Broadly neutralizing antibodies against HIV

Abstract

This disclosure relates to monoclonal human antibodies or binding fragments thereof targeting the CD4 binding site of human immunodeficiency virus HIV-1, pharmaceutical compositions comprising such monoclonal human antibodies or binding fragments thereof, kits comprising such antibodies or binding fragments thereof, and the use of said monoclonal antibodies or binding fragments thereof, the pharmaceutical compositions, and the kits as medicines and for the treatment or prevention of diseases caused by human immunodeficiency virus HIV-1.

Description

Technical Field

[0001] This invention relates to monoclonal human antibodies or binding fragments thereof targeting the CD4 binding site of human immunodeficiency virus HIV-1, pharmaceutical compositions comprising such monoclonal human antibodies or binding fragments thereof, kits comprising such antibodies or binding fragments thereof, and the use of said monoclonal human antibodies or binding fragments thereof, said pharmaceutical compositions, and said kits as medicines and for the treatment or prevention of diseases caused by human immunodeficiency virus HIV-1. Background Technology

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

[0003] While these results highlight the significant clinical potential of bNAb, pre-existing and re-emerging HIV-1 resistance leads to treatment failure and may strongly limit the use of bNAb in humans. Therefore, strategies to prevent and overcome viral escape are crucial for the effective implementation of bNAb-mediated approaches for HIV-1 prevention and treatment (Gruell, H., and Klein, F. (2018). Antibody-mediated prevention and treatment of HIV-1 infection. Retrovirology 15, 73).

[0004] In recent years, potent bNAbs have been isolated from HIV-1 infected donors, targeting various vulnerable epitopes on the HIV-1 envelope (Env) trimer. These epitopes include the CD4 binding site (CD4b), V1 / V2 loop, V3 glycan patch, membrane-proximal outer region, and the interface between the gp120 and gp41Env subunits.

[0005] Of these sites, CD4b is of particular interest because CD4 acts as the main receptor for viral entry. Most potent CD4b bNAbs are characterized by the use of the immunoglobulin heavy chain gene fragment IGVH1-2*02, high levels of somatic hypermutation, a five-residue complementarity-determining region 3 (CDRL3) of the light chain, and mimicry of the Env-CD4 interaction.

[0006] Named after the prototype antibody VRC01 (Wu, X., Yang, ZY, Li, Y., Hogerkorp, CM, Schief, WR, Seaman, MS, Zhou, T., Schmidt, SD, Wu, L., Xu, L., et al. (2010). Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science 329, 856-861.), these antibodies are called 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 fragment. However, compared to VH1-2 derived bNAbs, VH1-46 bNAbs reported to date have lower potency and breadth, limiting their potential for clinical application. For example, one of the best antibodies in this class, CH235.12, showed >10-fold less breadth and potency than VRC01 class bNAb N6 in in vitro studies against a large group of HIV-1 Env strains (Bonsignori, M., Zhou, T., Sheng, Z., Chen, L., Gao, F., Joyce, MG, Ozorowski, G., Chuang, GY, Schramm, CA, Wiehe, K., et al. (2016). Maturation Pathway from Germline to Broad HIV-1 Neutralizer of a CD4-Mimic Antibody. Cell 165, 449-463.).

[0008] Therefore, all CD4b bNAb that have entered clinical trials are members of the VRC01 class (3BNC117, N6, VRC01, and VRC07-523). However, although escape from VRC01 is associated with a decrease in viral fitness, the effect of VRC01 monotherapy is only transient, and this is evident in clinical trials (Scheid, JF, Horwitz, JA, Bar-On, Y., Kreider, EF, Lu, CL, Lorenzi, JC, Feldmann, A., Braunschweig, M., Nogueira, L., Oliveira, T., et al. (2016). HIV-1 antibody 3BNC117 suppresses viral rebound in humans during treatment interruption. Nature 535, 556-560.) and animal models of HIV-1 infection (Klein, F., Halper-Stromberg, A., Horwitz JA, Gruell, H., Scheid JF, Bournazos S., Mouquet H., Spatz LA, Diskin R., Abadir A., ​​et al. (2012). HIV The rapid emergence of viral escape variants is associated with the theory of a combination of broadly neutralizing antibodies in humanized mice. Nature 492(7427),118-22.

[0009] Due to these limitations of the known bNAbs currently under investigation, there is still a need for HIV antibodies that target the CD4 binding site, with potency and breadth exceeding those of known classic VH1-2 or VH1-46-derived bNAbs, possessing strong neutralizing activity against VRC01 escape variants, and effectively limiting viral escape and maintaining viral suppression when tested in HIV-1 infected organisms.

[0010] Therefore, one 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, combined 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 significantly limits the development of escape mutations and maintains efficacy against viruses exhibiting such escape mutations. Another object of the present invention is 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 targeting the CD4 binding site that has favorable in vivo pharmacokinetic properties. Summary of the Invention

[0011] These objectives are achieved through aspects of the invention described below.

[0012] According to a first aspect of the present invention, a monoclonal human antibody or a binding fragment thereof targeting the CD4 binding site of human immunodeficiency virus HIV-1 is provided, wherein the antibody amino acid sequence comprises V H Gene fragments 1-46 and V κ 3-20 gene fragments, wherein the antibody comprises 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, wherein X in any of SEQ ID No. 47 to SEQ ID No. 50 may be any amino acid or no amino acid, or an antibody sequence that is at least 80% identical to it.

[0013] According to a preferred embodiment of the first aspect of the invention, the antibody of option b) of the first aspect of the invention comprises a deletion of 2aa in FWR1.

[0014] According to another preferred embodiment of the first aspect of the invention, the antibody or its binding fragment exhibits broad neutralizing activity, exemplified by the neutralization of at least 11, preferably all 12, HIV-1 isolates from the global reference panel as described in de Camp et al., J Virol. 2014 Mar; 88(5):2489–2507, when tested at an antibody concentration of up to 25 μg / ml in a TZM-bl cell pseudovirus neutralization assay.

[0015] According to another preferred embodiment of the first aspect of the invention, the antibody or its binding fragment exhibits broad neutralizing activity, exemplified by neutralization of at least 89.9% (107 of 119), preferably at least 92.4% (110 of 119), and more preferably at least 96.6% (115 of 119) of the pseudoviruses contained in the 119-multiclade virus panel described in Schoofs et al., Immunity, 2019 Jun 18; 50(6):1513-1529.e9, when tested in a TZM-bl cell pseudovirus neutralization assay at antibody concentrations of up to 20 μg / ml.

[0016] According to a preferred embodiment of the first aspect of the invention, the antibody or its binding fragment, when tested in a TZM-bl cell pseudovirus neutralization assay at an antibody concentration of up to 25 μg / ml, exhibits a neutralizing potency (geometric mean IC50 of the neutralizing strain) 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, even more preferably less than 0.048 μg / ml, even more preferably 0.035 μg / ml, against the neutralizing strains of the global reference group described in de Camp et al., J Virol. 2014 Mar; 88(5):2489–2507. 50 ).

[0017] According to a preferred embodiment of the first aspect of the invention, the antibody or its binding fragment, when tested in a TZM-bl cell pseudovirus neutralization assay at an antibody concentration of up to 20 μg / ml, exhibits 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, and even more preferably less than 0.05 μg / ml against neutralizing strains of the 119-multiphylogenetic group described in Schoofs et al., Immunity, 2019 Jun 18; 50(6):1513-1529.e9. 50 ).

[0018] According to another preferred embodiment of the first aspect of the invention, when the antibody or its binding fragment is tested in a TZM-bl cell pseudovirus neutralization assay, it is effective against HIV-1 pseudovirus 89-F1_2_25(89-F1_2_25). env (GenBank: HM215349.1), exhibiting a neutralizing potency of less than 0.05 μg / ml, preferably less than 0.02 μg / ml, and even more preferably less than 0.01 μg / ml (IC50). 50 ).

[0019] According to another preferred embodiment of the first aspect of the invention, when the antibody or binding fragment is tested in a TZM-bl cell pseudovirus neutralization assay, it is at an IC50 concentration of less than 0.1 μg / ml, preferably less than 0.05 μg / ml. 50 The concentration neutralized all YU2 pseudovirus variants containing the YU2 envelope gene (GenBank:M93258.1) with one of the envelope mutations N279K, N280Y, G458D, G459D, or G471R (based on the HIV-1HXB2 envelope gene; GenBank:K03455 encoding residues).

[0020] According to a preferred embodiment of the first aspect of the invention, an initial subcutaneous injection of 1 mg of the antibody or its binding fragment thereof into humanized mice infected with HIV-1 NL4-3 / YU2 as described in Zhang et al., J Virol, 2002 Jun; 76(12):6332-43, followed by regular subcutaneous injections of 0.5 mg of the antibody or its binding fragment thereof every 3 to 4 days thereafter, resulting in a reduction of at least 0.8 log in plasma HIV-1 RNA load compared to the start of treatment in at least 70% of treated mice that had at least 5000 copies / ml of plasma HIV-1 RNA load at the start of treatment, as measured after 4 weeks of treatment, preferably after 6 weeks of treatment, and even more preferably after 8 weeks of treatment. 10 Preferably at least 1.0 log 10 .

[0021] According to a more preferred embodiment of the above-described embodiment of the first aspect of the invention, humanized mice are pretreated for 4 weeks by an initial subcutaneous injection of 1 mg of 3BNC117 or VRC01 or a combination thereof, followed by regular subcutaneous injections of 0.5 mg of 3BNC117 or VRC01 or a combination thereof every 3 to 4 days thereafter.

[0022] According to a preferred embodiment of the first aspect of the invention, an initial subcutaneous injection of 1 mg of the antibody or a binding fragment thereof into humanized mice infected with HIV-1 NL4-3 / YU2 as described in Zhang et al., J Virol, 2002 Jun; 76(12):6332-43, followed by regular subcutaneous injections of 0.5 mg of the antibody or a binding fragment thereof every 3 to 4 days thereafter, not for at least 4 weeks, results in the occurrence of one or more mutations in the CD4 binding sites (loop D, CD4 binding loop, β23 chain, V5 loop, and β24) that mediate resistance to the applied antibody.

[0023] According to another preferred embodiment of the first aspect of the invention, 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 10 days after injection.

[0024] According to another preferred embodiment of the first aspect of the invention, the antibody or its binding fragment does not contain CDRH3 having a length of 16 or 19 amino acids.

[0025] According to a preferred embodiment of the first aspect of the invention, the antibody or its binding fragment does not contain CDRH3 having a length of 18, 20 or 21 amino acids.

[0026] According to a preferred embodiment of the first aspect of the invention, the antibody or its binding fragment does not contain or is not composed of the amino acid sequence of antibodies NC37, NC133, AC40, AC41 or AC72 as described in Freund et al., Sci. Transl. Med. 9, eaal 2144 (2017).

[0027] According to another preferred embodiment of the first aspect of the invention, the antibody or its binding fragment comprises the heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3 amino acid sequences of an antibody comprising the group consisting of: 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. 10), SEQ ID No. 11 and the light chain amino acid sequence of SEQ ID No. 12), SEQ ID No. 13 and the light chain amino acid sequence of SEQ ID No. 14. SEQ ID No. 14 (composed of the light chain amino acid sequence), 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 SEQ ID No. 14 and the light chain amino acid sequence of SEQ ID No. 28), SEQ ID No. 14 (composed of the heavy chain amino acid sequence of SEQ ID No. 15 and the light chain amino acid sequence of SEQ ID No. 26), SEQ ID No. 14 (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), SEQ ID No. 14 (composed of the heavy chain amino acid sequence of SEQ ID No. 15 and the light chain amino acid sequence of SEQ ID No. 26), SEQ ID No. 14 (composed of the heavy chain amino acid sequence of SEQ ID No. 15 and the light chain amino acid sequence of SEQ ID No (Sequences 9-89 are 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 are 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 are 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), and 1-29 are composed of the heavy chain amino acid sequence of SEQ ID No. 29 and the light chain amino acid sequence of SEQ ID No. 30.37 (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), 3-78 (composed of the heavy chain amino acid sequence of SEQ ID No. 45 and the light chain amino acid sequence of SEQ ID No. 48), SEQ ID No. 39 (composed of the heavy chain amino acid sequence of SEQ ID No. 49 and the light chain amino acid sequence of SEQ ID No. 40), SEQ ID No. 41 (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), SEQ ID No. 43 (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), SEQ ID No. 45 (composed of the heavy chain amino acid sequence of SEQ ID No. 49 and the light chain amino acid sequence of SEQ ID No. 40), SEQ ID No. 49 ... The light chain amino acid sequence of No. 46 is preferably composed of the heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3 amino acid sequences of an 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, it is the heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3 amino acid sequences of an antibody from the group consisting of 1-18, 1-55, and 2-12. Even more preferably, it is the heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3 amino acid sequences of antibody 1-18 or 2-12. Particularly preferred are the heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3 amino acid sequences of antibody 1-18.

[0028] According to another preferred embodiment of the first aspect of the invention, the antibody or its binding fragment comprises a combination of a heavy chain and a light chain selected from the group consisting of: 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 ...4), 2-27 (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), 2-23 (composed of the heavy chain amino acid sequence of SEQ ID No. 11 and the light chain amino acid sequence of (1) 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. 16). (Sequence No. 30 is composed of the light chain amino acid sequence), 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. 34).38 (composed of 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), 3-78 (composed of the heavy chain amino acid sequence of SEQ ID No. 45 and the light chain amino acid sequence of SEQ ID No. 40), SEQ ID No. 49 (composed of the heavy chain amino acid sequence of SEQ ID No. 49 and the light chain amino acid sequence of SEQ ID No. 40), SEQ ID No. 49 (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), SEQ ID No. 43 (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), SEQ ID No. 45 (composed of the heavy chain amino acid sequence of SEQ ID No. 49 and the light chain amino acid sequence of SEQ ID No. 40), SEQ ID No. 49 ... The light chain amino acid sequence of No. 46 is preferably a combination of the heavy and light chains of an 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 a combination of the heavy and light chains of an antibody from the group consisting of 1-18, 1-55, and 2-12; even more preferably a combination of the heavy and light chains of antibodies 1-18 or 2-12; and particularly preferably a combination of the heavy and light chains of antibodies 1-18.

[0029] According to a second aspect of the invention, a pharmaceutical composition is provided comprising a monoclonal human antibody or a binding fragment thereof as described in the first aspect of the invention, and at least one pharmaceutically acceptable excipient.

[0030] According to a preferred embodiment of a second aspect of the invention, the pharmaceutical composition is a vaccine composition for use in human subjects.

[0031] According to a third aspect of the present invention, a kit is provided comprising a monoclonal human antibody or a binding fragment thereof as described in the first aspect of the present invention, and a container.

[0032] According to a fourth aspect of the invention, a monoclonal human antibody or a binding fragment thereof according to a first aspect of the invention, a pharmaceutical composition according to a second aspect of the invention, or a kit according to a third aspect of the invention are provided as pharmaceuticals, preferably as vaccines.

[0033] According to a fifth aspect of the invention, the use of a monoclonal human antibody or a binding fragment thereof according to a first aspect of the invention, a pharmaceutical composition according to a second aspect of the invention, or a kit according to a third aspect of the invention is provided for the treatment or prevention of disease caused by human immunodeficiency virus HIV-1 in human subjects, preferably for the treatment or prevention of acquired immunodeficiency syndrome (AIDS) in human subjects. Attached Figure Description

[0034] Figure 1The antibodies of the present invention demonstrate neutralizing activity against a group of 12 global reference pseudovirus strains (as described in de Camp et al., J Virol. 2014 Mar; 88(5):2489–2507) with different envelope amino acid sequences when tested in a TZM-bl cell pseudovirus neutralization assay. The tested antibodies exhibit high potency against the neutralizing strains and neutralize at least 92% (11 / 12) and up to all tested pseudoviruses.

[0035] Figure 2 The results showed that after pre-culturing with an increased amount of competitive antibody (x-axis), antibody 1-18 in the competitive ELISA... Figure 2 A), 1-55 ( Figure 2 B), and 2-12 ( Figure 2 C) Regarding BG505 SOSIP.664 The binding of HIV-1Env protein. Antibodies tested after pre-culturing with CD4 binding site-targeting antibodies 3BNC117, VRC01, and N6 showed reduced binding to BG505. SOSIP.664 The binding of the envelope protein indicates that the antibody shares an epitope that overlaps with the CD4 binding site.

[0036] Figure 3 The antibody demonstrated neutralizing activity (IC50) against 119 pseudoviruses with different HIV-1 envelope amino acid sequences (as described in Schoofs et al., Immunity, 2019 Jun 18; 50(6):1513-1529.e9) when tested in a TZM-bl cell pseudovirus neutralization assay. 50 Data are shown for antibodies 1-18, 1-55, and 2-12, VH1-2-derived CD4 binding site antibodies (3BNC117, VRC01, and N6) from late-stage clinical trials, and V3 ring-targeting antibodies (10-1074, PGT121).

[0037] Figure 4 The neutralizing activity of antibodies 1-18, 1-55, and 2-12 compared to other VH1-46-derived HIV-1 neutralizing antibodies is shown. Results against a total of 62 HIV-1 pseudoviruses are available in the antibody neutralization database CATNAP (Yoon et al., 2015).

[0038] Figure 5 The neutralizing activity of the antibody against pseudoviruses with different envelope amino acid sequences is shown, as determined by the TZM-bl cell pseudovirus assay.

[0039] Figure 6The left panel shows the neutralizing activity of antibodies 1-18, 1-55, and 2-12 against a group of HIV-1 YU2 pseudoviruses with different envelope sequences (a single amino acid mutation at the CD40 binding site), as determined in the TZM-bl cell pseudovirus neutralization assay. A comparison with antibody 8ANC131, derived from CD4 binding site antibodies 3BNC117, VRC01, N6, and VH1-46, is also shown. (Based on HIV-1...) HXB2 The mutated envelope residues were numbered using the reference strain.

[0040] Figure 7 Showing HIV-1 infection YU2 HIV-1 RNA plasma copy number (top) and their log in humanized mice (NL4-3 virus with an envelope gene replaced by YU2) 10 Changes (bottom, compared to baseline (HIV-1 RNA plasma copy number on day -1)). Mice were subcutaneously treated with a loading dose of 1 mg per antibody, followed by subcutaneous injections of 0.5 mg per antibody every 3–4 days. Mice treated with known CD4b antibodies 3BNC117, VRC01, or a combination of both antibodies showed a transient decrease in viremia, followed by a rapid viral rebound. In contrast, mice treated with antibodies 1–18 of the present invention alone showed a sustained decrease in viremia during the 8-week treatment period. The black dashed line represents the mean log 10 change.

[0041] Figure 8 The results showed that after 4 weeks of antibody treatment, through the action of... Figure 7 The HIV-1 sequence obtained from plasma by single-genome sequencing of the selected mice shown is corresponding to... Figure 7 The mouse ID is shown on the left. Sequence alignment with the YU2 wild-type sequence is shown at the top. Amino acid sequences identical to the YU2 wild-type sequence are indicated by dots, and mutations or deletions compared to the YU2 wild-type sequence are indicated by single-letter amino acid codes or dashed lines, respectively. In mice treated with known CD4b antibodies 3BNC117, VRC01, or combinations thereof, mutations in the CD4 binding epitopes (loop D, β23 chain, V5 loop) compared to the YU2 wild-type sequence were associated with viral rebound, while no such mutations were observed after 4 weeks of treatment alone with the antibody 1-18 of this invention.

[0042] Figure 9 Pretreatment with CD4 binding site antibodies 3BNC117, VRC01, or a combination of both (e.g.) Figure 7 (As shown) HIV-1 infection YU2 HIV-1 RNA plasma copy number (top) and their logarithmic values ​​in humanized mice of NL4-3 / YU210 Changes (bottom, compared to baseline (HIV-1 RNA plasma copy number on day 28)). After 4 weeks of treatment, antibody 1-18 was added to the previous treatment regimen and the treatment regimen was continued. A 1 mg loading dose of 1-18 was administered subcutaneously, followed by 0.5 mg every 3 to 4 days. Viral rebound occurred despite pretreatment with antibodies targeting other CD4 binding sites and circulation of viral variants with mutations at the CD4 binding site (see...). Figure 8 Treatment with antibody 1-18 resulted in sustained viral suppression in 18 / 19 mice.

[0043] Figure 10 The image shows the serum concentration of human IgG in NRG mice as determined by ELISA after a single intravenous injection of 0.5 mg of antibody on day 0 (arrows indicate this). Antibodies 3BNC117, VRC01, and 45-46 have known CD4 binding sites. G54W In comparison, antibodies 1-18, 1-55, and 2-12 of the present invention show a slower decrease in antibody concentration, which is more similar to V3 ring-targeting antibody 10-1074, which has a longer half-life in the human body than 3BNC117 (Mendoza et al., 2018).

[0044] Figure 11 The antibody demonstrates selective neutralizing activity (IC50) against pseudoviruses with different envelope amino acid sequences, as determined in the TZM-bl cell pseudovirus assay. 50 The pseudovirus group was selected as a representative of the diversity of the global HIV-1 pandemic (de Camp et al., 2014). The antibody of the present invention was compared with VH1-46-derived CD4 binding site antibody 8ANC131.

[0045] Figure 12 The antibody demonstrated neutralizing activity (IC50) against the 6545.v4.c1 and 89-F1_2_25 HIV-1 pseudovirus strains, as determined in the TZM-bl cell pseudovirus assay. 50 The antibody of the present invention was compared with HIV-1 neutralizing CD4 binding site antibodies N6, 3BNC117, VRC01, VRC07, VRC07-523-LS, 8ANC131, NIH45-46 and NIH45-46G54W.

[0046] Figure 13 This shows that compared to baseline (day 0), the HIV-1 infection rate has decreased. BAL HIV-1 RNA plasma copy number log in humanized mice (NL4-3 virus with an envelope gene replaced by the HIV-1 strain BAL) 10Changes. Mice were subcutaneously treated with a loading dose of 1 mg per antibody, followed by subcutaneous injection of 0.5 mg per antibody every 3–4 days. Mice treated with CD4 binding site antibody 3BNC117 or VRC01 showed a modest, transient decrease in viremia, followed by a rapid viral rebound. In contrast, mice treated with antibodies 1–18 of the present invention showed a sustained decrease in viremia during the 6-week treatment period. Untreated control mice. Black stripes represent the mean log compared to baseline. 10 Variations. Numbers represent individual mice by their respective IDs. The table on the right shows the maximum mean log observed for each treatment group. 10 HIV-1 RNA changes, day 21 log 10 HIV-1 RNA changes and log on day 42 10 HIV-1 RNA changes. Detailed Implementation

[0047] The inventors have dedicated themselves to solving the problems of this invention and have successfully discovered a new and useful human monoclonal antibody against HIV-1 that overcomes the shortcomings and deficiencies of known antibodies.

[0048] In this article, the inventors describe the new V H 1-46 and V κ 3-20-derived CD4 binding site antibodies have higher potency and broader binding range than classic V antibodies. H 1-46 and V H 1-2 derived bNAb. The observed high activity is thought to be due to common structural properties. These properties are based on V H 1-46 and V κ Combinations of 3-20 derived sequence analogs, defined by a common sequence of residues that show essential reactivity for their activity.

[0049] Of particular interest is that, compared to two of the most late-stage clinical trials, CD4b bNAb 3BNC117 and VRC01, the antibody according to the invention effectively limited viral escape and maintained neutralizing activity and complete viral inhibition against the VRC01 class of escape variants when tested in HIV-1-infected humanized mice. Therefore, the antibody of the present invention, comprising an antibody-mediated strategy for the effective treatment and prevention of HIV-1 infection, is a very promising candidate.

[0050] Therefore, the present invention provides a monoclonal human antibody or a binding fragment thereof that targets the CD4 binding site of human immunodeficiency virus HIV-1, wherein the antibody sequence comprises V H Gene fragments 1-46 and V κ 3-20 gene fragments, wherein the antibody comprises: a) a heavy chain sequence

[0051]

[0052] and light chain sequence

[0053]

[0054] or b) heavy chain sequence

[0055]

[0056] and light chain sequence

[0057]

[0058] X in any of SEQ ID No. 47 to SEQ ID No. 50 can be any amino acid or no amino acid, or an antibody amino acid sequence that is at least 80% identical to it.

[0059] In the context of this invention, the antibodies generated and described herein may be used and claimed as complete monoclonal human antibodies or any functional fragment or binding fragment thereof. Preferably, the monoclonal human antibody or any type of functional fragment or binding fragment thereof shall contain at least the complementarity-determining regions (CDRs) 1 to 3 of the heavy chain and the CDRs 1 to 3 of the light chain of the human monoclonal antibody.

[0060] The CDR region of the antibody sequence described in this article is preferably defined according to the IMGT numbering scheme, which is an adaptation of the Chothia numbering scheme (ImMunoGeneTics information). Lefranc et al., NAR 27:209-212 (1999); http: / / www.imgt.org).

[0061] In a preferred embodiment, the antibody is a monoclonal antibody or fragment thereof that maintains 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 may be an antibody containing the Fc domain of any human IgG isotype (e.g., IgG1, IgG2, IgG3, or IgG4).

[0062] Optionally, the antigen-binding compound consists of Fab, Fab', Fab'-SH, F(ab)2, Fv, a bispecific antibody, a single-chain antibody fragment, or a multispecific antibody containing multiple different antibody fragments, or contains Fab, Fab', Fab'-SH, F(ab)2, Fv, a bispecific antibody, a single-chain antibody fragment, or a multispecific antibody containing multiple different antibody fragments.

[0063] In this invention, an antibody or binding fragment targeting the CD4 binding site of HIV-1 refers to an antibody that binds to the CD4 binding site region within the gp120 envelope glycoprotein of HIV-1 with at least a 10-fold, more preferably at least 50-fold, and particularly preferably at least 100-fold increased affinity compared to unrelated epitopes, proteins, or protein regions. Generally, the term CD4 binding site herein refers to the CD4 binding site region within the gp120 envelope glycoprotein of HIV-1.

[0064] Furthermore, in this invention, V is included. H Gene fragments 1-46 and V κ The antibody amino acid sequences of gene fragments 3-20 refer to the antibody amino acid sequences based on and / or derived from the gene fragments, respectively. How to determine which V... H or V κ The gene fragment is used to assemble the amino acid sequence of the antibody. Although mutations in this gene fragment typically occur during the assembly of natural antibodies, by requiring the use of V... H Gene fragments 1-46 and V κ Gene fragments 3-20 are used to form specific antibodies, and those skilled in the art can easily see which primary sequences are required.

[0065] Therefore, monoclonal antibodies or their binding fragments should preferably be understood as containing components that are naturally derived from V. H Gene fragments 1-46 and V κ A sequence of combinations of 3-20 gene fragments. This preferably includes mutations of the degree of natural occurrence of said gene fragments.

[0066] To understand the significance and impact of these two fragments on the overall structural basis of the antibody for which protection is sought, it should be noted that V H 1-46 are responsible for 104 of the 131 amino acids in the heavy chain according to the common sequence No. 1, and for 96 of the 126 amino acids in the heavy chain according to the common sequence No. 3.

[0067] Similarly, V κ 3-20 comprise 96 of the 108 amino acids in the light chain according to common sequence No. 2, and 98 of the 111 amino acids in the light chain according to common sequence No. 4.

[0068] Based on the number of functional antibodies that the inventor can identify, two sets of shared sequences can also be formulated to include V. H Gene fragments 1-46 and V κAntibodies targeting the CD4 binding site of HIV-1 and gene fragments 3-20. These two groups appear to represent different primary sequence pathways to achieve highly efficient binding to the CD4 binding site, limit viral escape, and achieve broad and effective viral neutralization.

[0069] The first set of sequences consists of a common sequence No. 1 according to SEQ ID NO. 47 for the heavy chain and a common sequence No. 2 according to SEQ ID NO. 48 for the light chain. Many individualized sequences conforming to these common sequences have been studied, and the results obtained make it reasonable to suggest that combinations of common sequences with given fragments can provide effective structural guidance for obtaining a set of functional antibodies. Furthermore, two representative antibody groups conforming to the first set of sequences are antibodies 1-18 and 1-55, which have been studied in more detail.

[0070] The second set of sequences consists of the common sequence No. 3 according to SEQ ID NO. 49 for the heavy chain and the common sequence No. 2 according to SEQ ID NO. 50 for the light chain. Similarly, this paper has investigated numerous instances of individualized sequences as common sequences in the second set, and the results obtained make it reasonable that combinations of these common sequences with given fragments can provide effective structural guidance for obtaining functional antibodies. Furthermore, a representative antibody group consistent with the second set of sequences is antibody 2-12, which has also been studied in more detail.

[0071] Regarding the shared sequences described herein, X or Xaa in the amino acid sequence can represent any amino acid or no amino acid. However, based on the requirement that the antibody for which protection is sought should contain V... H Gene fragments 1-46 and V κ Additional requirements for gene fragments 3-20, as understood by those skilled in the art, are that the selection is more limited by the gene fragments on which the various antibodies are based.

[0072] Typically, the monoclonal human antibodies or their binding fragments described herein further comprise antibody amino acid sequences that are at least 80% identical to the sequences defined above, provided they still target the CD4 binding site of human immunodeficiency virus HIV-1. This means sequences containing antibody amino acid sequences with trivial mutations that do not interfere with structural folding and antibody affinity for the CD4 binding site.

[0073] For technicians, determining whether an antibody exhibiting a certain degree of identity targets the CD4 binding site of human immunodeficiency virus HIV-1 based on the above or common knowledge is a trivial task.

[0074] The determination of the percentage of identity between two sequences according to the present invention is accomplished using a mathematical algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA (1993) 90: 5873-5877). This algorithm is based on the BLASTN and BLASTP procedures of Altschul et al. (J. Mol. Biol. (1990) 215: 403-410). BLAST nucleotide retrieval is performed using the BLASTN procedure. To obtain vacancy alignment for comparison purposes, Gapped BLAST is used as described by Altschul et al. (Nucleic Acids Res. (1997) 25: 3389-3402). When using the BLAST and Gapped BLAST procedures, the default parameters of the respective procedures are used.

[0075] According to a preferred embodiment of the invention, the antibody amino acid sequence forms part of the invention and consists of, or comprises, a nucleic acid sequence having at least 85% identity, more preferably at least 90% identity, or even more preferably at least 95% identity with the sequences defined above and disclosed herein.

[0076] According to a preferred embodiment of the present invention, the antibody of option b) of the first aspect of the present invention comprises a deletion of 2aa in FWR1.

[0077] According to another preferred embodiment of the invention, the monoclonal human antibody or its binding fragment exhibits broad neutralizing activity, exemplified by the neutralization of at least 11, preferably all 12, HIV-1 isolates from the global reference group described in de Camp et al., J Virol. 2014 Mar; 88(5):2489–2507, when tested at antibody concentrations of up to 25 μg / ml in a TZM-bl cell pseudovirus neutralization assay.

[0078] The TZM-bl cell pseudovirus neutralization assay is a highly standardized assay commonly used in the art and to which this invention pertains, for analyzing the neutralizing efficacy of antibodies against various HIV-1 strains. In short, the antibody and viral strain are cultured together before the addition of TZM-bl target cells. These cells, upon successful infection, exhibit luciferase activity, resulting in a detectable luminescence signal in the presence of luciferin after cell lysis. Neutralizing antibodies prevent infection and thus prevent luminescence. The efficacy of neutralizing antibodies is determined by the antibody concentration required to reduce viral infectivity to a specific amount.

[0079] The standardized method for establishing and using 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 specification, alone and in combination with common prior art, enables those skilled in the art to establish and determine the conclusive readout of the TZM-bl cell pseudovirus neutralization assay used herein.

[0080] The global reference group described in de Camp et al., J Virol. 2014 Mar; 88(5):2489–2507, comprises a representative selection of 12 HIV-1 viral variants. The spectrum of HIV-1 serum neutralizing activity observed with this 12-virus group is generally considered to be very close to that observed with subtype-matched viruses. Furthermore, this group is highly sensitive for the detection of many known broadly neutralizing antibodies. Studies using this group allow for reliable prediction of the neutralizing width and / or potency of a given antibody.

[0081] According to another preferred embodiment of the first aspect of the invention, the antibody or its binding fragment exhibits broad neutralizing activity, exemplified by neutralization of at least 89.9% (107 of 119), preferably at least 92.4% (110 of 119), and more preferably at least 96.6% (115 of 119) of the pseudoviruses contained in the 119-multiphylogenetic group as described in Schoofs et al., Immunity, 2019 Jun 18; 50(6):1513-1529.e9, when tested in a TZM-bl cell pseudovirus neutralization assay at antibody concentrations of up to 20 μg / ml.

[0082] A more comprehensive group, cited in Schoofs et al., 2019 or Schoofs et al., 2019, was used to precisely identify breadth and potency by testing a large number of different pseudoviruses (i.e., HIV-1 strains). This large group is generally considered representative of all major circulating HIV-1 clades and provides detailed information on the neutralizing capacity of the antibodies tested.

[0083] According to a preferred embodiment of the first aspect of the invention, when the antibody or its binding fragment is tested in a TZM-bl cell pseudovirus neutralization assay at an antibody concentration of up to 25 μg / ml, it exhibits a neutralizing potency (geometric mean IC50) 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, even more preferably less than 0.048 μg / ml, even more preferably 0.035 μg / ml against the neutralizing strains of the global reference group described in de Camp et al., J Virol. 2014 Mar; 88(5):2489–2507. 50 ).

[0084] The neutralizing efficacy defined in this paper preferably considers only those viral variants that can be definitively identified as being neutralized by the corresponding antibodies. Based on the selection of definitively neutralizing variants, the geometric mean IC50 was determined among the neutralizing strains. 50 .

[0085] According to a preferred embodiment of the first aspect of the invention, when the antibody or its binding fragment is tested in a TZM-bl cell pseudovirus neutralization assay at an antibody concentration of up to 20 μg / ml, it exhibits a neutralizing potency (geometric mean IC50) 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 against the neutralizing strains of the 119-multiphylogenetic group described in Schoofs et al., Immunity, 2019 Jun 18; 50(6):1513-1529.e9. 50 ).

[0086] According to another preferred embodiment of the first aspect of the invention, when the antibody or its binding fragment is tested in a TZM-bl cell pseudovirus neutralization assay, it exhibits a neutralizing potency (IC50) of less than 0.05 μg / ml, preferably less than 0.02 μg / ml, more preferably up to 0.01 μg / ml, and even more preferably less than 0.01 μg / ml against HIV-1 pseudovirus 89-F1_2_25 (89-F1_2_25env gene, GenBank: HM215349.1). 50 ).

[0087] According to another preferred embodiment of the first aspect of the invention, when the antibody or its binding fragment is tested in a TZM-bl cell pseudovirus neutralization assay, it exhibits a neutralizing potency (IC50) of less than 10 μg / ml, preferably less than 1 μg / ml, more preferably less than 0.5 μg / ml, even more preferably less than 0.05 μg / ml, even more preferably at most 0.01 μg / ml, and particularly preferably less than 0.01 μg / ml against HIV-1 pseudovirus 6546.v4.c1 (6546.v4.c1 env gene, GenBank: HM215332.1). 50 ).

[0088] HIV-1 pseudovirus 89-F1_2_25 appears to be one of the most difficult strains to neutralize with antibodies targeting known CD4 binding sites. In fact, no previously known antibody targeting the CD4 binding site has been shown to have an IC50 value below 0.194 μg / ml. 50 The virus was successfully neutralized. However, the antibody described in this invention is capable of neutralizing the virus strain with much higher potency.

[0089] HIV-1 pseudovirus 6545.v4.c1 also appears to be an extremely difficult strain to neutralize by antibodies targeting known CD4 binding sites. No previously known antibodies targeting CD4 binding sites have been shown to have an IC50 concentration below 0.091 μg / ml. 50 The virus was successfully neutralized. However, the antibody described in this invention is capable of neutralizing the virus strain with much higher potency.

[0090] According to a preferred embodiment of the invention, when the antibody or binding fragment is tested in a TZM-bl cell pseudovirus neutralization assay, it is at an IC50 concentration of less than 0.1 μg / ml, preferably less than 0.05 μg / ml. 50 The concentration neutralized all YU2 pseudovirus variants containing the YU2 envelope gene (GenBank:M93258.1) with one of the envelope mutations N279K, N280Y, G458D, G459D, or G471R (based on the HIV-1HXB2 envelope gene; GenBank:K03455 encoding residues).

[0091] Mutations N279K, N280Y, G458D, and G459D have been associated with the development of viral rebound (i.e., treatment failure) during treatment with CD4-binding site antibodies in in vivo models of HIV-1 infection and have demonstrated the development of viral resistance to the administered CD4-binding site antibodies (Klein et al., Nature, 2012 Dec 6; 492(7427):118-22; Horwitz et al., ProcNatl Acad Sci USA, 2013 Oct 8; 110(41):16538-43). Furthermore, the antibody according to the invention is superior to known CD4-binding site antibodies of the prior art because the aforementioned mutations in the YU2 envelope gene do not eliminate neutralization and do not act as escape mutations against the antibody according to the invention.

[0092] According to another preferred embodiment of the invention, an initial subcutaneous injection of 1 mg of the antibody or its binding fragment thereof into humanized mice infected with HIV-1 NL4-3 / YU2 as described in Zhang et al., J Virol, 2002 Jun; 76(12):6332-43, followed by regular subcutaneous injections of 0.5 mg of the antibody or its binding fragment thereof every 3 to 4 days thereafter, resulting in a reduction of at least 0.8 log in plasma HIV-1 RNA load compared to the start of treatment in at least 70% of treated mice that had at least 5000 copies / ml of plasma HIV-1 RNA load at the start of treatment, as measured after 4 weeks of treatment, preferably after 6 weeks of treatment, and even more preferably after 8 weeks of treatment. 10 Preferably at least 1.0 log 10 .

[0093] According to another preferred embodiment of the invention, an initial subcutaneous injection of 1 mg of the antibody or its binding fragment thereof into humanized mice infected with HIV-1 NL4-3 / BAL, followed by regular subcutaneous injections of 0.5 mg of the antibody or its binding fragment thereof every 3 to 4 days thereafter, results in a reduction of at least 1.0 log in plasma HIV-1 RNA load in at least 60% of the treated mice, which had at least 30,000 copies / ml of plasma HIV-1 RNA load at the start of treatment, compared to the start of treatment, when measured after 4 weeks of treatment, or even more preferably after 6 weeks of treatment. 10 Preferably at least 1.5 log 10 Or even better, at least 1.75 log 10 .

[0094] According to a more preferred embodiment of the aforementioned implementation scheme, humanized mice are pretreated for 4 weeks by an initial subcutaneous injection of 1 mg of 3BNC117 or VRC01 or a combination thereof, followed by regular subcutaneous injections of 0.5 mg of 3BNC117 or VRC01 or a combination thereof every 3 to 4 days thereafter.

[0095] Humanized mice infected with HIV-1 NL4-3 / YU2 (YU2 env in the NL4-3 backbone, as described by Zhang et al., J Virol, 2002; 76:6332–6343) provide a well-established model in the field for studying the antiviral activity of in vivo neutralizing HIV-1 antibodies. These mice maintain stable levels of viremia (i.e., HIV-1 RNA copy number in plasma) and show a ratio of HIV-1 sequence diversity in the env gene to that observed in humans (Klein et al., Nature, 2012 Dec6; 492(7427):118-22).

[0096] This model has also been used to study the effects of monotherapy with CD4 binding site antibodies (Freund et al., PLoSPathog, 2015, Oct 30; 11(10):e1005238; Klein et al., Nature, 2012 Dec 6; 492(7427):118-22; Horwitz et al., Proc Natl Acad Sci USA, 2013 Oct 8; 110(41):16538-43; Freund et al., SciTransl Med, 2017 Jan 18; 9(373).pii:eaal2144). Antibodies according to the present invention, administered as monotherapy, resulted in a sustained inhibition of HIV-1 viral load in treated mice, thus outperforming other studies of CD4 binding site antibodies that only observed a transient decrease in HIV-1 viral load during antibody monotherapy. Furthermore, 1-18 exhibits superior in vivo activity compared to other CD4 binding site antibodies, and in this respect, it also achieves sustained viral suppression in mice after viral rebound occurred during pretreatment with CD4 binding site antibodies 3BNC117, VRC01, or a combination of both.

[0097] According to a preferred embodiment of the first aspect of the invention, an initial subcutaneous injection of 1 mg of the antibody or a binding fragment thereof into humanized mice infected with HIV-1 NL4-3 / YU2 as described in Zhang et al., J Virol, 2002 Jun; 76(12):6332-43, followed by subcutaneous injection of 0.5 mg of the antibody or a binding fragment thereof every 3 to 4 days thereafter, for a period not exceeding 4 weeks, results in the occurrence of one or more mutations in the CD4 binding site epitopes (loop D, CD4 binding loop, β23 chain, V5 loop, and β24 chain) that mediate resistance to the applied antibody.

[0098] Preferably, resistance to an antibody applied in the context of the preceding embodiments can be defined as the IC50 of the applied antibody in both the TZM-bl and pseudovirus assays when tested with HIV-1 pseudoviruses containing the mutated viral sequence. 50 The concentration should be at least 2.5 μg / ml.

[0099] Studies of other CD4 binding site antibodies as monotherapy have demonstrated the development of escape mutations that lead to antibody resistance and / or viral rebound (i.e., treatment failure) (Freund et al., PLoS Pathog, 2015, Oct 30; 11(10):e1005238; Klein et al., Nature, 2012 Dec 6; 492(7427):118-22; Horwitz et al., Proc Natl Acad Sci USA, 2013 Oct 8; 110(41):16538-43; Freund et al., Sci Transl Med, 2017 Jan 18; 9(373).pii:eaal2144). Compared to these CD4 binding site antibodies, the antibodies of the present invention have the advantage of preventing the development of mutations in the CD4 binding site epitopes, thus preventing treatment failure and maintaining antiviral activity.

[0100] According to another preferred embodiment of the first aspect of the invention, 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 10 days after injection.

[0101] The pharmacokinetic properties of HIV-1 neutralizing antibodies can vary. In humans, known CD4-binding site antibodies appear to have shorter half-lives than antibodies targeting the V3 ring (Mendoza et al., Nature, 2018 Sep; 561(7724):479-484), and this has also been observed in mouse models (Klein et al., Nature, 2012 Dec 6; 492(7427):118-22; Horwitz et al., Proc Natl Acad Sci USA, 2013 Oct 8; 110(41):16538-43). The antibodies of this invention are superior to other CD4-binding site antibodies because they maintain high serum levels in vivo for a longer period of time.

[0102] According to a preferred embodiment of the first aspect of the invention, the antibody or its binding fragment does not contain CDRH3 having a length of 16 or 19 amino acids and / or the antibody or its binding fragment contains CDRH3 having a length of 18, 20 or 21 amino acids.

[0103] According to a preferred embodiment of the first aspect of the invention, the antibody or its binding fragment does not contain or is not composed of the amino acid sequence of antibodies NC37, NC133, AC40, AC41 or AC72 as described in Freund et al., Sci. Transl. Med. 9, eaal 2144 (2017).

[0104] According to a preferred embodiment of a first aspect of the invention, the antibody or its binding fragment comprises the heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3 amino acid sequences of an antibody comprising the group consisting of: 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. 10), SEQ ID No. 11 and the light chain amino acid sequence of SEQ ID No. 12), SEQ ID No. 13 and the light chain amino acid sequence of SEQ ID No. 14. SEQ ID No. 14 (composed of the light chain amino acid sequence), 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 SEQ ID No. 14 and the light chain amino acid sequence of SEQ ID No. 28), SEQ ID No. 14 (composed of the heavy chain amino acid sequence of SEQ ID No. 15 and the light chain amino acid sequence of SEQ ID No. 26), SEQ ID No. 14 (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), SEQ ID No. 14 (composed of the heavy chain amino acid sequence of SEQ ID No. 15 and the light chain amino acid sequence of SEQ ID No. 26), SEQ ID No. 14 (composed of the heavy chain amino acid sequence of SEQ ID No. 15 and the light chain amino acid sequence of SEQ ID No (Sequences 9-89 are 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 are 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 are 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), and 1-29 are composed of the heavy chain amino acid sequence of SEQ ID No. 29 and the light chain amino acid sequence of SEQ ID No. 30.37 (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), 3-78 (composed of the heavy chain amino acid sequence of SEQ ID No. 45 and the light chain amino acid sequence of SEQ ID No. 48), SEQ ID No. 39 (composed of the heavy chain amino acid sequence of SEQ ID No. 49 and the light chain amino acid sequence of SEQ ID No. 40), SEQ ID No. 41 (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), SEQ ID No. 43 (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), SEQ ID No. 45 (composed of the heavy chain amino acid sequence of SEQ ID No. 49 and the light chain amino acid sequence of SEQ ID No. 40), SEQ ID No. 49 ... The light chain amino acid sequence of No. 46 is preferably composed of the heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3 amino acid sequences of an 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, it is the heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3 amino acid sequences of an antibody from the group consisting of 1-18, 1-55, and 2-12. Even more preferably, it is the heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3 amino acid sequences of antibody 1-18 or 2-12. Particularly preferred are the heavy chain CDR1 to CDR3 and light chain CDR1 to CDR3 amino acid sequences of antibody 1-18.

[0105] According to a specific preferred embodiment of the present invention, the antibody or its binding fragment comprises a combination of a heavy chain and a light chain of an antibody selected from the group consisting of: 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 ...4), 2-27 (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), 2-23 (composed of the heavy chain amino acid sequence of SEQ ID No. 11 and the light chain amino acid sequence (1) 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. 16). (Sequence No. 30 is composed of the light chain amino acid sequence), 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. 34).38 (composed of the light chain amino acid sequence of SEQ ID No. 39), 2-12 (composed of the heavy chain amino acid sequence of SEQ ID No. 49 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), 3-78 (composed of the heavy chain amino acid sequence of SEQ ID No. 45 and the light chain amino acid sequence of SEQ ID No. 40), SEQ ID No. 49 (composed of the heavy chain amino acid sequence of SEQ ID No. 49 and the light chain amino acid sequence of SEQ ID No. 40), SEQ ID No. 49 (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), SEQ ID No. 43 (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), SEQ ID No. 45 (composed of the heavy chain amino acid sequence of SEQ ID No. 49 and the light chain amino acid sequence of SEQ ID No. 40), SEQ ID No. 49 ... The light chain amino acid sequence of No. 46 is preferably a combination of the heavy and light chains of an 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 a combination of the heavy and light chains of an antibody from the group consisting of 1-18, 1-55, and 2-12; even more preferably a combination of the heavy and light chains of antibodies 1-18 or 2-12; and particularly preferably a combination of the heavy and light chains of antibodies 1-18.

[0106] In the specification of this application, antibody names may be used. It should be noted that antibodies consist of heavy and light chains, which also form part of this specification. If antibodies are referenced by their names or SEQ ID No., it should be understood that these references are interchangeable.

[0107] The present invention further relates to a pharmaceutical composition comprising a monoclonal human antibody or a binding fragment thereof as defined and further described herein, and at least one pharmaceutically acceptable excipient. Preferably, the pharmaceutical composition is a vaccine composition for human subjects.

[0108] The present invention also includes a kit comprising a monoclonal human antibody or a binding fragment thereof as defined and further described herein, and a container thereof.

[0109] In one aspect, the present invention also relates to monoclonal human antibodies or binding fragments thereof as defined and further described herein, pharmaceutical compositions and kits as described herein for use as medicines, and preferably for use as vaccines.

[0110] In another aspect, the present invention also relates to the use of monoclonal human antibodies or binding fragments thereof as defined and further described herein, pharmaceutical compositions and kits as described herein 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.

[0111] In another aspect, the present invention also relates to a method for treating a patient in a human subject suffering from a disease caused by human immunodeficiency virus HIV-1, preferably for 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 invention or a pharmaceutical composition of the invention is administered to the patient.

[0112] In another aspect, the present invention also relates to the use of the monoclonal human antibody or its binding fragment or the pharmaceutical composition of the present invention in the preparation of a medicament for treating diseases caused by human immunodeficiency virus HIV-1 in human subjects, preferably for treating or preventing acquired immunodeficiency syndrome (AIDS) in human subjects.

[0113] All embodiments of the invention described herein are considered to be possible in any combination unless those skilled in the art consider such combination to have no technical significance.

[0114] Example

[0115] A) Experimental methods

[0116] Isolation of monoclonal antibody sequences

[0117] Blood and leukocyte-cleared samples were obtained according to protocols (Protocols 13-364 and 16-054) approved by the Institutional Review Board of the University of Cologne, with written informed consent provided by participants. Peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation and stored at -150°C in 90% FBS and 10% DMSO. B cells were isolated from PBMCs by magnetic cell dissociation and labeled on ice for 30 minutes with anti-human CD19-AF700, anti-human IgG-APC, DAPI (BD), and HIV-1 Env bait protein. The HIV-1 Env bait protein was BG505. SOSIP.664 -GFP (Sliepen et al., 2015) or biotinylated (EZ-Link Sulfo NHSBioting and Labeling Kit, Thermo Fisher) YU2 gp140 (Yang et al., 2000), labeled with streptavidin-PE. Sorting of Env-reactive CD19 as previously described. + IgG + DAPI -Single cells (Ehrhardt et al., 2019). Sorted cells were cultured for 1 minute at 65°C with random hexamer primer NP-40 and RNase-free H2O. cDNA was then generated using SuperScript IV in the presence of RT buffer, dNTPs, DTT, H2O, RNasin, and RNaseOUT. Antibody sequences 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) for single-cell analysis.

[0118] Antibody sequence analysis

[0119] Sequences of the second PCR product with a minimum length of 240 nucleotides and an average Phred score ≥28 were annotated using IgBLAST (Ye et al., 2013), and trimmed from the frame region (FWR) 1 of the variable region to the end of the J gene. Base calls with Phred scores <16 were masked, and sequences with more than 15 masked nucleotides, frameshifts, or stop codons were excluded from further analysis. To analyze the potential clonalness of the sequences, all heavy chain producing sequences were grouped according to the same V gene, and their CDRH3 pairwise Levenshtein distances were determined. Individual sequences sharing the same V gene and having a minimum CDRH3 identity of 75% were classified as clones. After 10 rounds of random sequence input, the results that produced the fewest unassigned (non-clonal) sequences were selected for further analysis. All clones were manually revalidated to identify common mutations. Sequences initially assigned to different clones but sharing the same VDJ gene and amino acid and / or silent nucleotide mutations were then classified as subclones. IgBLAST was used to calculate nucleotide sequence identity with the phylogenetic line.

[0120] Monoclonal antibody production

[0121] To clone single-cell-derived antibodies, the first PCR product of single-cell PCR was used as a template and amplified using Q5 high-fidelity polymerase and specific forward and reverse primers (Tiller et al., 2008) with nucleotide sequences similar to the respective V and J regions for subsequent sequence-independent cloning (SLIC) of the expression vector. The PCR product was cloned into a human antibody expression vector (IgG1, κ, or λ chain) via SLIC assembly as previously described (von Boehmer et al., 2016). Antibodies were generated in HEK293-6E cells by transfection with polyethyleneimine. After 5–7 days, antibodies were purified from the supernatant obtained from protein G culture and subsequent elution from the column using 0.1 M glycine (pH 3.0). After buffer neutralization, the buffer was replaced with PBS, and the mixture was filtered-sterilized and stored at 4°C.

[0122] Fake virus production

[0123] As previously described, pseudoviruses were generated in HEK293T cells by co-transfection with the pSG3ΔEnv plasmid (Doria-Rose et al., 2017; Sarzotti-Kelsoe et al., 2014; Hraber et al., 2017; Seaman et al., 2010). To generate the YU2 pseudoviral mutant set, point mutations were introduced into the plasmid encoding the YU2 envelope gene using site-directed mutagenesis.

[0124] TZM-bl cell neutralization test

[0125] Neutralization assays were performed as previously described (Sarzotti-Kelsoe et al., 2014; Seaman et al., 2010). Murine leukemia virus (MuLV) pseudoviruses were used to determine nonspecific activity. Antibodies were tested in duplicate. For assays of pseudovirus mutants and the global reference group, bioluminescence was determined after the addition of luciferin / lysis buffer (10 mM mgCl2, 0.3 mM ATP, 0.5 mM Coenzyme A, 17 mM IGEPAL (all Sigma-Aldrich), and 1 mM D-luciferin (GoldBio) in Tris-HCl).

[0126] HIV-1 infected humanized mice

[0127] As previously described (Klein et al., 2012) and modified, humanized mice were generated. NOD.Cg-Rag1 tm1mom Il2rg tm1Wjl / SzJ(NRG) mice were injected intrahepatically with human CD34 within 5 days of birth and 3-6 hours after sublethal radiation.+ Humanized from hematopoietic umbilical cord blood and / or placental tissue stem cells. Replicating recombinant HIV-1 cells were harvested from the supernatant of transfected HEK293T cells. YU2 (YU2 env in NL4-3 skeleton (Zhang et al., 2002)) or HIV-1 BAL (BAL env in NL4-3 skeleton) infects humanized mice via intraperitoneal attack.

[0128] HIV-1 viral load assay

[0129] Plasma RNA was extracted from EDTA plasma samples using the MinElute Virus Mini Spin Kit, which includes an on-column DNase I digestion step. Viral load was determined by quantitative real-time PCR using previously described pol-specific primers (Horwitz et al., 2013). qPCR was performed on a LightCycler 480II using the Taqman RNA-to-Ct 1-Step Kit. Viral load was quantified using a standard curve comprising samples derived from known copy numbers in each qPCR run. The limit of accuracy for determining qPCR was 384 copies / ml. Logarithmic values ​​for viral loads <384 copies / ml were calculated assuming a copy number of 383 copies / ml. 10 change.

[0130] Antibody therapy in HIV-1 infected humanized mice

[0131] The antibody, diluted in PBS, was administered subcutaneously. Following a 1 mg loading dose, 0.5 mg doses were administered every 3-4 days.

[0132] Single-genome sequencing of HIV-1 in humanized mice

[0133] To determine the development of potential escape mutations in the HIV-1 env gene during antibody treatment in HIV-1-infected humanized mice, extracted plasma RNA was used to generate cDNA using SuperScript IV and antisense primer YB383 (Horwitz et al., 2017), followed by RNase H culture. Limiting-dilution nested PCR was then performed to amplify single-genomic env cDNA using Taq polymerase and primers YB383 and YB50 for the first PCR and primers YB49 and YB52 for the second PCR (Schoofs et al., 2019). Positive PCR reactions with dilutions <30% PCR efficiency were sequenced using Illumina dye sequencing, as previously described (Kryazhimskiy et al., 2014; Schoofs et al., 2016) and modified. PCR products were sequenced and readout assembled on an Illumina MiSeq after tagging, addition of indices and adapters via limited-cycle PCR, and purification (Gaebler et al., 2019). Full-length env sequences with fewer than 10 ambiguities (<75% nucleotide identity in readout) were analyzed.

[0134] Identify competing ELISAs for HIV-1 Env binding

[0135] Using the EZ Link Sulfo NHS Biotinylated and Labeled Kit (Thermo Fisher), antibodies 1-18, 1-55, and 2-12 were biotinylated according to the manufacturer's instructions, and then the buffer was replaced with PBS. High-binding ELISA plates were coated overnight at 4°C with 2 μg / ml anti-6XHis-tagged antibody. Wells were blocked at 37°C with 3% BSA in PBS for 60 min, and then blocked at 37°C with 2 μg / ml BG505 in PBS. SOSIP.664 - His (Sanders et al., 2013) cultured for 60 min. The competitive antibody was cultured for 60 min at a 1:3 dilution starting at 32 μg / ml in PBS at room temperature. The biotinylated antibody of interest was diluted to 0.5 μg / ml in PBS with 3% BSA and cultured for 60 min at room temperature, followed by a 1:5,000 dilution of peroxidase-streptavidin in PBS with 1% BSA / 0.05% Tween 20. The absorbance at 415 nm was determined using a microplate reader after the addition of ABTS solution. The plate was washed with 0.05% Tween 20 in PBS between steps.

[0136] In vivo antibody pharmacokinetic analysis

[0137] NRG mice were intravenously injected with 0.5 mg of antibody in 200 μl PBS. The total serum concentration of human IgG was determined by ELISA as previously described (Klein et al., 2012) with slight modifications. Briefly, highly binding ELISA plates were coated overnight at room temperature with anti-human IgG at a concentration of 2.5 μg / mL. The wells were then blocked with blocking buffer (2% BSA, 1 μM EDTA, and 0.1% Tween 20 in PBS). Human IgG1κ standards (in duplicate) and serially diluted serum samples in PBS were cultured for 90 min at room temperature, followed by 90 min at room temperature with HRP-conjugated anti-human IgG diluted 1:1,000 in blocking buffer. After adding ABTS, the optical density at 415 nm was determined using a microplate reader. The plates were washed with 0.05% Tween 20 in PBS between steps. Serum samples obtained prior to antibody injection were used to confirm the absence of human serum IgG at baseline.

[0138] B) Specific examples of the antibodies of the present invention

[0139] Example I - Isolation of broadly potent and potent VH1-46-derived HIV-1 neutralizing antibodies from an elite neutralizer for HIV-1 infection

[0140] Human B cells responsive to HIV-1 envelope proteins were isolated from HIV-1 infected individuals who had previously been identified as having excellent serum neutralizing activity against HIV-1 in in vitro experiments.

[0141] Therefore, soluble HIV-1 Env protein (YU2) labeled with a fluorescent dye was used. gp140 Or BG505 SOSIP.664 The isolated B cells were cultured and sorted individually (Ehrhardt et al., 2019). This method enabled the subsequent amplification of both heavy and light antibody gene fragments from a single HIV-1 Env-reactive B cell via PCR. This allowed the PCR products to be cloned into expression vectors for recombinant production of the corresponding antibodies encoded by a single B cell, enabling functional assays of the antibodies.

[0142] Sequence analysis of HIV-1-Env-responsive B cells identified expanded VH1-46-derived B cell clones. One of these clones is characterized by a 6-amino acid (aa) insertion into the heavy chain CDR1 region, represented as a shared sequence in the heavy chain (SEQ ID: 47) and light chain (SEQ ID: 48). Three additional B cell clones were identified, sharing similarities with each other and with the aforementioned clones. These additional clones do not carry the 6aa CDRH1 insertion but show a 2aa deletion in frame region 1 of their light chain. These clones are represented as separate shared sequences in the heavy chain (SEQ ID: 49) and light chain (SEQ ID: 50).

[0143] A total of 23 antibodies representing the identified VH1-46-derived B-cell clones were generated as monoclonal antibodies. To determine their overall neutralizing potency and breadth, the neutralizing activity of these antibodies was tested in a TZM-bl cell neutralization assay against a reference group of 12 HIV-1 pseudoviral strains, referred to as the "global group." Figure 1 This pseudovirus group was previously designed to represent the diversity of the global HIV-1 epidemic and to enable standardized evaluation of the activity of neutralizing antibodies.

[0144] Notably, all isolated VH1-46-derived antibodies exhibited high neutralizing activity against at least 11 of the 12 HIV-1 reference strains. Figure 1 The VH1-46-derived antibodies represented by SEQ IDs 47 and 48 demonstrated activity against all tested strains. In addition to their neutralizing width, the tested VH1-46-derived antibodies also exhibited high neutralizing potency. Neutralizing potency is typically defined by a 50% inhibitory concentration (IC50). 50 This indicates that when tested against the "global group," all tested VH1-46-derived antibodies showed high activity against neutralizing viral strains, with a geometric mean IC50. 50 Below 0.2 μg / ml Figure 1 ).

[0145] Example II - Identification of the CD4 binding site as a target of the antibody of the present invention

[0146] To determine the epitopes of the antibodies according to the present invention, the effects of representative antibodies 1-18, 1-55, and 2-12 on BG505 were investigated in the presence of antibodies with known specificity. SOSIP.664 The binding of HIV-1 envelope trimer was investigated. Interference from CD4 binding site antibodies 3BNC117, N6, and VRC01 was also detected. Figure 2 This indicates that the antibody of the present invention targets the CD4 binding site and the epitope that overlaps with the epitope of other antibodies that bind to the CD4 binding site.

[0147] Example III - High potency and breadth of antibodies 1-18, 1-55, and 2-12

[0148] To confirm the neutralizing potency and breadth of isolated VH1-46-derived HIV-1 antibodies, representative antibodies 1-18, 1-55, and 2-12 were individually tested against an expanded group of 119 HIV-1 pseudoviruses, as previously tested by Schoofs et al., 2019. This pseudovirus group represents the evolution of the pseudovirus group described in detail in Seaman et al., 2010. It provides a representativeness of the genetic and global diversity of HIV-1 Env variants. It includes HIV-1 variants from different clades or subtypes, including variants isolated from self-spreading / initial virus and those that are difficult to neutralize.

[0149] Notably, when tested on the 119-pseudovirus multi-clade group, the VH1-46-derived antibody of the present invention exhibited high neutralizing potency and broad band ( ). Figure 3 Specifically, when tested at antibody concentrations up to 20 μg / ml, antibody 1-18 neutralized 96.6% of the tested pseudoviruses and showed a geometric mean IC50 of 0.048 μg / ml against the neutralized pseudoviruses. 50 ( Figure 3 This antibody exhibits greater width and potency than other HIV-1 neutralizing antibodies that also target the CD4 binding site, derived from the VH1-2 gene fragment, and are in late-stage clinical trials (3BNC117 and VRC01). Figure 3 Furthermore, antibody 1-18 in the 119-multi-clade group exhibited higher potency than another VH1-2-derived CD4-binding antibody, N6, in clinical trials. Figure 3 ).

[0150] Furthermore, when compared with other VH1-46-derived HIV-1 neutralizing antibodies targeting the CD4 binding site, the representative VH1-46-derived antibody of this invention exhibited higher potency and wider band for results against a total of 62 identical HIV-1 pseudoviruses (Yoon et al., 2015). Figure 4 ).

[0151] Example IV - Neutralizing activity of pseudoviruses that poorly neutralize antibodies against other CD4 binding sites

[0152] Although CD4 binding site antibodies can achieve high levels of neutralization width (i.e., activity against a large number of different HIV-1 Env variants), antibody-resistant HIV-1 variants exist. Therefore, it is important to identify novel CD4 binding site antibodies that are highly active against such HIV-1 variants.

[0153] Compared to CD4-binding site antibodies (N6, 3E3NC117, VRC01) in late-stage clinical trials, the antibodies of this invention exhibit significantly higher potency (i.e., they have lower IC50 values). 50 Neutralization of several strains (representative members 1-18, 1-55 and 2-12 are shown in...) Figure 5 ).

[0154] For example, when tested in the TZM-bl pseudovirus neutralization assay at concentrations up to 20 μg / ml, pseudoviruses including the HIV-1 Env protein of strain 89-F1_2_25 were not neutralized by CD4 binding site antibodies (N6, 3BNC117, VRC01) used in clinical trials (i.e., these antibodies did not achieve an IC50 against that specific virus). 50 ()( 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 were completely incapable of neutralizing the virus (i.e., IC50). 50 >20 μg / ml). Among the three antibodies that neutralize this virus, NC37 has the highest IC50 value. 50 The concentration was 17.7 μg / ml, VRC 16 The IC50 of VRC13, an antibody derived from the VH1-69 gene, was 3.12 μg / ml. 50 The concentration was 0.19 μg / ml. In contrast, the representative antibodies of this invention (1-18, 1-55, and 2-12) showed very high potency (IC50). 50 The concentrations of 0.007, 0.014, and 0.006 μg / ml, respectively, neutralized HIV-1 pseudovirus 89-F1_2_25. The same applies to all other antibodies of this invention (IC50 of all antibodies). 50 Between 0.006 and 0.025 μg / ml, see [reference needed]. Figure 12 Another viral strain highly resistant to antibodies against the common CD4 binding site is strain 6545.v4.c1. This strain is resistant to 43 CD4 binding site IgG antibodies listed in the CATNAP database of neutralizing antibodies (Yoon et al., 2015), which has a 75% resistance rate. Most importantly, antibodies against late-stage CD4 binding sites are ineffective (IC50). 50 <10 μ / ml) to neutralize this strain (IC50 of N6) 50 : 16.23; IC 3BNC117 50 >20; VRC01 IC 50 >20; VRC07-523-LS IC 50 >20). Conversely, all antibodies of the present invention showed IC50 values ​​between 0.008 and 9.37 μg / ml.50 This strain is highly neutralizing. Figure 12 ).

[0155] Therefore, the antibody of the present invention provides a solution to the poor neutralization of viruses 89-F1_2_25 and 6545.v4.c1 via CD4 binding site antibodies. When tested against 89-F1_2_25, the antibody of the present invention is about 1 log stronger than the aforementioned optimal CD4 binding site antibody against this virus. 10 .

[0156] Example V - Neutralizing activity against pseudoviruses with CD4 binding site mutations that confer resistance to antibodies against other CD4 binding sites.

[0157] Antibody resistance mediated by the development of CD4 binding site escape mutations leads to treatment failure of antibody therapy for HIV-1 infection (Klein et al., 2012). Therefore, it is crucial to identify CD4 binding site antibodies that are unaffected by these escape mutations to provide therapeutic options for HIV-1 variants carrying these mutations.

[0158] In TZM-bl cells and assays, CD4 binding site antibodies were tested against YU2 pseudovirus variants with specific CD4 binding site mutations. Figure 6 Mutations at the CD4 binding site affect the activity of previously known antibodies targeting the CD4 binding site. Figure 6 For example, the CD4 binding site antibodies VRC01 and 8ANC131 have an IC50 response against wild-type YU2 pseudovirus. 50 The concentrations were 0.107 μg / ml and 0.256 μg / ml, respectively, but no neutralization was observed for the ring D mutant in any test (i.e., no IC50 at test antibody concentrations up to 2.5 μg / ml). 50 Another CD4 binding site antibody, N6, is affected by the mutation N279K. Figure 6 Conversely, the representative antibodies of the present invention tested (1-18, 1-55, and 2-12) maintained high neutralizing activity against the tested YU2 pseudovirus variant. Figure 6 ).

[0159] Therefore, the present invention provides a solution for resistance to CD4 binding site antibodies against viral variants with mutations in the CD4 binding site.

[0160] Example VI - Maintaining viral suppression via antibody monotherapy in an in vivo model of HIV-1 infection

[0161] HIV-1 infection YU2The humanized mice developed by Zhang et al. (2002) provide a model for studying the antiviral activity of antibodies that neutralize HIV-1 in vivo. To generate humanized mice, immunodeficient NRG mice were irradiated on the first day after birth and injected intrahepatically with artificial blood CD34. + Stem cells. This leads to the development of human lymphocytes that can be infected by replicating HIV-1. These mice maintained stable levels of viremia (i.e., HIV-1 RNA copy number in plasma) and showed a ratio of similar HIV-1 sequence diversity observed in humans (Klein et al., 2012). 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 (Klein et al., 2012; NC37: Freund et al., 2017). In all these studies, only a transient effect on viral load was observed, and viral rebound (i.e., viremia returning to baseline levels) developed rapidly. Furthermore, this viral rebound was associated with the development of mutations in the antibody target site. Therefore, there is a need for novel CD4-binding antibody capable of maintaining viral suppression when administered as a monotherapy.

[0162] When treating HIV-1 with CD4 binding site antibodies 3BNC117, VRC01, or a combination thereof YU2 When humanized mice were infected (subcutaneously administered a 1 mg loading dose of antibody, followed by 0.5 mg subcutaneously every 3–4 days), only a transient decrease in HIV-1 RNA copy number was observed, and in most mice, viremia returned to baseline levels within 2–3 weeks. Figure 7 Conversely, when mice were treated with the representative antibody 1-18 of the present invention according to the same dosing regimen, viral suppression was observed in all treated mice during the 8-week treatment period compared to the viral copy number at the start of treatment. Figure 7 In ≥80% of mice, HIV-1 RNA plasma copy number decreased to levels below the precision limit of the assay used (384 copies / ml).

[0163] Therefore, the antibody of the present invention is able to target HIV-1 even when administered as a single therapy. YU2 It effectively maintains viral suppression in infected humanized mice.

[0164] Furthermore, when HIV-1 is treated with CD4 binding site antibodies 3BNC117 or VRC01 as described above... BALWhen infected with humanized mice, only a slight and transient decrease in HIV-1 RNA copy number was observed, and in most mice, viremia returned to baseline levels within 2 weeks. Figure 13 Conversely, when mice were treated with the representative antibody 1-18 of the present invention according to the same dosing regimen, viral suppression was observed in all treated mice during the 6-week treatment period compared to the viral copy number at the start of treatment. Figure 13 ).

[0165] Therefore, the antibody of the present invention is able to target HIV-1 even when administered as a single therapy. BAL It effectively maintains viral suppression in infected humanized mice.

[0166] Example VII - Preventing the development of viral mutations at the CD4 binding site during antibody monotherapy

[0167] Viral rebound during antibody monotherapy targeting CD4 binding sites is often associated with the development of viral sequence mutations at the antibody target site (179NC75: Freund et al., 2015; 3BNC117: Horwitz et al., 2013; 45-46). G54W (Klein et al., 2012; NC37: Freund et al., 2017). Therefore, there is a need for novel CD4 binding site antibodies that can prevent the development of mutations in CD4 binding site antibodies to prevent antibody resistance mediated by such mutations.

[0168] As described in Example IV, mutations in the CD4 binding site (loop D, β23 chain / V5 loop) were observed in all test mice treated with 3BNC117, VRC01, or 3BNC117+VRC01. Figure 8 Conversely, in the mice described in Example IV, which maintained detectable viremia after four weeks of antibody treatment with antibodies 1-18 of the present invention, mutations at these sites were found in only one of the 10 sequences. Figure 7 It is noteworthy that when this mutation (G459D) was tested in the YU2 pseudovirus group, the activity of 1-18 was unaffected. Figure 6 ).

[0169] Example VIII - Maintaining viral suppression via antibody monotherapy in an in vivo model of HIV-1 infection following viral rebound after VRC01 therapy.

[0170] Failure of antibody monotherapy using CD4 binding site antibodies in HIV-1-infected humanized mice has led to viral rebound and is associated with viral resistance to the administered antibody (Freund et al., 2015; Horwitz et al., 2013; Klein et al., 2012; Freund et al., 2017). Therefore, there is a need for novel CD4 binding site antibodies to provide in vivo therapeutic options after failure of previous CD4 binding site therapy.

[0171] After observing viral rebound during 4 weeks of treatment with 3BNC117, VRC01, or a combination thereof (as described in Example VI), increasing the antibody 1-18 of the present invention to the treatment regimen (subcutaneous administration of a 1 mg loading dose, followed by subcutaneous administration of 0.5 mg every 3 to 4 days) resulted in a sustained reduction in viremia in 95% (18 / 19) of the treated mice. Figure 9 Therefore, the antibody of the present invention provides an option for controlling HIV-1 in vivo, even after pretreatment with antibodies targeting other CD4 binding sites has failed.

[0172] Example IX - Excellent in vivo half-life compared to other CD4 binding site antibodies

[0173] Not only do the neutralizing potency and width of HIV-1 neutralizing antibodies vary, but their in vivo half-lives also differ. For example, the V3 ring-targeting antibody 10-1074 has a longer half-life than the CD4-binding antibody 3BNC117 when administered to HIV-1 infected individuals (Mendoza et al., 2018). Therefore, new antibodies targeting the CD4 binding site should possess favorable in vivo pharmacokinetic properties compared to currently available CD4-binding antibodies.

[0174] To determine their pharmacokinetic properties, representative antibodies of the present invention (1-18, 1-55, and 2-12) were individually injected intravenously into NRG mice. Antibodies binding to the CD4 binding site 3BNC117, VRC01, and 45-46 were also included. G54W In contrast, the antibodies tested according to the present invention showed a slower decrease in serum IgG concentration, more similar to 10-1074, as determined by total human IgG ELISA. Figure 10 Therefore, compared with other antibodies targeting the CD4 binding site of HIV-1, the antibodies of the present invention exhibited favorable pharmacokinetic properties.

[0175] References in the Implementation Examples section:

[0176] Anderson,J.P.,et al.(2000).Testing the hypothesis of a recombinantorigin of human immunodeficiency virus type 1subtype E.J.Virol.74,10752-10765.

[0177] deCamp,A.,et al.(2014).Global Panel of HIV-1Env Reference Strains forStandardized Assessments of Vaccine-Elicited Neutralizing Antibodies.J,Virol.88,2489-2507.

[0178] Doria-Rose,N.A.,et al.(2017).Mapping Polyclonal HIV-1AntibodyResponses via Next-Generation Neutralization Fingerprinting.PLoS Pathog 13,e1006148.

[0179] Dosenovic,P.,et al.(2019).Anti-idiotypic antibodies elicit anti-HIV-1-specific B cell responses.J.Exp.Med.

[0180] Ehrhardt et al.,(2019)Polyclonal and convergent antibody response toEbola virus vaccine rVSV-ZEBOV.Nat Med.25,1589-1600.

[0181] Freund,N.T.,et al.(2015).ANew Glycan-Dependent CD4-Binding SiteNeutralizing Antibody Exerts Pressure on HIV-1In Vivo.PLoS Pathog.11,:e1005238.

[0182] Freund,N.T.,et al.(2017).Coexistence of potent HIV-1 broadlyneutralizing antibodies and antibody-sensitive viruses in a viremiccontroller.Sci Transl Med.9,eaal2144

[0183] Gaebler,C.,et al.(2019).Combination of quadruplex qPCR and next-generation sequencing for qualitative and quantitative analysis of the HIV-1latent reservoir.J.Exp.Med.

[0184] Horwitz,J.A.,et al.(2013).HIV-1 suppression and durable control bycombining single broadly neutralizing antibodies and antiretroviral drugs inhumanized mice.Proc.Natl.Acad.Sci.USA110,16538-16543.

[0185] Horwitz,J.A.,et al.(2017).Non-neutralizing Antibodies Alter theCourse of HIV-1 Infection In Vivo.Cell 170,637-648 e610.

[0186] Hraber,P.,et al.(2017).Panels of HIV-1 Subtype C Env ReferenceStrains for Standardized Neutralization Assessments.J.Virol.91.

[0187] Klein,F.,et al.(2012).HIV therapy by a combination of broadlyneutralizing antibodies in humanized mice.Nature 492,118-122.

[0188] Kreer,C.,et al.(2019).openPrimeR for multiplex amplification ofhighly diverse templates.bioRxiv.https: / / doi.org / 10.1101 / 847574

[0189] Kryazhimskiy,S.,et al.(2014).Microbial evolution.Global epistasismakes adaptation predictable despite sequence-level stochasticity.Science344,1519-1522.

[0190] Mendoza,P.,et al.(2018).Combination therapy with anti-HIV-1antibodies maintains viral suppression.Nature 561,479-484.

[0191] Pietzsch,J.,et al.(2010).Human anti-HIV-neutralizing antibodiesfrequently target a conserved epitope essential for viralfitness.J.Exp.Med.207,1995-2002.

[0192] Sanders,R.W.,et al.(2013).Anext-generation cleaved,soluble HIV-1 Envtrimer,BG505 SOSIP.664 gp140,expresses multiple epitopes for broadlyneutralizing but not non-neutralizing antibodies.PLoS Pathog.9,e1003618.

[0193] Sarzotti-Kelsoe,M.,et al.(2014).Optimization and validation of theTZM-bl assay for standardized assessments of neutralizing antibodies againstHIV-1.J.Immunol.Methods 409,131-146.

[0194] Schoofs,T.,et al.(2016).HIV-1 therapy with monoclonal antibody3BNC117elicits host immune responses against HIV-1.Science 352,997-1001.

[0195] Seaman,M.S.,et al.(2010).Tiered categorization of a diverse panel ofHIV-1Env pseudoviruses for assessment of neutralizing antibodies.J.Virol.84,1439-1452.

[0196] Sliepen,K.,et al.(2015).Engineering and Characterization of aFluorescent Native-Like HIV-1 Envelope Glycoprotein Trimer.Biomolecules 5,2919-2934.

[0197] Tiller,T.,et al.(2008).Efficient generation of monoclonal antibodiesfrom single human B cells by single cell RT-PCR and expression vectorcloning.J.Immunol.Methods 329,112-124.

[0198] von Boehmer,L.,et al.(2016).Sequencing and cloning of antigen-specific antibodies from mouse memory B cells.Nat.Protoc.11,1908-1923.

[0199] Yang,X.,et al..(2000).Characterization of stable,soluble trimerscontaining complete ectodomains of human immunodeficiency virus type 1envelope glycoproteins.J.Virol.74,5716-5725.

[0200] Ye,J.,et al.(2013).IgBLAST:an immunoglobulin variable domain sequenceanalysis tool.Nucleic Acids Res.41,W34-40.

[0201] Yoon,H.,et al.(2015).CATNAP:a tool to compile,analyze and tallyneutralizing antibody panels.Nucleic Acids Res.43,W213-219.

[0202] Zhang,YJ,et al.(2002).Envelope-dependent,cyclophilin-independent effects of glycosaminoglycans on human immunodeficiency virus type 1attachment and infection.J.Virol.76,6332-6343. sequence list <110> University of Cologne <120> Broadly neutralizing antibodies against HIV <130> UK0702P-WO <150> EP19212986.4 <151> 2019-12-02 <160> 80 <170> BiSSAP 1.3.6 <210> 1 <211> 131 <212> PRT <213> Homo sapiens <220> <223> Heavy chains of antibody 1-18 <400> 1 Gln Gly Arg Leu Phe Gln Ser Gly Ala Glu Val Lys Arg Pro Gly Ala 1 5 10 15 Ser Val Arg Ile Ser Cys Arg Ala Asp Asp Asp Pro Tyr Thr Asp Asp 20 25 30 Asp Thr Phe Thr Lys Tyr Trp Thr His Trp Ile Arg Gln Ala Pro Gly 35 40 45 Gln Arg Pro Glu Trp Leu Gly Val Ile Ser Pro His Phe Ala Arg Pro 50 55 60 Ile Tyr Ser Tyr Lys Phe Arg Asp Arg Leu Thr Leu Thr Arg Asp Ser 65 70 75 80 Ser Leu Thr Ala Val Tyr Leu Glu Leu Lys Gly Leu Gln Pro Asp Asp 85 90 95 Ser Gly Ile Tyr Phe Cys Ala Arg Asp Pro Phe Gly Asp Arg Ala Pro 100 105 110 His Tyr Asn Tyr His Met Asp Val Trp Gly Gly Gly Thr Ala Val Ile 115 120 125 Val Ser Ser 130 <210> 2 <211> 108 <212> PRT <213> Homo sapiens <220> <223> Light chain of antibody 1-18 <400> 2 Glu Val Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Asp Arg Val Ile Leu Ser Cys Arg Ala Ser Gln Gly Leu Asp Ser Ser 20 25 30 His Leu Ala Trp Tyr Arg Phe Lys Arg Gly Gln Ile Pro Thr Leu Val 35 40 45 Ile Phe Gly Thr Ser Asn Arg Ala Arg Gly Thr Pro Asp Arg Phe Ser 50 55 60 It should be noted that in the original text, there seems to be a misspelling in the tag " ", which is likely to be " ". This has been corrected in the translation for consistency.Gly Ser Gly Ser Gly Ala Asp Phe Thr Leu Thr Ile Ser Arg Val Glu 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Tyr Gly Gly Thr Pro 85 90 95 Ile Thr Phe Gly Gly Gly Thr Thr Leu Asp Lys Lys 100 105 <210> 3 <211> 131 <212> PRT <213> Homo sapiens <220>[[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​ Thr Gly Ile Tyr Phe Cys Ala Arg Asp Pro Phe Gly Asp Met Tyr Pro 100 105 110 His Tyr Asn Tyr His Met Asp Val Trp Gly Gly Gly Thr Thr Val Ile 115 120 125 Val Ser Ala 130 <210> 4 <211> 108 <212> PRT <213> Homo sapiens <220> <223> Light chain of antibody 1-21 <400> 4 Glu Val Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Asp Arg Val Ile Leu Ser Cys Lys Ala Ser Glu Gly Leu Ser Ser Ser 20 25 30 Asp Leu Ala Trp Tyr Arg Phe Lys Gly Gly Gln Ile Pro Thr Leu Val 35 40 45 Ile Phe Gly Thr Ser Thr Arg Ala Arg Gly Thr Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Val Glu 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Tyr Gly Gly Thr Pro 85 90 95 Ile Thr Phe Gly Gly Gly Thr Thr Leu Asp Lys Lys 100 105 <210> 5 <211> 131 <212> PRT <213> Homo sapiens <220> <223> Heavy chain of antibody 1-33 <400> 5 Gln Gly Arg Leu Phe Gln Ser Gly Thr Glu Val Lys Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Arg Ala Asp Asp Asp Pro Tyr Thr Asp Asp 20 25 30 Asp Thr Phe Thr Lys Tyr Tyr Thr His Trp Ile Arg Gln Ala Pro Gly 35 40 45 Gln Arg Pro Glu Trp Leu Gly Val Ile Ser Pro His Phe Ala Arg Pro 50 55 60 Ile Tyr Ser Tyr Lys Phe Arg Asp Arg Leu Thr Leu Thr Arg Asp Ser 65 70 75 80 Ser Leu Thr Ala Val Tyr Phe Glu Leu Arg Gly Leu Gln Pro Asp Asp 85 90 95 Thr Gly Ile Tyr Phe Cys Ala Arg Asp Pro Phe Gly Asp Met Tyr Pro 100 105 110 His Tyr Asn Tyr His Met Asp Val Trp Gly Gly Gly Thr Thr Val Ile 115 120 125 Val Ser Ala 130 <210> 6 <211> 108 <212> PRT <213> Homo sapiens <220> <223> Light chain of antibody 1-33 <400> 6 Glu Val Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Asp Arg Val Ile Leu Ser Cys Lys Ala Ser Glu Gly Leu Ser Ser Ser 20 25 30 Asp Leu Ala Trp Tyr Arg Phe Lys Gly Gly Gln Ile Pro Thr Leu Val 35 40 45 Ile Phe Gly Thr Ser Asn Arg Ala Arg Gly Thr Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Arg Val Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Tyr Gly Gly Thr Pro 85 90 95 Ile Thr Phe Gly Gly Gly Thr Thr Leu Asp Lys Lys 100 105 <210> 7 <211> 131 <212> PRT <213> Homo sapiens <220> <223> Heavy chain of antibody 1-54 <400> 7 Gln Gly Arg Leu Phe Gln Ser Gly Thr Glu Val Lys Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Arg Ala Asp Asp Asp Pro Tyr Thr Asp Asp 20 25 30 Asp Thr Phe Thr Lys Tyr Tyr Thr His Trp Ile Arg Gln Ala Pro Gly 35 40 45 Gln Arg Pro Glu Trp Leu Gly Val Ile Ser Pro His Phe Ala Arg Pro 50 55 60 Ile Tyr Ser Tyr Lys Phe Gln Asp Arg Leu Thr Leu Thr Arg Asp Ser 65 70 75 80 Ser Leu Thr Ala Val Tyr Phe Glu Leu Arg Asp Leu Gln Ser Asp Asp 85 90 95 Thr Gly Ile Tyr Phe Cys Ala Arg Asp Pro Phe Gly Asp Met Tyr Pro 100 105 110 His Tyr Asn Ser His Met Asp Val Trp Gly Gly Gly Thr Thr Val Ile 115 120 125 Val Ser Ala 130 <210> 8 <211> 108[[ID==42]] <212> PRT <213> Homo sapiens <220> <223> Light chain of antibody 1-54 <400> 8 Glu Val Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Asp Arg Val Ile Leu Ser Cys Lys Ala Ser Glu Gly Leu Ser Ser Ser 20 25 30 Asp Leu Ala Trp Tyr Arg Phe Lys Ser Gly Gln Ile Pro Thr Leu Val 35 40 45 Ile Phe Gly Ala Ser Asn Arg Ala Arg Gly Thr Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Arg Val Glu[[ID=十八]] 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Tyr Gly Gly Thr Pro 85 90 95 Ile Thr Phe Gly Gly Gly Thr Thr Leu Asp Lys Lys 100 105 <210> 9 <211> 131 <212> PRT <213> Homo sapiens <220> <223> Heavy chain of antibody 1-55 <400> 9 Gln Arg Arg Leu Phe Gln Ser Gly Thr Glu Val Lys Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Arg Ala Asp Asp Asp Pro Tyr Thr Asp Asp 20 25 30 Asp Thr Phe Thr Lys Tyr Tyr Thr His Trp Ile Arg Gln Ala Pro Gly 35 40 45 Gln Pro Pro Glu Trp Leu Gly Val Ile Ser Pro His Phe Ala Arg Pro 50 55 60 Ile Tyr Ser Tyr Lys Phe Arg Asp Arg Leu Thr Leu Thr Arg Asp Ser 65 70 75 80 Ser Leu Thr Ala Val Tyr Phe Glu Leu Arg Gly Leu Gln Pro Asp Asp 85 90 95 Thr Gly Ile Tyr Phe Cys Ala Arg Asp Pro Phe Gly Asp Met Tyr Pro 100 105 110 His Tyr Asn Tyr His Met Asp Val Trp Gly Gly Gly Thr Thr Val Ile 115 120 125 Val Ser Ala 130 <210> 10 <211> 108 <212> PRT <213> Homo sapiens <220> <223> Light chain of antibody 1-55 <400> 10 Glu Ala Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Asp Arg Val Ile Leu Ser Cys Lys Ala Ser Glu Gly Leu Ser Ser Ser 20 25 30 Asp Leu Ala Trp Tyr Arg Phe Lys Gly Gly Gln Ile Pro Thr Leu Val 35 40 45 Ile Phe Gly Ala Ser Asn Arg Ala Arg Gly Thr Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Arg Val Glu 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Tyr Gly Gly Thr Pro 85 90 95 Ile Thr Phe Gly Gly Gly Thr Thr Leu Asp Lys Lys 100 105 <210> 11 <211> 131 <212> PRT <213> Homo sapiens <220> <223> Heavy chain of antibody 2-10 <400> 11 Gln Gly Arg Leu Phe Gln Ser Gly Thr Glu Val Lys Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Arg Ala Asp Asp Asp Pro Tyr Thr Asp Asp 20 25 30 Asp Thr Phe Thr Lys Tyr His Thr His Trp Ile Arg Gln Ala Pro Gly 35 40 45 Gln Arg Pro Glu Trp Leu Gly Val Ile Ser Pro His Tyr Ala Arg Pro 50 55 60 Ile Tyr Ser Tyr Lys Phe Gln Asp Arg Leu Thr Leu Thr Arg Asp Ser 65 70 75 80 Ser Leu Thr Ala Val Tyr Phe Glu Leu Arg Gly Leu Gln Pro Asp Asp 85 90 95 Thr Gly Ile Tyr Phe Cys Ala Arg Asp Pro Phe Gly Asn Met Tyr Pro 100 105 110 His Tyr Asn Tyr His Met Asp Val Trp Gly Gly Gly Thr Thr Val Ile 115 120 125 Val Ser Ala 130 <210> 12 <211> 108 <212> PRT <213> Homo sapiens <220> <223> Light chain of antibody 2-10 <400> 12 Glu Val Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Asp Arg Val Ile Leu Ser Cys Lys Ala Ser Glu Gly Leu Ser Ser Ser 20 25 30 Asp Leu Ala Trp Tyr Arg Phe Lys Arg Gly Gln Ile Pro Thr Leu Val 35 40 45 Ile Phe Gly Ala Ser Thr Arg Ala Arg Gly Thr Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Arg Val Glu 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Tyr Gly Gly Thr Pro 85 90 95 Ile Thr Phe Gly Gly Gly Thr Thr Leu Asp Lys Lys 100 105 <210> 13 <211> 131 <212> PRT <213> Homo sapiens <220> <223> Heavy chain of antibody 2-22 <400> 13 Gln Gly Arg Leu Phe Gln Ser Gly Ala Glu Val Lys Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Arg Ala Asp Asp Asp Pro Tyr Thr Asp Asp 20 25 30 Asp Thr Phe Thr Lys Tyr Tyr Thr His Trp Ile Arg Gln Ala Pro Gly 35 40 45 Gln Arg Pro Glu Trp Leu Gly Val Ile Ser Pro His Phe Ala Arg Pro 50 55 60 Ile Tyr Ser Tyr Lys Phe Arg Asp Arg Leu Thr Leu Thr Arg Asp Ser 65 70 75 80 Ser Leu Thr Ala Val Tyr Phe Glu Leu Arg Gly Leu Gln Pro Asp Asp 85 90 95 Thr Gly Ile Tyr Phe Cys Ala Arg Asp Pro Phe Gly Asp Met Tyr Pro 100 105 110 His Tyr Asn Tyr His Met Asp Val Trp Gly Gly Gly Thr Thr Val Ile 115 120 125 Val Ser Ala 130 <210> 14 <211> 108 <212> PRT <213> Homo sapiens <220> <223> Light chain of antibody 2-22 <400> 14 Glu Val Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Asp Arg Val Ile Leu Ser Cys Lys Ala Ser Glu Gly Leu Ser Ser Ser 20 25 30 Asp Leu Ala Trp Tyr Arg Phe Lys Gly Gly Gln Ile Pro Thr Leu Val 35 40 45 Ile Phe Ala Thr Ser Asn Arg Ala Arg Gly Thr Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Lys Val Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Tyr Gly Gly Thr Pro 85 90 95 Ile Thr Phe Gly Gly Gly Thr Thr Leu Asp Lys Lys 100 105 <210> 15 <211> 131 <212> PRT <213> Homo sapiens <220> <223> Heavy chain of antibody 2-27 <400> 15 Gln Gly Arg Leu Phe Gln Ser Gly Thr Glu Val Lys Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Arg Ala Asp Asp Asp Pro Tyr Thr Asp Asp 20 25 30 Asp Thr Phe Thr Lys Tyr Tyr Thr His Trp Ile Arg Gln Ala Pro Gly 35 40 45 Gln Arg Pro Glu Trp Leu Gly Val Ile Ser Pro His Tyr Ala Arg Pro 50 55 60 Ile Tyr Ser Tyr Lys Phe Arg Asp Arg Leu Thr Leu Thr Arg Asp Ser 65 70 75 80 Ser Leu Thr Ala Val Tyr Phe Glu Leu Arg Gly Leu Gln Pro Asp Asp 85 90 95 Thr Gly Ile Tyr Phe Cys Ala Arg Asp Pro Phe Gly Asp Met Tyr Pro 100 105 110 His Tyr Asn Tyr His Met Asp Val Trp Gly Gly Gly Thr Thr Val Ile 115 120 125 Val Ser Ala 130 <210> 16 <211> 108 <212> PRT <213> Homo sapiens <220> <223> Light chain of antibody 2-27 <400> 16 Glu Val Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Asp Arg Val Ile Leu Ser Cys Lys Ala Ser Glu Gly Leu Ser Ser Ser 20 25 30 Asp Leu Ala Trp Tyr Arg Phe Lys Gly Gly Gln Ile Pro Thr Leu Val 35 40 45 Ile Phe Gly Ala Ser Asn Arg Ala Arg Gly Thr Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Arg Val Glu 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Tyr Gly Gly Thr Pro 85 90 95 Ile Thr Phe Gly Gly Gly Thr Thr Leu Asp Lys Lys 100 105 <210> 17 <211> 131 <212> PRT <213> Homo sapiens <220> <223> Heavy chain of antibody 2-47 <{400}> 17 Gln Arg Arg Leu Phe Gln Ser Gly Thr Glu Val Lys Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Arg Ala Asp Asp Asp Pro Tyr Thr Asp Asp 20 25 30 Asp Thr Phe Thr Lys Tyr Tyr Thr His Trp Ile Arg Gln Ala Pro Gly 35 40 45 Gln Arg Pro Glu Trp Leu Gly Val Ile Ser Pro His Tyr Ala Arg Pro 50 55 60 Ile Tyr Ser Tyr Lys Phe Gln Asp Arg Leu Thr Leu Thr Arg Asp Ser 65 70 75 80 Ser Leu Thr Ala Val Tyr Phe Glu Leu Arg Gly Leu Gln Pro Asp Asp 85 90 95 Thr Gly Ile Tyr Phe Cys Ala Arg Asp Pro Phe Gly Asp Met Tyr Pro 100 105 110 His Tyr Asn Tyr His Met Asp Val Trp Gly Gly Gly Thr Thr Leu Ile 115 120 125 Val Ser Ala 130 <210> 18 <211> 108 <212> PRT <213> Homo sapiens <220> <223> Light chain of antibody 2-47 <400> 18 Glu Val Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly (1)(5)(10)(15) Asp Arg Val Ile Leu Ser Cys Lys Ala Ser Glu Gly Leu Ser Ser Ser (20)(25)(30) Asp Leu Ala Trp Tyr Arg Phe Lys Ser Gly Gln Ile Pro Thr Leu Val (35)(40)(45) Ile Phe Gly Ala Ser Asn Arg Ala Arg Gly Thr Pro Asp Arg Phe Ala (50)(55)(60) Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Arg Val Glu<{} (65)(70)(75)(80) Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Tyr Gly Gly Thr Pro (85)(90)(95) Ile Thr Phe Gly Gly Gly Thr Thr Leu Asp Lys Lys (100)(105) <210> 19 <211> 131 <212> PRT <213> Homo sapiens<{} <220> <223> Heavy chain of antibody 3-59<{} <400> 19 Gln Arg Arg Leu Phe Gln Ser Gly Thr Glu Val Lys Arg Pro Gly Ala (1)(5)(10)(15) Ser Val Arg Ile Ser Cys Arg Ala Asp Asp Asp Pro Tyr Thr Asp Asp 20 25 30 Asp Thr Phe Thr Lys Tyr Trp Thr His Trp Ile Arg Gln Ala Pro Gly 35 40 45 Gln Arg Pro Glu Trp Leu Gly Val Ile Ser Pro His Phe Ala Arg Pro 50 55 60 Ile Tyr Ser Tyr Lys Phe Gln Asp Arg Leu Thr Leu Thr Arg Asp Ser 65 70 75 80 Ser Leu Thr Gly Val Tyr Leu Glu Leu Lys Gly Leu Gln Leu Asp Asp 85 90 95 Ser Gly Ile Tyr Phe Cys Ala Arg Asp Pro Phe Gly Asp Arg Ala Pro 100 105 110 His Tyr Asn Tyr His Met Asp Val Trp Gly Gly Gly Thr Ala Val Ile 115 120 125 Val Ser Ser 130 <210> 20 <211> 108 <212> PRT <213> Homo sapiens <220> <223> Light chain of antibody 3-59 <400> 20 Glu Val Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Asp Arg Val Ser Phe Ser Cys Arg Ala Ser Glu Gly Leu Asp Thr Ser 20 25 30 Gln Leu Ala Trp Tyr Arg Phe Lys Arg Gly Gln Ile Pro Thr Leu Val 35 40 45 Ile Phe Ala Thr Ser Asn Arg Ala Arg Gly Thr Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Ala Asp Phe Thr Leu Thr Ile Asn Arg Val Glu 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Tyr Gly Ala Thr Pro 85 90 95 Ile Thr Phe Gly Gly Gly Thr Thr Leu Asp Lys Lys 100 105 <210> 21 <211> 131 <212> PRT <213> Homo sapiens <220> <223> Heavy chain of antibody 5-18 <400> 21 Gln Arg Arg Leu Phe Gln Ser Gly Thr Glu Val Lys Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Arg Ala Asp Asp Asp Pro Tyr Thr Asp Asp 20 25 30 Asp Thr Phe Thr Lys Tyr Tyr Thr His Trp Ile Arg Gln Ala Pro Gly 35 40 45 Gln Arg Pro Glu Trp Leu Gly Val Ile Ser Pro His Phe Ala Arg Pro 50 55 60 Ile Tyr Ser Tyr Lys Phe Gln Asp Arg Leu Thr Leu Thr Arg Asp Ser 65 70 75 80 Ser Leu Thr Ala Val Tyr Phe Glu Leu Arg Gly Leu Gln Pro Asp Asp 85 90 95 Thr Gly Ile Tyr Phe Cys Ala Arg Asp Pro Phe Gly Asp Met Tyr Pro 100 105 110 His Tyr Asn Tyr His Met Asp Val Trp Gly Gly Gly Thr Thr Val Ile 115 120 125 Val Ser Ala 130 <210> 22 <211> 108 <212> PRT <213> Homo sapiens <220> <223> Light chain of antibody 5-18 <400> 22 Glu Val Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Asp Arg Val Ile Leu Ser Cys Lys Ala Ser Glu Gly Leu Ser Ser Ser 20 25 30 Asp Leu Ala Trp Tyr Arg Phe Lys Gly Gly Gln Ile Pro Thr Leu Val 35 40 45 Ile Phe Gly Thr Ser Thr Arg Ala Arg Gly Thr Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Val Glu 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Tyr Gly Gly Thr Pro 85 90 95 Ile Thr Phe Gly Gly Gly Thr Thr Leu Asp Lys Lys 100 105 <210> 23 <211> 131 <212> PRT <213> Homo sapiens <220> <223> Heavy chain of antibody 8-10 <400> 23 Gln Gly Arg Leu Phe Gln Ser Gly Ala Glu Val Lys Arg Pro Gly Ala 1 5 10 15 Ser Val Arg Ile Ser Cys Arg Ala Asp Asp Asp Pro Tyr Thr Asp Asp 20 25 30 Asp Thr Phe Thr Lys Tyr Trp Thr His Trp Ile Arg Gln Ala Pro Gly 35 40 45 Gln Arg Pro Glu Trp Leu Gly Val Ile Ser Pro His Phe Ala Arg Pro 50 55 60 Ile Tyr Ser Tyr Lys Phe Arg Asp Arg Leu Thr Leu Thr Arg Asp Ser 65 70 75 80 Ser Leu Thr Ala Val Tyr Leu Glu Leu Lys Gly Leu Gln Pro Asp Asp 85 90 95 Ser Gly Ile Tyr Phe Cys Ala Arg Asp Pro Phe Gly Asp Arg Ala Pro 100 105 110 His Tyr Asn Tyr His Met Asp Val Trp Gly Gly Gly Thr Ala Val Ile 115 120 125 Val Ser Ser 130 <210> 24 <211> 108 <212> PRT <213> Homo sapiens <220> <223> Light chain of antibody 8-10 <400> 24 Glu Val Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Asp Arg Val Ile Leu Ser Cys Arg Ala Ser Gln Gly Leu Asp Ser Ser 20 25 30 His Leu Ala Trp Tyr Arg Phe Lys Arg Gly Gln Ile Pro Thr Leu Val 35 40 45 Ile Phe Gly Thr Ser Asn Arg Ala Arg Gly Thr Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Ala Asp Phe Thr Leu Thr Ile Ser Arg Val Glu 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Tyr Gly Gly Thr Pro 85 90 95 Ile Thr Phe Gly Gly Gly Thr Thr Leu Asp Lys Lys 100 105 <210> 25 <211> 131 <212> PRT <213> Homo sapiens <220> <223> Heavy chain of antibody 9-23 <400> 25 Gln Ala His Leu Phe Gln Ser Gly Ala Glu Leu Lys Arg Pro Gly Ala 1 5 10 15 Ser Val Arg Ile Ser Cys Arg Ala Asp Asp Asp Pro Tyr Thr Asp Asp 20 25 30 Asp Thr Phe Thr Lys Tyr Tyr Thr His Trp Ile Arg Gln Ala Pro Gly 35 40 45 Gln Arg Pro Glu Trp Leu Gly Val Ile Ser Pro His Phe Ala Arg Pro 50 55 60 Ile Tyr Ser Tyr Lys Phe Gln Asp Arg Leu Thr Leu Thr Arg Asp Ser 65 70 75 80 Ser Leu Thr Ala Val Tyr Leu Glu Leu Arg Ser Leu Arg Leu Asp Asp 85 90 95 Thr Gly Ile Tyr Tyr Cys Ala Arg Asp Pro Phe Gly Glu Arg Ala Pro 100 105 110 His Tyr Asn Tyr His Met Asp Val Trp Gly Ala Gly Thr Thr Val Ile 115 120 125 Val Ser Ser 130 <210> 26 <211> 108 <212> PRT <213> Homo sapiens <220> [[ID=二十]]<223> Light chain of antibody 9-23 <400> 26 Glu Val Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly 1 5 10 15 Asp Arg Val Val Leu Ser Cys Arg Ala Ser Glu Gly Leu Asp Ser Ser 20 25 30 Gln Leu Ala Trp Tyr Arg Phe Lys Asp Gly Gln Ile Pro Arg Leu Val 35 40 45 Leu Phe Gly Val Ser Asn Arg Ala Arg Gly Thr Pro Asp Arg Phe Ser 50 55 60 Gly Gly Gly Ser Gly Ala Asp Phe Thr Leu Thr Ile Ser Arg Val Glu 65 70 75 80 Arg Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Phe Gly Ala Thr Pro 85 90 95 Ile Thr Phe Gly Gly Gly Thr Arg Leu Asp Met Asn 100 105 <210> 27 <211> 131 <212> PRT <213> Homo sapiens <220> <223> Heavy chain of antibody 10-7 <400> 27 Gln Gly Arg Phe Phe Gln Ser Gly Ala Glu Val Lys Arg Pro Gly Ala 1 5 10 15 Ser Val Arg Ile Ser Cys Arg Ala Asp Asp Asp Pro Tyr Thr Asp Asp 20 25 30 Asp Thr Phe Thr Lys Tyr Trp Thr His Trp Ile Arg Gln Ala Pro Gly 35 40 45 Gln Arg Pro Glu Trp Leu Gly Val Ile Ser Pro His Phe Ala Arg Pro 50 55 60 Ile Tyr Ser Tyr Lys Phe Arg Asp Arg Leu Thr Leu Thr Arg Asp Ser 65 70 75 80 Ser Leu Thr Ala Val Tyr Leu Glu Leu Gln Gly Leu Gln Pro Asp Asp 85 90 95 Ser Gly Ile Tyr Tyr Cys Ala Arg Asp Pro Phe Gly Asp Arg Ala Pro 100 10​​​​​​​​​​​​ <212> PRT <213> Homo sapiens <220> <223> antibody 10⁻⁷ light chain <400> 28 Glu Val Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Asp Arg Val Ile Leu Ser Cys Arg Ala Ser Glu Gly Leu Asp Pro Thr 20 25 30 His Leu Ala Trp Tyr Arg Phe Lys Arg Gly Gln Ile Pro Thr Leu Val 35 40 45 Ile Phe Gly Thr Ser Asn Arg Ala Arg Gly Thr Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Glu Ala Asp Phe Thr Leu Thr Ile Thr Arg Val Glu 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Tyr Gly Gly Thr Pro 85 90 95 Ile Thr Phe Gly Gly Gly Thr Thr Leu Asp Lys Lys 100 105 <210> 29 <211> 123 <212> PRT <213> Homo sapiens <220> <223> Heavy chain of antibody 8-52 <400> 29 Gln Leu Val Gln Trp Gly Gly Gly Val Lys Arg Pro Gly Ala Ser Val 1 5 10 15 Arg Ile Ser Cys Gln Cys Pro Glu Asp Thr Phe Thr Lys Tyr Tyr Ile 20 25 30 His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Leu Gly Met 35 40 45 Val Ser Pro His Gly Gly Arg Pro Phe His Thr Ser Glu Phe Arg Asp 50 55 60 Arg Leu Thr Met Thr Arg Asp Ile His Glu Thr Thr His His Met Val 65 70 75 80 Leu Ser Gly Leu Gly Val Ala Asp Ser Gly Thr Tyr Phe Cys Ala Arg 85 90 95 Asp Pro Leu Gly Glu Lys Ser Pro Ala Tyr Ser His His Met Asp Val 100 105 110 Trp Gly Gly Gly Ala Thr Val Ile Val Ser Ser 115 120 <210> 30 <211> 111 <212> PRT <213> Homo sapiens <220> <223> Light chain of antibody 8-52 <400> 30 Ala Val Val Leu Thr Gln Ser Pro Val Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Thr Ala Ile Leu Ser Cys Arg Ala Ser His Gly Leu Asp Thr Arg 20 25 30 His Val Thr Trp Phe Gln Gln Lys Arg Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Phe Ala Thr Tyr Arg Arg Ala Ser Gly Val Ser Asp Arg Phe Arg 50 55 60 Ala Thr Asp Ser Gly Ser Ala Thr Asp Phe Asn Leu Thr Ile Thr Ala 65 70 75 80 Val Glu Pro Ala Asp Phe Ala Thr Tyr Phe Cys Gln Thr Tyr Gly Ala 85 90 95 Ile Thr Pro Ile Thr Phe Gly Gly Gly Thr Lys Val Asp Leu Lys 100 105 110 <210> 31 <211> 123 <212> PRT <213> Homo sapiens <220> <223> Heavy chain of antibody 9-89 <400> 31 Gln Leu Val Gln Trp Gly Gly Gly Val Lys Arg Pro Gly Ala Ser Val 1 5 10 15 Arg Ile Ser Cys Gln Cys Pro Glu Asp Thr Phe Thr Lys Tyr Tyr Ile 20 25 30 His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Leu Gly Met 35 40 45 Val Ser Pro His Gly Gly Arg Pro Phe His Thr Ser Glu Phe Arg Asp 50 55 60 Arg Leu Thr Met Thr Arg Glu Ile His Glu Thr Thr His His Met Val 65 70 75 80 Leu Ser Gly Leu Gly Val Gly Asp Ser Gly Thr Tyr Phe Cys Ala Arg 85 90 95 Asp Pro Leu Gly Glu Lys Ser Pro Ala Tyr Ser His His Met Asp Val[[ID=X17]] 100 105 110 Trp Gly Gly Gly Ala Thr Val Ile Val Ser Ser 115 120 <210> 32 <211> 111 <212> PRT <213> Homo sapiens <220> <223> Light chain of antibody 9-89 <400> 32 Ala Val Val Leu Thr Gln Ser Pro Val Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Ser Ala Val Leu Ser Cys Arg Ala Ser His Gly Leu Asp Thr Arg 20 25 30 His Val Thr Trp Phe Gln Gln Lys Arg Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Phe Ala Thr Ser Arg Arg Ala Ser Gly Val Ser Asp Arg Phe Arg 50 55 60 Ala Thr Asp Gly Gly Ser Ala Thr Asp Phe Asn Leu Thr Ile Thr Ala 65 70 75 80 Val Glu Pro Ala Asp Phe Ala Thr Tyr Tyr Cys Gln Thr Tyr Gly Ala 85 90 95 Ile Thr Pro Ile Thr Phe Gly Gly Gly Thr Lys Leu Asp Leu Lys 100 105 110 <210> 33 <211> 126 <212> PRT <213> Homo sapiens <220> <223> Heavy chain of antibody 9-71 <400> 33 Gln Leu Ala Gln Ser Gly Gly Gly Val Lys Lys Pro Gly Ala Ser Val 1 5 10 15 Lys Ile Ser Cys Val Thr Pro Glu Ser Thr Phe Thr Lys Tyr Trp Leu 20 25 30 His Trp Val Arg Gln Ala Pro Gly Gln Gly Phe Glu Trp Leu Gly Val 35 40 (此处原文“45”有误,推测为“41”,若有误请按正确原文修改)41 Val Ser Pro His Gly Gly Arg Pro Met Phe Ala Asn Lys Phe Arg Asp 50 55 60 Arg Leu Thr Leu Thr Arg Asp Ile His Thr Thr Thr His Tyr Met Glu 65 70 75 80 Leu Arg Gly Leu Thr Ser Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Arg 85 90 95 Asp Ser Phe Gly Glu Thr Phe Arg His Ser Gly Asp Gln Pro Tyr Gln 100 105 110 Met Asp Val Trp Gly Gly Gly Thr Asn Ile Val Val Ser Ser 115 12​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Ser Tyr Gly Ser Ile Thr 85 90 95 Pro Leu Ile Phe Gly Gly Gly Thr Arg Val Asp Val Lys 100 105 <210> 35 <211> 125 <212> PRT <213> Homo sapiens <220> <223> Heavy chain of antibody 1-23 <400> 35 Gln Leu Val Gln Ser Gly Gly Gly Val Arg Arg Pro Gly Ala Ser Val 1 5 10 15 Lys Ile Ser Cys Glu Thr Pro Glu Tyr Thr Phe Thr Lys Tyr Trp Leu 20 25 30 His Trp Leu Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Met Gly Val 35 40 45 Val Ser Pro His Gly Gly Arg Pro Met Phe Ala Phe Glu Phe Arg Asp 50 55 600001367Arg Leu Thr Leu Thr Arg Asp Ile His Thr Thr Thr His Tyr Met Glu 65 70 75 80 Leu Arg Gly Leu Thr Ser Asp Asp Thr Ala Val Tyr Phe Cys Ala Arg 85 90 95 Asp Ser Phe Gly Glu Thr Phe Arg Gly His Asp Gln Pro Tyr Gln Met 100 105 110 Asp Val Trp Gly Gly Gly Thr Thr Val Val Val Ser Ser 115 120 125 <210> 36 <211> 109 <212> PRT <213> Homo sapiens <220> <223> Light chain of antibody 1-23 <400> 36 Glu Pro Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Gly Ala Thr Leu Ser Cys Arg Ala Arg Gln Gly Phe Ser Ala Asp 20 25 30 His Val Ala Trp Phe Gln Lys Lys Pro Gly Arg Pro Pro Arg Leu Leu 35 40 45 Ile Phe Glu Ala Ser Arg Arg Ala Ser Gly Thr Pro Glu Arg Phe Ser 50 55 60[[ID=3?]] Gly Gly Gly Ser Gly Pro Glu Tyr Thr Leu Thr Ile Thr Arg Val Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Ser Tyr Gly Ser Ile Thr 85 90 95 Pro Leu Val Phe Gly Gly Gly Thr Arg Val Asp Val Lys 100 105 It should be noted that there seems to be a formatting issue with the "3?" in the ID=37 tag in the original text. It's likely a typo, but I've left it as is in the translation for consistency with the original. <210> 37 <211> 125 <212> PRT <213> Homo sapiens <220> <223> Heavy chain of antibody 1-29 <400> 37 Gln Leu Val Gln Ser Gly Gly Gly Val Arg Arg Pro Gly Ala Ser Val 1 5 10 15 Lys Val Ser Cys Glu Thr Pro Glu Tyr Thr Phe Thr Lys Tyr Trp Leu 20 25 30 His Trp Leu Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Met Gly Val 35 40 45 Val Ser Pro His Gly Gly Arg Pro Met Phe Ala Phe Glu Phe Arg Asp 50 55 60 Arg Leu Thr Leu Thr Arg Asp Ile His Thr Thr Thr His Tyr Met Glu 65 70 75 80 Leu Arg Gly Leu Thr Ser Asp Asp Thr Ala Val Tyr Phe Cys Ala Arg 85 90 95 Asp Ser Phe Gly Glu Thr Phe Arg Gly His Asp Gln Pro Tyr Gln Met 100 105 110 Asp Leu Trp Gly Gly Gly Thr Thr Val Val Val Ser Ser 115 120 125 <210> 38 <211> 109 <212> PRT <213> Homo sapiens <220> <223> Light chain of antibody 1-29 <400> 38 Glu Pro Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Gly Val Thr Leu Ser Cys Arg Ala Arg Gln Gly Phe Ser Ala Asp 20 25 30 His Val Ala Trp Phe Gln Lys Lys Pro Gly Arg Pro Pro Arg Leu Leu 35 40 45 Ile Phe Glu Thr Ser Arg Arg Ala Ser Gly Thr Pro Glu Arg Phe Ser 50 55 60 Gly Gly Gly Ser Gly Pro Glu Tyr Thr Leu Thr Ile Thr Arg Val Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Ser Tyr Gly Ser Ile Thr 85 90 95 Pro Leu Val Phe Gly Gly Gly Thr Arg Val Asp Val Lys 100 105 <210> 39 <211> 125 <212> PRT <213> Homo sapiens <220> <223> Heavy chain of antibody 2-12 <400> 39 Gln Leu Val Gln Ser Gly Gly Gly Val Arg Arg Pro Gly Ala Ser Val 1 5 10 15 Lys Ile Ser Cys Glu Thr Pro Glu Asp Thr Phe Thr Lys Tyr Trp Leu 20 25 30 His Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Val 35 40 45 Val Ser Pro His Gly Gly Arg Pro Met Phe Ala Phe Glu Phe Arg Asp 50 55 60 Arg Leu Thr Leu Thr Arg Asp Ile His Thr Thr Thr His Phe Met Glu 65 70 75 80 Leu Arg Gly Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85 90 95 Asp Pro Phe Gly Glu Thr Phe Arg Gly His Asp Gln Pro Tyr Arg Met 100 105 110 Asp Val Trp Gly Gly Gly Thr Thr Ile Val Val Ser Ser 115 120 125 <210> 40 <211> 109 <212> PRT <213> Homo sapiens <220> <223> Light chain of antibody 2-12 <400> 40 Glu Pro Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Gly Ala Thr Leu Ser Cys Arg Ala Arg Gln Gly Phe Ser Ala Asp 20 25 30 His Val Ala Trp Phe Gln Lys Lys Pro Gly Arg Pro Pro Arg Leu Leu 35 40 45 Ile Phe Glu Ala Ser Arg Arg Ala Ser Gly Thr Pro Glu Arg Phe Ser 50 55 60 [[ID=ll]]Gly Ser Gly Ser Gly Pro Glu Tyr Thr Leu Thr Ile Thr Arg Val Glu 65 70 75 80 Ala Glu Asp Phe Ala Val Tyr Tyr Cys Gln Ser Tyr Gly Ser Ile Thr 85 90 95 Pro Leu Val Phe Gly Gly Gly Thr Arg Val Asp Val Lys 100 105 <210> 41 <211> 125 <212> PRT <213> Homo sapiens <220> <223> Heavy chain of antibody 2-21 <400> 41 Gln Leu Val Gln Ser Gly Gly Gly Val Lys Arg Pro Gly Ala Ser Val 1 5 10 15 Lys Ile Ser Cys Glu Thr Pro Glu Tyr Thr Phe Thr Lys Tyr Trp Leu 20 25 30 His Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Val 35 40 45 Val Ser Pro His Gly Gly Arg Pro Met Phe Ala Phe Glu Phe Arg Asp 50 55 60 Arg Leu Thr Leu Thr Arg Asp Ile His Thr Thr Thr His Tyr Met Glu 65 70 75 80 Leu Gly Gly Leu Thr Leu Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85 90 95 Asp Pro Phe Gly Glu Thr Phe Arg Gly Arg Glu Gln Pro Tyr Gln Met 100 105 110 Asp Val Trp Gly Gly Gly Thr Thr Ile Val Val Thr Ser 115 120 125 <210> 42 <211> 109 <212> PRT <213> Homo sapiens <220> <223> Light chain of antibody 2-21 <400> 42 Glu Ala Leu Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Gly Ala Thr Leu Ser Cys Arg Ala Arg Gln Gly Phe Ser Ala Asp 20 25 30 His Val Ala Trp Phe Gln Lys Lys Pro Gly Arg Pro Pro Arg Leu Leu 35 40 45 Ile Phe Glu Ala Ser Arg Arg Ala Ser Gly Thr Pro Glu Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Pro Glu Tyr Thr Leu Thr Ile Thr Arg Val Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Ser Tyr Gly Ser Ile Thr 85 90 95 Pro Leu Val Phe Gly Gly Gly Thr Arg Val Asp Val Lys 100 105 <210> 43 <211> 125 <212> PRT <213> Homo sapiens <220> <223> Heavy chain of antibody 3-07 <400> 43 Gln Leu Val Gln Ser Gly Gly Gly Val Arg Arg Pro Gly Ala Ser Val 1 5 10 15 Lys Ile Ser Cys Glu Thr Pro Glu Asp Thr Phe Thr Lys Tyr Trp Leu 20 25 30 His Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Val 35 40 45 Val Ser Pro His Gly Gly Arg Pro Met Phe Ala Phe Glu Phe Arg Asp 50 55 60 Arg Leu Thr Leu Thr Arg Asp Ile His Thr Thr Thr His Phe Met Glu 65 70 75 80 Leu Gly Gly Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85 90 95 Asp Pro Phe Gly Glu Thr Phe Arg Gly His Asp Gln Pro Tyr Arg Met 100 105 110 Asp Val Trp Gly Gly Gly Thr Thr Ile Val Val Ser Ser 115 120 125 <210> 44 <211> 109 <212> PRT <213> Homo sapiens <220> <223> Light chain of antibody 3-07 <400> 44 Glu Pro Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Gly Ala Thr Leu Ser Cys Arg Ala Arg Gln Gly Phe Ser Ala Asp 20 25 30 His Val Ala Trp Phe Gln Lys Lys Pro Gly Arg Pro Pro Arg Leu Leu 35 40 45 Ile Phe Glu Ala Ser Arg Arg Ala Ser Gly Thr Pro Glu Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Pro Glu Tyr Thr Leu Thr Ile Thr Arg Val Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Ser Tyr Gly Ser Ile Thr 85 90 95 Pro Leu Val Phe Gly Gly Gly Thr Arg Val Asp Val Lys 100 105 <210> 45 <211> 125 <212> PRT <213> Homo sapiens ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ 115 120 125 <210> 46 <211> 109 <212> PRT <213> Homo sapiens <220> <223> Light chain of antibody 3-78 <400> 46 Glu Pro Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Arg Gln Gly Phe Ser Ala Asp 20 25 30 His Val Ala Trp Phe Gln Lys Lys Pro Gly Arg Pro Pro Arg Leu Leu 35 40 45 Ile Phe Glu Ala Ser Arg Arg Ala Ser Gly Thr Pro Glu Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Pro Glu Tyr Thr Leu Thr Ile Thr Arg Val Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Ser Tyr Gly Ser Ile Thr 85 90 95 Pro Leu Val Phe Gly Gly Gly Thr Arg Val Asp Val Lys 100 105 <210> 47 <211> 131 <212> PRT <213> Homo sapiens <220> <221> SITE <222> 2, 3, 4, 9, 11, 19, 39, 50, 61, 71, 84, 87, 90, 91, 93, 94, 97, 101, 109, 110, 111, 116, 123, 126, 127, 131 <223> Xaa can represent any amino acid or no amino acid <400> 47 Gln Xaa Xaa Xaa Phe Gln Ser Gly Xaa Glu Xaa Lys Arg Pro Gly Ala 1 5 10 15 Ser Val Xaa Ile Ser Cys Arg Ala Asp Asp Asp Pro Tyr Thr Asp Asp 20 25 30 Asp Thr Phe Thr Lys Tyr Xaa Thr His Trp Ile Arg Gln Ala Pro Gly 35 40 45 Gln Xaa Pro Glu Trp Leu Gly Val Ile Ser Pro His Xaa Ala Arg Pro 50 55 60 Ile Tyr Ser Tyr Lys Phe Xaa Asp Arg Leu Thr Leu Thr Arg Asp Ser 65 70 75 80 Ser Leu Thr Xaa Val Tyr Xaa Glu Leu Xaa Xaa Leu Xaa Xaa Asp Asp 85 90 95 Xaa Gly Ile Tyr Xaa Cys Ala Arg Asp Pro Phe Gly Xaa Xaa Xaa Pro 100 105 110 His Tyr Asn Xaa His Met Asp Val Trp Gly Xaa Gly Thr Xaa Xaa Ile 115 120 125 Val Ser Xaa 130 <210> 48 <211> 108 <212> PRT <213> Homo sapiens <220> <221> SITE <222> 2, 13, 20, 21, 24, 30, 31, 32, 33, 41, 46, 51, 52, 54, 64, 66, 69, 70, 77, 78, 81, 92, 94, 104, 107, 108 <223> Xaa can represent any amino acid or no amino acid <220> <221> SITE <222> 49 [[ID=2(]]<223> Xaa can be Leu or Ile <400> 48 Glu Xaa Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Xaa Ser Pro Gly 1 5 10 15 Asp Arg Val Xaa Xaa Ser Cys Xaa Ala Ser Glx Gly Leu Xaa Xaa Xaa 20 25 30 Xaa Leu Ala Trp Tyr Arg Phe Lys Xaa Gly Gln Ile Pro Xaa Leu Val 35 40 45 Xaa Phe Xaa Xaa Ser Xaa Arg Ala Arg Gly Thr Pro Asp Arg Phe Xaa[[ID=]] 50 55 60 Gly Xaa Gly Ser Xaa Xaa Asp Phe Thr Leu Thr Ile Xaa Xaa Val Glx 65 70 75 80 Xaa Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Xaa Gly Xaa Thr Pro It should be noted that there is an error in the original text where "[[ID=2(]]" should be corrected to "" for accurate translation.85 90 95 Ile Thr Phe Gly Gly Gly Thr Xaa Leu Asp Xaa Xaa 100 105 <210> 49 <211> 126 <212> PRT <213> Homo sapiens <220> <221> SITE <222> 3, 5, 10, 11, 17, 18, 21, 22, 25, 31, 35, 41, 43, 46, 48, 57, 58, 59, 60, 61, 68, 71, 74, 78, 80, 82, 85, 86, 87, 89, 90, 91, 93, 98, 99, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 115, 120, 121, 122, 123, 125 <223> Xaa can represent any amino acid or no amino acid <220> <221> SITE <222> 32 <223> Xaa can be Leu or Ile <400> 49 Gln Leu Xaa Gln Xaa Gly Gly Gly Val Xaa Xaa Pro Gly Ala Ser Val 1 5 10 15 Xaa Xaa Ser Cys Xaa Xaa Pro Glu Xaa Thr Phe Thr Lys Tyr Xaa Xaa 20 25 30 His Trp Xaa Arg Gln Ala Pro Gly Xaa Gly Xaa Glu Trp Xaa Gly Xaa 35 40 45 Val Ser Pro His Gly Gly Arg Pro Xaa Xaa Xaa Xaa Xaa Phe Arg Asp 50 55 60 Arg Leu Thr Xaa Thr Arg Xaa Ile His Xaa Thr Thr His Xaa Met Xaa 65 70 75 80 Leu Xaa Gly Leu Xaa Xaa Xaa Asp Xaa Xaa Xaa Tyr Xaa Cys Ala Arg 85 90 95 Asp Xaa Xaa Gly Glu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Met Asp Xaa Trp Gly Gly Gly Xaa Xaa Xaa Xaa Val Xaa Ser 115 120 125 <210> 50 <211> 111 <212> PRT <213> Homo sapiens <220> <221> SITE <222> 1, 2, 3, 9, 13, 18, 19, 20, 26, 27, 29, 30, 31, 32, 34, 35, 39, 40, 41, 43, 44, 51, 52, 53, 57, 59, 60, 61, 64, 65, 66, 67, 68, 69, 71, 72, 73, 74, 75, 79, 80, 83, 84, 88, 90, 93, 96, 101, 107, 108, 110 <223> Xaa can represent any amino acid or no amino acid <220> <221> SITE <222> 100 <223> Xaa can be Leu or Ile <400> 50 Thank You Thank You Thr Gln Ser Pro Thank You Thr Leu Ser Thr Gln Ser Pro Gly 1 5 10 15 Glu Zhang Zhang Leu Ser Cys Arg Ala Zhang Gly Zhang Zhang Y 20 25 30 His No. Trp Phe Gln No. Gly No. Pro Arg Leu Leu 35 40 45 Ile Phe More Thanks Arg Arg Ala Thanks Gly Thanks Arg Phe Thanks 50 55 60 Yes Yes Yes Yes Yes Ser Yes Yes Yes Leu Thr Island Yes 65 70 75 80 Val Glu Mole Asp Phe Ala Mole Tyr Mole Cys Gln Mole Tyr Gly Mole 85 90 95 Thr Pro Module Phe Gly Gly Gly Thr Module Asp Module Lys 100 105 110 <210> 51 <211> 11 <212> PRT <213> Personal Information Photo1 <220> <223> HIV‐1 gp120 (YU2 protein) protein (aa 274–284) <400> 51 Ser Glu Asn Phe Thr Asn Asn Ala Lys Thr Ile 1 5 10 <210> 52 <211> 14 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> The β23 / V5 loop (aa 458-471) of HIV-1 gp120 (YU2 wild-type sequence) <400> 52 Gly Gly Lys Asp Thr Asn Gly Thr Glu Ile Phe Arg Pro Gly 1 5 10 <210> 53 <211> 6 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> V1 loop (aa 159-164) of HIV-1 gp120 (YU2 wild-type sequence) <400> 53 Phe Asn Ile Thr Thr Ser 1 5 <210> 54 <211> 19 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> V3 loop (aa 299-321) of HIV-1 gp120 (YU2 wild-type sequence) <400> 54 Asn Asn Thr Arg Lys Ser Ile Asn Ile Gly Pro Gly Arg Ala Leu Tyr 1 5 10 15 Thr Thr Gly <210> 55 <211> 13 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> The CD4-binding loop (aa 363-375) of HIV-1 gp120 (YU2 wild-type sequence) <400> 55 Pro Ser Ser Gly Gly Asp Pro Glu Ile Val Thr His Ser 1 5 10 <210> 56 <211> 9 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> The β20 / β21 loop (aa 425-433) of HIV-1 gp120 (YU2 wild-type sequence) <400> 56 Asn Met Trp Gln Glu Val Gly Lys Ala 1 5 <210> 57 <211> 11 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> T278I / N279K mutant from mouse 1021 with loop D <400> 57 Ser Glu Asn Phe Ile Lys Asn Ala Lys Thr Ile 1 5 10 <210> 58 <211> 11 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> A281T mutant of loop D from mice 1021 and 1186 <400> 58 Ser Glu Asn Phe Thr Asn Asn Thr Lys Thr Ile 1 5 10 <210> 59 <211> 11 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> A281D mutant of loop D from mice 1021, 1211 and 1087 <400> 59 Ser Glu Asn Phe Thr Asn Asn Asp Lys Thr Ile 1 5 10 <210> 60 <211> 12 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> The G459D / T462del / N463del / G471E mutant from the β23 / V5 loop of mouse 1021 <400> 60 Gly Asp Lys Asp Gly Thr Glu Ile Phe Arg Pro Glu 1 5 10 <210> 61 <211> 14 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> G459D / G471E mutant from the β23 / V5 loop of mouse 1021 <400> 61 Gly Asp Lys Asp Thr Asn Gly Thr Glu Ile Phe Arg Pro Glu 1 5 10 <210> 62 <211> 11 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> K282E mutants of loop D from mice 1135 and 1140 <400> 62 Ser Glu Asn Phe Thr Asn Asn Ala Glu Thr Ile 1 5 10 <210> 63 <211> 14 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> G459D / G464E mutants from the β23 / V5 loop of mice 1135, 1140, and 940 <400> 63 Gly Asp Lys Asp Thr Asn Glu Thr Glu Ile Phe Arg Pro Gly 1 5 10 <210> 64 <211> 14 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> G459D mutants of the β23 / V5 loop from mouse numbers 1140, 1130, 940, 1087, 1186, 1214, and 1069. <400> 64 Gly Asp Lys Asp Thr Asn Gly Thr Glu Ile Phe Arg Pro Gly 1 5 10 <210> 65 <211> 14 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> G459E mutant from mouse 1211 with β23 / V5 loop <400> 65 Gly Glu Lys Asp Thr Asn Gly Thr Glu Ile Phe Arg Pro Gly 1 5 10 <210> 66 <211> 11 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> K282R mutant of loop D from mouse 1211 <400> 66 Ser Glu Asn Phe Thr Asn Asn Ala Arg Thr Ile 1 5 10 <210> 67 <211> 11 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> N280D mutants of loop D from mice 1050, 1130, 1161, and 1087 <400> 67 Ser Glu Asn Phe Thr Asn Asp Ala Lys Thr Ile 1 5 10 <210> 68 <211> 14 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> G459V mutant from mouse 1050 with β23 / V5 loop <400> 68 Gly Val Lys Asp Thr Asn Gly Thr Glu Ile Phe Arg Pro Gly 1 5 10 <210> 69 <211> 14 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> G459V / K460E mutant from the β23 / V5 loop of mouse 1050 <400> 69 Gly Val Glu Asp Thr Asn Gly Thr Glu Ile Phe Arg Pro Gly 1 5 10 <210> 70 <211> 11 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> N279S mutant of loop D from mouse 1130 <400> 70 Ser Glu Asn Phe Thr Ser Asn Ala Lys Thr Ile 1 5 10 <210> 71 <211> 14 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> G459D / T465N mutant from the β23 / V5 loop of mouse 1130 <400> 71 Gly Asp Lys Asp Thr Asn Gly Asn Glu Ile Phe Arg Pro Gly 1 5 10 <210> 72 <211> 14 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> The G459D / T465N / G471E mutant from the β23 / V5 loop of mouse 1130 <400> 72 Gly Asp Lys Asp Thr Asn Gly Asn Glu Ile Phe Arg Pro Glu 1 5 10 <210> 73 <211> 11 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> N279Y mutant of ring D from mouse 1210 <400> 73 Ser Glu Asn Phe Thr Tyr Asn Ala Lys Thr Ile 1 5 10 <210> 74 <211> 11 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> T278A / A281T mutant from mouse 1210 with loop D <400> 74 Ser Glu Asn Phe Ala Asn Asn Thr Lys Thr Ile 1 5 10 <210> 75 <211> 11 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> N279K mutant of loop D from mouse 940 <400> 75 Ser Glu Asn Phe Thr Lys Asn Ala Lys Thr Ile 1 5 10 <210> 76 <211> 11 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> S274F mutant of loop D from mice 940 and 1087 <400> 76 Phe Glu Asn Phe Thr Asn Asn Ala Lys Thr Ile 1 5 10 <210> 77 <211> 14 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> G459D / K460E mutant from the β23 / V5 loop of mouse 1186 <400> 77 Gly Asp Glu Asp Thr Asn Gly Thr Glu Ile Phe Arg Pro Gly 1 5 10 <210> 78 <211> 11 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> S274Y mutant of loop D from mouse 1214 <400> 78 Tyr Glu Asn Phe Thr Asn Asn Ala Lys Thr Ile 1 5 10 <210> 79 <211> 9 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> M426I mutant from mouse 1069 with β20 / β21 loop <400> 79 Asn Ile Trp Gln Glu Val Gly Lys Ala 1 5 <210> 80 <211> 14 <212> PRT <213> Human Immunodeficiency Virus 1 <220> <223> G471R mutant from mouse 1069 with β23 / V5 loop <400> 80 Gly Gly Lys Asp Thr Asn Gly Thr Glu Ile Phe Arg Pro Arg 1 5 10

Claims

1. A monoclonal human antibody or a binding fragment thereof targeting the CD4 binding site of human immunodeficiency virus HIV-1, wherein the antibody or the binding fragment thereof comprises a combination of heavy chain variable domains HCDR1, HCDR2, and HCDR3 and light chain variable domains LCDR1, LCDR2, and LCDR3, wherein the CDRs are defined according to the IMGT numbering scheme, and wherein the heavy chain variable domain is the amino acid sequence of SEQ ID NO: 1 and the light chain variable domain is the amino acid sequence of SEQ ID NO:

2.

2. The monoclonal human antibody or its binding fragment according to claim 1, wherein the antibody is an IgG1, IgG2, IgG3 or IgG4 antibody.

3. The monoclonal human antibody or its binding fragment according to claim 1 or 2, wherein the binding fragment comprises Fab, Fab', Fab'-SH, F(ab)2, Fv or a single-chain antibody fragment.

4. The monoclonal human antibody or its binding fragment according to claim 1 or 2, wherein the binding fragment is composed of Fab, Fab', Fab'-SH, F(ab)2, Fv or a single-chain antibody fragment.

5. A monoclonal human antibody or a binding fragment thereof targeting the CD4 binding site of human immunodeficiency virus HIV-1, wherein the antibody or the binding fragment thereof comprises a combination of a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain is the amino acid sequence of SEQ ID NO: 1 and the light chain variable domain is the amino acid sequence of SEQ ID NO:

2.

6. The monoclonal human antibody or its binding fragment according to claim 5, wherein the antibody is an IgG1, IgG2, IgG3 or IgG4 antibody.

7. The monoclonal human antibody or its binding fragment according to claim 5 or 6, wherein the binding fragment comprises Fab, Fab', Fab'-SH, F(ab)2, Fv or a single-chain antibody fragment.

8. The monoclonal human antibody or its binding fragment according to claim 5 or 6, wherein the binding fragment is composed of Fab, Fab', Fab'-SH, F(ab)2, Fv or a single-chain antibody fragment.

9. A pharmaceutical composition comprising a monoclonal human antibody or a binding fragment thereof according to any one of claims 1 to 8; and at least one pharmaceutically acceptable excipient.

10. A kit comprising a monoclonal human antibody or a binding fragment thereof according to any one of claims 1 to 8 and a container.

11. The monoclonal human antibody or its binding fragment according to any one of claims 1 to 8, the pharmaceutical composition according to claim 9, or the kit according to claim 10, characterized in that, Use for the preparation of a medicine for the treatment or prevention of acquired immunodeficiency syndrome (AIDS) in human subjects.

12. The monoclonal human antibody or its binding fragment according to any one of claims 1 to 8, the pharmaceutical composition according to claim 9, or the kit according to claim 10, characterized in that, Use for preparing a medicine for treating human subjects infected with HIV-1.

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