution (Thermo Fisher Scientific 002024), and absorbance was measured at 415 nm and 695 nm using a microplate reader (Tecan).

[0097] fake virus preparation Pseudoviruses were generated in HEK293T cells by co-transfection with the pSG3ΔEnv plasmid, as previously described (Doria-Rose et al., PLoS Pathog. Jan 4, 2017; 13(1):e1006148. doi: 10.1371 / journal.ppat; Sarzotti-Kelsoe et al., J Immunol Methods. Jul 2014; 409:131-46. doi:10.1016 / j.jim.2013.11.022; Hraber et al., J Virol. Sep 12, 2017; 91(19):e00991-17. doi: 10.1128 / JVI.00991-17.; Seaman et al., J Virol. Feb 2010; 84(3):1439-52. doi:10.1128 / JVI.02108-09).

[0098] TZM-bl cell neutralization assay Neutralization assays were performed as previously described (Sarzotti-Kelsoe et al., 2014, ibid.; Seaman et al., 2010, ibid.). Nonspecific activity was determined using murine leukemia virus (MuLV) pseudovirus. Two copies of the test antibody were used. For the global reference group, bioluminescence was measured after adding luciferin / lysis buffer (a solution of 10 mM MgCl2, 0.3 mM ATP, 0.5 mM Coenzyme A, 17 mM IGEPAL (all from Sigma-Aldrich) and 1 mM D-luciferin (GoldBio) in Tris-HCl).

[0099] B) Specific embodiments of the antibody of the present invention Example I - Isolation of a broadly and potent VH4-34-derived target V3 from well-neutralized individuals infected with HIV-1 HIV-1 neutralizing antibodies at glycan sites Human B cells that are responsive to HIV-1 envelope proteins were isolated from individuals infected with HIV-1, who had previously been identified in in vitro assays as having abnormal serum neutralizing activity against HIV-1.

[0100] Therefore, isolated B cells were mixed with soluble HIV-1 Env protein (YU2) labeled with a fluorescent dye. gp140 Or BG505 SOSIP.664 The cells were incubated together and then single-cell sorted (Giesemann et al., 2021, ibid.). This method enables the subsequent amplification of heavy and light antibody gene segments from a single HIV-1 Env-reactive B cell via PCR. This allows the PCR products to be cloned into expression vectors to recombinantly prepare the corresponding antibodies encoded by a single B cell, thereby enabling functional testing of the antibodies.

[0101] Sequence analysis of HIV-1-Env-reactive B cells identified expanded VH4-34-derived B cell clones. To determine the target epitopes of the isolated mAbs, the binding activity of all antibodies of this invention against the HIV-1 envelope trimer of BG505.SOSIP.664 was tested in the presence of reference antibodies with known epitopes (as described in Schoofs et al., Immunity, June 18, 2019; 50(6):1513-1529.e9 and Schommers et al., Cell, February 6, 2020; 180(3):471-489.e22). Figure 5 The binding activity of all antibodies of this invention is selectively inhibited by V3 glycan bNAbs BG18 and / or 10-1074, indicating that the V3 glycan site is a binding epitope for these bNAbs. Figure 5 ).

[0102] Antibody 007 represents this B-cell clone. To determine overall neutralizing potency and breadth, the neutralizing activity of antibody 007, as well as other antibodies of the present invention, against a reference group of 12 HIV-1 pseudovirus strains was tested in a TZM-bl cell neutralization assay. This reference group was referred to as the "global group" (GZM-bl cell neutralization assay). Figure 1 This group of pseudoviruses was previously designed to represent the diversity of the global HIV-1 epidemic and to enable standardized assessment of the activity of neutralizing antibodies.

[0103] It is noteworthy that 007 and other antibodies of the present invention exhibited high neutralizing activity in 100% (12 / 12) of the strains included in this group. Figure 1 Neutralizing efficacy is typically determined by a 50% inhibitory concentration (IC50). 50 (This indicates that) 007 showed high activity when tested against the "global group," with a geometric mean IC50 against neutralized viral strains. 50 It is 0.008 μg / ml ( Figure 1 The remaining antibodies of this invention also exhibit very high neutralizing activity and, unlike prior art V3 glycan site antibodies, are capable of neutralizing each of the viral strains shown in the group.

[0104] Example II - High potency and broad efficacy of antibody 007 To confirm the neutralizing potency and breadth of 007, an expanded group of antibodies was tested against 119 HIV-1 pseudoviruses, as previously described in Schoofs et al., 2019. This group of pseudoviruses represents the development of a group of pseudoviruses detailed in the following literature: Seaman et al., J Virol. Feb. 2010; 84(3):1439-52. doi: 10.1128 / JVI.02108-09. It provides a representativeness of the genetic and global diversity of HIV-1 Env variants. It includes HIV-1 variants from different clades or subtypes, including variants isolated from spreading / foundational viruses and difficult-to-neutralize viruses.

[0105] It is worth noting that when tested on multiple clades of the 119 pseudovirus, 007 exhibited high efficacy and breadth. Figure 2 Specifically, when tested at antibody concentrations up to 10 μg / ml, antibody 007 neutralized 66% of the tested pseudoviruses and showed a geometric mean IC50 of 0.01 μg / ml against the neutralized pseudoviruses. 50 For this neutralized pseudovirus, results are available for a total of 114 identical HIV-1 pseudoviruses (Yoon et al., 2015, ibid.). Figure 2 This breadth and potency is unprecedented and surpasses that observed in other HIV-1 neutralizing antibodies that also target the V3 glycan site and are in advanced stages of clinical testing, such as 10-1074 or PGT121. Figure 2 Furthermore, antibody 007 in the 119 multi-clade group exhibits greater breadth and potency than the best V3 glycan site bNAb in the prior art. Figure 2 and Figure 3 ).

[0106] Example III - Neutralizing activity against pseudoviruses that are poorly neutralized by antibodies targeting other V3 glycan sites Although the V3 glycan site bNAb can achieve high levels of neutralizing breadth (i.e., activity against a large number of different HIV-1 Env variants), antibody-resistant HIV-1 variants exist. Therefore, it is important to identify novel V3 glycan site antibodies with high activity against such HIV-1 variants. Thus, we assayed the antibodies described in this invention against a group of nine (… Figure 4 The neutralizing characteristics of HIV-1 variants, which are resistant to neutralization of the V3 glycan site bNAb.

[0107] Figure 4The test virus strains shown are part of the 119 multiclade pseudovirus group and the “global group” (e.g., Schoofs et al., Immunity, June 18, 2019; 50(6):1513-1529.e9; de Camp et al., J Virol. March 2014; 88(5): 2489–2507; Seaman et al., J Virol. February 2010; 84(3):1439-52. doi: 10.1128 / JVI.02108-09. Antibodies 007, 01_C10, 01_G04, and 03_A07 neutralized resistant pseudoviral variants with significantly higher breadth and potency compared to V3 glycan site antibodies in advanced phases of clinical trials and previously disclosed best V3 glycan site bNAbs (10-1074, BG18, PGT121, PGT128, PGT130, and PGT135). Figure 4 ).

[0108] For example, when tested at concentrations up to 10 μg / ml in the TZM-bl pseudovirus neutralization assay, 100% of the 12 pseudovirus groups were neutralized by 007, with a very effective IC50 of 0.013 μg / ml. 50 Similar strong results were observed in terms of neutralizing breadth and potency for antibodies 01_C10, 01_G04, and 03_A07. In contrast, the most extensive and potent V3 glycan site reference bNAb described to date neutralized only 0%–8% of 12 pseudovirus groups. Figure 4 ).

[0109] Therefore, the antibody of the present invention provides a solution to poor virus neutralization with resistance to the neutralization of V3 glycan site bNAb.

Claims

1. An antibody or antigen-binding fragment thereof targeting the V3 glycan site of human immunodeficiency virus HIV-1, wherein the antibody or antigen-binding fragment thereof comprises a combination of a variable region heavy chain comprising heavy chain CDR1 to CDR3 and a variable region light chain comprising the amino acid sequence of light chain CDR1 to CDR3 of an antibody selected from the group consisting of: 007 (containing the CDR-H1 amino acid sequence of SEQ ID No. 1, the CDR-H2 amino acid sequence of SEQ ID No. 2, the CDR-H3 amino acid sequence of SEQ ID No. 3, the CDR-L1 amino acid sequence of SEQ ID No. 4, the CDR-L2 amino acid sequence of SEQ ID No. 5, and the CDR-L3 amino acid sequence of SEQ ID No. 6). 03_A07 (containing the CDR-H1 amino acid sequence of SEQ ID No. 7, the CDR-H2 amino acid sequence of SEQ ID No. 8, the CDR-H3 amino acid sequence of SEQ ID No. 9, the CDR-L1 amino acid sequence of SEQ ID No. 10, the CDR-L2 amino acid sequence of SEQ ID No. 11, and the CDR-L3 amino acid sequence of SEQ ID No. 12). 01_G04 (containing the CDR-H1 amino acid sequence of SEQ ID No. 13, the CDR-H2 amino acid sequence of SEQ ID No. 14, the CDR-H3 amino acid sequence of SEQ ID No. 15, the CDR-L1 amino acid sequence of SEQ ID No. 16, the CDR-L2 amino acid sequence of SEQ ID No. 17, and the CDR-L3 amino acid sequence of SEQ ID No. 18), and 01_C10 (containing the CDR-H1 amino acid sequence of SEQ ID No. 19, the CDR-H2 amino acid sequence of SEQ ID No. 20, the CDR-H3 amino acid sequence of SEQ ID No. 21, the CDR-L1 amino acid sequence of SEQ ID No. 22, the CDR-L2 amino acid sequence of SEQ ID No. 23, and the CDR-L3 amino acid sequence of SEQ ID No. 24).

2. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises a combination of a variable region heavy chain amino acid sequence and a variable region light chain amino acid sequence of an antibody selected from the group consisting of: 007 (contains or is composed of the following amino acid sequences respectively:) (Amino acid sequences of SEQ ID No. 25 and SEQ ID No. 26) 03_A07 (contains or is composed of the following amino acid sequences: the amino acid sequence of SEQ ID No. 27 and the amino acid sequence of SEQ ID No. 28, respectively). 01_G04 (containing or composed of the following amino acid sequences: the amino acid sequence of SEQ ID No. 29 and the amino acid sequence of SEQ ID No. 30, respectively). 01_C10 (contains or consists of the following amino acid sequences respectively: the amino acid sequence of SEQ ID No. 31 and the amino acid sequence of SEQ ID No. 32).

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the amino acid sequence contained therein is an antibody selected from the group consisting of 007, 03_A07 and 01-G04, preferably an antibody selected from the group consisting of 007 and 03_A07, more preferably antibody 007.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof further comprises an Fc domain, a CH1 domain, a CL1 domain, a hinge domain or any combination thereof, optionally wherein the antibody is an IgG1 isotype.

5. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4 and at least one pharmaceutically acceptable excipient.

6. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition is a vaccination composition for human subjects.

7. The pharmaceutical composition according to claim 5 or 6, wherein the pharmaceutical composition further comprises at least one antibody targeting the CD4 binding site of HIV-1.

8. A kit comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4, and a container.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the pharmaceutical composition according to any one of claims 5 to 7, or the kit according to claim 8, wherein they are used as pharmaceuticals.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the pharmaceutical composition according to any one of claims 5 to 7, or the kit according to claim 8, for use in the treatment or prevention of said human immunodeficiency virus HIV-1 infection in mammalian subjects.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the pharmaceutical composition according to any one of claims 5 to 7, or the kit according to claim 8, for use in the treatment or prevention of disease caused by said human immunodeficiency virus HIV-1 in mammalian subjects.

12. The antibody or antigen-binding fragment thereof, pharmaceutical composition or kit used according to any one of claims 9 to 11, wherein the use is in human subjects.

13. The antibody or antigen-binding fragment thereof, pharmaceutical composition or kit used according to any one of claims 9 to 12, wherein the use is for the treatment or prevention of acquired immunodeficiency syndrome (AIDS).

14. A nucleic acid encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4, wherein optionally the nucleic acid is codon-optimized for expression in a host cell.

15. An expression vector comprising a nucleic acid according to claim 14 that is functionally associated with an expression control sequence, optionally wherein the expression vector is a viral vector.

16. A host cell comprising the nucleic acid according to claim 14 or the expression vector according to claim 15.

17. A method for generating an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4, said method comprising: (a) Culture the host cells according to claim 16 under conditions that allow expression of the antibody or its antigen-binding fragment, and (b) Recover the antibody or its antigen-binding fragment.

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