Broad spectrum neutralizing antibodies targeting CD4 binding sites on HIV Env

Abstract

The present disclosure relates to monoclonal human antibodies, or binding fragments thereof, directed against the CD4 binding 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, 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 binding fragments thereof targeting the CD4 binding 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, and monoclonal antibodies or binding fragments thereof used as pharmaceuticals and for treating, curing or preventing diseases caused by human immunodeficiency virus HIV-1, as well as pharmaceutical compositions and kits. 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 treatments 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 cyclic glycan plaques, the proximal outer region of the membrane, fusion peptides, silencing surfaces, and the interface between the gp120 and gp41 Env subunits.

[0003] Of these sites, CD4bs are of particular interest because CD4 acts as the primary receptor for viral entry. BNAbs encoded by the immunoglobulin heavy chain gene segment IGHV1-2 typically mimic the CD4 contact residues of viral envelope proteins, exhibiting high levels of somatic hypermutation and depending on a five-amino acid light chain complementarity-determining region (CDRL3). These antibodies are also known as VRC01 class antibodies and contain most of the most potent and broadly potent CD4bs bNAbs identified to date. Due to their significant antiviral activity, several CD4bs bNAbs have entered advanced clinical trials. Representative members of the VRC01 class include VRC01, VRC07, VRC07-523-LS, 3BNC117, NIH45-46, N6, and N49-P7.

[0004] Other CD4bs bNAbs are encoded by the immunoglobulin heavy chain gene segment IGHV1-46 and do not require the five-amino acid CDRL3. Representative members of this antibody class are 8ANC131, CH235, and 1B2530. These antibodies generally exhibit lower potency and breadth when compared to VRC01 class antibodies. An exception is the recently identified bNAb 1-18, which has shown high levels of potency and breadth against a large multiclade HIV-1 pseudovirus reference group.

[0005] In preclinical animal models, bNAb exhibits robust anti-infective protection even at low serum concentrations and has shown 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). Although recent antibody-mediated prophylaxis (AMP) efficacy trials using CD4bs bNAb VRC01 have not demonstrated overall efficacy in preventing HIV-1 infection, subgroup analyses showed efficacy of VRC01 in preventing infection with VRC01-sensitive strains (IC80 < 1 μg / ml). Therefore, the identification and development of highly effective CD4bs bNAb are highly relevant to HIV-1 prevention. These bNAb need to possess enhanced neutralizing potency, broad coverage, and restricted viral escape pathways. Therefore, the isolation of novel CD4bs bNAb with antiviral activity exceeding that of known anti-HIV-1 bNAb remains urgently needed.

[0006] The purpose of this 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.

[0007] Furthermore, another object of the present invention is to provide a novel human monoclonal antibody against HIV-1 that limits the emergence of viral escape variants and maintains effectiveness against such variants. Summary of the Invention

[0008] These objectives have been achieved through the various aspects of the invention as specified below.

[0009] According to a first aspect of the invention, an antibody or antigen-binding fragment thereof targeting the CD4 binding site of human immunodeficiency virus HIV-1 is provided, wherein the antibody or antigen-binding fragment thereof comprises a combination of heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3 amino acid sequences from the group consisting of: 04_A06 (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), 01_D03 (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 SEQ ID No. 10). SEQ ID No. 11 (CDR-L2 amino acid sequence and SEQ ID No. 12 (CDR-L3 amino acid sequence)), 04_B09 (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)), 05_C07 (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)), and 01_B01 (containing SEQ ID No. 13 (CDR-H1 amino acid sequence), SEQ ID No. 24 (CDR-L3 amino acid sequence)). The amino acid sequences of CDR-H1 in SEQ ID No. 25, CDR-H2 in SEQ ID No. 26, CDR-H3 in SEQ ID No. 27, CDR-L1 in SEQ ID No. 28, CDR-L2 in SEQ ID No. 29, and CDR-L3 in SEQ ID No. 30 are listed.

[0010] 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: 04_A06 (composed of the heavy chain amino acid sequence of SEQ ID No. 31 and the light chain amino acid sequence of SEQ ID No. 41), 01_D03 (composed of the heavy chain amino acid sequence of SEQ ID No. 33 and the light chain amino acid sequence of SEQ ID No. 34), 04_B09 (composed of the heavy chain amino acid sequence of SEQ ID No. 35 and the light chain amino acid sequence of SEQ ID No. 36), 05_C07 (composed of the heavy chain amino acid sequence of SEQ ID No. 37 and the light chain amino acid sequence of SEQ ID No. 38), and 01_B01 (composed of the heavy chain amino acid sequence of SEQ ID No. 39 and the light chain amino acid sequence of SEQ ID No. 40).

[0011] According to a preferred embodiment of the first aspect of the present invention or the foregoing embodiments of the first aspect of the present invention, the amino acid sequence contained in the antibody or its antigen-binding fragment is an antibody selected from the group consisting of 04_A06, 01_D03, 04_B09 and 05_C07, preferably an antibody selected from the group consisting of 04_A06, 01_D03 and 04_B09, more preferably an antibody selected from the group consisting of 04_A06 and 01_D03, and particularly preferably antibody 04_A06.

[0012] 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.

[0013] According to a preferred embodiment of the second aspect of the invention, the pharmaceutical composition is a vaccination composition for human subjects.

[0014] 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.

[0015] According to a fourth 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 as a medicine.

[0016] 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.

[0017] 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.

[0018] In a preferred embodiment of the fifth or sixth aspect of the invention, the use is in human subjects.

[0019] 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).

[0020] 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.

[0021] 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.

[0022] According to a ninth aspect of the invention, a host cell is provided, which contains a nucleic acid according to a seventh aspect of the invention or an expression vector according to an eighth aspect of the invention.

[0023] 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

[0024] 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); The antibodies 04_A06 and 01_D03, as described herein, neutralize 100% (12 / 12) of the “global group” strains and show high levels of potency.

[0025] Figure 2 The neutralizing activity (IC50) of antibody 04_A06 of the present invention against a multi-clade group 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 described in Schoofs et al., Immunity, June 18, 2019; 50(6):1513-1529.e9); shows that antibody 04_A06 is the most potent and broadest range of CD4bs bNAbs (3BNC117, 1-18, N49P7, N6, VRC07 and VRC07-) in the prior art. 523-LS The data were compared; for presentation in this figure, 99 pseudovirus strains from 119 multi-clade groups were selected, with neutralization data for the reference antibody already published; neutralization data for the reference bNAb were obtained 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 Search within ).

[0026] Figure 3 The invention's antibody 04_A06 demonstrated neutralizing activity (IC50) against multiple clades of 119 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 described in Schoofs et al., Immunity, June 18, 2019; 50(6):1513-1529.e9); for presentation in this figure, 76 pseudovirus strains (A) or 99 pseudovirus strains (B) from 119 multiphylogenetic clades were selected, with neutralization data for reference antibodies published; (A) shows the neutralizing activity of 04_A06 compared to other anti-HIV-1 bNAb targeting a variety of different known epitopes, and (B) shows the neutralizing activity of antibody 04_A06 compared to the most potent and broadest anti-HIV-1 bNAb in the art; neutralization data for reference bNAb were retrieved from the antibody neutralization database CATNAP (Yoon et al., 2015, ibid.).

[0027] Figure 4 The neutralizing activity (IC50) of antibody 04_A06 of the present invention against a multi-clade group (subtype C group) of 100 clades C HIV-1 pseudoviruses with different HIV-1 envelope amino acid sequences was demonstrated when tested in a TZM-bl cell pseudovirus neutralization assay. 50(as described in Hraber et al., J Virol., 12 September 2017; 91 (19):e00991-17); data for antibody 04_A06 compared with the most potent and broadest range of CD4bs bNAbs in the art (3BNC117, 1-18, N6, VRC01 and VRC07-523-LS) are shown; neutralization data for bNAbs were retrieved from the antibody neutralization database CATNAP (Yoon et al., 2015, ibid.).

[0028] Figure 5 The neutralizing activity (IC50) of antibody 04_A06 of the present invention against a group of nine pseudoviruses with different HIV-1 envelope amino acid sequences was demonstrated when tested in a TZM-bl cell pseudovirus neutralization assay. 50 The pseudoviruses in this group are known to exhibit excellent resistance to CD4bs bNAb; the viral strains in this group are part of the 119 polyphyletic clades and subtypes of the C pseudovirus group (as described in Schoofs et al., Immunity, 18 June 2019; 50(6):1513-1529.e9; and Hraber et al., J Virol., 12 September 2017; 91(19):e00991-17); antibody 04_A06 is shown to be effective against the most potent and broadest CD4bs bNAb described to date (VRC07). -523-LS Data compared to N6, 3BNC117, 1-18, and VRC01; neutralization data for bNAb were retrieved from the antibody neutralization database CATNAP (Yoon et al., 2015, ibid.).

[0029] Figure 6 The neutralizing activity (IC50) of antibody 04_A06 of the present invention against a group of seven pseudoviruses with different HIV-1 envelope amino acid sequences was demonstrated when tested in a TZM-bl cell pseudovirus neutralization assay. 50The virus strains tested were selected from a reference pseudovirus group called the “CD4bs bNAb resistance group” and described in Zhou et al., PLoS Pathog. 13 June 2019; 15(6):e1007819. doi: 10.1371 / journal.ppat.1007819. PMID: 31194843; PMCID: PMC6592578 12 September 2017; 91(19):e00991-17; The pseudoviruses of the published reference group showed excellent resistance to CD4bs bNAb; Data on antibody 04_A06 were shown compared with the most effective and broadest CD4bs bNAb in the art (VRC07, N6, NIH45-46, 1-18, VRC01, N49P7, 3BNC117); All antibodies were tested in duplicate in independent assays.

[0030] Figure 7 The neutralizing activity (IC50) of antibody 01_D03 of the present invention against a group of eight pseudoviruses with different HIV-1 envelope amino acid sequences was demonstrated when tested in a TZM-bl cell pseudovirus neutralization assay. 50 The viral strains in this group are part of the 119 multiphylogenetic clade (as described in Schoofs et al., Immunity, June 18, 2019; 50(6):1513-1529.e9); the selected pseudoviruses in this group are insensitive to CD4bs bNAb neutralization; insensitivity to antibody neutralization is defined as IC50. 50 >1 μg / ml; Data for antibody 01_D03 are shown compared to the most potent and broadest CD4bs bNAb of the prior art (VRC07-523-LS, N6, VRC07, N49P7, 1-18); All antibodies were tested in duplicate in independent assays, and neutralization data for bNAb were retrieved from the antibody neutralization database CATNAP (Yoon et al., 2015, ibid.).

[0031] Figure 8 The neutralizing activity (IC50) of antibody 04_A06 of the present invention against a group of 185 pseudoviruses with different HIV-1 envelope amino acid sequences was demonstrated when tested in a TZM-bl cell pseudovirus neutralization assay. 50 and IC 80The viral strains of this pseudovirus group were generated from viral sequences obtained from antibody-mediated prophylaxis experiments (e.g., Corey L, Two Randomized Trials of Neutralizing Antibodies to Prevent HIV-1 Acquisition. N Engl J Med. 2021 Mar 18; 384(11):1003-1014. doi: 10.1056 / NEJMoa2031738. PMID: 33730454; PMCID: PMC8189692). Data for antibody 04_A06 compared to reference bNAb VRC01 are shown; neutralization data for other reference bNAbs are not publicly available; neutralization data for reference bNAb VRC01 are from Corey L, Two Randomized Trials of Neutralizing Antibodies to Prevent HIV-1 Acquisition. N Engl J Med. 2021 Mar 18; 384(11):1003-1014. doi: 10.1056 / NEJMoa2031738. PMID: 33730454; PMCID: PMC8189692). Med. 18 March 2021; 384(11):1003-1014. Doi: 10.1056 / NEJMoa2031738. PMID: 33730454; PMCID: PMC8189692.

[0032] Figure 9 It shows HIV-1 infection YU2 HIV-1 RNA plasma copy number in humanized mice (top figure) and its logistic change (bottom figure, compared to baseline (HIV-1 RNA plasma copy number on day -1)) (the envelope gene of NL4-3 virus replaces the envelope gene of YU2); mice were subcutaneously treated with a loading dose of 1 mg antibody, followed by subcutaneous injection of 0.5 mg antibody every 3-4 days; mice treated with known CD4bs antibodies VRC01 and VRC07 showed a transient reduction in viremia, followed by a rapid viral rebound; in contrast, mice treated with only the antibody 04_A06 of this invention showed a sustained reduction in viremia throughout the 84-day treatment period; the black line represents the mean logistic change.

[0033] Figure 10 This illustrates HIV-1 infection pretreated with CD4 binding site antibody VRC01. YU2 HIV-1 RNA plasma copy number in humanized mice (NL4-3 / YU2) (top figure) and its logistic changes (bottom figure, compared with baseline (HIV-1 RNA plasma copy number on day 28)) Figure 8(as shown); four weeks after treatment, antibody 04_A06 was added to the ongoing prior treatment regimen; 04_A06 was administered subcutaneously at a loading dose of 1 mg, followed by 0.5 mg every 3 to 4 days; although viral rebound occurred after pretreatment with CD4 binding site antibody VRC01, treatment with antibody 04_A06 resulted in sustained viral suppression in all treated animals. Detailed Implementation

[0034] The inventors dedicated themselves to solving the problems of this invention and successfully discovered a novel and useful human monoclonal antibody against HIV-1 that overcomes the shortcomings and deficiencies of known antibodies.

[0035] In this paper, the inventors describe novel CD4-binding site antibodies with potency and breadth exceeding those of known CD4bs bNAbs (such as antibodies 1-18 disclosed in WO 2021 / 110764A1 or others). These properties open up unprecedented and new possibilities for antibody-based therapies and the prevention of HIV-1-related diseases.

[0036] Therefore, in a first aspect, the present invention provides an antibody or antigen-binding fragment thereof targeting the CD4 binding site of human immunodeficiency virus HIV-1, wherein the antibody or antigen-binding fragment thereof comprises a combination of heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3 amino acid sequences from the group consisting of: 04_A06 (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), 01_D03 (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 SEQ ID No. 10, respectively). SEQ ID No. 11 (CDR-L2 amino acid sequence and SEQ ID No. 12 CDR-L3 amino acid sequence), 04_B09 (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), 05_C07 (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), and 01_B01 (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. 24 CDR-L3 amino acid sequence), and SEQ ID No. 18 CDR-L3 amino acid sequence. The amino acid sequences of CDR-H1 (SEQ ID No. 25), CDR-H2 (SEQ ID No. 26), CDR-H3 (SEQ ID No. 27), CDR-L1 (SEQ ID No. 28), CDR-L2 (SEQ ID No. 29), and CDR-L3 (SEQ ID No. 30) are listed.

[0037] 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.

[0038] 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 ).

[0039] 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:

[0040] The CDRL3 sequences of antibodies 04_A06 and 04_B09 could not be accurately determined using IMGT software. This is believed to be due to specific sequences not conforming to the IMGT numbering scheme and the criteria for defining CDR3 sequences. The CDRL3 sequences of 04_A06 and 04_B09 described herein are defined based on the assumption of amino acid substitutions from FGPG to FAPG in the J region motif (F / WGXG) and compared to gene sequences encoding CD4bs bNAb from other IGHV1-2 and IGKV1-33.

[0041] The CDRL3 sequences of antibodies 01_D03, 05_C07, and 01_B01 could not be accurately determined using IMGT software. This is believed to be due to specific sequences not conforming to the IMGT numbering scheme and the criteria for defining CDR3 sequences. The CDRL3 sequence of 01_D03 is defined in this paper based on the assumption of amino acid substitutions from FGPG to FPPV in the J region motif (F / WGXG) and compared with gene sequences encoding CD4bs bNAbs from other IGHV1-2 and IGKV1-33. The CDRL3 sequences of 05_C07 and 01_B01 are defined in this paper based on the assumption of amino acid substitutions from FGPG to FPPA in the J region motif (F / WGXG) and compared with gene sequences encoding CD4bs bNAbs from other IGHV1-2 and IGKV1-33.

[0042] Furthermore, a preferred embodiment of the first aspect of the invention provides an antibody or antigen-binding fragment targeting the CD4 binding site of HIV-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 selected from the group consisting of: 04_A06 (composed of the heavy chain amino acid sequence of SEQ ID No. 31 and the light chain amino acid sequence of SEQ ID No. 32, or preferably composed of the heavy chain amino acid sequence of SEQ ID No. 31 and the light chain amino acid sequence defined in SEQ ID No. 41), 01_D03 (composed of the heavy chain amino acid sequence of SEQ ID No. 33 and the light chain amino acid sequence of SEQ ID No. 34), 04_B09 (composed of the heavy chain amino acid sequence of SEQ ID No. 35 and the light chain amino acid sequence of SEQ ID No. 36), 05_C07 (composed of the heavy chain amino acid sequence of SEQ ID No. 37 and the light chain amino acid sequence of SEQ ID No. 38), and 01_B01 (composed of the heavy chain amino acid sequence of SEQ ID No. 33 and the light chain amino acid sequence of SEQ ID No. 34). It consists of the heavy chain amino acid sequence of SEQ ID No. 39 and the light chain amino acid sequence of SEQ ID No. 40.

[0043] 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 04_A06, 01_D03, 04_B09, 05_C07 and 01_B01 described herein are as follows:

[0044] 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 04_A06 (SEQ ID No. 31). 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_D03 (SEQ ID No. 33). 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 04_B09 (SEQ ID No. 35). 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 05_C07 (SEQ ID No. 37). According to an embodiment of the present invention, the antibody or its antigen-binding fragment comprises the heavy chain variable region amino acid sequence of antibody 01_B01 (SEQ ID No. 39).

[0045] According to an embodiment of the invention, the antibody or its antigen-binding fragment comprises the light chain variable region amino acid sequence of antibody 04_A06 as defined in SEQ ID No. 32. According to an alternative and preferred embodiment of the invention, the antibody or its antigen-binding fragment comprises the light chain variable region amino acid sequence of antibody 04_A06 as defined in SEQ ID No. 41. According to this embodiment, the light chain variable region amino acid sequence of antibody 04_A06 may preferably be defined herein as SEQ ID NO: 41. According to an embodiment of the invention, the antibody or its antigen-binding fragment comprises the light chain variable region amino acid sequence of antibody 01_D03 (SEQ ID No. 34). According to an embodiment of the invention, the antibody or its antigen-binding fragment comprises the light chain variable region amino acid sequence of antibody 04_B09 (SEQ ID No. 36). According to an embodiment of the invention, the antibody or its antigen-binding fragment comprises the light chain variable region amino acid sequence of antibody 05_C07 (SEQ ID No. 38). According to an embodiment of the invention, the antibody or its antigen-binding fragment comprises the light chain variable region amino acid sequence of antibody 01_B01 (SEQ ID No. 40).

[0046] 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. 31 and the light chain variable region amino acid sequence of SEQ ID No. 32. 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. 31 and the light chain variable region amino acid sequence of SEQ ID No. 41. 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. 33 and the light chain variable region amino acid sequence of SEQ ID No. 34. 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. 35 and the light chain variable region amino acid sequence of SEQ ID No. 36. 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. 37 and the light chain variable region amino acid sequence of SEQ ID No. 38. 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. 39 and the light chain variable region amino acid sequence of SEQ ID No. 40.

[0047] According to a specific embodiment of the present invention, the antibody is composed of two heavy chains of sequence SEQ ID No. 31 and two light chains of sequence SEQ ID No. 32, or the antibody is composed of two heavy chains of sequence SEQ ID No. 31 and two light chains of sequence SEQ ID No. 41, or the antibody is composed of two heavy chains of sequence SEQ ID No. 33 and two light chains of sequence SEQ ID No. 34, or the antibody is composed of two heavy chains of sequence SEQ ID No. 35 and two light chains of sequence SEQ ID No. 36, or the antibody is composed of two heavy chains of sequence SEQ ID No. 37 and two light chains of sequence SEQ ID No. 38, or the antibody is composed of two heavy chains of sequence SEQ ID No. 39 and two light chains of sequence SEQ ID No. 40.

[0048] 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 04_A06, 01_D03, 04_B09 and 05_C07, preferably from the group consisting of 04_A06, 01_D03 and 04_B09, more preferably from the group consisting of 04_A06 and 01_D03, and particularly preferably antibody 04_A06.

[0049] 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 04_A06. 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_D03. 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 04_B09. 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 05_C07. 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_B01.

[0050] 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).

[0051] Optionally, the antigen-binding compound consists of or contains the following substances: Fab, Fab', Fab'-SH, F(ab)2, Fv, biantibody, single-chain antibody fragment, or multispecific antibody containing multiple different antibody fragments.

[0052] In this invention, an antibody or binding fragment targeting the CD4 binding site of HIV-1 means an antibody that binds to the CD4 binding site region within the gp120 envelope glycoprotein of HIV-1 with at least 10-fold, more preferably at least 50-fold, and particularly preferably at least 100-fold increased affinity compared to unrelated epitopes, proteins, or protein regions. Generally, the term CD4 binding site herein refers to the CD4 binding site region within the gp120 envelope glycoprotein of HIV-1.

[0053] This invention also provides and covers monoclonal human antibodies or binding fragments comprising at least 80% identical antibody amino acid sequences to those defined and disclosed herein, provided they still target the CD4 binding site of human immunodeficiency virus HIV-1. This means sequences comprising small mutations in the antibody amino acid sequence that do not interfere with structural folding and antibody affinity for the CD4 binding site.

[0054] 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 preferred that the antibody amino acid sequence, which is at least 80% identical to the sequence defined herein, does not carry mutations in the CDR, more preferably not in the antibody's CDR or frame region, if no mutations are present at all.

[0055] Determining whether antibodies exhibiting a certain degree of similarity target the CD4 binding site of human immunodeficiency virus HIV-1, based on the above information or common knowledge, is a simple task for technicians.

[0056] 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.

[0057] According to a preferred embodiment of the invention, the antibody amino acid sequence forms part of the invention and consists of or comprises a nucleic acid sequence that is at least 85% identical, more preferably at least 90% identical, and even more preferably at least 95% identical to the sequence 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 sequence disclosed herein.

[0058] 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 art and in the technical field of this invention to analyze the neutralizing potency of antibodies against various HIV-1 strains. Briefly, the antibody and viral strain are incubated together before the addition of TZM-bl target cells. These cells, upon successful infection, exhibit luciferase activity, resulting in a detectable luminescent signal in the presence of luciferin after cell lysis. Neutralizing antibodies prevent infection, thereby preventing the generation of luminescence. The potency of neutralizing antibodies is determined by the antibody concentration required to reduce viral infectivity to a specific amount.

[0059] Exemplary and standardized methods for establishing and adopting this assay are described in Sarzotti-Kelsoe et al.; J Immunol Methods The details are disclosed and described in detail in July 2014; O: 131-146. doi:10.1016 / j.jim.2013.11.022. Preferably, the neutralizing activity is determined using the method 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.

[0060] 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 .

[0061] 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 04_A06 (composed of the heavy chain amino acid sequence of SEQ ID No. 31 and the light chain amino acid sequence as defined in SEQ ID No. 32, or preferably composed of the heavy chain amino acid sequence of SEQ ID No. 31 and the light chain amino acid sequence as defined in SEQ ID No. 41).

[0062] 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.

[0063] In the description of this application, antibody names may be used. It should be noted that antibodies consist of heavy and light chains, which also form part of this specification. If an antibody is referred to by its name or by the SEQ ID No., it should be understood that these references are interchangeable.

[0064] 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. Preferably, the pharmaceutical composition according to the second aspect of the invention is a vaccination composition for human subjects.

[0065] Preferably, the pharmaceutical composition according to the second aspect of the invention may further comprise one or more antibodies that target different epitopes within HIV-1, preferably targeting the HIV-1 envelope (Env) trimer, and more preferably targeting the V3 glycan site.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] In a preferred embodiment of the fifth or sixth aspect of the invention, the use is in human subjects.

[0071] 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).

[0072] 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.

[0073] 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 preparation 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.

[0074] 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.

[0075] 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.

[0076] According to a ninth aspect of the invention, a host cell is provided, which contains a nucleic acid according to a seventh aspect of the invention or an expression vector according to an eighth aspect of the invention.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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. + lgG + 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: 10.1016 / j.jim.2020.112752 (two PCRs) were used for amplification. 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 clonability of the sequences, all productive heavy chain sequences were grouped by the same V gene, and their CDRH3 pairwise Levenshtein distances were determined.

[0081] Individual sequences sharing the same V gene and exhibiting a minimum CDRH3 identity of 75% were grouped as clones. 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 re-validated 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 as subclones. Nucleotide sequence identity with the phylogenetic line was calculated using IgBLAST.

[0082] 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.) with expression vector overhangs for subsequent sequence-independent ligation-independent cloning (SLIC). The PCR product was cloned into human antibody expression vectors (IgG1, κ, or λ chains) via SLIC assembly, 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). After neutralization in buffer, buffer exchange to PBS, and filtration sterilization, the antibody is stored at 4°C.

[0083] fake virus preparation Pseudoviruses were generated in HEK293T cells by co-transfection with the pSG3AEnv 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).

[0084] 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).

[0085] Humanized mice infected with HIV-1 Humanized mice were prepared as previously described (Klein et al., 2012), but with modifications. Human CD34 was administered intrahepatically within five days of birth and 3–6 hours after sublethal radiation exposure. + Hematopoietic cord blood and / or placental tissue stem cells, NOD.Cg- Rag1 tm1mom ll2rg tm1Wjl / SzJ (NRG) mice were humanized using replicative recombinant HIV-1. YU2 (YU2 in the NL4-3 skeleton) env Humanized mice were infected by intraperitoneal attack (Zhang et al., J Virol. 2002, 76(12):6332-43. doi: 10.1128 / jvi.76.12.6332-6343.2002), and the recombinant virus was harvested from the supernatant of transfected HEK293T cells.

[0086] HIV-1 viral load measurement Plasma RNA was extracted from EDTA plasma samples using the MinElute Virus Miniature Spin Kit, including an on-column DNase I digestion step. Viral load was determined by quantitative real-time PCR using previously described pol-specific primers (Horwitz et al., Proc Natl Acad Sci US A. 2013 Oct 8; 110(41):16538-43. doi: 10.1073 / pnas.1315295110). qPCR was performed on a QuantStudio 5 using the Taqman RNA-to-Ct 1-Step-Kit. Viral load was quantified by a standard curve derived from samples with known copy numbers from each qPCR run. The accuracy limit of qPCR was determined to be 784 copies / ml.

[0087] Antibody therapy in humanized mice infected with HIV-1 The antibody, diluted in PBS, is administered subcutaneously. Following a 1 mg loading dose, 0.5 mg doses are administered every 3–4 days.

[0088] B) Specific embodiments of the antibody of the present invention Example I - Isolation of widely and effectively VH1-2 derived HIV-1 from well-neutralized individuals infected with HIV-1 and antibodies 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.

[0089] 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.

[0090] Sequence analysis of HIV-1-Env-responsive B cells identified expanded VH1-2-derived B cell clones. One of these clones is characterized by a twelve-amino acid (aa) insertion and a one-amino acid deletion in the heavy chain CDR1 / FWR1 region. Antibody 04_A06 represents this B cell clone. Another clone among these contains a 6-aa CDRH1 / FWRH1 and a 4-aa FWRH3 insertion. Antibody 01_D03 is representative of this B cell clone.

[0091] To determine the overall neutralizing potency and extent, the neutralizing activity of isolated antibody 04_A06, along with other antibodies, 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." 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.

[0092] It is noteworthy that all antibodies of the present invention (i.e., 04_A06, 01_D03, 04_B09, 05_C07, and 01_B01) exhibited high to medium neutralizing activity against 100% (12 / 12) of the strains included in the group. Figure 1 Neutralizing efficacy is typically determined by a 50% inhibitory concentration (IC50). 50(This indicates that) when tested against the “global group,” all antibodies of this invention exhibited a geometric mean IC50 of less than 0.05 μg / ml. 50 Among them, 04_A06 and 01_D03 are highly active, with a geometric mean IC50 against neutralized viral strains. 50 The concentration was 0.038 μg / ml or 0.021 μg / ml. Figure 1 ).

[0093] Example II - High potency and broad-spectrum antibody 04_A06 To confirm the neutralizing potency and breadth of 04_A06, an expanded group of antibodies was tested against 119 HIV-1 pseudoviruses, as previously tested in Schoofs et al., 2019. This group of pseudoviruses represents the evolution of a group of pseudoviruses detailed in Seaman et al., 2010. 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.

[0094] It is worth noting that when tested on the 119 pseudovirus multi-clade, 04_A06 exhibited high efficacy and breadth (…). Figure 2 Specifically, when tested at antibody concentrations up to 10 μg / ml, antibody 04_A06 neutralized 99% of the tested pseudoviruses and showed a geometric mean IC50 of 0.036 μg / ml against the neutralized pseudoviruses. 50 ( Figure 2 This breadth and potency is superior to other HIV-1 neutralizing antibodies that target the same CD4 binding site and are in advanced stages of clinical testing (e.g., 3BNC117, VRC01, and VRC07). -523-LS The breadth and effectiveness observed in ) Figure 2 Furthermore, antibody 04_A06 showed higher potency than N6 in clade 119, which is another VH1-2-derived CD4-binding antibody currently under clinical testing. Figure 2 ).

[0095] Furthermore, when compared with other HIV-1 neutralizing antibodies and other CD4b bNAbs, these antibodies provide protection against a total of 76 ( ). Figure 3 A) and 99 kinds ( Figure 3 B) Results of the same HIV-1 pseudovirus (Yoon et al., 2015, ibid.), while 04_A06 showed the highest level of efficacy and / or breadth.

[0096] Example III - High potency and broad spectrum of antibody 04_A06 against clade C HIV-1 pseudovirus Group M HIV-1 viruses can be divided into nine clades and different recombinants. HIV-1 viruses from clade C are responsible for approximately half of all current HIV-1 infections and represent the most common lineage worldwide. Therefore, clade C viruses have been studied in many different antibody and vaccine studies. To confirm the antiviral activity of antibody 04_A06 against clinically relevant HIV-1 clades, we determined the neutralization profile of the antibody against a published reference group consisting of 100 HIV-1 pseudoviruses and difficult-to-neutralize viruses isolated from different stages of infection. The so-called subtype group C is described in detail in Hraber et al., J Virol., 2017 Sep 12; 91(19):e00991-17.

[0097] It is worth noting that when tested against 100 strains of subtype C pseudovirus group, 04_A06 exhibited high efficacy and broad coverage. Figure 4 Specifically, when tested at antibody concentrations up to 10 μg / ml, antibody 04_A06 neutralized 99% of the tested pseudoviruses and showed a geometric mean IC50 of 0.057 μg / ml against the neutralized pseudoviruses. 50 ( Figure 4 The breadth and potency of 04_A06 are superior to the best CD4bs bNAb (3BNC117, 1-18, N6, VRC01, VRC07) studied to date for subtype C pseudovirus group. -523-LS The breadth and effectiveness observed () Figure 4 CD4bs reference bNAb neutralization data were obtained from the CATNAP database (Yoon et al., 2015, ibid.).

[0098] Example IV - Neutralizing activity against pseudoviruses that are poorly neutralized by antibodies targeting other CD4 binding sites Although CD4 binding site antibodies can achieve high levels of broad neutralization (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 CD4 binding site antibodies with high activity against such HIV-1 variants. Thus, we assayed antibody 04_A06 described in this invention against a group of nine (… Figure 5 ) and seven kinds ( Figure 6 Neutralizing characteristics of HIV-1 variants that are resistant to neutralization of CD4bs bNAb.

[0099] Figure 5The test virus strain shown is part of the 119 multiphylogenetic clades and subtypes of the C pseudovirus group (as described in Schoofs et al., Immunity, 18 June 2019; 50(6):1513-1529.e9, Hraber et al., J Virol., 12 September 2017; 91(19):e00991-17). Figure 6 The test virus strain shown was selected from the reference pseudovirus group known as the “CD4bs bNAb resistance group” and described in Zhou et al., PLoS Pathog. 13 June 2019; 15(6):e1007819. Doi: 10.1371 / journal.ppat.1007819.PMID: 31194843; PMCID: PMC6592578 12 September 2017; 91(19):e00991-17.

[0100] Antibodies targeting CD4 binding sites in advanced phases of clinical trials and previously published CD4bs-like bNAb (VRC07) -523-LS Compared to the best antibodies among N6, 3BNC117, 1-18, and VRC01, antibody 04_A06 neutralizes resistant pseudoviral variants with a much higher breadth and / or potency. Figure 5 and Figure 6 ).

[0101] For example, when tested at concentrations up to 10 μg / ml in the TZM-bl pseudovirus neutralization assay, 100% of the nine-pseudovirus group and 86% of the seven-pseudovirus group were neutralized by O4_A06. In contrast, the most extensive and potent CD4bs reference bNAb described to date neutralized only 0%–67% of the nine-pseudovirus group. Figure 5 ) and 0%-57% of the seven pseudovirus groups ( Figure 6 Furthermore, compared to the reference CD4bs bNAb, 04_A06 showed the highest efficacy against strains of seven pseudovirus groups (IC). 50 (0.153 μg / ml) Figure 6 ).

[0102] Therefore, the antibody of the present invention provides a solution to poor virus neutralization with resistance to CD4bs bNAb.

[0103] Example V - Neutralizing activity of antibody 01_D03 against pseudoviruses insensitive to CD4bs bNAb. In addition to resistant viral variants, there exist variants that are insensitive to the neutralization of most CD4bs bNAbs. Clinical trials have demonstrated that the sensitivity of viral variants is critical to the success of bNAb-mediated treatment or prevention strategies. The inventors investigated the neutralizing characteristics of antibody O1_D03 against a virome composed of eight pseudoviral strains known to be insensitive to the neutralization of most CD4bs bNAbs. Figure 7 ).

[0104] The viral strains in this group are part of the 119 multiclade pseudovirus group (as described in Schoofs et al., Immunity, 18 June 2019; 50(6):1513-1529.e9). (via IC) 50 >1 μg / ml defines insensitivity to neutralization by a given antibody. Antibody 01_D03 neutralized 88% (7 / 8) of viral strains from eight pseudovirus groups, while the most extensive and effective reference bNAb described to date against CD4bs neutralized only 25%–75% ( Figure 7 Therefore, the antibody of the present invention provides a solution for viruses that are poorly neutralized and insensitive to most other CD4bs bNAb.

[0105] Example VI - Neutralizing activity against pseudoviruses generated by antibody-mediated prophylaxis assays Recent antibody-mediated prophylaxis (AMP) trials have provided evidence for the concept that bNAb administration can, in principle, effectively prevent HIV-1 infection in humans. In this trial, the prophylactic efficacy of CD4bs bNAb VRC01 largely depended on the sensitivity of circulating viral strains to the antibody. VRC01 showed 75% prophylactic efficacy against neutralizing viral strains, with an IC50 value of [missing information]. 80 Less than 1 μg / ml (as described in Corey L, Two Randomized Trials of Neutralizing Antibodies to Prevent HIV-1 Acquisition. N Engl J Med. 2021 Mar 18; 384(11):1003-1014. Doi: 10.1056 / NEJMoa2031738. PMID: 33730454; PMCID: PMC8189692).

[0106] Antibody 04_A06 neutralized up to 98% of the 185 pseudovirus strains generated from viral sequences obtained by the AMP assay. Figure 8 In contrast, VRC01 only showed a maximum breadth of 83% against the tested pseudovirus groups. Furthermore, 04_A06 against the GeoMean IC virus group... 50(0.084 μg / ml) is approximately 10 times that of VRC01 (0.839 μg / ml). Therefore, 04_A06 can be considered a promising bNAb candidate for future antibody-based prophylaxis strategies.

[0107] Example VII - Maintaining viral suppression via antibody monotherapy in an in vivo HIV-1 infection model HIV-1 infection YU2 Humanized mice (Zhang et al., 2002, ibid.) provide a model for studying the antiviral activity of antibodies that neutralize HIV-1 in vivo. To generate humanized mice, immunodeficient NRG mice were irradiated on the first day after birth and injected intrahepatically with artificial blood CD34. + Stem cells. This leads to the development of human lymphocytes that can be infected with replicating HIV-1. These mice maintained stable levels of viremia (i.e., HIV-1 RNA copy number in plasma) and showed a similar rate of HIV-1 sequence diversification as observed in humans (Klein et al., Nature. Dec. 6, 2012; 492(7427):118-22. doi: 10.1038 / naturel 1604). This mouse model has previously been used to determine the antiviral activity of several CD4-binding site antibodies administered in vivo as a single therapy (179NC75: Freund et al., PLoS Pathog. 2015 Oct 30; 11 (10):e1005238. doi: 10.1371 / journal.ppat.1005238; 3BNC117: Horwitz et al., Proc Natl Acad Sci US A. 2013 Oct 8; 110(41):16538-43. doi: 10.1073 / pnas.1315295110; 45-46). G54W : Klein et al., 2012, ibid.; NC37: Freund et al., Sci Transl Med. Jan 18, 2017; 9(373):eaal2144. doi:10.1126 / scitranslmed.aal2144).

[0108] In all these studies, only transient effects on viral load were observed, and viral rebound (i.e., recovery of viremia to baseline levels) developed rapidly. Furthermore, this viral rebound was associated with the development of mutations at the antibody target site. Therefore, there is a need for novel CD4-binding site antibodies that can maintain viral suppression when administered as monotherapy.

[0109] When humanized mice infected with HIV-1 YU2 were treated with CD4 binding site antibodies VRC01 or VRC07 (subcutaneous (sc) 1 mg loading dose of each antibody, followed by 0.5 mg subcutaneously every 3–4 days), only a transient decrease in HIV-1 RNA copy number was observed, and most mice recovered viremia to baseline levels within 2–4 weeks. Figure 9 In contrast, when mice were treated with the representative antibody 04_A06 of the present invention according to the same dosing regimen, viral suppression was observed in all treated mice compared with the viral copy number at the start of treatment, lasting for 84 days. Figure 9 In all mice (100%), HIV-1 RNA plasma copy number decreased to levels below the accuracy limit of the assay used (784 copies / ml).

[0110] Therefore, even when administered as a monotherapy, 04_A06 was able to effectively induce and maintain viral suppression in humanized mice infected with HIV-1 YU2.

[0111] Example VIII - In an in vivo HIV-1 infection model following viral rebound after VRC01 therapy, antibody monoclonal antibody was used. One therapy to maintain viral suppression Failure of antibody monotherapy using CD4-binding site antibodies in humanized mice infected with HIV-1 has led to viral rebound and is associated with viral resistance to the administered antibody (Freund et al., 2015, ibid.; Horwitz et al., 2013, ibid.; Klein et al., 2012, ibid.; Freund et al., 2017, ibid.). Therefore, there is a need for novel CD4-binding site antibodies that provide an in vivo treatment option after previous failures of CD4-binding site therapies.

[0112] After observing viral rebound during four weeks of treatment with VRC01 (as described in Example VII), adding the antibody 04_A06 of the present invention (1 mg subcutaneous loading dose, followed by 0.5 mg subcutaneously every 3 to 4 days) to the treatment regimen resulted in a sustained reduction in viremia in all (100%) treated mice. Figure 10 Therefore, even after pretreatment with antibodies targeting other CD4 binding sites fails, antibody 04_A06 provides an option for controlling HIV-1 in vivo.

Claims

1. An antibody or an antigen-binding fragment thereof targeting the CD4 binding site of human immunodeficiency virus (HIV-1), wherein the antibody or antigen-binding fragment thereof comprises a combination of the heavy chain CDR1 to CDR3 and the light chain CDR1 to CDR3 amino acid sequences of an antibody selected from the group consisting of: 04_A06 (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), 01_D03 (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). 04_B09 (possessing 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). 05_C07 (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), and 01_B01 (containing the CDR-H1 amino acid sequence of SEQ ID No. 25, the CDR-H2 amino acid sequence of SEQ ID No. 26, the CDR-H3 amino acid sequence of SEQ ID No. 27, the CDR-L1 amino acid sequence of SEQ ID No. 28, the CDR-L2 amino acid sequence of SEQ ID No. 29, and the CDR-L3 amino acid sequence of SEQ ID No. 30).

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: 04_A06 (composed of the heavy chain amino acid sequence of SEQ ID No. 31 and the light chain amino acid sequence of SEQ ID No. 41). 01_D03 (composed of the heavy chain amino acid sequence of SEQ ID No. 33 and the light chain amino acid sequence of SEQ ID No. 34). 04_B09 (composed of the heavy chain amino acid sequence of SEQ ID No. 35 and the light chain amino acid sequence of SEQ ID No. 36). 05_C07 (composed of the heavy chain amino acid sequence of SEQ ID No. 37 and the light chain amino acid sequence of SEQ ID No. 38), and 01_B01 (composed of the heavy chain amino acid sequence of SEQ ID No. 39 and the light chain amino acid sequence of SEQ ID No. 40).

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 04_A06, 01_D03, 04_B09 and 05_C07, preferably an antibody selected from the group consisting of 04_A06, 01_D03 and 04_B09, more preferably an antibody selected from the group consisting of 04_A06 and 01_D03, and particularly preferably antibody 04_A06.

4. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3 and at least one pharmaceutically acceptable excipient.

5. The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition is a vaccination composition for human subjects.

6. A kit comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3, and a container.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the pharmaceutical composition according to any one of claims 4 or 5, or the kit according to claim 6, are used as pharmaceuticals.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the pharmaceutical composition according to any one of claims 4 or 5, or the kit according to claim 6, for use in the treatment or prevention of said human immunodeficiency virus HIV-1 infection in mammalian subjects.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the pharmaceutical composition according to any one of claims 4 or 5, or the kit according to claim 6, for use in the treatment or prevention of disease caused by said human immunodeficiency virus HIV-1 in mammalian subjects.

10. The antibody or antigen-binding fragment thereof, pharmaceutical composition or kit for use according to any one of claims 8 or 9, wherein the use is for use in human subjects.

11. The antibody or antigen-binding fragment thereof, pharmaceutical composition or kit for use according to any one of claims 8 to 10, wherein the use is for the treatment or prevention of acquired immunodeficiency syndrome (AIDS).

12. A nucleic acid, wherein the nucleic acid encodes an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3.

13. An expression vector comprising the nucleic acid of claim 12 that is functionally associated with an expression control sequence.

14. A host cell comprising the nucleic acid according to claim 12 or the expression vector according to claim 13.

15. A method for preparing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, said method comprising: (a) Culture the host cells according to claim 14 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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