Broadly neutralizing antibodies against HIV

Abstract

To provide a novel human monoclonal antibody against HIV-1.SOLUTION: The present disclosure relates to monoclonal human antibodies or binding fragments thereof that target the CD4 binding site of the human immunodeficiency virus HIV-1, a pharmaceutical composition comprising such monoclonal human antibodies or binding fragments thereof, a kit comprising such antibodies or binding fragments thereof, and the monoclonal antibodies or binding fragments thereof, the pharmaceutical composition, and the kit for use as a medicament and in the treatment or prevention of a disease caused by the human immunodeficiency virus HIV-1.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to monoclonal human antibodies or binding fragments thereof against the CD4 binding site of human immunodeficiency virus HIV-1, pharmaceutical compositions containing such monoclonal human antibodies or binding fragments thereof, kits comprising such antibodies or binding fragments thereof, and monoclonal antibodies or binding fragments thereof, pharmaceutical compositions, and kits used as medicaments and for the treatment or prevention of diseases caused by human immunodeficiency virus HIV-1.

Background Art

[0002] Broadly neutralizing antibodies (bNAbs) targeting the HIV-1 envelope protein (Env) can prevent infection in animal models and are being investigated for passive immunization in clinical trials. Furthermore, bNAbs have been demonstrated to suppress viremia and delay viral rebound after interruption of antiretroviral therapy (ART) in HIV-1-infected individuals.

[0003] These results highlight the significant clinical potential of bNAbs, but prior and de novo HIV-1 resistance can cause treatment failure and may strongly limit the application of bNAbs in humans. Therefore, strategies to prevent and overcome viral escape are important for effectively implementing bNAb-mediated approaches for the prevention and treatment of HIV-1 (Non-Patent Document 1).

[0004] In recent years, potent bNAbs targeting distinct vulnerable epitopes on the HIV-1 envelope (Env) trimer have been isolated from HIV-1-infected donors. These epitopes include the CD4 binding site (CD4bs), V1 / V2 loop, V3 loop glycan patch, membrane-proximal external region, and the interface between the gp120 and gp41 Env subunits.

[0005] Among these sites, CD4 serves as a major receptor for virus entry, so the CD4bs is of particular interest. Most of the most potent CD4bs bNAbs utilize the immunoglobulin heavy chain gene segment IGHV1-2 * utilization, high levels of somatic hypermutation, a 5-residue light chain complementarity-determining region 3 (CDRL3), and mimicry of the Env-CD4 interaction.

[0006] These antibodies, named after the prototype antibody VRC01 (Non-Patent Document 2), are referred to as VRC01-class bNAbs. Other members of this class include 3BNC117, NIH45-46, N49-P7, N6, and VRC07-523.

[0007] Other bNAbs that mimic CD4 binding are derived from the VH1-46 gene segment. However, compared to VH1-2-derived bNAbs, the VH1-46 bNAbs reported to date have low potency and breadth that limit their potential for clinical use. For example, CH235.12, one of the best antibodies in this class, is not as broad and has less than one-tenth the potency of the VRC01-class bNAb N6 when tested in vitro against a large panel of HIV-1 Env strains (Non-Patent Document 3).

[0008] Thus, all CD4bs bNAbs that have advanced to clinical trials are members of the VRC01 class (3BNC117, N6, VRC01, and VRC07-523). However, while escape from VRC01 is associated with a decrease in viral fitness, the effect of monotherapy with VRC01-class antibodies is only transient and is associated with the rapid emergence of viral escape mutants in both clinical trials (Non-Patent Document 4) and animal models of HIV-1 infection (Non-Patent Document 5). associated.

Prior Art Documents

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

Problems to be Solved by the Invention

[0010] Due to the drawbacks of currently investigated known bNAbs, when tested in HIV-1 infected organisms, it exceeds the potency and breadth of known classical VH1-2-derived or VH1-46-derived bNAbs, has strong neutralizing activity against VRC01-class escape mutants, effectively limits viral escape, and maintains viral suppression. There is still a need for HIV antibodies that target the CD4 binding site.

[0011] Therefore, an object of the present invention is to provide a novel human monoclonal antibody against HIV-1 that has broad neutralizing activity against a wide selection of different viral strains in combination with high neutralizing potency against such viral strains. A further object of the present invention is to provide a novel human monoclonal antibody against HIV-1 that shows significant limitation of the occurrence of escape mutations and maintains effectiveness against viruses showing such escape mutations. It is also an object of the present invention to provide a novel human monoclonal antibody against HIV-1 that confers complete viral suppression in infected individuals without significant viral rebound during antibody monotherapy. Furthermore, an object of the present invention is to provide a novel human monoclonal antibody against the CD4 binding site that has favorable pharmacokinetic properties in vivo.

[0012] These objects were solved by the aspects of the present invention described below.

Means for Solving the Problems

[0013] According to a first aspect of the present invention, there is provided a monoclonal human antibody or a binding fragment thereof against the CD4 binding site of human immunodeficiency virus HIV-1, wherein the antibody amino acid sequence contains the V H 1-46 gene segment and the V κ 3-20 gene segment, and the antibody contains either a) the heavy chain amino acid sequence of SEQ ID NO: 47 and the light chain amino acid sequence of SEQ ID NO: 48, or b) the heavy chain amino acid sequence of SEQ ID NO: 49 and the light chain amino acid sequence of SEQ ID NO: 50, and X in any of SEQ ID NOs: 47 to 50 may be any amino acid or may be absent of an amino acid, or the antibody sequence is at least 80% identical to the sequence.

[0014] According to a preferred embodiment of the first aspect of the present invention, the antibody of alternative b) of the first aspect of the present invention contains a 2aa deletion in FWR1.

[0015] According to another preferred embodiment of the first aspect of the present invention, when testing preferably all 12 strains at an antibody concentration of up to 25 μg / ml in the TZM-bl cell pseudovirus neutralization assay, the antibody or its binding fragment neutralizes at least 11 of the 12 HIV-1 isolate reference strains of the global reference panel described in de Camp et al., J Virol. 2014 Mar; 88(5): 2489-2507 and exhibits broad neutralizing activity exemplified by such neutralization.

[0016] According to yet another preferred embodiment of the first aspect of the present invention, when the antibody or its binding fragment is tested at an antibody concentration of up to 20 μg / ml in the TZM-bl cell pseudovirus neutralization assay, it shows broad neutralizing activity exemplified by neutralization of at least 89.9% (107 out of 119), preferably at least 92.4% (110 out of 119), more preferably at least 96.6% (115 out of 119) of the pseudoviruses included in the 119-multiclade virus panel described in Schoofs et al., Immunity, 2019 Jun 18;50(6):1513-1529.e9.

[0017] According to a preferred embodiment of the first aspect of the present invention, when the antibody or its binding fragment is tested at an antibody concentration of up to 25 μg / ml in the TZM-bl cell pseudovirus neutralization assay, it has a neutralizing potency of less than 0.3 μg / ml, preferably less than 0.2 μg / ml, more preferably less than 0.15 μg / ml, even more preferably less than 0.1 μg / ml, even more preferably less than 0.05 μg / ml, still more preferably 0.048 μg / ml, particularly preferably 0.035 μg / ml, against the neutralizing strains of the global reference panel described in de Camp et al., J Virol. 2014 Mar; 88(5): 2489-2507. (geometric mean IC of the neutralizing strain). 50 )

[0018] According to a preferred embodiment of the first aspect of the present invention, when the antibody or its binding fragment is tested at an antibody concentration of up to 20 μg / ml in the TZM-bl cell pseudovirus neutralization assay, it has a neutralizing potency of less than 0.2 μg / ml, preferably less than 0.1 μg / ml, more preferably less than 0.08 μg / ml, even more preferably less than 0.05 μg / ml, against the neutralizing strains of the 119-multiclade virus panel described in Schoofs et al., Immunity, 2019 Jun 18;50(6):1513-1529.e9. 50 (geometric mean IC of the neutralizing strain).

[0019] According to another preferred embodiment of the first aspect of the present invention, when tested in a TZM-bl pseudovirus neutralization assay, the antibody or its binding fragment has a neutralizing potency (IC env ) of less than 0.05 μg / ml, preferably less than 0.02 μg / ml, and even more preferably less than 0.01 μg / ml against the HIV-1 pseudovirus 89-F1_2_25 (89-F1_2_25 50 ; GenBank: HM215349.1).

[0020] According to yet another preferred embodiment of the first aspect of the present invention, when tested in a TZM-bl pseudovirus neutralization assay, the antibody or binding fragment has an IC 50 concentration of less than 0.1 μg / ml, preferably less than 0.05 μg / ml, and neutralizes all of the YU2 pseudovirus variants containing any one of the envelope mutations N279K, N280Y, G458D, G459D, or G471R (residues numbered according to the HIV-1 HXB2 envelope gene; GenBank: K03455) in the YU2 envelope gene (GenBank: M93258.1).

[0021] According to a preferred embodiment of the first aspect of the present invention, following an initial subcutaneous injection of 1 mg of the antibody or its binding fragment, 3 to 4 days later, for humanized mice infected with HIV-1 NL4-3 / YU2 as described in Zhang et al., J Virol, 2002 Jun;76(12):6332-43, performing regular subcutaneous injections of 0.5 mg of the antibody or its binding fragment every 3 to 4 days results in at least a 0.8 log 10 , preferably at least a 1.0 log 10 decrease in the plasma HIV-1 RNA load in at least 70% of the treated mice having an HIV-1 RNA load of at least 5000 copies / ml plasma at the start of treatment, as measured 4 weeks after treatment, preferably 6 weeks after treatment, and even more preferably 8 weeks after treatment, compared to the start of treatment.

[0022] According to a more preferred embodiment of the above embodiment of the first aspect of the present invention, a humanized mouse is first treated for 4 weeks with a single initial subcutaneous injection of 1 mg of 3BNC117 or VRC01, or a combination of both, followed by a regular subcutaneous injection of 0.5 mg of 3BNC117 or VRC01, or a combination of both, every 3 to 4 days, 3 to 4 days later.

[0023] According to a preferred embodiment of the first aspect of the present invention, following an initial subcutaneous injection of 1 mg of an antibody or its binding fragment, 3 to 4 days later, a regular subcutaneous injection of 0.5 mg of the antibody or its binding fragment is administered every 3 to 4 days to humanized mice infected with HIV-1 NL4-3 / YU2 as described in Zhang et al., J Virol, 2002 Jun;76(12):6332-43, for at least 4 weeks, without resulting in the occurrence of one or more mutations in the CD4 binding sites (loop D, CD4 binding loop, beta23 strand, V5 loop, and beta24) that mediate resistance to the administered antibody.

[0024] According to another preferred embodiment of the first aspect of the present invention, an intravenous injection of 0.5 mg of an antibody or its binding fragment into NRG mice results in a detectable serum level of the antibody or its binding fragment of at least 50 μg IgG / ml serum 10 days after the injection.

[0025] According to another preferred embodiment of the first aspect of the present invention, the antibody or its binding fragment does not contain a CDRH3 having a length of 16 or 19 amino acids.

[0026] According to a preferred embodiment of the first aspect of the present invention, the antibody or its binding fragment contains a CDRH3 having a length of 18, 20, or 21 amino acids.

[0027] According to a preferred embodiment of the first aspect of the present invention, the antibody or its binding fragment is the antibody NC37 described in Freund et al., Sci. Transl. Med. 9, eaal2144 (2) It does not contain the amino acid sequence of NC133, AC40, AC41 or AC72, or is not composed only of these.

[0028] According to another preferred embodiment of the first aspect of the present invention, the antibody or its binding fragment is 1-18 (composed of the heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 2), 1-21 (composed of the heavy chain amino acid sequence of SEQ ID NO: 3 and the light chain amino acid sequence of SEQ ID NO: 4), 1-33 (composed of the heavy chain amino acid sequence of SEQ ID NO: 5 and the light chain amino acid sequence of SEQ ID NO: 6), 1-54 (composed of the heavy chain amino acid sequence of SEQ ID NO: 7 and the light chain amino acid sequence of SEQ ID NO: 8), 1-55 (composed of the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 10), 2-10 (composed of the heavy chain amino acid sequence of SEQ ID NO: 11 and the light chain amino acid sequence of SEQ ID NO: 12), 2-22 (composed of the heavy chain amino acid sequence of SEQ ID NO: 13 and the light chain amino acid sequence of SEQ ID NO: 14), 2-27 (composed of the heavy chain amino acid sequence of SEQ ID NO: 15 and the light chain amino acid sequence of SEQ ID NO: 16), 2-47 (composed of the heavy chain amino acid sequence of SEQ ID NO: 17 and the light chain amino acid sequence of SEQ ID NO: 18), 3-59 (composed of the heavy chain amino acid sequence of SEQ ID NO: 19 and the light chain amino acid sequence of SEQ ID NO: 20), 3-60 (composed of the heavy chain amino acid sequence of SEQ ID NO: 21 and the light chain amino acid sequence of SEQ ID NO: 22), 3-61 (composed of the heavy chain amino acid sequence of SEQ ID NO: 23 and the light chain amino acid sequence of SEQ ID NO: 24), 3-62 (composed of the heavy chain amino acid sequence of SEQ ID NO: 25 and the light chain amino acid sequence of SEQ ID NO: 26), 3-63 (composed of the heavy chain amino acid sequence of SEQ ID NO: 27 and the light chain amino acid sequence of SEQ ID NO: 28), 3-64 (composed of the heavy chain amino acid sequence of SEQ ID NO: 29 and the light chain amino acid sequence of SEQ ID NO: 30), 3-65 (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), 3-66 (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), 3-67 (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), 3-68 (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), 3-69 (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), 3-70 (composed of the heavy chain amino acid sequence of SEQ ID NO: 41 and the light chain amino acid sequence of SEQ ID NO: 42), 3-71 (composed of the heavy chain amino acid sequence of SEQ ID NO: 43 and the light chain amino acid sequence of SEQ ID NO: 44), 3-72 (composed of the heavy chain amino acid sequence of SEQ ID NO: 45 and the light chain amino acid sequence of SEQ ID NO: 46), 3-73 (composed of the heavy chain amino acid sequence of SEQ ID NO: 47 and the light chain amino acid sequence of SEQ ID NO: 48), 3-74 (composed of the heavy chain amino acid sequence of SEQ ID NO: 49 and the light chain amino acid sequence of SEQ ID NO: 50), 3-75 (composed of the heavy chain amino acid sequence of SEQ ID NO: 51 and the light chain amino acid sequence of SEQ ID NO: 52), 3-76 (composed of the heavy chain amino acid sequence of SEQ ID NO: 53 and the light chain amino acid sequence of SEQ ID NO: 54), 3-77 (composed of the heavy chain amino acid sequence of SEQ ID NO: 55 and the light chain amino acid sequence of SEQ ID NO: 56), 3-78 (composed of the heavy chain amino acid sequence of SEQ ID NO: 57 and the light chain amino acid sequence of SEQ ID NO: 58), 3-79 (composed of the heavy chain amino acid sequence of SEQ ID NO: 59 and the light chain amino acid sequence of SEQ ID NO: 60), 3-80 (composed of the heavy chain amino acid sequence of SEQ ID NO: 61 and the light chain amino acid sequence of SEQ ID NO: 62), 3-81 (composed of the heavy chain amino acid sequence of SEQ ID NO: 63 and the light chain amino acid sequence of SEQ ID NO: 64), 3-82 (composed of the heavy chain amino acid sequence of SEQ ID NO: 65 and the light chain amino acid sequence of SEQ ID NO: 66), 3-83 (composed of the heavy chain amino acid sequence of SEQ ID NO: 67 and the light chain amino acid sequence of SEQ ID NO: 68), 3-84 (composed of the heavy chain amino acid sequence of SEQ ID NO: 69 and the light chain amino acid sequence of SEQ ID NO: 70), 3-85 (composed of the heavy chain amino acid sequence of SEQ ID NO: 71 and the light chain amino acid sequence of SEQ ID NO: 72), 3-86 (composed of the heavy chain amino acid sequence of SEQ ID NO: 73 and the light chain amino acid sequence of SEQ ID NO: 74), 3-87 (composed of the heavy chain amino acid sequence of SEQ ID NO: 75 and the light chain amino acid sequence of SEQ ID NO: 76), 3-88 (composed of the heavy chain amino acid sequence of SEQ ID NO: 77 and the light chain amino acid sequence of SEQ ID NO: 78), 3-89 (composed of the heavy chain amino acid sequence of SEQ ID NO: 79 and the light chain amino acid sequence of SEQ ID NO: 80), 3-90 (composed of the heavy chain amino acid sequence of SEQ ID NO: 81 and the light chain amino acid sequence of SEQ ID NO: 82), 3-91 (composed of the heavy chain amino acid sequence of SEQ ID NO: 83 and the light chain amino acid sequence of SEQ ID NO: 84), 3-92 (composed of the heavy chain amino acid sequence of SEQ ID NO: 85 and the light chain amino acid sequence of SEQ ID NO: 86), 3-93 (composed of the heavy chain amino acid sequence of SEQ ID NO: 87 and the light chain amino acid sequence of SEQ ID NO: 88), 3-94 (composed of the heavy chain amino acid sequence of SEQ ID NO: 89 and the light chain amino acid sequence of SEQ ID NO: 90), 3-95 (composed of the heavy chain amino acid sequence of SEQ ID NO: 91 and the light chain amino acid sequence of SEQ ID NO: 92), 3-96 (composed of the heavy chain amino acid sequence of SEQ ID NO: 93 and the light chain amino acid sequence of SEQ ID NO: 94), 3-97 (composed of the heavy chain amino acid sequence of SEQ ID NO: 95 and the light chain amino acid sequence of SEQ ID NO: 96), 3-98 (composed of the heavy chain amino acid sequence of SEQ ID NO: 97 and the light chain amino acid sequence of SEQ ID NO: 98), 3-99 (composed of the heavy chain amino acid sequence of SEQ ID NO: 99 and the light chain amino acid sequence of SEQ ID NO: 100), 4-100 (composed of the heavy chain amino acid sequence of SEQ ID NO: 101 and the light chain amino acid sequence of SEQ ID NO: 102), 4-101 (composed of the heavy chain amino acid sequence of SEQ ID NO: 103 and the light chain amino acid sequence of SEQ ID NO: 104), 4-102 (composed of the heavy chain amino acid sequence of SEQ ID NO: 105 and the light chain amino acid sequence of SEQ ID NO: 106), 4-103 (composed of the heavy chain amino acid sequence of SEQ ID NO: 107 and the light chain amino acid sequence of SEQ ID NO: 108), 4-104 (composed of the heavy chain amino acid sequence of SEQ ID NO: 109 and the light chain amino acid sequence of SEQ ID NO: 110), 4-105 (composed of the heavy chain amino acid sequence of SEQ ID NO: 111 and the light chain amino acid sequence of SEQ ID NO: 112), 4-106 (composed of the heavy chain amino acid sequence of SEQ ID NO: 113 and the light chain amino acid sequence of SEQ ID NO: 114), 4-107 (composed of the heavy chain amino acid sequence of SEQ ID NO: 115 and the light chain amino acid sequence of SEQ ID NO: 116), 4-108 (composed of the heavy chain amino acid sequence of SEQ ID NO: 117 and the light chain amino acid sequence of SEQ ID NO: 118), 4-109 (composed of the heavy chain amino acid sequence of SEQ ID NO: 119 and the light chain amino acid sequence of SEQ ID NO: 120), 4-110 (composed of the heavy chain amino acid sequence of SEQ ID NO: 121 and the light chain amino acid sequence of SEQ ID NO: 122), 4-111 (composed of the heavy chain amino acid sequence of SEQ ID NO: 123 and the light chain amino acid sequence of SEQ ID NO: 124), 4-112 (composed of the heavy chain amino acid sequence of SEQ ID NO: 125 and the light chain amino acid sequence of SEQ ID NO: 126), 4-113 (composed of the heavy chain amino acid sequence of SEQ ID NO: 127 and the light chain amino acid sequence of SEQ ID NO: 128), 4-114 (composed of the heavy chain amino acid sequence of SEQ ID NO: 129 and the light chain amino acid sequence of SEQ ID NO: 130), 4-115 (composed of the heavy chain amino acid sequence of SEQ ID NO: 131 and the light chain amino acid sequence of SEQ ID NO: 132), 4-116 (composed of the heavy chain amino acid sequence of SEQ ID NO: 133 and the light chain amino acid sequence of SEQ ID NO: 134), 4-117 (composed of the heavy chain amino acid sequence of SEQ ID NO: 135 and the light chain amino acid sequence of SEQ ID NO: 136), 4-118 (composed of the heavy chain amino acid sequence of SEQ ID NO: 137 and the light chain amino acid sequence of SEQ ID NO: 138), or 4-119 (composed of the heavy chain amino acid sequence of SEQ ID NO: 139 and the light chain amino acid sequence of SEQ ID NO: 140). (comprising the light chain amino acid sequence of column and SEQ ID NO: 20), 5-18 (comprising the heavy chain amino acid sequence of SEQ ID NO: 21 and the light chain amino acid sequence of SEQ ID NO: 22), 8-10 (comprising the heavy chain amino acid sequence of SEQ ID NO: 23 and the light chain amino acid sequence of SEQ ID NO: 24), 9-23 (comprising the heavy chain amino acid sequence of SEQ ID NO: 25 and the light chain amino acid sequence of SEQ ID NO: 26), 10-7 (comprising the heavy chain amino acid sequence of SEQ ID NO: 27 and the light chain amino acid sequence of SEQ ID NO: 28), 8-52 (comprising the heavy chain amino acid sequence of SEQ ID NO: 29 and the light chain amino acid sequence of SEQ ID NO: 30), 9-89 (comprising the heavy chain amino acid sequence of SEQ ID NO: 31 and the light chain amino acid sequence of SEQ ID NO: 32), 9-71 (comprising the heavy chain amino acid sequence of SEQ ID NO: 33 and the light chain amino acid sequence of SEQ ID NO: 34), 1-23 (comprising the heavy chain amino acid sequence of SEQ ID NO: 35 and the light chain amino acid sequence of SEQ ID NO: 36), 1-29 (comprising the heavy chain amino acid sequence of SEQ ID NO: 37 and the light chain amino acid sequence of SEQ ID NO: 38), 2-12 (comprising the heavy chain amino acid sequence of SEQ ID NO: 39 and the light chain amino acid sequence of SEQ ID NO: 40), 2-21 (comprising the heavy chain amino acid sequence of SEQ ID NO: 41 and the light chain amino acid sequence of SEQ ID NO: 42), 3-07 (comprising the heavy chain amino acid sequence of SEQ ID NO: 43 and the light chain amino acid sequence of SEQ ID NO: 44), 3-78 (comprising the heavy chain amino acid sequence of SEQ ID NO: 45 and the light chain amino acid sequence of SEQ ID NO: 46), a single antibody from the group comprising, preferably a single antibody from the group comprising 1-18, 1-33, 1-55, 2-27, 1-23, 1-29, 2-12, 2-21, 3-07 and 3-78, more preferably a single antibody from the group comprising 1-18, 1-55 and 2-12, even more preferably antibody 1-18 or 2-12, particularly preferably comprising the amino acid sequences of heavy chain CDR1 - CDR3 and light chain CDR1 - CDR3 of antibody 1-18.

[0029] According to another preferred embodiment of the first aspect of the present invention, the antibody or its binding fragment is 1-18 (comprising the heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 2), 1-21 (comprising the heavy chain amino acid sequence of SEQ ID NO: 3 and the light chain amino acid sequence of SEQ ID NO: 4), 1-33 (comprising the heavy chain amino acid sequence of SEQ ID NO: 5 and the light chain amino acid sequence of SEQ ID NO: 6), 1-54 (comprising the heavy chain amino acid sequence of SEQ ID NO: 7 and the light chain amino acid sequence of SEQ ID NO: 8), 1-55 (comprising the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 10), 2-10 (comprising the heavy chain amino acid sequence of SEQ ID NO: 11 and the light chain amino acid sequence of SEQ ID NO: 12), 2-22 (comprising the heavy chain amino acid sequence of SEQ ID NO: 13 and the light chain amino acid sequence of SEQ ID NO: 14), 2-27 (comprising the heavy chain amino acid sequence of SEQ ID NO: 15 and the light chain amino acid sequence of SEQ ID NO: 16), 2-47 (comprising the heavy chain amino acid sequence of SEQ ID NO: 17 and the light chain amino acid sequence of SEQ ID NO: 18), 3-59 (comprising the heavy chain amino acid sequence of SEQ ID NO: 19 and the light chain amino acid sequence of SEQ ID NO: 20), 5-18 (comprising the heavy chain amino acid sequence of SEQ ID NO: 21 and the light chain amino acid sequence of SEQ ID NO: 22), 8-10 (comprising the heavy chain amino acid sequence of SEQ ID NO: 23 and the light chain amino acid sequence of SEQ ID NO: 24), 9-23 (comprising the heavy chain amino acid sequence of SEQ ID NO: 25 and the light chain amino acid sequence of SEQ ID NO: 26), 10-7 (comprising the heavy chain amino acid sequence of SEQ ID NO: 27 and the light chain amino acid sequence of SEQ ID NO: 28), 8-52 (comprising the heavy chain amino acid sequence of SEQ ID NO: 29 and the light chain amino acid sequence of SEQ ID NO: 30), 9-89 (comprising the heavy chain amino acid sequence of SEQ ID NO: 31 and the light chain amino acid sequence of SEQ ID NO: 32), 9-71 (comprising the heavy chain amino acid sequence of SEQ ID NO: 33 and the light chain amino acid sequence of SEQ ID NO: 34), 1-23 (comprising the heavy chain amino acid sequence of SEQ ID NO: 35 and the light chain amino acid sequence of SEQ ID NO: 36), 1-29 (comprising the heavy chain amino acid sequence of SEQ ID NO: 37 and the light chain amino acid sequence of SEQ ID NO: 38), 2-12 (comprising the heavy chain amino acid sequence of SEQ ID NO: 39 and the light chain amino acid sequence of SEQ ID NO: 40), 2-21 (comprising the heavy chain amino acid sequence of SEQ ID NO: 41 and the light chain amino acid sequence of SEQ ID NO: 42), 3-07 (comprising the heavy chain amino acid sequence of SEQ ID NO: 43 and the light chain amino acid sequence of SEQ ID NO: 44),One antibody selected from the group consisting of 3-78 (comprising the heavy chain amino acid sequence of SEQ ID NO: 45 and the light chain amino acid sequence of SEQ ID NO: 46), preferably one antibody from the group consisting of 1-18, 1-33, 1-55, 2-27, 1-23, 1-29, 2-12, 2-21, 3-07 and 3-78, more preferably one antibody from the group consisting of 1-18, 1-55 and 2-12, even more preferably antibody 1-18 or 2, -12, particularly preferably comprising the combination of the heavy and light chains of antibody 1-18.

[0030] According to a second aspect of the present invention, there is provided a pharmaceutical composition comprising a monoclonal human antibody according to the first aspect of the present invention or a binding fragment thereof and at least one pharmaceutically acceptable excipient.

[0031] According to a preferred embodiment of the second aspect of the present invention, the pharmaceutical composition is a vaccination composition for human subjects.

[0032] According to a third aspect of the present invention, there is provided a kit comprising a monoclonal human antibody according to the first aspect of the present invention or a binding fragment thereof and a container.

[0033] According to a fourth aspect of the present invention, there is provided a monoclonal human antibody according to the first aspect of the present invention or a binding fragment thereof, which is used as a medicament, preferably as a vaccine, the pharmaceutical composition according to the second aspect of the present invention, or the kit according to the third aspect of the present invention.

[0034] According to a fifth aspect of the present invention, there is provided a monoclonal human antibody according to the first aspect of the present invention or a binding fragment thereof, which is used for the treatment or prevention of diseases caused by human immunodeficiency virus HIV-1 in human subjects, preferably for the treatment or prevention of acquired immunodeficiency syndrome (AIDS) in human subjects, the pharmaceutical composition according to the second aspect of the present invention, or the kit according to the third aspect of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0035]

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Mode for Carrying Out the Invention

[0036] The inventors of the present invention have dedicated themselves to solving the problems of the present invention and have successfully found a novel and useful human monoclonal antibody against HIV-1 that overcomes the disadvantages and drawbacks of known antibodies.

[0037] In this specification, the inventors describe CD4-binding site antibodies derived from classical V H 1-46 and V H 1-2 that exceed the potency and breadth of bNAbs. The structural basis for the observed high activity is thought to be caused by common structural characteristics. These characteristics are based on a combination of sequence similarities derived from V H 1-46 and V κ 3-20 and are further defined by a consensus sequence that indicates residues critical for their activity. H 1-46 and V κ 3-20 and are further defined by a consensus sequence that indicates residues critical for their activity.

[0038] Particularly interesting is that compared to the two most clinically advanced CD4bs bNAbs, 3BNC117 and VRC01, the antibodies according to the present invention effectively limit viral escape and maintain both neutralizing activity against VRC01-class escape mutants and complete virus suppression when tested in HIV-1-infected humanized mice. Thus, the antibodies of the present invention include very promising candidates for antibody-mediated strategies for the effective treatment and prevention of HIV-1 infection.

[0039] Accordingly, the present invention provides a monoclonal human antibody or a binding fragment thereof against the CD4 binding site of the human immunodeficiency virus HIV-1, wherein the antibody sequence comprises V H 1-46 gene segment and V κ 3-20 gene segment, and the antibody a) a heavy chain sequence QXXXFQSGXEXKRPGASVXISCRADDDPYTDDDTFTKYXTHWIRQAPGQXPEWLGVISPHXARPIYSYKFXDRLTLTRDSSLTXVYXELXXLXXDDXGIYXCARDPFGXXXPHYNXHMDVWGXGTXXIVSX (Consensus Sequence No. 1; Sequence No. 47) and a light chain sequence EXVLTQSPAILSXSPGDRVXXSCXASZGLXXXXLAWYRFKXGQIPXLVJFXXSXRARGTPDRFXGXGSXXDFTLTIXXVZXEDFATYYCQRXGXTPITFGGGTXLDXX (Consensus Sequence No. 2; Sequence No. 48), or b) a heavy chain sequence QLXQXGGGVXXPGASVXXSCXXPEXTFTKYXJHWXRQAPGXGXEWXGXVSPHGGRPXXXXXFRDRLTXTRXIHXTTHXMXLXGLXXXDXXXYXCARDXXGEXXXXXXXXXXXMDXWGGGX XXXVXS (Consensus Sequence No. 3; Sequence No. 49) and a light chain sequence XXXLTQSPXTLSXSPGEXXXLSCRAXXGXXXXHXXWFQXXXGXXPRLLIFXXXRRAXGXXXRFXXXXXXSXXXXXLTIXXVEXXDFAXYXCQXYGXITPJXFGGGTXXDXK (Consensus Sequence No. 4; Sequence No. 50), comprising X in any of Sequence Nos. 47 to 50 may be any amino acid or may be absent of amino acid, or provides a monoclonal human antibody or a binding fragment thereof that is at least 80% identical to the sequence.

[0040] Within the context of the present invention, the antibodies made and described herein are used as full monoclonal human antibodies, or as any functional or binding fragment thereof, and may be recited in the claims. Preferably, the monoclonal human antibody, or any kind of functional or binding fragment thereof, must include at least the complementarity determining regions (CDRs) 1-3 of the heavy chain and CDRs 1-3 of the light chain of the human monoclonal antibody.

[0041] The CDR regions of the antibody sequences described herein are preferably defined according to the numbering scheme of IMGT, which is an adaptation of the Chothia numbering scheme (ImMunoGeneTics information system (trademark); Lefranc et al., NAR 27:209-212 (1999); http: / / www.imgt.org).

[0042] In a preferred embodiment, the antibody is a monoclonal antibody or a fragment thereof that retains the binding specificity and the ability to neutralize infectious pathogens. In a preferred embodiment, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. For example, the antibody can be an antibody that includes the Fc domain of any human IgG isotype (e.g., IgG1, IgG2, IgG3, or IgG4).

[0043] Optionally, the antigen-binding compound consists of, or comprises, only Fab, Fab’, Fab’-SH, F(ab)2, Fv, diabody, single-chain antibody fragment, or multispecific antibodies comprising a plurality of different antibody fragments.

[0044] Within the present invention, an antibody or binding fragment thereto against 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 and has an affinity increased by at least 10-fold, more preferably at least 50-fold, and particularly preferably at least 100-fold as compared to an unrelated epitope, protein, or protein region. Generally, the term CD4 binding site as used herein refers to the CD4 binding site region within the gp120 envelope glycoprotein of HIV-1.

[0045] Furthermore, within the present invention, H the antibody amino acid sequences containing the V κ 1-46 gene segment and the V H 3-20 gene segment mean antibody amino acid sequences based on and / or derived from the above gene segments, respectively. Methods for determining which V κ or V H gene segments are used for assembling the antibody amino acid sequences are generally known in the art. Mutations in the above gene segments generally occur in the assembly of natural antibodies, but it will be readily apparent to those skilled in the art what primary sequence requirements are defined by requiring the use of the V κ 1-46 gene segment and the V

[0046] Therefore, a monoclonal antibody or its binding fragment is preferably understood to include a sequence that can be naturally derived from a combination of the V H 1- 46 gene segment and the V κ 3-20 gene segment. This may preferably include mutagenesis to the extent naturally present in the above gene segments.

[0047] To understand the significance and influence of these two segments on the overall structural basis of the antibody recited in the claims, V HIt should be noted that 1-46 bears 104 out of a total of 131 amino acids in the heavy chain according to Consensus Sequence No. 1 and 96 out of a total of 126 amino acids in the heavy chain according to Consensus Sequence No. 3.

[0048] Similarly, V κ 3-20 occupies 96 out of a total of 108 amino acids in the light chain according to Consensus Sequence No. 2 and 98 out of a total of 111 amino acids in the light chain according to Consensus Sequence No. 4.

[0049] Based on the number of functional antibodies that the inventors were able to identify, it became further possible to assemble two sets of consensus sequences for antibodies against the CD4 binding site of HIV-1. These two sets appear to be different primary sequence approaches for achieving high-efficiency binding to the CD4 binding site, limiting virus escape, and the breadth and potency of virus neutralization. H 1-46 gene segment and V κ 3-20 gene segment. For antibodies against the CD4 binding site of HIV-1, it became further possible to assemble two sets of consensus sequences. These two sets appear to be different primary sequence approaches for achieving high-efficiency binding to the CD4 binding site, limiting virus escape, and the breadth and potency of virus neutralization.

[0050] The first set of sequences consists of Consensus Sequence No. 1 according to SEQ ID NO: 47 for the heavy chain and Consensus Sequence No. 2 according to SEQ ID NO: 48 for the light chain. Many individualized sequences compatible with these consensus sequences have been studied herein, and the results obtained therefrom lend credence to the fact that the combination of the consensus sequences and the defined segments provides sufficient structural guidance to obtain a series of functional antibodies. Furthermore, two representative ones of a series of antibodies compatible with the first set of sequences are antibody 1-18 and antibody 1-55, which have been studied in more detail.

[0051] The second set of sequences consists of consensus sequence number 3 according to sequence number 49 for the heavy chain and consensus sequence number 2 according to sequence number 50 for the light chain. Again, many individualized sequences are studied herein as examples of the second set of consensus sequences, and the results obtained thereby also lend credence to the fact that combinations of these consensus sequences with defined segments provide sufficient structural guidance to obtain functional antibodies. Further, one representative of a series of antibodies that conform to the second set of sequences is antibody 2-12, which has also been studied in more detail.

[0052] With respect to the consensus sequences described herein, X or Xaa within the amino acid sequence can represent any amino acid or non-amino acid. However, based on the additional requirement that the antibodies recited in the claims are to include the V H 1-46 gene segment and the V κ 3-20 gene segment, it will be apparent to those skilled in the art that the options are more restricted by these gene segments that form the basis underlying each antibody.

[0053] In general, the monoclonal human antibodies or binding fragments thereof described herein further include antibody amino acid sequences that are at least 80% identical to the sequences defined above, as long as they are still directed against the CD4 binding site of the human immunodeficiency virus HIV-1. This means including sequences having minor variations in the antibody amino acid sequence that do not interfere with the structural folding and affinity of the antibody for the CD4 binding site.

[0054] For those skilled in the art, it is a trivial task to determine whether an antibody showing a certain degree of identity is directed against the CD4 binding site of the human immunodeficiency virus HIV-1 based on the above or general common sense.

[0055] The determination of percent identity between two sequences is accomplished in accordance with the present invention using the mathematical algorithms of Karlin and Altschul (Proc. Natl. Acad. Sci. USA (1993) 90:5873-5877). Such algorithms are the basis of the BLASTN and BLASTP programs of Altschul et al. (J. Mol. Biol. (1990) 215:403-410). BLAST nucleotide searches are performed using the BLASTN program. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described by Altschul et al. (Nucleic Acids Res. (1997) 25:3389-3402). When utilizing the BLAST and Gapped BLAST programs, the default parameters of each program are used. BLAST nucleotide searches are performed using the BLASTN program. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described by Altschul et al. (Nucleic Acids Res. (1997) 25:3389-3402). When utilizing the BLAST and Gapped BLAST programs, the default parameters of each program are used. are utilized.

[0056] According to a preferred embodiment of the invention, antibody amino acid sequences consisting of or comprising only nucleic acid sequences that are at least 85% identical, more preferably at least 90% identical, and even more preferably at least 95% identical to the sequences defined above and disclosed herein form part of the invention.

[0057] According to a preferred embodiment of the invention, the antibody of alternative b) of the first aspect of the invention comprises a 2aa deletion in FWR1.

[0058] According to another preferred embodiment of the invention, a monoclonal human antibody or a binding fragment thereof preferably exhibits broad neutralizing activity exemplified by the neutralization of at least 11 of the 12 HIV- reference strains of the global reference panel described in de Camp et al., J Virol. 2014 Mar; 88(5): 2489-2507, when testing all 12 strains preferably at an antibody concentration of up to 25 μg / ml in a TZM-bl cell pseudovirus neutralization assay. strains.

[0059] The TZM-bl cell-based pseudovirus neutralization assay is a very standardized assay commonly used in the art and in the art of the present invention for the analysis of the neutralizing potency of antibodies against various HIV-1 strains. Briefly, the antibody and the virus strain are incubated together before the addition of TZM-bl target cells. These cells exhibit luciferase activity upon successful infection, resulting in a luminescence signal detectable in the presence of luciferin after cell lysis. Neutralizing antibodies can prevent infection and thus the occurrence of luminescence. The potency of a neutralizing antibody is determined by the concentration of the antibody required to reduce viral infectivity by a specific amount.

[0060] The standardized method for setting up and employing this assay is disclosed and described in detail in Sarzotti-Kelsoe et al.; J Immunol Methods. 2014 July ; 0: 131-146. doi:10.1016 / j.jim.2013.11.022. This description alone, and in combination with general prior art, enables one of ordinary skill in the art to establish and determine the definitive readout of the TZM-bl cell-based pseudovirus neutralization assay used herein.

[0061] The global reference panel described in de Camp et al., J Virol.2014 Mar; 88(5): 2489-2507 includes 12 selected representative HIV-1 virus variants. The spectrum of HIV-1 serum neutralizing activity seen in this 12-virus panel is generally accepted as closely approximating the activity seen with subtype-matched viruses. Furthermore, this panel is highly sensitive for the detection of many known broadly neutralizing antibodies. Studies performed with this panel enable a reliable prediction of the neutralization breadth and / or potency of a given antibody.

[0062] According to another preferred embodiment of the first aspect of the present invention, when the antibody or its binding fragment is tested at an antibody concentration of up to 20 μg / ml in the TZM-bl cell pseudovirus neutralization assay, at least 89.9% (107 out of 119), preferably at least 92.4% (110 out of 119), even more preferably at least 96.6% (115 out of 119) of the pseudoviruses contained in the 119 multiclade virus panel described in Schoof et al., Immunity, 2019 Jun 18;50(6):1513-1529.e9, as exemplified by neutralization of the pseudoviruses, showing broad neutralizing activity.

[0063] The more comprehensive panel by Schoof et al., 2019, and those referenced therein, helps to accurately identify breadth and potency by testing a large number of different pseudoviruses (i.e., HIV-1 strains). This large panel is generally accepted as representative of all major circulating HIV-1 clades and provides detailed information regarding the neutralizing capacity of the antibodies tested.

[0064] According to another preferred embodiment of the first aspect of the present invention, when the antibody or its binding fragment is tested at an antibody concentration of up to 25 μg / ml in the TZM-bl cell pseudovirus neutralization assay, against the neutralizing strains of the global reference panel described in de Camp et al., J Virol. 2014 Mar; 88(5): 2489-2507, less than 0.3 μg / ml, preferably less than 0.2 μg / ml, more preferably less than 0.15 μg / ml, even more preferably less than 0.1 μg / ml, even more preferably less than 0.05 μg / ml, still more preferably 0.048 μg / ml, even more preferably 0.035 μg / ml of neutralizing potency (geometric mean IC ) is shown. 50 )

[0065] The neutralizing potency defined in this specification preferably takes into account only those viral variants that can be positively determined to be neutralized by each antibody. Then, based on the selection of positively neutralized variants, the geometric mean IC 50 is determined for the neutralized strain.

[0066] According to a preferred embodiment of the first aspect of the present invention, when the antibody or its binding fragment is tested at an antibody concentration of up to 20 μg / ml in the TZM-bl cell pseudovirus neutralization assay, against the neutralizing strains of the 119-multiclade virus panel described in Schoofs et al., Immunity, 2019 Jun 18;50(6):1513-1529.e9, it has a neutralizing potency (geometric mean IC 50 ) of less than 0.2 μg / ml, preferably less than 0.1 μg / ml, more preferably less than 0.08 μg / ml, and even more preferably less than 0.05 μg / ml.

[0067] According to another preferred embodiment of the first aspect of the present invention, when the antibody or its binding fragment is tested in the TZM-bl pseudovirus neutralization assay, it has a neutralizing potency (IC 50 ) of less than 0.05 μg / ml, preferably less than 0.02 μg / ml, more preferably at most 0.01 μg / ml, and even more preferably less than 0.01 μg / ml against the HIV-1 pseudovirus 89-F1_2_25 (89-F1_2_25 env gene; GenBank: HM215349.1).

[0068] According to another preferred embodiment of the first aspect of the present invention, when the antibody or its binding fragment is tested in the TZM-bl pseudovirus neutralization assay, it has a neutralizing potency (IC50 ) is shown.

[0069] HIV-1 pseudovirus 89-F1_2_25 appears to be one of the most difficult strains to neutralize with known CD4-binding site antibodies. In fact, none of the previously known antibodies targeting the CD4-binding site have achieved neutralization of the above pseudovirus at an IC 50 value of less than 0.194 μg / ml. However, the antibodies according to the present invention can neutralize the above strain with much higher potency. has not yet been demonstrated.

[0070] HIV-1 pseudovirus 6545.v4.c1 also appears to be a very difficult strain to neutralize with CD4-binding site antibodies. None of the previously known antibodies targeting the CD4-binding site have achieved neutralization of the above pseudovirus at an IC 50 value of less than 0.091 μg / ml. However, there are antibodies according to the present invention that can neutralize the above strain with much higher potency.

[0071] According to a preferred embodiment of the present invention, the antibody or binding fragment, when tested in a TZM-bl pseudovirus neutralization assay, has an IC 50 concentration of less than 0.1 μg / ml, preferably less than 0.05 μg / ml, and neutralizes all of the YU2 pseudovirus variants containing any one of the envelope mutations N279K, N280Y, G458D, G459D, or G471R (HIV-1 HXB2 envelope gene; residues numbered according to GenBank: K03455) in the YU2 envelope gene (GenBank: M93258.1).

[0072] The mutations N279K, N280Y, G458D, and G459D are in the context of HIV-1 infection It is associated with the occurrence of viral rebound (i.e., treatment failure) during treatment with a CD4-binding site antibody in a vivo model, indicating the occurrence of viral resistance to the administered CD4-binding site antibody (Non-Patent Document 5, Horwitz et al, Proc Natl Acad Sci U S A, 2013 Oct 8;110(41):16538-43). Also in this case, the antibody according to the present invention is superior to known CD4-binding site antibodies of the prior art in that the mutations in the YU2 envelope gene described above do not abolish neutralization and do not act as escape mutations against the antibody according to the present invention.

[0073] According to another preferred embodiment of the present invention, following an initial subcutaneous injection of 1 mg of an antibody or its binding fragment, 3 to 4 days later, a humanized mouse infected with HIV-1 NL4-3 / YU2 described in Zhang et al., J Virol, 2002 Jun;76(12):6332-43 is subcutaneously injected regularly with 0.5 mg of the antibody or its binding fragment every 3 to 4 days. When measured 4 weeks after treatment, preferably 6 weeks after treatment, and more preferably 8 weeks after treatment, in at least 70% of the treated mice having an HIV-1 RNA load of at least 5000 copies / ml plasma at the start of treatment, compared to the start of treatment, there is a decrease in the HIV-1 RNA load in plasma of at least 0.8 log 10 , preferably at least 1.0 log 10 .

[0074] According to another preferred embodiment of the present invention, following an initial subcutaneous injection of 1 mg of an antibody or its binding fragment, 3 to 4 days later, a humanized mouse infected with HIV-1 NL4-3 / BAL is subcutaneously injected regularly with 0.5 mg of the antibody or its binding fragment every 3 to 4 days. When measured 4 weeks after treatment, and more preferably 6 weeks after treatment, in at least 60% of the treated mice having an HIV-1 RNA load of at least 30000 copies / ml plasma at the start of treatment, compared to the start of treatment, there is at least 1.0 log 10 , preferably at least 1.5 log10 , and even more preferably at least 1.75 log 10 resulting in a decrease in the HIV-1 RNA load in plasma.

[0075] According to a more preferred embodiment of the above embodiment, the humanized mouse is first treated for 4 weeks with a single initial subcutaneous injection of 1 mg of 3BNC117 or VRC01, or a combination of both, and then, 3 to 4 days later, a regular subcutaneous injection of 0.5 mg of 3BNC117 or VRC01, or a combination of both, is performed every 3 to 4 days.

[0076] HIV-1 Humanized mice infected with NL4-3 / YU2 (YU2 env in the NL4-3 backbone described in Zhang et al., J Virol, 2 002;76: 6332-6343) provide a well-established model in the art for studying the antiviral activity of neutralizing HIV-1 antibodies in vivo. These mice can maintain a stable level of viremia (i.e., the number of HIV-1 RNA copies in plasma) and can exhibit an HIV-1 sequence diversification rate similar to that observed in humans in the env gene (Non-Patent Document 5).

[0077] ​This model has also been used to examine the efficacy of monotherapy with CD4-binding site antibodies (Freund et al., PLoS Pathog, 2015, Oct 30;11(10):e1005238, Non-Patent Document 5, Horwitz et al, Proc Natl Acad Sci U S A, 2013 Oct 8;110(41):16538-43, Freund et al., Sci Transl Med, 2017 Jan 18;9(373). pii: eaal2144). The antibody according to the present invention administered as monotherapy results in the maintenance of HIV-1 virus load suppression in the treated mice, and thus is superior to other studied CD4-binding site antibodies in which only a transient decrease in the HIV-1 virus load is observed during antibody monotherapy. Furthermore, the in vivo activity of 1-18 is superior to other CD4-binding site antibodies in that maintenance of virus suppression is achieved even after virus rebound occurs during prior treatment of mice with the CD4-binding site antibodies 3BNC117, VRC01, or combinations thereof.

[0078] According to a preferred embodiment of the first aspect of the present invention, following an initial subcutaneous injection of 1 mg of the antibody or its binding fragment, 3 to 4 days later, a humanized mouse infected with HIV-1 NL4-3 / YU2 as described in Zhang et al., J Virol, 2002 Jun;76(12):6332-43 is subcutaneously injected regularly with 0.5 mg of the antibody or its binding fragment every 3 to 4 days for at least 4 weeks, without causing the occurrence of one or more mutations in the CD4-binding sites (loop D, CD4-binding loop, beta 23 strand, V5 loop, and beta 24 strand) that mediate resistance to the administered antibody.

[0079] Preferably, the resistance to the administered antibody used in the context of the previous embodiment can be defined as resulting in an IC of at least 2.5 μg / ml of the administered antibody in the TZM-bl neutralization pseudovirus assay when the HIV-1 pseudovirus generated with the viral sequence containing this mutation is tested. 50 to result in.

[0080] The emergence of escape mutations that result in antibody resistance and / or are associated with viral rebound (i.e., treatment failure) has been demonstrated in antibody monotherapy studies of other CD4-binding site antibodies (Freund et al., PLoS Pathog, 2015, Oct 30;11(10):e1005238, Non-Patent Document 5, Horwitz et al, Proc Natl Acad Sci U S A, 2013 Oct 8;110(41):16538-43, Freund et al., Sci Transl Med, 2017 Jan 18;9(373). pii: eaal2144). Compared with these CD4-binding site antibodies, the antibodies of the present invention are excellent in that they can prevent the occurrence of mutations in the CD4-binding site epitope, thus preventing treatment failure and maintaining antiviral activity.

[0081] According to another preferred embodiment of the first aspect of the present invention, intravenous injection of 0.5 mg of an antibody or a binding fragment into NRG mice results in a detectable serum level of the antibody or its binding fragment of at least 50 μg IgG / ml serum 10 days after injection.

[0082] HIV-1 neutralizing antibodies can vary in their pharmacokinetic properties. In humans, currently known CD4-binding site antibodies appear to have a shorter half-life than antibodies targeting the V3 loop (Mendoza et al., Nature, 2018 Sep;561(7724):479-484), and this has also been observed in mouse models (Non-Patent Document 5, Horwitz et al, Proc Natl Acad Sci U S A, 2013 Oct 8;110(41):16538-43). Compared with other CD4-binding site antibodies, the antibodies of the present invention are excellent in maintaining higher serum levels in vivo for a longer period. This has also been observed in mouse models (Non-Patent Document 5, Horwitz et al, Proc Natl Acad Sci U S A, 2013 Oct 8;110(41):16538-43). Compared with other CD4-binding site antibodies, the antibodies of the present invention are excellent in maintaining higher serum levels in vivo for a longer period. in that respect.

[0083] According to a preferred embodiment of the first aspect of the present invention, the antibody or its binding fragment does not contain a CDRH3 having a length of 16 or 19 amino acids, and / or the antibody or its binding fragment contains a CDRH3 having a length of 18, 20 or 21 amino acids.

[0084] According to a preferred embodiment of the first aspect of the present invention, the antibody or its binding fragment does not contain the amino acid sequences of the antibodies NC37, N C133, AC40, AC41 or AC72 described in Freund et al., Sci. Transl. Med. 9, eaal2144 (2017), or is not composed only of these.

[0085] According to a preferred embodiment of the first aspect of the present invention, the antibody or its binding fragment is 1-18 (composed of the heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 2), 1-21 (composed of the heavy chain amino acid sequence of SEQ ID NO: 3 and the light chain amino acid sequence of SEQ ID NO: 4), 1-33 (composed of the heavy chain amino acid sequence of SEQ ID NO: 5 and the light chain amino acid sequence of SEQ ID NO: 6), 1-54 (composed of the heavy chain amino acid sequence of SEQ ID NO: 7 and the light chain amino acid sequence of SEQ ID NO: 8), 1-55 (composed of the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 10), 2-10 (composed of the heavy chain amino acid sequence of SEQ ID NO: 11 and the light chain amino acid sequence of SEQ ID NO: 12), 2-22 (composed of the heavy chain amino acid sequence of SEQ ID NO: 13 and the light chain amino acid sequence of SEQ ID NO: 14), 2-27 (composed of the heavy chain amino acid sequence of SEQ ID NO: 15 and the light chain amino acid sequence of SEQ ID NO: 16), 2-47 (composed of the heavy chain amino acid sequence of SEQ ID NO: 17 and the light chain amino acid sequence of SEQ ID NO: 18), 3-59 (composed of the heavy chain amino acid sequence of SEQ ID NO: 19 and the light chain amino acid sequence of SEQ ID NO: 20), 5-18 (composed of the heavy chain amino acid sequence of SEQ ID NO: 21 and the light chain amino acid sequence of SEQ ID NO: 22), 8-10 (composed of the heavy chain amino acid sequence of SEQ ID NO: 23 and the light chain amino acid sequence of SEQ ID NO: 24), 9-23 (composed of the heavy chain amino acid sequence of SEQ ID NO: 25 and the light chain amino acid sequence of SEQ ID NO: 26), 10-7 (composed of the heavy chain amino acid sequence of SEQ ID NO: 27 and the light chain amino acid sequence of SEQ ID NO: 28), 8-52 (composed of the heavy chain amino acid sequence of SEQ ID NO: 29 and the light chain amino acid sequence of SEQ ID NO: 30), 9-89 (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), 9-71 (composed of the heavy chain amino acid sequence of SEQ ID NO: 33 and the light chain amino acid sequence of SEQ ID NO: 3, 1-23 (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), 1-29 (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), 2-12 (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), 2-21 (composed of the heavy chain amino acid sequence of SEQ ID NO: 41 and the light chain amino acid sequence of SEQ ID NO: 42), 3-07 (composed of the heavy chain amino acid sequence of SEQ ID NO: 43 and the light chain amino acid sequence of SEQ ID NO: 44),One antibody from the group comprising 3-78 (consisting of the heavy chain amino acid sequence of SEQ ID NO: 45 and the light chain amino acid sequence of SEQ ID NO: 46), preferably one antibody from the group comprising 1-18, 1-33, 1-55, 2-27, 1-23, 1-29, 2-12, 2-21, 3-07 and 3-78, more preferably one antibody from the group comprising 1-18, 1-55 and 2-12, even more preferably antibody 1-18 or 2-12, particularly preferably comprising the amino acid sequences of heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3 of antibody 1-18.,

[0086] According to a particularly preferred embodiment of the present invention, the antibody or its binding fragment is 1-18 (consisting of the heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 2), 1-21 (consisting of the heavy chain amino acid sequence of SEQ ID NO: 3 and the light chain amino acid sequence of SEQ ID NO: 4), 1-33 (consisting of the heavy chain amino acid sequence of SEQ ID NO: 5 and the light chain amino acid sequence of SEQ ID NO: 6), 1-54 (consisting of the heavy chain amino acid sequence of SEQ ID NO: 7 and the light chain amino acid sequence of SEQ ID NO: 8), 1-55 (consisting of the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 10 ) one antibody selected from the group consisting of 2-10 (comprising the heavy chain amino acid sequence of SEQ ID NO: 11 and the light chain amino acid sequence of SEQ ID NO: 12), 2-22 (comprising the heavy chain amino acid sequence of SEQ ID NO: 13 and the light chain amino acid sequence of SEQ ID NO: 14), 2-27 (comprising the heavy chain amino acid sequence of SEQ ID NO: 15 and the light chain amino acid sequence of SEQ ID NO: 16), 2-47 (comprising the heavy chain amino acid sequence of SEQ ID NO: 17 and the light chain amino acid sequence of SEQ ID NO: 18), 3-59 (comprising the heavy chain amino acid sequence of SEQ ID NO: 19 and the light chain amino acid sequence of SEQ ID NO: 20), 5-18 (comprising the heavy chain amino acid sequence of SEQ ID NO: 21 and the light chain amino acid sequence of SEQ ID NO: 22), 8-10 (comprising the heavy chain amino acid sequence of SEQ ID NO: 23 and the light chain amino acid sequence of SEQ ID NO: 24), 9-23 (comprising the heavy chain amino acid sequence of SEQ ID NO: 25 and the light chain amino acid sequence of SEQ ID NO: 26), 10-7 (comprising the heavy chain amino acid sequence of SEQ ID NO: 27 and the light chain amino acid sequence of SEQ ID NO: 28), 8-52 (comprising the heavy chain amino acid sequence of SEQ ID NO: 29 and the light chain amino acid sequence of SEQ ID NO: 30), 9-89 (comprising the heavy chain amino acid sequence of SEQ ID NO: 31 and the light chain amino acid sequence of SEQ ID NO: 32), 9-71 (comprising the heavy chain amino acid sequence of SEQ ID NO: 33 and the light chain amino acid sequence of SEQ ID NO: 34), 1-23 (comprising the heavy chain amino acid sequence of SEQ ID NO: 35 and the light chain amino acid sequence of SEQ ID NO: 36), 1-29 (comprising the heavy chain amino acid sequence of SEQ ID NO: 37 and the light chain amino acid sequence of SEQ ID NO: 38), 2-12 (comprising the heavy chain amino acid sequence of SEQ ID NO: 39 and the light chain amino acid sequence of SEQ ID NO: 40), 2-21 (comprising the heavy chain amino acid sequence of SEQ ID NO: 41 and the light chain amino acid sequence of SEQ ID NO: 42), 3-07 (comprising the heavy chain amino acid sequence of SEQ ID NO: 43 and the light chain amino acid sequence of SEQ ID NO: 44), 3-78 (comprising the heavy chain amino acid sequence of SEQ ID NO: 45 and the light chain amino acid sequence of SEQ ID NO: 46); preferably one antibody from the group consisting of antibodies 1-18, 1-33, 1-55, 2-27, 1-23, 1-29, 2-12, 2-21, 3-07 and 3-78; more preferably one antibody from the group consisting of antibodies 1-18, 1-55 and 2-12; even more preferably antibody 1-18 or 2-12; particularly preferably the combination of the heavy chain and light chain of antibody 1-18.

[0087] In the description of the present application, the name of an antibody may be used. It is pointed out that an antibody consists of a heavy chain and a light chain, which also form part of this description. When referring to an antibody by its name or with respect to a sequence number, it should be understood that these reference methods have the same meaning.

[0088] The present invention further relates to a pharmaceutical composition comprising a monoclonal human antibody or a binding fragment thereof according to the present invention as defined and further described herein, and at least one pharmaceutically acceptable excipient. Preferably, the pharmaceutical composition is a vaccination composition for a human subject.

[0089] The present invention also encompasses a kit comprising a monoclonal human antibody or a binding fragment thereof according to the present invention as defined and further described herein, and a container.

[0090] In one aspect, the present invention also relates to a monoclonal human antibody or a binding fragment thereof according to the present invention as defined and further described herein for use as a medicament, preferably for use as a vaccine, the pharmaceutical composition described herein, and the kit.

[0091] In another aspect, the present invention also relates to a monoclonal human antibody or a binding fragment thereof according to the present invention as defined and further described herein for use in the treatment or prevention of 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, the pharmaceutical composition described herein, and the kit.

[0092] In another aspect, the present invention also relates to a method of treating a patient suffering from a disease caused by human immunodeficiency virus HIV-1 in a human subject, preferably a method for use in the treatment or prevention of acquired immunodeficiency syndrome (AIDS) in a human subject wherein an effective amount of a monoclonal human antibody or a binding fragment thereof according to the present invention, or the pharmaceutical composition of the present invention, is administered to the patient.

[0093] In another aspect, the present invention also relates to the use of a monoclonal human antibody or a binding fragment thereof according to the present invention, or a pharmaceutical composition of the present invention, in the manufacture of a medicament for the treatment of a disease caused by human immunodeficiency virus HIV-1 in a human subject, preferably for the treatment or prevention of acquired immunodeficiency syndrome (AIDS) in a human subject.

[0094] All embodiments of the invention described herein can be combined in any combination, provided that those skilled in the art do not consider such combinations to be without technical significance.

Examples

[0095] A) Experimental method Isolation of monoclonal antibody sequences Samples of blood and leukapheresis were obtained based on protocols (protocols 13-364 and 16-054) approved by the ethics review committee of the University of Cologne, and the participants provided written informed consent. Peripheral blood mononuclear cells (PBMCs) were isolated 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 an HIV-1 Env bait protein. The HIV-1 Env bait protein was either BG505 SOSIP.664 -GFP (Sliepen et al., 2015), or biotinylated (EZ-Link Sulfo NHS Bioting and Labeling Kit, Thermo Fisher) YU2 labeled with streptavidin-PE gp140 (Yang et al., 2000). Env-reactive CD19 + IgG + DAPI -Single cells were sorted as previously described (Ehrhardt et al., 2019). The sorted cells were incubated with random hexamer primers, NP-40, and RNase-free H2O at 65 °C for 1 minute. Subsequently, cDNA was generated using SuperScript IV in the presence of RT Buffer, dNTP, DTT, H2O, RNasin, and RNaseOUT. Antibody sequences for single-cell analysis were amplified by semi-nested PCR using Taq polymerase and previously described primers CG_RT (Ozawa et al., 2006, first PCR), IgG_Internal RT (Tiller et al., 2008, second PCR), and OPT5 / oPR-primer mix (Kreer et al., 2019, both PCRs).

[0096] Antibody sequence analysis The sequences of the second PCR products with an average Phred score of 28 or higher and a minimum length of 240 nucleotides were annotated with IgBLAST (Ye et al., 2013), and the variable region framework region (FWR) 1 to the end of the J gene were trimmed. Base calls with a Phred score of less than 16 were masked, and sequences with more than 15 masked nucleotides, frameshifts, or stop codons were excluded from further analysis. To analyze the sequences for potential clonality, all productive heavy-chain sequences were grouped by the same V gene, and the Levenshtein distance for each pair of their CDRH3s was determined. Individual sequences were grouped into clones that shared the same V gene and had a minimum CDRH3 identity of 75%. After 10 rounds with randomized input of the sequences, the result with the fewest number of unassigned (non-clonal) sequences was selected for further analysis. All clones were manually re-verified by the researchers to identify shared mutations. Initially assigned to different clones However, sequences sharing the same VDJ genes and amino acid and / or silent nucleotide mutations were subsequently grouped into subclones. Nucleotide sequence identity to the germline was calculated using IgBLAST.

[0097] Monoclonal antibody production For cloning of single cell-derived antibodies, the first PCR product of single cell PCR was used as a template, and specific forward and reverse primers similar to the respective nucleotide sequences of the V and J regions containing Q5 high-fidelity polymerase and expression vector overhangs for subsequent sequence and ligation-independent cloning (SLIC) (Tiller et al., 2008) were used for amplification. The PCR products were cloned into human antibody expression vectors (IgG1, kappa, or lambda chain) by SLIC assembly as previously described (von Boehmer et al., 2016). Antibodies were produced in HEK293-6E cells by transfection using polyethyleneimine. After 5 to 7 days, antibodies were purified from the supernatant after protein G incubation and subsequent elution from the chromatography column using 0.1 M glycine (pH = 3.0). After buffer neutralization, buffer exchange into PBS, and filter sterilization, the antibodies were stored at 4°C. Pseudovirus production

[0098] Pseudoviruses were produced in HEK293T cells by co-transfection with the pSG3ΔEnv plasmid as previously described (Doria-Rose et al., 2017, Sarzotti-Kelsoe et al., 2014, Hraber et al., 2017, Seaman et al., 2010). To generate the YU2 pseudovirus mutant panel, site-directed mutagenesis was used to introduce point mutations into the plasmid encoding the YU2 envelope gene.

[0099] ​​TZM-bl cell neutralization assay The neutralization assay was performed as previously described (Sarzotti-Kelsoe et al., 2014, Seaman et al., 2010). Mouse leukemia virus (MuLV) pseudotyped virus was used to determine non-specific activity. Antibodies were tested in duplicate. In assays of pseudovirus mutants and global reference panels, bioluminescence was determined after adding luciferin / lysis buffer (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). Specific activity was determined. Antibodies were tested in duplicate. In assays of pseudovirus mutants and global reference panels, bioluminescence was determined after adding luciferin / lysis buffer (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). In assays of pseudovirus mutants and global reference panels, bioluminescence was determined after adding luciferin / lysis buffer (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).

[0100] HIV-1-infected humanized mice Humanized mice were generated with modifications to a previous description (Non-Patent Document 5). NOD.Cg-Rag1 Il2rg / SzJ (NRG) mice were humanized by intrahepatic injection of sublethal irradiated human CD34 hematopoietic umbilical cord blood and / or placental tissue stem cells within 5 days after birth, 3 to 6 hours after irradiation. The humanized mice were infected by intraperitoneal challenge with replication-competent recombinant HIV-1 (YU2 env in the NL4-3 backbone (Zhang et al., 2002)) or HIV-1 (BAL env in the NL4-3 backbone) collected from the supernatant of transfected HEK293T cells. tm1mom Il2rg tm1Wjl / SzJ (NRG) mice were humanized by intrahepatic injection of sublethal irradiated human CD34 hematopoietic umbilical cord blood and / or placental tissue stem cells within 5 days after birth, 3 to 6 hours after irradiation. The humanized mice were infected by intraperitoneal challenge with replication-competent recombinant HIV-1 (YU2 env in the NL4-3 backbone (Zhang et al., 2002)) or HIV-1 (BAL env in the NL4-3 backbone) collected from the supernatant of transfected HEK293T cells. + / SzJ (NRG) mice were humanized by intrahepatic injection of sublethal irradiated human CD34 hematopoietic umbilical cord blood and / or placental tissue stem cells within 5 days after birth, 3 to 6 hours after irradiation. The humanized mice were infected by intraperitoneal challenge with replication-competent recombinant HIV-1 (YU2 env in the NL4-3 backbone (Zhang et al., 2002)) or HIV-1 (BAL env in the NL4-3 backbone) collected from the supernatant of transfected HEK293T cells. YU2 (YU2 env in the NL4-3 backbone (Zhang et al., 2002)) or HIV-1 s BAL (BAL env in the NL4-3 backbone) by intraperitoneal challenge.

[0101] Measurement of HIV-1 virus load Plasma RNA was extracted from EDTA plasma samples using the MinElute Virus Mini Spin Kit, which includes an on-column DNase I digestion step. The viral load was determined by quantitative real-time PCR using pol-specific primers described previously (Horwitz et al., 2013). qPCR was performed on a LightCycler 480 II using the Taqman RNA-to-Ct 1-Step-Kit. The viral load was quantified by including a standard curve derived from samples with known copy numbers for each qPCR run and. The limit of precision of qPCR was determined to be 384 copies / ml. The Log 10 change for viral loads less than 384 copies / ml was calculated assuming a copy number of 383 copies / ml.

[0102] Antibody Therapy of HIV-1 Infected Humanized Mice Antibodies diluted in PBS were injected subcutaneously. Following a loading dose of 1 mg, doses of 0.5 mg were injected every 3 to 4 days.

[0103] Single Genome Sequencing of HIV-1 in Humanized Mice To determine the occurrence of potential escape mutations in the HIV-1 env gene during antibody therapy of HIV-1 infected humanized mice, the extracted plasma RNA was used to generate cDNA using SuperScript IV and the antisense primer YB383 (Horwitz et al., 2017), followed by RNase H incubation. Subsequently, limiting dilution nested PCR was performed using Taq polymerase and the primers YB383 and YB50 for the first PCR and YB49 and YB52 for the second PCR to amplify single genome env cDNA (Schoofs et al., 2019). Positive PCR reactions at dilution rates with a PCR efficiency of less than 30% were sequences using Illumina dye sequencing, with modifications to that described previously (Kryazhimskiy et al., 2014, Schoofs et al., 2016). After tagging, index and adapter addition by limited-cycle PCR, and purification, PCR products were sequenced on an Illumina MiSeq and reads were assembled (Gaebler et al., 2019). Full-length env sequences with less than 10 ambiguities (less than 75% nucleotide identity across reads) were analyzed.

[0104] ELISA to determine competition for HIV-1 Env binding Antibody 1-18, antibody 1-55, and antibody 2-12 were biotinylated using the EZ Link Sulfo NHS Biotin and Labeling Kit (Thermo Fisher) according to the manufacturer's instructions, followed by buffer exchange into PBS. The wells were coated with anti-6xHis tag antibody at 2 μg / ml overnight at 4°C. The wells were blocked with 3% BSA in PBS at 37°C for 60 minutes and then treated with BG505. SOSIP.664 The plates were incubated with ABTS-His (Sanders et al., 2013) at 2 μg / ml in PBS for 60 minutes at 37°C. Competing antibodies were incubated at a 1:3 dilution starting at 32 μg / ml in PBS for 60 minutes at room temperature. The biotinylated antibody of interest was diluted to 0.5 μg / ml in 3% BSA in PBS and incubated for 60 minutes at room temperature, followed by the addition of peroxidase-streptavidin diluted 1:5000 in 1% BSA / 0.05% Tween 20 in PBS. Absorbance at 415 nm was determined on a microplate reader after the addition of ABTS solution. The plate was washed with 0.05% Tween 20 in PBS between each step.

[0105] In vivo antibody pharmacokinetic analysis NRG mice were intravenously injected with 0.5 mg of antibody in 200 μl PBS. Total serum human IgG concentrations were determined by ELISA as previously described (Non-Patent Document 5) with minor modifications. Briefly, high-binding ELISA plates were coated with anti-human IgG at a concentration of 2.5 μg / ml overnight at room temperature. Subsequently, wells were blocked with blocking buffer (2% BSA, 1 μM EDTA, and 0.1% Tween 20 in PBS). Serial dilutions (in duplicate) of human IgG1 kappa standards and serum samples in PBS were incubated for 90 minutes at room temperature, followed by incubation with HRP-conjugated anti-human IgG diluted 1:1000 in blocking buffer for 90 minutes at room temperature. Following the addition of ABTS, the optical density at 415 nm was determined using a microplate reader. Plates were washed with 0.05% Tween 20 in PBS between each step. Serum samples obtained before antibody injection were used to confirm the absence of baseline human serum IgG.

[0106] B) Specific Examples of Antibodies of the Invention Example I Isolation of Broadly Potent VH1-46-Derived HIV-1 Neutralizing Antibodies from HIV-1-Infected Elite Neutralizers Human B cells reactive to HIV-1 envelope proteins were isolated from HIV-1 infected individuals previously identified as having exceptional serum neutralizing activity against HIV-1 in in vitro assays.

[0107] For this purpose, isolated B cells were transfected with fluorescent dye-labeled soluble HIV-1 Env protein (YU2 gp140 or BG505 SOSIP.664 ) and single-cell sorting was performed (Ehrhardt et al., 2019). This approach allowed the identification of This allowed for the subsequent amplification of heavy and light antibody gene segments from individual HIV-1 Env-reactive B cells, which allowed for the cloning of PCR products into expression vectors for recombinant production of the corresponding antibodies encoded by individual B cells, allowing functional testing of the antibodies.

[0108] Analysis of the sequences of HIV-1-Env-reactive B cells allowed the identification of expanded VH1-46-derived B cell clones. One of these clones was characterized by a six-amino acid (aa) insertion into the heavy chain CDR1 region, and this clone is represented by the consensus sequences of the heavy chain (SEQ ID NO: 47) and the light chain (SEQ ID NO: 48). Three additional B cell clones were identified, and they share similarity with each other and with previously described clones. These additional clones do not have the 6-aa CDRH1 insertion but show a 2-aa deletion in framework region 1 of their light chains. These clones are represented by separate consensus sequences for the heavy chain (SEQ ID NO: 49) and the light chain (SEQ ID NO: 50).

[0109] A total of 23 antibodies representative of the identified VH1-46-derived B cell clones were produced as monoclonal antibodies. To determine their overall neutralizing potency and breadth, these antibodies were tested for their neutralizing activity in a TZM-bl cell neutralization assay against a reference panel of 12 HIV-1 pseudovirus strains referred to as the "global panel" (Figure 1). This panel of pseudoviruses was previously designed to represent the diversity of the global HIV-1 epidemic and to allow for a standardized assessment of the activity of neutralizing antibodies.

[0110] Notably, all of the isolated VH1-46-derived antibodies showed high neutralizing activity against at least 11 of the 12 HIV-1 reference strains (Figure 1). The VH1-46-derived antibodies represented by SEQ ID NO: 47 and SEQ ID NO: 48 demonstrated activity against all of the strains tested. In addition to their neutralizing breadth, the VH1-46-derived antibodies tested also demonstrated high neutralizing potency. Neutralizing potency is usually represented by the 50% inhibitory concentration (IC 50 ). When tested against the "global panel", all of the VH1-46-derived antibodies tested were highly active with a geometric mean IC 50 of 0.2 μg / ml or less against the neutralized virus strains (Figure 1).

[0111] Example II Identification of the CD4 binding site as a target of the antibody of the present invention To determine the epitope of the antibody according to the present invention, the binding of representative antibodies 1-18, 1-55, and 2-12 to the HIV-1 envelope trimer of BG505 SOSIP.664 in the presence of antibodies with known specificities was examined. Interference with the CD4 binding site antibodies 3BNC117, N6, and VRC01 was detected (Figure 2). This indicates that the antibody of the present invention targets an epitope that overlaps with the CD4 binding site and the epitopes of other antibodies that bind to the CD4 binding site.

[0112] Example III High-potency and broad antibodies 1-18, 1-55, and 2-12 To confirm the neutralizing potency and breadth of the isolated VH1-46-derived HIV-1 antibodies, representative antibodies 1-18, 1-55, and 2-12 were individually tested against an expanded panel of 119 HIV-1 pseudoviruses as previously tested in Schoofs et al., 2019. This panel of pseudoviruses represents the development of the panel of pseudoviruses described in detail in Seaman et al., 2010. This provides a representation of the genetic and overall diversity of HIV-1 Env variants. This includes HIV-1 variants of different clades or subtypes, including variants isolated from transmitted / founder viruses and viruses that are difficult to neutralize. In particular, the VH1-46-derived antibodies of the present invention tested demonstrated high neutralizing potency and breadth when tested against the 119-pseudovirus multiclade panel (Figure 3). Specifically, when tested at antibody concentrations up to 20 μg / ml, antibody 1-18 neutralized 96.6% of the pseudoviruses tested, with a geometric mean IC of 0.048 μg / ml against the neutralized pseudoviruses

[0113] Notably, the VH1-46-derived antibodies of the present invention tested demonstrated high neutralizing potency and breadth when tested against the 119-pseudovirus multiclade panel (Figure 3). Specifically, when tested at antibody concentrations up to 20 μg / ml, antibody 1-18 neutralized 96.6% of the pseudoviruses tested, with a geometric mean IC of 0.048 μg / ml against the neutralized pseudoviruses 50is shown (Figure 3). This breadth and potency targets the CD4 binding site, is derived from the VH1-2 gene segment, and is higher than that seen in other HIV-1 neutralizing antibodies (3BNC117 and VRC01) that have advanced to clinical trials (Figure 3). Furthermore, the potency of antibody 1-18 in the 119-multiclade panel was higher than that of N6, another VH1-2-derived CD4 binding site antibody in clinical trials (Figure 3).

[0114] Furthermore, results against a total of 62 identical HIV-1 pseudoviruses were available (Yoon et al., 2015). When compared to other VH1-46-derived HIV-1 neutralizing antibodies that target the CD4 binding site, representative VH1-46-derived antibodies of the present invention demonstrated higher potency and breadth (Figure 4). When compared to other VH1-46-derived HIV-1 neutralizing antibodies that target the CD4 binding site, representative VH1-46-derived antibodies of the present invention demonstrated higher potency and breadth (Figure 4).

[0115] Example IV Neutralizing Activity Against Pseudoviruses Not Sufficiently Neutralized by Other CD4 Binding Site Antibodies CD4 binding site antibodies can achieve a high level of neutralizing breadth (i.e., are active against a number of different HIV-1 Env variants), but antibody-resistant HIV-1 variants exist. Therefore, it is important to identify novel CD4 binding site antibodies that are highly active against such HIV-1 variants.

[0116] Compared to CD4 binding site antibodies (N6, 3BNC117, VRC01) that have advanced to clinical trials, the antibodies of the present invention neutralize several strains with much higher potency (i.e., they have lower IC 50 (shown in Figure 5 for representative members 1-18, 1-55, and 2-12).

[0117] For example, a pseudovirus containing the HIV-1 Env protein of virus strain 89-F1_2_25, when tested at a concentration of up to 20 μg / ml in the TZM-bl pseudovirus neutralization assay, is not neutralized by CD4 binding site antibodies (N6, 3BNC117, VRC01) in clinical trials (i.e., these antibodies have an IC 50do not reach) (Figure 5). Furthermore, among the 30 CD4-binding site IgG antibodies listed in the neutralizing antibody database CATNAP (Yoon et al., 2015), 89.7%( 26 / 29) of the antibodies do not neutralize this virus at all (i.e., IC 50 > 20 μg / ml). Among the three antibodies that neutralize this virus, NC37 has an IC 50 of 17.7 μg / ml, VRC 16 has an IC 50 of 3.12 μg / ml, and the VH1-69 gene-derived antibody VRC13 has an IC 50 of 0.19 μg / ml. In contrast, representative antibodies of the present invention (1-18, 1-55, and 2-12), like all other antibodies of the present invention (IC 50 ranging from 0.006 μg / ml to 0.025 μg / ml across all antibodies, see Figure 12), have very high potency (IC 50 of 0.007 μg / ml, 0.014 μg / ml, and 0.006 μg / ml, respectively) to neutralize the HIV-1 pseudovirus 89-F1_2 _25. Another virus strain that is highly resistant to common CD4-binding site antibodies is the 6545.v4.c1 strain. This strain is resistant to 75% of the 43 CD4-binding site IgG antibodies listed in the neutralizing antibody database CATNAP (Yoon et al., 2015) . Most importantly, none of the advanced CD4-binding site antibodies can potently neutralize this strain (IC 50 < 10 μ / ml) (IC 50 of N6: 16.23; 3BNC117: over 20; VRC01: over 20; VRC07-523-LS: over 20). In contrast, all antibodies of the present invention potently neutralize this strain with IC 50 ranging from 0.008 μg / ml to 9.37 μg / ml (Figure 12).

[0118] Therefore, the antibodies of the present invention provide a solution to the insufficient neutralization of viruses 89-F1_2_25 and 6545.v4.c1 by CD4 binding site antibodies. When tested against 89-F1_2_25, the antibodies of the present invention are approximately 1 log 10 more potent than the best CD4 binding site antibodies against this virus described previously.

[0119] Example V Neutralization Activity Against Pseudoviruses with CD4 Binding Site Mutations That Confer Resistance to Other CD4 Binding Site Antibodies Antibody resistance mediated by the emergence of CD4 binding site escape mutations results in treatment failure of antibody therapy for HIV-1 infection (Non-Patent Document 5). Therefore, identifying CD4 binding site antibodies that are not affected by these escape mutations is important for providing treatment options for HIV-1 variants carrying these mutations.

[0120] CD4 binding site antibodies were tested in a TZM-bl cell neutralization assay against a panel of YU2 pseudovirus mutants with specific CD4 binding site mutations (Figure 6). Mutations in the CD4 binding site affected the activity of previously known CD4 binding site antibodies (Figure 6). For example, the IC 50 values of CD4 binding site antibodies VRC01 and 8ANC131 against wild-type YU2 pseudovirus were 0.107 μg / ml and 0.256 μg / ml, respectively, but no neutralization was observed against any of the mutants with loop D mutations tested (i.e., no IC 50 value was obtained up to the tested antibody concentration of 2.5 μg / ml). Another CD4 binding site antibody, N6, was affected by the N279K mutation (Figure 6). In contrast, the representative antibodies of the present invention tested (1-18, 1-55, and 2-12) maintained high neutralization activity against the YU2 pseudovirus mutants tested (Figure 6).

[0121] Therefore, the antibodies of the present invention provide a solution to the resistance of CD4 binding site antibodies to virus mutants with mutations in the CD4 binding site.

[0122] Example VI Maintenance of virus suppression by antibody monotherapy in an in vivo model of HIV-1 infection HIV-1 YU2 Humanized mice infected with HIV-1 (Zhang et al., 2002) provide a model for studying the antiviral activity of neutralizing HIV-1 antibodies in vivo. To generate humanized mice, immunodeficient NRG mice are irradiated within the first day of life and injected intrahepatically with human hematopoietic CD34 + stem cells. This results in the development of human lymphocytes that can be infected with replicable HIV-1. These mice can maintain stable levels of viremia (i.e., the number of HIV-1 RNA copies in plasma) and exhibit an HIV-1 sequence diversification rate similar to that observed in humans (Non-Patent Document 5). This mouse model has previously been used to determine the antiviral activity of several CD4-binding site antibodies administered as monotherapy in vivo (179NC75: Freund et al., 2015; 3BNC117: Horwitz et al, 2013; 45-46 G54W : Non-Patent Document 5; NC37: Freund et al., 2017). In all of these studies, only a transient effect on virus load was observed, and viral rebound (i.e., return to the baseline level of viremia) occurred rapidly. Furthermore, this viral rebound was associated with the occurrence of mutations at the antibody target site. Therefore, there is a need for novel CD4-binding site antibodies that can maintain virus suppression when administered as monotherapy.

[0123] HIV-1 YU2When infected humanized mice were treated with the CD4-binding site antibodies 3BNC117, VRC01, or combinations thereof (administered a loading dose of 1 mg per antibody subcutaneously (s.c.), followed by 0.5 mg s.c. every 3 to 4 days), only a transient decrease in the HIV-1 RNA copy number was observed, and viremia returned to baseline levels in most mice within 2 to 3 weeks (Figure 7). In contrast, when mice were treated according to the same dosing scheme with a representative antibody 1-18 of the present invention, virus suppression was observed in all treated mice over an 8-week treatment period compared to the virus copy number at the start of treatment (Figure 7). In more than 80% of the mice, the plasma HIV-1 RNA copy number decreased to levels below the limit of accuracy of the assay used (384 copies / ml).

[0124] Therefore, the antibody of the present invention can effectively maintain virus suppression in HIV-1 YU2 infected humanized mice even when administered as monotherapy.

[0125] Furthermore, as described above, when HIV-1 BAL infected humanized mice were treated with the CD4-binding site antibodies 3BNC117 or VRC01, only a slight and transient decrease in the HIV-1 RNA copy number was observed, and viremia returned to baseline levels in most mice within 2 weeks (Figure 13). In contrast, when mice were treated according to the same dosing scheme with a representative antibody 1-18 of the present invention, virus suppression was observed in all treated mice over a 6-week treatment period compared to the virus copy number at the start of treatment (Figure 13).

[0126] Therefore, the antibody of the present invention can effectively maintain virus suppression in HIV-1 BAL infected humanized mice even when administered as monotherapy.

[0127] Example VII Prevention of the Occurrence of Virus Mutations at the CD4-Binding Site during Antibody Monotherapy Viral rebound during antibody monotherapy using CD4-binding site antibodies is typically associated with the emergence of viral sequence variants at the antibody target site (179NC75: Freund et al., 2015; 3BNC117: Horwitz et al, 2013; 45-46 G54W : Non-Patent Document 5; NC37: Freund et al., 2017). Thus, a novel CD4-binding site antibody that can prevent the occurrence of mutations in the CD4-binding site antibody is required to prevent antibody resistance mediated by such mutations.

[0128] Mutations in the CD4-binding site (Loop D, beta23 strand / V5 loop) were observed in all test mice receiving 3BNC117, VRC01, or 3BNC117+VRC01 therapy, as described in Example IV (Figure 8). In contrast, mutations in these sites were found in only 1 out of 10 sequences in the mice of the experiment described in Example IV where detectable viremia remained 4 weeks after antibody therapy with antibody 1-18 of the present invention (Figure 7). Notably, when this mutation (G459D) was tested with the YU2 pseudovirus panel, the activity of 1-18 was not affected (Figure 6).

[0129] Example VIII Maintenance of viral suppression by antibody monotherapy in an in vivo model of HIV-1 infection after viral rebound from VRC01-class therapy Failure of antibody monotherapy using CD4-binding site antibodies in HIV-1-infected humanized mice results in viral rebound and is associated with viral resistance to the administered antibody (Freund et al., 2015, Horwitz et al, 2013, Non-Patent Document 5, Freund et al., 2017 ). Thus, a novel CD4-binding site antibody that provides an option for in vivo treatment after previous failure of CD4-binding site antibody therapy is needed.

[0130] Following viral rebound observed during 4 weeks of treatment with 3BNC117, VRC01, or a combination thereof (described in Example VI), addition of the antibody 1-18 of the invention to the treatment regimen (administering a loading dose of 1 mg s.c. followed by 0.5 mg s.c. every 3 to 4 days) resulted in a sustained decrease in viremia in 95% (18 / 19) of the treated mice (Figure 9). Thus, the antibodies of the invention provide an option for in vivo control of HIV-1 even after failure of pretreatment with other CD4-binding site antibodies.

[0131] Example IX Preferred in vivo half-life compared to other CD4-binding site antibodies HIV-1 neutralizing antibodies can vary not only in neutralizing potency and breadth but also in their in vivo half-life. For example, the V3 loop-targeting antibody 10-1074 has a longer half-life than the CD4-binding site antibody 3BNC117 when administered to HIV-1-infected individuals (Mendoza et al., 2018). Thus, novel antibodies targeting the CD4-binding site should have favorable pharmacokinetic properties in vivo compared to currently available CD4-binding site antibodies.

[0132] To determine their pharmacokinetic properties, representative antibodies of the invention (1-18, 1-55, and 2-12) were individually injected intravenously into NRG mice. The CD4-binding site antibodies 3BNC117, VRC01, and 45-46 G54W Compared to, the antibodies of the invention tested showed a slower decline in their serum IgG concentrations as determined by total human IgG ELISA and were more similar to those of 10-1074 (Figure 10). Thus, the antibodies of the invention tested demonstrate favorable pharmacokinetic properties compared to other CD4-binding site antibodies targeting HIV-1.

[0133] References in the Examples section: Anderson, J.P., et al. (2000). Testing the hypothesis of a recombinant origin of human immunodeficiency virus type 1 subtype E. J. Virol. 74, 10752-10765. deCamp, A., et al. (2014). Global Panel of HIV-1 Env Reference Strains for Standardized Assessments of Vaccine-Elicited Neutralizing Antibodies. J, Virol. 88, 2489-2507. Doria-Rose, N.A., et al. (2017). Mapping Polyclonal HIV-1 Antibody Responses via Next-Generation Neutralization Fingerprinting. PLoS Pathog 13, e1006148. Dosenovic, P., et al. (2019). Anti-idiotypic antibodies elicit anti-HIV-1- specific B cell responses. J. Exp. Med. Ehrhardt et al., (2019) Polyclonal and convergent antibody response to Ebola virus vaccine rVSV-ZEBOV. Nat Med. 25,1589-1600. Freund, N.T., et al. (2015). A New Glycan-Dependent CD4-Binding Site Neutralizing Antibody Exerts Pressure on HIV-1 In Vivo. PLoS Pathog. 11, :e1005238. Freund, N.T., et al. (2017). Coexistence of potent HIV-1 broadly neutralizing antibodies and antibody-sensitive viruses in a viremic controller. Sci Transl Med. 9, eaal2144 Gaebler, C., et al. (2019). Combination of quadruplex qPCR and next- generation sequencing for qualitative and quantitative analysis of the HIV-1 latent reservoir. J. Exp. Med. Horwitz, J.A., et al. (2013). HIV-1 suppression and durable control by combining single broadly neutralizing antibodies and antiretroviral drugs in humanized mice. Proc. Natl. Acad. Sci. USA 110, 16538-16543. Horwitz, J.A., et al. (2017). Non-neutralizing Antibodies Alter the Course of HIV-1 Infection In Vivo. Cell 170, 637-648 e610. Hraber, P., et al. (2017). Panels of HIV-1 Subtype C Env Reference Strains for Standardized Neutralization Assessments. J. Virol. 91. Klein, F., et al. (2012). HIV therapy by a combination of broadly neutralizing antibodies in humanized mice. Nature 492, 118-122. Kreer, C., et al. (2019). openPrimeR for multiplex amplification of highly diverse templates. bioRxiv. https: / / doi.org / 10.1101 / 847574 Kryazhimskiy, S., et al. (2014). Microbial evolution. Global epistasis makes adaptation predictable despite sequence-level stochasticity. Science 344, 1519-1522. Mendoza, P., et al. (2018). Combination therapy with anti-HIV-1 antibodies maintains viral suppression. Nature 561, 479-484. Pietzsch, J., et al. (2010). Human anti-HIV-neutralizing antibodies frequently target a conserved epitope essential for viral fitness. J. Exp. Med. 207, 1995- 2002. Sanders, R.W., et al. (2013). A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies. PLoS Pathog. 9, e1003618. Sarzotti-Kelsoe, M., et al. (2014). Optimization and validation of the TZM-bl assay for standardized assessments of neutralizing antibodies against HIV-1. J. Immunol. Methods 409, 131-146. Schoofs, T., et al. (2016). HIV-1 therapy with monoclonal antibody 3BNC117 elicits host immune responses against HIV-1. Science 352, 997-1001. Seaman, M.S., et al. (2010). Tiered categorization of a diverse panel of HIV-1 Env pseudoviruses for assessment of neutralizing antibodies. J. Virol. 84, 1439-1452. Sliepen, K., et al. (2015). Engineering and Characterization of a Fluorescent Native-Like HIV- 1 Envelope Glycoprotein Trimer. Biomolecules 5, 2919-2934. Tiller, T., et al. (2008). Efficient generation of monoclonal antibodies from single human B cells by single cell RT- PCR and expression vector cloning. J. Immunol. Methods 329, 112-124. von Boehmer, L., et al. (2016). Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nat. Protoc. 11, 1908-1923. Yang, X., et al.. (2000). Characterization of stable, soluble trimers containing complete ectodomains of human immunodeficiency virus type 1 envelope glycoproteins. J. Virol. 74, 5716-5725. Ye, J., et al. (2013). IgBLAST: an immunoglobulin variable domain sequence analysis tool. Nucleic Acids Res. 41, W34-40. Yoon, H., et al. (2015). CATNAP: a tool to compile, analyze and tally neutralizing antibody panels. Nucleic Acids Res. 43, W213-219. Zhang, Y.J., et al. (2002). Envelope-dependent, cyclophilin-independent effects of glycosaminoglycans on human immunodeficiency virus type 1 attachment and infection. J. Virol. 76, 6332-6343.

Claims

1. A monoclonal human antibody or a binding fragment thereof against the CD4 binding site of human immunodeficiency virus HIV-1, wherein the antibody amino acid sequence is V Ｈ 1-46 gene segment and said V κ 3-20 gene segment, and wherein the antibody is a) a heavy chain amino acid sequence QXXXXFQSGXEXKRPGASVXISC RADDDPYTDDDTF TKYXTHWIRQAPG QXP EWLGVISP HXARP IYSYKFXDRLTLTRDSSLTVYXELXXXXDDXGIYXCARDPFGXXXXPHYNXHMDVWGGXTXIVSX (Consensus SEQ ID NO: 1; SEQ ID NO: 47), and a light chain amino acid sequence EXVL TQSPAILSXSPGDRVXXXSCXASZGLXXXXLAWYRFKXGQIPXL VJFXXXSXRA RGTPDRFXGXG SX XDFTLTIXXVZ XEDFATYYCQRXGXTPI TFGGGGXTLDXX (Consensus SEQ ID NO: 2; SEQ ID NO: 48), or b) a heavy chain amino acid sequence QLXQXG GGGVX XP GASVX XSCXXPEXTFTKY XJH WXRQAPGXGXEWXGVSPHGG RPXXXXXXFRD RLTXTRXIHXTTHXMXLG LXXXXDXXXYXCARDXXXXGEX XXXXXXXXXXMDXWGGGGXXX XVXS (Consensus SEQ ID NO: 3; SEQ ID NO: 49), and a light chain amino acid sequence XXXLTQSPXTLSXSPGEXXXXLSCRAXXGXXXXHXXXXWFQXXXGXXXPRLLIFXXXRR AX GXXXRFXXX XXXXXXXXXXSX XXXXXXXLTIXXVEX XDFAXYXCQXYG XITPJXF GGGGXTXXDK (Consensus SEQ ID NO: 4; SEQ ID NO: 50), and X in any of SEQ ID NOs: 47 to 50 may be any amino acid or may be absent, or a monoclonal human antibody or a binding fragment thereof, wherein the antibody amino acid sequence is at least 80% identical to the sequence.

2. The monoclonal human antibody or a binding fragment thereof according to claim 1, wherein the antibody of alternative b) of claim 1 contains a 2aa deletion in FWR1, and / or the antibody or a binding fragment thereof does not contain a CDRH3 having a length of 16 or 19 amino acids, and / or the antibody or a binding fragment thereof contains a CDRH3 having a length of 18, 20 or 21 amino acids.

3. When the antibody or a binding fragment thereof is preferably tested on all 12 strains at an antibody concentration of up to 25 μg / ml in a TZM-bl cell pseudovirus neutralization assay, de Camp et The monoclonal human antibody or binding fragment according to claim 1 or 2, which exhibits broad neutralizing activity exemplified by neutralization of at least 11 out of 12 HIV-1 reference panel strains of the global reference panel described in al., J Virol. 2014 Mar; 88(5): 2489-2507 The monoclonal human antibody or binding fragment according to claim 1 or 2, which exhibits broad neutralizing activity exemplified by neutralization of at least 11 out of 12 HIV-1 reference panel strains of the global reference panel described in al., J Virol. 2014 Mar; 88(5): 2489-2507

4. When the antibody or its binding fragment is tested at an antibody concentration of up to 20 μg / ml in the TZM-bl cell pseudovirus neutralization assay, at least 89.9% (107 out of 119), preferably at least 92.4% (110 out of 119), more preferably at least 96.6% (115 out of 119) of the pseudoviruses contained in the 119 multi-clade virus panel described in Schoofs et al., Immunity, 2019 Jun The monoclonal human antibody or binding fragment according to any one of claims 1 to 3, which exhibits broad neutralizing activity exemplified by neutralization of the pseudoviruses contained in the 119 multi-clade virus panel described in Schoofs et al., Immunity, 2019 Jun 18;50(6):1513-1529.e9

5. When the antibody or its binding fragment is tested at an antibody concentration of up to 25 μg / ml in the TZM-bl cell pseudovirus neutralization assay, it has a neutralizing potency of less than 0.3 μg / ml, preferably less than 0.2 μg / ml, more preferably less than 0.15 μg / ml, even more preferably less than 0.1 μg / ml, even more preferably less than 0.05 μg / ml, still more preferably 0.048 μg / ml or less, particularly preferably 0.035 μg / ml or less, against the neutralizing strains of the global reference panel described in de Camp et al., J Virol. 2014 Mar; 88(5): 2489-2507 (geometric mean IC ５０ ). The monoclonal human antibody or binding fragment according to any one of claims 1 to 4, which shows).

6. When the antibody or its binding fragment is tested at an antibody concentration of up to 20 μg / ml in the TZM-bl cell pseudovirus neutralization assay, Schoofs et al., Immunity, 2019 For the neutralizing strain of the 119 multiclade virus panel described in Jun 18;50(6):1513-1529.e9, the neutralizing potency is less than 0.2 μg / ml, preferably less than 0.1 μg / ml, preferably less than 0.08 μg / ml, and even more preferably less than 0.05 μg / ml (geometric mean IC of the neutralizing strain ５０ ), the monoclonal human antibody or binding fragment according to any one of claims 1 to 5.

7. When the antibody or its binding fragment is tested in a TZM-bl pseudovirus neutralization assay, it has a neutralizing potency (IC ｅｎｖ 50) of less than 0.05 μg / ml, preferably less than 0.02 μg / ml, and even more preferably less than 0.01 μg / ml against the HIV-1 pseudovirus 89-F1_2_25 (89-F1_2_25 ５０ ; GenBank: HM215349.1). The monoclonal human antibody or binding fragment according to any one of claims 1 to 6

8. When the antibody or binding fragment is tested in a TZM-bl pseudovirus neutralization assay, an IC ５０ concentration of less than 0.1 μg / ml, preferably less than 0.05 μg / ml, and neutralizes all of the YU2 pseudovirus variants containing the YU2 envelope gene (GenBank: M93258.1) having one of the envelope mutations N279K, N280Y, G458D, G459D, or G471R (HIV-1 HXB2 envelope gene; residues numbered according to GenBank: K03455), the monoclonal human antibody or binding fragment according to any one of claims 1 to 7.

9. Following an initial subcutaneous injection of 1 mg of the antibody or its binding fragment, 3 to 4 days later, for humanized mice infected with HIV-1 NL4-3 / YU2 as described in Zhang et al., J Virol, 2002 Jun;76(12):6332-43, regular subcutaneous injection of 0.5 mg of the antibody or its binding fragment every 3 to 4 days is carried out. When measured 4 weeks after treatment, preferably 6 weeks after treatment, and even more preferably 8 weeks after treatment, in at least 70% of the treated mice having an HIV-1 RNA load of at least 5000 copies / ml plasma at the start of treatment, compared to the start of treatment, there is a decrease in the HIV-1 RNA load in the plasma of at least 0.8 log １０ , preferably at least 1.0 log １０ The monoclonal human antibody or binding fragment according to any one of claims 1 to 8, which brings about a decrease in the HIV-1 RNA load in the plasma.

10. The monoclonal human antibody or binding fragment according to claim 9, wherein the humanized mouse is first treated with a single initial subcutaneous injection of 1 mg of 3BNC117 or VRC01, or a combination of both, for 4 weeks, and then, 3 to 4 days later, regular subcutaneous injections of 0.5 mg of 3BNC117 or VRC01, or a combination of both, are administered every 3 to 4 days

11. Following an initial subcutaneous injection of 1 mg of the antibody or its binding fragment, 3 to 4 days later, Subcutaneous injection of 0.5 mg of the antibody or its binding fragment every 3 to 4 days into humanized mice infected with HIV-1 NL4-3 / YU2 as described in Zhang et al., J Virol, 2002 Jun;76(12):6332-43 does not result in the occurrence of one or more mutations in the CD4 binding site epitopes (loop D, CD4 binding loop, beta23 strand, V5 loop, and beta24 strand) that mediate resistance to the administered antibody for at least 4 weeks, the monoclonal human antibody or binding fragment according to any one of claims 1 to 10.

12. Intravenous injection of 0.5 mg of the antibody or its binding fragment into NRG mice results in a detectable serum level of at least 50 μg IgG / ml serum of the antibody or its binding fragment 10 days after injection, the monoclonal human antibody or binding fragment according to any one of claims 1 to 11.

13. The antibody or its binding fragment is 1-18 (composed of the heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 2), 1-21 (composed of the heavy chain amino acid sequence of SEQ ID NO: 3 and the light chain amino acid sequence of SEQ ID NO: 4), 1-33 (composed of the heavy chain amino acid sequence of SEQ ID NO: 5 and the light chain amino acid sequence of SEQ ID NO: 6), 1-54 (composed of the heavy chain amino acid sequence of SEQ ID NO: 7 and the light chain amino acid sequence of SEQ ID NO: 8), 1-55 (composed of the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 10), 2-10 (composed of the heavy chain amino acid sequence of SEQ ID NO: 11 and the light chain amino acid sequence of SEQ ID NO: 12), 2-22 (composed of the heavy chain amino acid sequence of SEQ ID NO: 13 and the light chain amino acid sequence of SEQ ID NO: 14), 2-27 (composed of the heavy chain amino acid sequence of SEQ ID NO: 15 and the light chain amino acid sequence of SEQ ID NO: 16), 2-47 (composed of the heavy chain amino acid sequence of SEQ ID NO: 17 and the light chain amino acid sequence of SEQ ID NO: 18), 3-59 (composed of the heavy chain amino acid sequence of SEQ ID NO: 19 and the light chain amino acid sequence of SEQ ID NO: 20), 5-18 (composed of the heavy chain amino acid sequence of SEQ ID NO: 21 and the light chain amino acid sequence of SEQ ID NO: 22), 8-10 (composed of the heavy chain amino acid sequence of SEQ ID NO: 23 and the light chain amino acid sequence of SEQ ID NO: 24), 9-23 (composed of the heavy chain amino acid sequence of SEQ ID NO: 25 and the light chain amino acid sequence of SEQ ID NO: 26), 10-7 (composed of the heavy chain amino acid sequence of SEQ ID NO: 27 and the light chain amino acid sequence of SEQ ID NO: 28), 8-52 (composed of the heavy chain amino acid sequence of SEQ ID NO: 29 and the light chain amino acid sequence of SEQ ID NO: 30), 9-89 (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), 9-71 (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), 1-23 (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), 1-29 (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), 2-12 (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), 2-21 (composed of the heavy chain amino acid sequence of SEQ ID NO: 41 and the light chain amino acid sequence of SEQ ID NO: 42), 3-07 (composed of the heavy chain amino acid sequence of SEQ ID NO: 43 and the light chain amino acid sequence of SEQ ID NO: 44),One antibody from the group comprising 3-78 (consisting of the heavy chain amino acid sequence of SEQ ID NO: 45 and the light chain amino acid sequence of SEQ ID NO: 46), preferably one antibody from the group comprising 1-18, 1-33, 1-55, 2-27, 1-23, 1-29, 2-12, 2-21, 3-07 and 3-78, more preferably one antibody from the group comprising 1-18, 1-55 and 2-12, even more preferably antibody 1-18 or 2-12, particularly preferably the monoclonal human antibody or binding fragment according to any one of claims 1 to 12, comprising the amino acid sequences of heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3 of antibody 1-18.

14. The antibody or its binding fragment is 1-18 (comprising the heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 2), 1-21 (comprising the heavy chain amino acid sequence of SEQ ID NO: 3 and the light chain amino acid sequence of SEQ ID NO: 4), 1-33 (comprising the heavy chain amino acid sequence of SEQ ID NO: 5 and the light chain amino acid sequence of SEQ ID NO: 6), 1-54 (comprising the heavy chain amino acid sequence of SEQ ID NO: 7 and the light chain amino acid sequence of SEQ ID NO: 8), 1-55 (comprising the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 10), 2-10 (comprising the heavy chain a comprising the amino acid sequence and the light chain amino acid sequence of SEQ ID NO: 12), 2-22 (comprising the heavy chain amino acid sequence of SEQ ID NO: 13 and the light chain amino acid sequence of SEQ ID NO: 14), 2-27 (comprising the heavy chain amino acid sequence of SEQ ID NO: 15 and the light chain amino acid sequence of SEQ ID NO: 16), 2-47 (comprising the heavy chain amino acid sequence of SEQ ID NO: 17 and the light chain amino acid sequence of SEQ ID NO: 18), 3-59 (comprising the heavy chain amino acid sequence of SEQ ID NO: 19 and the light chain amino acid sequence of SEQ ID NO: 20), 5-18 (comprising the heavy chain amino acid sequence of SEQ ID NO: 21 and the light chain amino acid sequence of SEQ ID NO: 22), 8-10 (comprising the heavy chain amino acid sequence of SEQ ID NO: 23 and the light chain amino acid sequence of SEQ ID NO: 24), 9-23 (comprising the heavy chain amino acid sequence of SEQ ID NO: 25 and the light chain amino acid sequence of SEQ ID NO: 26), 10-7 (comprising the heavy chain amino acid sequence of SEQ ID NO: 27 and the light chain amino acid sequence of SEQ ID NO: 28), 8-52 (comprising the heavy chain amino acid sequence of SEQ ID NO: 29 and the light chain amino acid sequence of SEQ ID NO: 30), 9-89 (comprising the heavy chain amino acid sequence of SEQ ID NO: 31 and the light chain amino acid sequence of SEQ ID NO: 32), 9-71 (comprising the heavy chain amino acid sequence of SEQ ID NO: 33 and the light chain amino acid sequence of SEQ ID NO: 34), 1-23 (comprising the heavy chain amino acid sequence of SEQ ID NO: 35 and the light chain amino acid sequence of SEQ ID NO: 36), 1-29 (comprising the heavy chain amino acid sequence of SEQ ID NO: 37 and the light chain amino acid sequence of SEQ ID NO: 38), 2-12 (comprising the heavy chain amino acid sequence of SEQ ID NO: 39 and the light chain amino acid sequence of SEQ ID NO: 40), 2-21 (comprising the heavy chain amino acid sequence of SEQ ID NO: 41 and the light chain amino acid sequence of SEQ ID NO: 42), 3-07 (comprising the heavy chain amino acid sequence of SEQ ID NO: 43 and the light chain amino acid sequence of SEQ ID NO: 44), 3-78 (comprising the heavy chain amino acid sequence of SEQ ID NO: 4⑤ and the light chain amino acid sequence of SEQ ID NO: 46), one antibody selected from the group consisting of, preferably one antibody from the group comprising 1-18, 1-33, 1-55, 2-27, 1-23, 1-29, 2-12, 2-21, 3-07 and 3-78, more preferably one antibody from the group comprising 1-18, 1-55 and 2-12, even more preferably antibody 1-18 or 2-12, particularly preferably the combination of the heavy chain and light chain of antibody 1-18, the monoclonal human antibody or binding fragment according to any one of claims 1 to 13.

15. A pharmaceutical composition comprising a monoclonal human antibody or a binding fragment thereof according to any one of claims 1 to 14 and at least one pharmaceutically acceptable excipient, preferably wherein the pharmaceutical composition is a vaccination composition for a human subject.

16. A kit comprising a monoclonal human antibody or a binding fragment thereof according to any one of claims 1 to 14 and a container.

17. A monoclonal human antibody or a binding fragment thereof according to any one of claims 1 to 14, the pharmaceutical composition according to claim 15, or the kit according to claim 16, which is used as a medicine, preferably used as a vaccine.

18. A monoclonal human antibody or a binding fragment thereof according to any one of claims 1 to 14, the pharmaceutical composition according to claim 15, or the kit according to claim 16, which is used for the treatment or prevention of diseases caused by human immunodeficiency virus HIV-1 in a human subject, preferably used for the treatment or prevention of acquired immunodeficiency syndrome (AIDS) in a human subject.

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