HK40017754B - Multispecific antibodies that target hiv gp120 and cd3 - Google Patents

Description

Multispecific antibodies targeting HIV GP120 and CD3

Cross Reference to Related Applications

This application claims priority to U.S. provisional patent application serial No. 62/523,141, filed on 21/6/2017, which is incorporated herein by reference in its entirety.

Sequence listing

The present application contains a sequence listing, filed electronically in ASCII format and incorporated by reference herein in its entirety. The ASCII copy was created at 20.6.2018 under the name 35648-0054WO1_SL. Txt and was 164,114 bytes in size.

Technical Field

The present disclosure relates to antibodies for the treatment and prevention of Human Immunodeficiency Virus (HIV) infection. In particular, provided herein are multispecific antibodies including broadly neutralizing anti-HIV antibodies, and methods of using these antibodies to reduce HIV replication and to treat and prevent HIV infection.

Background

Human Immunodeficiency Virus (HIV) infection and related diseases are a major public health problem worldwide. Most currently approved therapies for HIV infection target viral reverse transcriptase, protease, and integrase, but resistance of HIV to these existing drugs, long-term toxicity, and inadequate patient compliance with daily dosing regimens have been problems associated with these therapies. Therefore, it is important to discover and develop new HIV drugs.

WO2012/030904 describes human anti-HIV antibodies derived from memory B cells of HIV infected donors, which are capable of inhibiting infection of HIV-1 species from multiple clades. However, the therapeutic use of these antibodies is limited due to problems with immunogenicity, pharmacokinetics, antigen specificity, effector function, and manufacture. Thus, there is a need in the art for novel anti-HIV antibodies with advantageous properties for therapeutic use.

Disclosure of Invention

The present disclosure provides, inter alia, compositions and methods for treating or preventing HIV. More specifically, provided herein are multispecific antibodies that target the Human Immunodeficiency Virus (HIV) envelope (Env) glycoprotein GP120 (GP 120) and a second antigen (e.g., cluster of differentiation 3 (CD 3); anti-IgA receptor (CD 89)), and uses thereof.

In one aspect, the disclosure provides multispecific antibodies that bind to human immunodeficiency virus-1 (HIV-1) envelope (Env) glycoprotein gp120 (gp 120) and human CD3 (e.g., human CD3 epsilon). The antibody comprises a first antigen binding domain comprising a first heavy chain variable domain (VH) and a first light chain variable domain (VL). The first antigen binding domain binds gp120 and comprises a first VH-Complementarity Determining Region (CDR) 1 comprising an amino acid sequence of SEQ ID NO 1; a first VH-CDR2 comprising the amino acid sequence of SEQ ID NO. 2; a first VH-CDR3 comprising the amino acid sequence of SEQ ID NO. 3; a first VL-CDR1 comprising the amino acid sequence of SEQ ID NO. 4; a first VL-CDR2 comprising the amino acid sequence of SEQ ID NO. 5; and a first VL-CDR3 comprising the amino acid sequence of SEQ ID NO 6. The antibody further comprises a second antigen-binding domain that binds human CD3 (e.g., human CD3 epsilon). In certain embodiments, the anti-gp 120 antibody binds to a polypeptide comprising SEQ ID NO:21 or a protein consisting thereof. In some cases, the anti-gp 120 antibody binds to a polypeptide comprising SEQ ID NO:38 or a protein consisting thereof. In some cases, the anti-gp 120 antibody binds to free HIV-1 virus. In some cases, the anti-gp 120 antibody binds to HIV-1 infected cells. In some cases, the anti-gp 120 antibody binds to both free HIV-1 virus and HIV-1 infected cells. In certain instances, the anti-gp 120 antibody binds to at least two different HIV-1 strains (e.g., group M, group N, group O, or group P). In one embodiment, the anti-gp 120 antibody binds pwito.c/2474 (accession No. JN944948 and NIH AIDS Reagent Program catalog No. 11739). In another embodiment, the anti-gp 120 antibody binds pCH058.C/2960 (accession number JN944940 and NIH AIDS Reagent Program catalog number 700010058).

In another aspect, the disclosure provides multispecific antibodies that bind to human immunodeficiency virus-1 (HIV-1) envelope (Env) glycoprotein gp120 (gp 120) and IgA receptor CD 89. The antibody comprises a first antigen binding domain comprising a first heavy chain variable domain (VH) and a first light chain variable domain (VL). The first antigen binding domain binds gp120 and comprises a heavy chain variable region comprising SEQ ID NO:1, a first VH-Complementarity Determining Region (CDR) 1 comprising the amino acid sequence of SEQ ID NO:2, a first VH-CDR2 comprising the amino acid sequence of SEQ ID NO:3, a first VH-CDR3 comprising the amino acid sequence of SEQ ID NO:4, a first VL-CDR1 comprising the amino acid sequence of SEQ ID NO:5 and a first VL-CDR2 comprising the amino acid sequence of SEQ ID NO:6, or a second VL-CDR3 of the amino acid sequence of seq id no. The antibody also comprises a second antigen-binding domain that binds CD89 (e.g., human CD89/FCAR; uniProtKB-P24071). In certain embodiments, the anti-gp 120 antibody binds to a polypeptide comprising SEQ ID NO:21 or a protein consisting thereof. In some cases, the anti-gp 120 antibody binds to a polypeptide comprising SEQ ID NO:38 or a protein consisting thereof. In some cases, the anti-gp 120 antibody binds to free HIV-1 virus. In some cases, anti-gp 120 antibodies bind to HIV-1 infected cells. In some cases, anti-gp 120 antibodies bind to free HIV-1 virus and HIV-1 infected cells. In certain instances, the anti-gp 120 antibody binds to at least two different HIV-1 strains (e.g., group M, group N, group O, or group P). In one embodiment, the anti-gp 120 antibody binds pwito.c/2474 (accession No. JN944948 and NIH AIDS Reagent Program catalog No. 11739). In another embodiment, the anti-gp 120 antibody binds to pCH058.C/2960 (accession number JN944940 and NIH AIDS Reagent Program catalog number 700010058).

In some embodiments of both aspects above, the first VH comprises a VH sequence identical to SEQ ID NO:7 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the first VL comprises an amino acid sequence identical to SEQ ID NO:8 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In certain instances of these embodiments, the nucleic acid sequence of SEQ ID NO:8 at one or more of positions 66, 67A and 67C (Kabat numbering). In certain embodiments, the first VL comprises an amino acid sequence identical to SEQ ID NO:81 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In certain embodiments, the first VL comprises an amino acid sequence identical to SEQ ID NO:82 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In certain embodiments, the first VL comprises an amino acid sequence identical to SEQ ID NO:83 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In certain embodiments, the first VL comprises an amino acid sequence identical to SEQ ID NO:84 are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some cases, the VH described above is directly linked to SEQ ID NO: 56-65. In other cases, the VH described above is identical to SEQ ID NO:66-75, to each other. In some cases, the VH described above is directly linked to a VH having SEQ ID NO:77 (e.g., substitutions that increase half-life and/or decrease effector function). In certain embodiments, the aforementioned VH is linked, directly or by intervening amino acids (e.g., a G-S linker sequence), to an amino acid sequence comprising a CH1 domain, a CH2 domain, and a CH3 domain from an IgG1 (e.g., a human IgG1, e.g., an IgG1m3 allotype), and an IgG3 hinge region (e.g., an "open" IgG3 hinge variant "IgG 3C-" described in WO 2017/096221).

In some embodiments of the above two aspects, the first antigen binding domain comprises a polypeptide having an amino acid sequence identical to SEQ ID NO:9, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the first antigen binding domain comprises a polypeptide having an amino acid sequence identical to SEQ ID NO:10 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the first antigen binding domain comprises a polypeptide having an amino acid sequence identical to SEQ ID NO:40, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the first antigen binding domain comprises a polypeptide having an amino acid sequence identical to SEQ ID NO:78 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the first antigen binding domain comprises a polypeptide having an amino acid sequence identical to SEQ ID NO:79, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In some embodiments, the first antigen binding domain comprises a polypeptide having an amino acid sequence identical to SEQ ID NO:80, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical.

In certain embodiments of the above two aspects, the first VH comprises SEQ ID NO:7 and the first VL comprises the amino acid sequence of SEQ ID NO: 8. In other embodiments, the first VH comprises SEQ ID NO:7 and the first VL comprises the amino acid sequence of SEQ ID NO:81, or a pharmaceutically acceptable salt thereof. In other embodiments, the first VH comprises SEQ ID NO:7 and the first VL comprises the amino acid sequence of SEQ ID NO: 82. In other embodiments, the first VH comprises SEQ ID NO:7 and the first VL comprises the amino acid sequence of SEQ ID NO:83 of the sequence listing. In other embodiments, the first VH comprises SEQ ID NO:7 and the first VL comprises the amino acid sequence of SEQ ID NO: 84. In some cases, the VH described above is identical to SEQ ID NO: 56-65. In other cases, the VH described above is identical to SEQ ID NO:66-75, to each other. In some cases, the VH described above can be directly linked to a VH having the amino acid sequence of SEQ ID NO:77 (e.g., substitutions that increase half-life and/or decrease effector function). In certain instances, the aforementioned VH is linked, directly or through one or more intermediate amino acids (e.g., a G-S linker sequence), to an amino acid sequence comprising the CH1, CH2, and CH3 domains of an IgG1 (e.g., a human IgG1, e.g., an IgG1m3 allotype), and an IgG3 hinge region (e.g., the "open" IgG3 hinge variant "IgG 3C-" described in WO 2017/096221).

In some embodiments of the above two aspects, the first antigen binding domain comprises a polypeptide having the amino acid sequence of SEQ ID NO:9, and comprises a light chain having the amino acid sequence set forth in SEQ ID NO:10, or a light chain of the amino acid sequence set forth in seq id No. 10. In some embodiments, the first antigen binding domain comprises a polypeptide having the amino acid sequence of SEQ ID NO:9 and comprises a light chain having the amino acid sequence set forth in SEQ ID NO:40, or a light chain of the amino acid sequence shown in seq id no. In some embodiments, the first antigen binding domain comprises a polypeptide having the amino acid sequence of SEQ ID NO:9, and comprises a light chain having the amino acid sequence set forth in SEQ ID NO:78 to the amino acid sequence shown in seq id no. In some embodiments, the first antigen binding domain comprises a polypeptide having the amino acid sequence of SEQ ID NO:9 and comprises a light chain having the amino acid sequence set forth in SEQ ID NO:79, or a pharmaceutically acceptable salt thereof. In some embodiments, the first antigen binding domain comprises a polypeptide having the amino acid sequence of SEQ ID NO:9, and comprises a light chain having the amino acid sequence set forth in SEQ ID NO:80, or a light chain of the amino acid sequence set forth in seq id no.

In certain embodiments, the multispecific antibody is a bispecific antibody.

In certain embodiments, the second antigen-binding domain binds human CD3 and comprises a second VH and a second VL. The second VH comprises a VH comprising SEQ ID NO:11, a second VH-CDR1 comprising the amino acid sequence of SEQ ID NO:12 and a second VH-CDR2 comprising the amino acid sequence of SEQ ID NO:13, and a second VH-CDR3 of the amino acid sequence of seq id No. 13. In some embodiments, the second VL comprises a vh comprising SEQ ID NO:14, a second VL-CDR1 comprising the amino acid sequence of SEQ ID NO:15 and a second VL-CDR2 comprising the amino acid sequence of SEQ ID NO:16, or a second VL-CDR3 of the amino acid sequence of seq id no.

In certain embodiments, the second antigen-binding domain binds human CD3 and comprises a second VH and a second VL, wherein the second VH comprises a VH comprising SEQ ID NO:11, a second VH-CDR1 comprising the amino acid sequence of SEQ ID NO:12 and a second VH-CDR2 comprising the amino acid sequence of SEQ ID NO:13, a second VH-CDR3 of the amino acid sequence of seq id no; and wherein the second VL comprises a vh comprising SEQ ID NO:14, a second VL comprising the amino acid sequence of SEQ ID NO:15 and a second VL-CDR2 comprising the amino acid sequence of SEQ ID NO:16, or a second VL-CDR3 of the amino acid sequence of seq id no.

In certain embodiments, the second antigen-binding domain binds to human CD89 and comprises a second VH and a second VL. The second VH comprises a VH comprising SEQ ID NO:98, a second VH-CDR1 comprising the amino acid sequence of SEQ ID NO:99 and a second VH-CDR2 comprising the amino acid sequence of SEQ ID NO:100, or a second VH-CDR3 of the amino acid sequence of seq id no. In some embodiments, the second VL comprises a vh comprising SEQ ID NO:103, a second VL-CDR1 comprising the amino acid sequence of SEQ ID NO:104 and a second VL-CDR2 comprising the amino acid sequence of SEQ ID NO:105, or a second VL-CDR3 of the amino acid sequence of seq id no.

In certain embodiments, the second antigen-binding domain binds human CD89 and comprises a second VH and a second VL, wherein the second VH comprises a VH comprising SEQ ID NO:98, a second VH-CDR1 comprising the amino acid sequence of SEQ ID NO:99 and a second VH-CDR2 comprising the amino acid sequence of SEQ ID NO:100, a second VH-CDR3 of the amino acid sequence of seq id no; and wherein the second VL comprises a vh comprising SEQ ID NO:103, a second VL-CDR1 comprising the amino acid sequence of SEQ ID NO:104 and a second VL-CDR2 comprising the amino acid sequence of SEQ ID NO:105, or a second VL-CDR3 of the amino acid sequence of seq id no.

In some embodiments, the second VH comprises a VH sequence identical to SEQ ID NO:17, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the second VL comprises a sequence identical to SEQ ID NO:18, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical.

In one embodiment, the second VH comprises SEQ ID NO:17, and the second VL comprises the amino acid sequence set forth in SEQ ID NO:18, or a pharmaceutically acceptable salt thereof.

In some embodiments, the second VH comprises a VH sequence identical to SEQ ID NO:96 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the second VL comprises a sequence identical to SEQ ID NO:101 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical.

In one embodiment, the second VH comprises SEQ ID NO:96, and the second VL comprises the amino acid sequence set forth in SEQ ID NO:101, or a pharmaceutically acceptable salt thereof.

In certain embodiments, the second antigen-binding domain comprises a polypeptide having an amino acid sequence that is identical to SEQ ID NO:19 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the second antigen-binding domain comprises a light chain variable region having a sequence identical to SEQ ID NO:20, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical.

In certain embodiments, the second antigen-binding domain comprises a polypeptide having an amino acid sequence that is identical to SEQ ID NO:97 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the second antigen-binding domain comprises a light chain variable region having a sequence identical to SEQ ID NO:102, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical.

In some embodiments, the antibody is a kappa-lambda antibody, an amphipathic and retargeting molecule (DART), "knob-in-hole" structural antibody (knob-in-hole), a strand exchange engineered domain antibody (SEEDbody), a bispecific T cell engager (BiTe), a CrossMab, fcab, diabodies (diabodies), a tandem diabodies (TandAb), or

In certain embodiments, the first antigen binding domain is fused, directly or by insertion of an amino acid sequence, to a first heavy chain constant region selected from the group consisting of human IgG1, human IgG2, human IgG3, human IgG4, human IgA1, and human IgA 2. In some cases, the first antigen-binding domain is fused, directly or by insertion of an amino acid sequence, to a first heavy chain constant region, wherein the constant region is from a human IgG1 (e.g., an IgG1m3 allotype), except that the IgG1 hinge region is replaced with an IgG3 hinge region (e.g., an "open" IgG3 hinge variant "IgG3C-" described in WO 2017/096221). In some embodiments, the second antigen-binding domain is fused, directly or by insertion of an amino acid sequence, to a second heavy chain constant region selected from the group consisting of human IgG1, human IgG2, human IgG3, human IgG4, human IgA1, and human IgA 2. In some cases, the second antigen-binding domain is fused, directly or by insertion of an amino acid sequence, to a second heavy chain constant region, wherein the constant region is from a human IgG1 (e.g., an IgG1m3 allotype), except that the IgG1 hinge region is replaced by an IgG3 hinge region (e.g., an "open" IgG3 hinge variant "IgG3C-" described in WO 2017/096221).

In a specific embodiment, the first heavy chain constant region is human IgG1 and the second heavy chain constant region is human IgG1.

In some embodiments, the first antigen binding domain is fused, directly or by insertion of an amino acid sequence, to a first light chain constant region that is a human λ constant region. In other embodiments, the second antigen-binding domain is fused, directly or by insertion of an amino acid sequence, to a second light chain constant region that is a human lambda constant region.

In some embodiments, the first heavy chain constant region comprises one of: F405L, F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, or F405Y amino acid mutation and the second heavy chain constant region comprises a K409R amino acid mutation. In one instance, the first heavy chain constant region comprises a F405L amino acid mutation and the second heavy chain constant region comprises a K409R amino acid mutation. In another instance, the first heavy chain constant region comprises a F405A amino acid mutation and the second heavy chain constant region comprises a K409R amino acid mutation. In another instance, the first heavy chain constant region comprises a F405D amino acid mutation and the second heavy chain constant region comprises a K409R amino acid mutation. In another instance, the first heavy chain constant region comprises a F405E amino acid mutation and the second heavy chain constant region comprises a K409R amino acid mutation. In another instance, the first heavy chain constant region comprises a F405H amino acid mutation and the second heavy chain constant region comprises a K409R amino acid mutation. In another instance, the first heavy chain constant region comprises the F405I amino acid mutation and the second heavy chain constant region comprises the K409R amino acid mutation. In another instance, the first heavy chain constant region comprises a F405K amino acid mutation and the second heavy chain constant region comprises a K409R amino acid mutation. In another instance, the first heavy chain constant region comprises a F405M amino acid mutation and the second heavy chain constant region comprises a K409R amino acid mutation. In another instance, the first heavy chain constant region comprises a F405N amino acid mutation and the second heavy chain constant region comprises a K409R amino acid mutation. In another instance, the first heavy chain constant region comprises a F405Q amino acid mutation and the second heavy chain constant region comprises a K409R amino acid mutation. In another instance, the first heavy chain constant region comprises a F405S amino acid mutation and the second heavy chain constant region comprises a K409R amino acid mutation. In another instance, the first heavy chain constant region comprises a F405T amino acid mutation and the second heavy chain constant region comprises a K409R amino acid mutation. In another instance, the first heavy chain constant region comprises a F405V amino acid mutation and the second heavy chain constant region comprises a K409R amino acid mutation. In another instance, the first heavy chain constant region comprises a F405W amino acid mutation and the second heavy chain constant region comprises a K409R amino acid mutation. In another instance, the first heavy chain constant region comprises a F405Y amino acid mutation and the second heavy chain constant region comprises a K409R amino acid mutation.

In another embodiment, the first heavy chain constant region comprises a K409R amino acid mutation and the second heavy chain constant region comprises one of: F405L, F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W or F405Y amino acid mutation. In one instance, the first heavy chain constant region comprises a K409R amino acid mutation and the second heavy chain constant region comprises a F405L amino acid mutation. In another instance, the first heavy chain constant region comprises a K409R amino acid mutation and the second heavy chain constant region comprises a F405A amino acid mutation. In one instance, the first heavy chain constant region comprises a K409R amino acid mutation and the second heavy chain constant region comprises a F405D amino acid mutation. In one instance, the first heavy chain constant region comprises a K409R amino acid mutation and the second heavy chain constant region comprises a F405E amino acid mutation. In one instance, the first heavy chain constant region comprises a K409R amino acid mutation and the second heavy chain constant region comprises a F405H amino acid mutation. In one instance, the first heavy chain constant region comprises a K409R amino acid mutation and the second heavy chain constant region comprises a F405I amino acid mutation. In one instance, the first heavy chain constant region comprises a K409R amino acid mutation and the second heavy chain constant region comprises a F405K amino acid mutation. In one instance, the first heavy chain constant region comprises a K409R amino acid mutation and the second heavy chain constant region comprises a F405M amino acid mutation. In one instance, the first heavy chain constant region comprises a K409R amino acid mutation and the second heavy chain constant region comprises a F405N amino acid mutation. In one instance, the first heavy chain constant region comprises a K409R amino acid mutation and the second heavy chain constant region comprises a F405Q amino acid mutation. In one instance, the first heavy chain constant region comprises a K409R amino acid mutation and the second heavy chain constant region comprises a F405S amino acid mutation. In one instance, the first heavy chain constant region comprises a K409R amino acid mutation and the second heavy chain constant region comprises a F405T amino acid mutation. In one instance, the first heavy chain constant region comprises a K409R amino acid mutation and the second heavy chain constant region comprises a F405V amino acid mutation. In one instance, the first heavy chain constant region comprises a K409R amino acid mutation and the second heavy chain constant region comprises a F405W amino acid mutation. In one instance, the first heavy chain constant region comprises a K409R amino acid mutation and the second heavy chain constant region comprises a F405Y amino acid mutation.

In certain embodiments, the effector function of the first heavy chain constant region and the second heavy chain constant region is reduced or eliminated (e.g., relative to the effector function of an antibody having wild-type IgG1 Fc).

In some embodiments, the first heavy chain constant region comprises a human IgG1 heavy chain constant region comprising an N297A mutation or an N297Q mutation, and/or the second heavy chain constant region comprises a human IgG1 heavy chain constant region comprising an N297A mutation or an N297Q mutation.

In another aspect, the disclosure provides bispecific antibodies that bind gp120 and human CD 3. The bispecific antibody comprises a first arm that binds gp 120. The first arm comprises a first heavy chain comprising a first heavy chain constant region comprising a first mutation that is any one of an F405T, F405V, F405W, or F405Y amino acid mutation in F405L, F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, or a K409R mutation; and a first VH comprising a VH comprising SEQ ID NO:1, VH-CDR1 comprising the amino acid sequence of SEQ ID NO:2 comprises and contains the amino acid sequence of SEQ ID NO:3, VH-CDR3 of the amino acid sequence of 3. In one instance, the first arm comprises a first heavy chain comprising a first heavy chain constant region comprising a first mutation that is one of a F405L or a K409R mutation. The first arm further comprises a first light chain comprising a first light chain constant region; and a first VL comprising a vh comprising SEQ ID NO:4, VL-CDR1 comprising the amino acid sequence of SEQ ID NO:5 and VL-CDR2 comprising the amino acid sequence of SEQ ID NO:6, VL-CDR3 of the amino acid sequence of seq id no. The bispecific antibody comprises a second arm that binds CD3 (e.g., human CD3 epsilon)). The second arm comprises a second heavy chain comprising a second heavy chain constant region comprising a second mutation that is any one of an F405L, F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, or F405Y amino acid mutation, or a K409R mutation; and a second VH comprising a VH comprising SEQ ID NO:11, a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:12 and a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:13, VH-CDR3 of the amino acid sequence of seq id No. 13. In one instance, the second arm comprises a second heavy chain comprising a second mutated second heavy chain constant region comprising one of the F405L or K409R mutations. The second arm comprises a second light chain comprising a second light chain constant region; and a second VL comprising a vh comprising SEQ ID NO:14, VL-CDR1 comprising the amino acid sequence of SEQ ID NO:15 and VL-CDR2 comprising the amino acid sequence of SEQ ID NO:16, VL-CDR3 of the amino acid sequence of seq id no. The first mutation and the second mutation are different mutations.

In another aspect, the disclosure provides bispecific antibodies that bind gp120 and human CD 89. The bispecific antibody comprises a first arm that binds gp 120. The first arm comprises a first heavy chain comprising a first heavy chain constant region comprising a first mutation that is any one of an amino acid mutation of F405L, F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, or F405Y, or a K409R mutation; and a first VH comprising a VH comprising SEQ ID NO:1, VH-CDR1 comprising the amino acid sequence of SEQ ID NO:2 and VH-CDR2 comprising the amino acid sequence of SEQ ID NO:3, VH-CDR3 of the amino acid sequence of 3. In one instance, the first arm comprises a first heavy chain comprising a first heavy chain constant region comprising a first mutation that is either a F405L or a K409R mutation. The first arm further comprises a first light chain comprising a first light chain constant region; and a first VL comprising a vh comprising SEQ ID NO:4, VL-CDR1 comprising the amino acid sequence of SEQ ID NO:5 and VL-CDR2 comprising the amino acid sequence of SEQ ID NO:6, VL-CDR3 of the amino acid sequence of seq id no. The bispecific antibody comprises a second arm that binds CD89 (e.g., human CD 89). The second arm comprises a second heavy chain comprising a second heavy chain constant region comprising a second mutation that is any one of an F405L, F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, or F405Y amino acid mutation, or a K409R mutation; a second VH comprising a VH comprising SEQ ID NO:98, VH-CDR1 comprising the amino acid sequence of SEQ ID NO:99 and a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:100, and VH-CDR3 of the amino acid sequence of seq id no. In one instance, the second arm comprises a second heavy chain comprising a second heavy chain constant region comprising a second mutation that is either a F405L or a K409R mutation. The second arm comprises a second light chain comprising a second light chain constant region; and a second VL comprising a vh comprising SEQ ID NO:103, VL-CDR1 comprising the amino acid sequence of SEQ ID NO:104 and a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:105, VL-CDR3 of the amino acid sequence. The first mutation and the second mutation are different mutations.

In certain embodiments of the two aspects above, the anti-gp 120 antibody arm binds to a polypeptide comprising or consisting of SEQ ID NO:21, and a protein consisting of the amino acid sequence shown in the sequence table 21. In some cases, the anti-gp 120 antibody arm binds to a polypeptide comprising or consisting of SEQ ID NO:38, or a pharmaceutically acceptable salt thereof. In some cases, the anti-gp 120 antibody arm binds to free HIV-1 virus. In some cases, anti-gp 120 antibodies bind to HIV-1 infected cells. In some cases, the anti-gp 120 antibody arm binds to free HIV-1 virus and HIV-1 infected cells. In certain instances, the anti-gp 120 antibody binds to at least two different HIV-1 strains (e.g., group M, group N, group O, or group P). In one embodiment, the anti-gp 120 antibody arm binds pwitto.c/2474 (accession No. JN944948 and NIH AIDS Reagent Program catalog No. 11739). In another embodiment, the anti-gp 120 antibody arm binds to pCH058.C/2960 (accession No. JN944940 and NIH AIDS Reagent Program catalog No. 700010058).

In some embodiments, the first VH comprises a VH sequence identical to SEQ ID NO:7 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the first VL comprises an amino acid sequence identical to SEQ ID NO:8 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In certain instances of these embodiments, SEQ ID NO:8 (Kabat numbering) is unchanged at one or more of positions 66, 67A and 67C (Kabat numbering). In certain embodiments, the first VL comprises an amino acid sequence identical to SEQ ID NO:81 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In certain embodiments, the first VL comprises an amino acid sequence identical to SEQ ID NO:82 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In certain embodiments, the first VL comprises an amino acid sequence identical to SEQ ID NO:83 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In certain embodiments, the first VL comprises an amino acid sequence identical to SEQ ID NO:84, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some cases, the VH described above is directly or through insertion of an amino acid (e.g., a G-S linker sequence) to a VH having the sequence of SEQ ID NO:77 (e.g., substitutions that increase half-life and/or decrease effector function).

In some embodiments, the first heavy chain comprises a heavy chain variable region identical to SEQ ID NO:9 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the first light chain comprises a heavy chain variable region identical to SEQ ID NO:10 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the first light chain comprises a heavy chain variable region identical to SEQ ID NO:40, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the first light chain comprises a heavy chain variable region identical to SEQ ID NO:78 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the first light chain comprises a heavy chain variable region identical to SEQ ID NO:79, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In some embodiments, the first light chain comprises a heavy chain variable region identical to SEQ ID NO:80 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical.

In certain embodiments, the second VH comprises a VH sequence identical to SEQ ID NO:17, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the second VL comprises a sequence identical to SEQ ID NO:18, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical.

In certain embodiments, the second VH comprises a VH sequence identical to SEQ ID NO:96 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the second VL comprises a sequence identical to SEQ ID NO:101 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical.

In some embodiments, the second heavy chain comprises a heavy chain identical to SEQ ID NO:19 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In certain embodiments, the second light chain comprises a sequence identical to SEQ ID NO:20 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical.

In some embodiments, the second heavy chain comprises a heavy chain identical to SEQ ID NO:97 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In certain embodiments, the second light chain comprises a sequence identical to SEQ ID NO:102 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical.

In one embodiment, the first VH comprises SEQ ID NO:7 and the first VL comprises the amino acid sequence of SEQ ID NO:8, and/or the second VH comprises the amino acid sequence of SEQ ID NO:17 and the second VL comprises the amino acid sequence of SEQ ID NO: 18.

In one embodiment, the first VH comprises SEQ ID NO:7 and the first VL comprises the amino acid sequence of SEQ ID NO:8, and/or the second VH comprises the amino acid sequence of SEQ ID NO:96 and the second VL comprises the amino acid sequence of SEQ ID NO:101, or a pharmaceutically acceptable salt thereof.

In another embodiment, the first VH comprises SEQ ID NO:7 and the first VL comprises the amino acid sequence of SEQ ID NO:81, and/or the second VH comprises the amino acid sequence of SEQ ID NO:17 and the second VL comprises the amino acid sequence of SEQ ID NO: 18.

In another embodiment, the first VH comprises SEQ ID NO:7 and the first VL comprises the amino acid sequence of SEQ ID NO:81, and/or the second VH comprises the amino acid sequence of SEQ ID NO:96 and the second VL comprises the amino acid sequence of SEQ ID NO:101, or a pharmaceutically acceptable salt thereof.

In further embodiments, the first VH comprises SEQ ID NO:7 and the first VL comprises the amino acid sequence of SEQ ID NO:82, and/or the second VH comprises the amino acid sequence of SEQ ID NO:17 and the second VL comprises the amino acid sequence of SEQ ID NO: 18.

In further embodiments, the first VH comprises SEQ ID NO:7 and the first VL comprises the amino acid sequence of SEQ ID NO:82, and/or the second VH comprises the amino acid sequence of SEQ ID NO:96 and the second VL comprises the amino acid sequence of SEQ ID NO:101, or a pharmaceutically acceptable salt thereof.

In another embodiment, the first VH comprises SEQ ID NO:7 and the first VL comprises the amino acid sequence of SEQ ID NO:83, and/or the second VH comprises the amino acid sequence of SEQ ID NO:17 and the second VL comprises the amino acid sequence of SEQ ID NO: 18.

In another embodiment, the first VH comprises SEQ ID NO:7 and the first VL comprises the amino acid sequence of SEQ ID NO:83, and/or the second VH comprises the amino acid sequence of SEQ ID NO:96 and the second VL comprises the amino acid sequence of SEQ ID NO:101, or a pharmaceutically acceptable salt thereof.

In yet another embodiment, the first VH comprises SEQ ID NO:7 and the first VL comprises the amino acid sequence of SEQ ID NO:84, and/or the second VH comprises the amino acid sequence of SEQ ID NO:17 and the second VL comprises the amino acid sequence of SEQ ID NO: 18.

In yet another embodiment, the first VH comprises SEQ ID NO:7 and the first VL comprises the amino acid sequence of SEQ ID NO:84, and/or the second VH comprises the amino acid sequence of SEQ ID NO:96 and the second VL comprises the amino acid sequence of SEQ ID NO:101, or a pharmaceutically acceptable salt thereof.

In another embodiment, the first heavy chain comprises SEQ ID NO:9 and the first light chain comprises the amino acid sequence of SEQ ID NO:10, and/or the second heavy chain comprises the amino acid sequence of SEQ ID NO:19 and the second light chain comprises SEQ ID NO: 20.

In yet another embodiment, the first VH comprises SEQ ID NO:7 and the first VL comprises the amino acid sequence of SEQ ID NO:84, and/or the second VH comprises the amino acid sequence of SEQ ID NO:96 and the second VL comprises the amino acid sequence of SEQ ID NO:101, or a pharmaceutically acceptable salt thereof.

In another embodiment, the first heavy chain comprises SEQ ID NO:9 and the first light chain comprises the amino acid sequence of SEQ ID NO:40, and/or the second heavy chain comprises the amino acid sequence of SEQ ID NO:19 and the second light chain comprises SEQ ID NO: 20.

In another embodiment, the first heavy chain comprises SEQ ID NO:9 and the first light chain comprises the amino acid sequence of SEQ ID NO:40, and/or the second heavy chain comprises the amino acid sequence of SEQ ID NO:97 and the second light chain comprises SEQ ID NO: 102.

In another embodiment, the first heavy chain comprises SEQ ID NO:9 and the first light chain comprises the amino acid sequence of SEQ ID NO:78, and/or the second heavy chain comprises the amino acid sequence of SEQ ID NO:19 and the second light chain comprises SEQ ID NO: 20.

In another embodiment, the first heavy chain comprises SEQ ID NO:9 and the first light chain comprises the amino acid sequence of SEQ ID NO:78, and/or the second heavy chain comprises the amino acid sequence of SEQ ID NO:97 and the second light chain comprises SEQ ID NO:102, or a pharmaceutically acceptable salt thereof.

In yet another embodiment, the first heavy chain comprises SEQ ID NO:9 and the first light chain comprises the amino acid sequence of SEQ ID NO:79, and/or the second heavy chain comprises the amino acid sequence of SEQ ID NO:19 and the second light chain comprises SEQ ID NO: 20.

In yet another embodiment, the first heavy chain comprises SEQ ID NO:9 and the first light chain comprises the amino acid sequence of SEQ ID NO:79, and/or the second heavy chain comprises the amino acid sequence of SEQ ID NO:97 and the second light chain comprises SEQ ID NO:102, or a pharmaceutically acceptable salt thereof.

In further embodiments, the first heavy chain comprises SEQ ID NO:9 and the first light chain comprises the amino acid sequence of SEQ ID NO:80, and/or the second heavy chain comprises the amino acid sequence of SEQ ID NO:19 and the second light chain comprises SEQ ID NO: 20.

In further embodiments, the first heavy chain comprises SEQ ID NO:9 and the first light chain comprises the amino acid sequence of SEQ ID NO:80, and/or the second heavy chain comprises the amino acid sequence of SEQ ID NO:97 and the second light chain comprises SEQ ID NO: 102.

In certain embodiments of all of the above aspects and embodiments, the antibody further comprises a cytotoxic agent, a radioisotope, a therapeutic agent, an antiviral agent, or a detectable label.

In a further aspect, the present disclosure provides a composition of nucleic acid molecules comprising a first light chain variable region or a first light chain encoding a first antigen binding domain of an antibody disclosed above. In another aspect, the present disclosure provides compositions comprising a nucleic acid molecule encoding a first heavy chain variable region or a first heavy chain of a first antigen binding domain of an antibody disclosed above. In yet another aspect, the present disclosure provides compositions comprising a nucleic acid molecule encoding a first light chain variable region or a first light chain of a second antigen-binding domain of an antibody disclosed above. In yet another aspect, the present disclosure provides compositions comprising a nucleic acid molecule encoding a second heavy chain variable region or a second heavy chain of a second antigen-binding domain of an antibody disclosed above.

In another aspect, the disclosure features a host cell comprising one or more of the foregoing nucleic acids. In some cases, the host cell contains all four chains of the bispecific antibody. In other cases, the host cell comprises a nucleic acid encoding the gp 120-binding arm of the bispecific antibody. In other cases, the host cell comprises a nucleic acid encoding the CD3 binding arm of the bispecific antibody. In still other cases, the host cell comprises a nucleic acid encoding the CD89 binding arm of the bispecific antibody. In some embodiments, the host cell is selected from the group consisting of E.coli, pseudomonas, bacillus, streptomyces, yeast (e.g., pichia, saccharomyces), CHO, YB/20, NS0, PER-C6, HEK-293T, NIH-3T3, heLa, BHK, hep G2, SP2/0, R1.1, B-W, L-M, COS 1, COS 7, BSC1, BSC40, BMT10 cells, plant cells, insect cells, and human cells in tissue culture.

In yet another aspect, a method of producing an antibody that binds gp120 and human CD3 (or an antibody that binds gp120 and human CD 89) is characterized. The method comprises culturing the above-described host cell under conditions such that the nucleic acid molecule is expressed and the antibody is produced.

In another aspect, a method of detecting gp120 and CD3 (or CD 89) -expressing cells in a sample is disclosed. The method comprises contacting the sample with an antibody described herein.

In yet another aspect, the present disclosure provides a pharmaceutical composition comprising an antibody described herein and a pharmaceutically acceptable excipient.

In further embodiments, the disclosure features kits comprising an antibody described herein and a) a detection reagent, b) a gp120 and/or CD3 and/or CD89 antigen, c) a notice reflective of approved use or sale for human administration, or d) a combination thereof.

Also provided are methods of treating or preventing human immunodeficiency virus infection in a human subject in need thereof. The method comprises administering to a human subject a therapeutically effective amount of an antibody or pharmaceutical composition disclosed herein. In some embodiments, the human immunodeficiency virus infection is an HIV-1 infection. In some embodiments, the virus in the patient has Env positive for N332 PNG. In certain embodiments, HIV is of clade B, G, a, AC, or AE.

In another aspect, the disclosure features an antibody that binds gp 120. The antibody comprises a VH and a VL. The VH comprises a VH comprising SEQ ID NO:1, VH-CDR1 comprising the amino acid sequence of SEQ ID NO:2, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:3, VH-CDR3 of the amino acid sequence of 3.VL comprises a vh comprising SEQ ID NO:4, VL-CDR1 comprising the amino acid sequence of SEQ ID NO:5 and VL-CDR2 comprising the amino acid sequence of SEQ ID NO:6, VL-CDR3 of the amino acid sequence of seq id no. In addition, the VL comprises a tyrosine, phenylalanine or threonine at position 67A (Kabat numbering), or a glycine at position 67 (Kabat numbering).

In certain embodiments, the anti-gp 120 antibody binds to a polypeptide comprising or consisting of SEQ ID NO:21, and a protein consisting of the amino acid sequence shown in the figure 21. In some cases, the anti-gp 120 antibody binds to a polypeptide comprising or consisting of SEQ ID NO: 38. In some cases, the anti-gp 120 antibody binds to free HIV-1 virus. In some cases, anti-gp 120 antibodies bind to HIV-1 infected cells. In some cases, anti-gp 120 antibodies bind to free HIV-1 virus and HIV-1 infected cells. In certain instances, the anti-gp 120 antibody binds to at least two different HIV-1 strains (e.g., group M, group N, group O, or group P). In one embodiment, the anti-gp 120 antibody binds pwito.c/2474 (accession No. JN944948 and NIH AIDS Reagent Program catalog No. 11739). In another embodiment, the anti-gp 120 antibody binds pCH058.C/2960 (accession number JN944940 and NIH AIDS Reagent Program catalog number 700010058).

In some embodiments, the VH comprises a sequence identical to SEQ ID NO:7 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the VH is linked, directly or by insertion of an amino acid sequence (e.g., a G-S linker), to a human IgG1 constant region (e.g., an IgG1m3 allotype) comprising 0-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions that reduce effector function and/or increase the pharmacokinetic half-life of the antibody. In some cases, the antibody has a hinge region from an IgG3 antibody (e.g., an "open" IgG 3C-hinge variant disclosed in WO 2017/096221) and CH1, CH2, and CH3 regions from a human IgG1 antibody (e.g., an IgG1m3 allotype).

In certain embodiments, the VL comprises a sequence identical to SEQ ID NO: 81. 82, 83 or 84, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical.

In some embodiments, the heavy chain comprises a heavy chain identical to SEQ ID NO:9 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical.

In certain embodiments, the light chain comprises a heavy chain variable region identical to SEQ ID NO: 40. 78, 79, or 80, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical.

In one embodiment, the antibody comprises a heavy chain comprising SEQ ID NO:9, and a light chain comprising the amino acid sequence set forth in SEQ ID NO:40, or a light chain of an amino acid sequence as set forth in any one of seq id nos.

In another embodiment, the antibody comprises a heavy chain variable region comprising SEQ ID NO:9, and a light chain comprising the amino acid sequence set forth in SEQ ID NO:78 to a heavy chain of any one of the amino acid sequences set forth in seq id no.

In yet another embodiment, the antibody comprises a heavy chain comprising SEQ ID NO:9, and a light chain comprising the amino acid sequence set forth in SEQ ID NO:79 of any one of the amino acid sequences set forth herein.

In another embodiment, the antibody comprises a heavy chain variable region comprising SEQ ID NO:9, and a light chain comprising the amino acid sequence set forth in SEQ ID NO:80, or a light chain of an amino acid sequence as set forth in any one of seq id nos.

In some embodiments, the above-disclosed antibodies further comprise a cytotoxic agent, a radioisotope, a therapeutic agent, an antiviral agent, or a detectable label.

In another aspect, the disclosure features a pharmaceutical composition comprising the antibody of this aspect and a pharmaceutically acceptable carrier.

In another aspect, the disclosure relates to one or more nucleic acids encoding an antibody of this aspect.

In another aspect, the present disclosure provides one or more vectors comprising one or more of the nucleic acids described above.

In yet another aspect, the disclosure features a host cell comprising one or more of the vectors described above.

In a further aspect, the disclosure provides methods for producing anti-gp 120 antibodies. The method comprises culturing the above-described host cell under conditions in which one or more nucleic acids are expressed and antibodies are produced.

Also featured are methods of treating or preventing human immunodeficiency virus infection in a human subject in need thereof. The method comprises administering to a human subject a therapeutically effective amount of an antibody or pharmaceutical composition of this aspect. In some embodiments, the human immunodeficiency virus infection is an HIV-1 infection. In some embodiments, HIV in the patient has Env positive for N332 PNG. In certain embodiments, HIV is of clade B, G, a, AC, or AE.

In another aspect, the disclosure features an antibody fragment that binds gp 120. The antibody fragment comprises VH and VL. The VH comprises a VH comprising SEQ ID NO:1, VH-CDR1 comprising the amino acid sequence of SEQ ID NO:2, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:3, VH-CDR3 of the amino acid sequence of seq id no. VL comprises a vh comprising SEQ ID NO:4, VL-CDR1 comprising the amino acid sequence of SEQ ID NO:5 and VL-CDR2 comprising the amino acid sequence of SEQ ID NO:6, VL-CDR3 of the amino acid sequence of seq id no. In addition, the VL comprises a tyrosine, phenylalanine or threonine at position 67A (Kabat numbering), or a glycine at position 67 (Kabat numbering).

In certain embodiments, the anti-gp 120 antibody fragment binds to a polypeptide comprising or consisting of SEQ ID NO:21, and a protein consisting of the amino acid sequence shown in the figure 21. In some cases, the anti-gp 120 antibody fragment binds to a polypeptide comprising or consisting of SEQ ID NO:38, or a pharmaceutically acceptable salt thereof. In some cases, the anti-gp 120 antibody fragment binds to free HIV-1 virus. In some cases, anti-gp 120 antibodies bind to HIV-1 infected cells. In some cases, anti-gp 120 antibody fragments bind to free HIV-1 virus and HIV-1 infected cells. In some cases, the anti-gp 120 antibody binds to at least two different HIV-1 strains (e.g., group M, group N, group O, or group P). In one embodiment, the anti-gp 120 antibody fragment binds pwito. C/2474 (accession No. JN944948 and NIH AIDS Reagent Program catalog No. 11739). In another embodiment, the anti-gp 120 antibody fragment binds to pCH058.C/2960 (accession number JN944940 and NIH AIDS Reagent Program catalog number 700010058).

In some embodiments, the VH comprises a sequence identical to SEQ ID NO:7 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical.

In certain embodiments, the VL comprises a sequence identical to SEQ ID NO: 81. 82, 83 or 84, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical.

In some embodiments, the antibody fragment is a Fab, F (ab) 2, fv, scFv, sc (Fv) 2, or diabody.

In some embodiments, the above-described antibody fragment further comprises a cytotoxic agent, a radioisotope, a therapeutic agent, an antiviral agent, or a detectable label.

In another aspect, the disclosure features a pharmaceutical composition comprising the antibody fragment of this aspect and a pharmaceutically acceptable carrier.

In another aspect, the disclosure relates to one or more nucleic acids encoding the antibody fragments of this aspect.

In another aspect, the present disclosure provides one or more vectors comprising one or more of the nucleic acids described above.

In yet another aspect, the disclosure features a host cell comprising one or more of the vectors described above.

In a further aspect, the disclosure provides methods of producing anti-gp 120 antibody fragments. The method comprises culturing the above-described host cell under conditions in which one or more nucleic acids are expressed and antibody fragments are produced.

In another aspect, the disclosure features a method of treating or preventing HIV in a human subject in need thereof. The method comprises administering to the human subject a therapeutically effective amount of the antibody fragment or pharmaceutical composition of this aspect. In some embodiments, the human immunodeficiency virus infection is an HIV-1 infection. In some embodiments, HIV in the patient has Env positive for N332 PNG. In certain embodiments, HIV is of clade B, G, a, AC, or AE.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Detailed Description

The multispecific antibodies described herein bind to the Human Immunodeficiency Virus (HIV) envelope (Env) protein gp120 (gp 120) and cluster of differentiation 3 (CD 3) (e.g., CD3 epsilon) and effectively kill HIV-infected cells. For example, a multispecific antibody is a bispecific antibody having two antigen-binding arms, wherein the bispecific antibody binds an antigen on an HIV-infected cell (e.g., gp 120) in one arm and binds an antigen on a T cell (e.g., CD 3) in the other arm, and a T cell (e.g., CD 4) can be conjugated to the antigen on the T cell ⁺ T cells and/or CD8 ⁺ T cells) are targeted to HIV-infected cells, resulting in killing of HIV-infected cells. Bispecific antibodies that bind to CD3 and gp120 can redirect CD8 ⁺ And CD4 ⁺ T cells to kill gp120 expressing cells (e.g., HIV infected cells). The T cells kill HIV infected cells regardless of their T cell receptor specificity. In one embodiment, the bispecific antibody is anti-gp 120X anti-CD 3The duobodies of the present disclosure have significant advantages over other bispecific platforms known in the art.A significant advantage of the platform over, for example, DART platforms is that the duobodies disclosed herein can recruit CD4 ⁺ T cells to kill target cells (e.g., HIV infected cells). No CD4 observed with DART ⁺ T cell mediated killing (see Sloan et al, PLOS Patholoens, 11 (11): e1005233.Doi:10.1371/journal. Ppat.1005233 (2015)). This is a significant advantage, since the target in HIV therapy is also CD4 ⁺ T cells. One problem with using antibodies that require innate effector cells for HIV therapeutic activity is that the effector cells may not be present in latently infected CD4 ⁺ Tissue in which T cells reside, and if CD4 ⁺ The T cell itself may be an effector cell, thenThis is not a problem.

HIV-1 is the major family of HIV and accounts for 95% of all infections worldwide. HIV-2 is found primarily in some Western Africa countries.

The HIV viruses are divided into specific groups, M, N, O and P, where M is the "main" group and contributes to the majority of HIV/AIDS worldwide. The M groups are further subdivided into subtypes (also called clades) that are prevalent in different geographical locations, according to their genetic sequence.

The M group "subtype" or "clade" is the HIV-1 group M subtype defined by the genetic sequence data. Examples of group M subtypes include subtypes A-K. Certain subtypes are known to be more virulent or resistant to different drug treatments. There are also "circulating recombinant forms" or CRFs derived from recombination between different subtypes of viruses, each assigned a number. For example, CRF12_ BF is a recombination between subtypes B and F. Subtype a is not very common in the west. Subtype B is the predominant form in Europe, america, japan, thailand and Australia. Subtype C is the predominant form in southern Africa, eastern Africa, india, nepal and parts of China. Subtype D is usually only seen in eastern and central africa. Subtype E was never identified as non-recombinant, it only recombined with subtype a as CRF01_ AE. Subtype F is found in the middle of Africa, south America and eastern Europe. Subtype G (and CRF02_ AG) is found in Africa and Central Europe. Subtype H is restricted to central Africa. Subtype I was originally used to describe the strain now designated CRF04_ cpx, where cpx was used for "complex" recombination of several subtypes. Subtype J is mainly seen in north africa, central africa and west africa, and the caribbean subtype K is limited to the democratic republic of congo and karilong. These subtypes are sometimes further divided into sub-subtypes, such as A1 and A2 or F1 and F2. In 2015, CRF19 strain, a recombinant of subtype a, subtype D and subtype G and subtype D proteases, was found in cuba, closely associated with the rapid development of aids.

The present disclosure specifically provides neutralizing antibodies (e.g., broadly neutralizing abs) that target gp120 polypeptides on the surface of HIV-infected cells. Neutralizing antibodies against viral envelope proteins can provide an adaptive immune defense against HIV-1 exposure by blocking infection of susceptible cells. Extensive neutralization indicated that the antibody could neutralize HIV-1 isolates from different clades. Thus, antibodies encompassed by the present disclosure have cross-clade binding activity.

gp120

The envelope glycoprotein gp120 (or gp 120) is a 120kDa glycoprotein that is part of the outer layer of HIV. It exists as a viral membrane spike (viral membrane spike) itself, consisting of three gp120 molecules linked together and anchored to the membrane by gp41 proteins. gp120 is important for viral infection because it facilitates HIV entry into host cells through interaction with cell surface receptors. These receptors include DC-SIGN, heparan sulfate proteoglycans and CD4 receptors. Binding to CD4 on helper T cells induces the initiation of a cascade of conformational changes in gp120 and gp41, which results in fusion of the virus to the host cell membrane.

gp120 is encoded by the HIV env gene. The env gene encodes a gene product of about 850 amino acids. The major env product is the protein gp160, which is cleaved in the endoplasmic reticulum by the cellular proteases furin to gp120 (about 480 amino acids) and gp41 (about 345 amino acids).

The amino acid sequence of an exemplary gp160 polypeptide of HIV clone WITO is provided below (V3 hypervariable loop bold, and N332 potential N-linked glycosylation site bold and underlined):

the amino acid sequence of an exemplary gp120 polypeptide is provided below:

the amino acid sequence of another exemplary gp120 polypeptide is provided below (see www. Bioafrica. Net/proteomics/ENV-gp120prot. Html):

genomic diversity between independent human immunodeficiency virus type 1 (HIV-1) isolates, in successive isolates from the same patient to a lesser extent, and even within single patient isolates, is a well-known feature of HIV-1. Although this sequence heterogeneity is distributed throughout the genome, most of the heterogeneity is located in the env genes. Comparison of predicted amino acid sequences from several different isolates revealed that sequence heterogeneity was clustered in five variable regions (designated V1 to V5) of the surface glycoprotein gp 120. The V3 region, although only 35 amino acids long, exhibits considerable sequence variability. Interestingly, despite this variability, the V3 region includes mediation and CD4 ⁺ Determinants of cellular interactions. The increase in gp120 variability results in higher levels of viral replication, indicating increased viral fitness in individuals infected with diverse HIV-1 variants. Variability in potential N-linked glycosylation sites (PNGS) also leads to increased viral adaptability. PNGS allows long-chain carbohydrates to bind to the hypervariable regions of gp 120. Thus, the number of PNGS in env may influence the virus' adaptability by providing more or less sensitivity to neutralizing antibodies.

The consensus sequence for the gp 120V 3 region is provided below (Milich et al, J Virol.,67 (9): 5623-5634 (1993)):

CTRPNNNTRKSIHIGPGRAFYTTGEIIGDIRQAHC(SEQ ID NO:22)。

anti-gp 120 antibodies

The disclosure features anti-gp 120 antibodies. In certain embodiments, these antibodies bind to HIV-1 antigens expressed on the surface of cells and eliminate or kill infected cells.

In certain embodiments, these antibodies are neutralizing antibodies (e.g., monoclonal) that target HIV-1. A "neutralizing antibody" is an antibody that can neutralize the ability of HIV to initiate and/or perpetuate an infection in a host and/or in a target cell in vitro. The present disclosure provides neutralizing monoclonal human antibodies, wherein the antibodies recognize an antigen from HIV, such as a gp120 polypeptide. In certain embodiments, a "neutralizing antibody" can inhibit entry of an HIV-1 virus (e.g., SF162 and/or JR-CSF) at a neutralization index of >1.5 or >2.0 (Kostrikis LG et al J.Virol.,70 (1): 445-458 (1996)).

In some embodiments, these antibodies are broadly neutralizing antibodies (e.g., monoclonal) targeting HIV-1. "broadly neutralizing antibody" refers to an antibody that neutralizes more than one HIV-1 virus species (from a diverse clade and different strains within a clade) in a neutralization assay. Broadly neutralizing antibodies can neutralize at least 2, 3, 4, 5, 6, 7, 8, 9 or more different HIV-1 strains belonging to the same or different clades. In particular embodiments, the broadly neutralizing antibody can neutralize multiple HIV-1 species belonging to at least 2, 3, 4, 5, or 6 different clades. In certain embodiments, the inhibitory concentration of the antibody can be less than about 0.0001. Mu.g/ml, less than about 0.001. Mu.g/ml, less than about 0.01. Mu.g/ml, less than about 0.1. Mu.g/ml, less than about 0.5. Mu.g/ml, less than about 1.0. Mu.g/ml, less than about 5. Mu.g/ml, less than about 10. Mu.g/ml, less than about 25. Mu.g/ml, less than about 50. Mu.g/ml or less than about 100. Mu.g/ml to neutralize about 50% of the input virus in a neutralization assay.

In one embodiment, the anti-gp 120 antibodies of the present disclosure are related to an antibody described as PGT-121LO6 in PCT application publication No. WO 2012/030904. Table 1 below provides relevant sequence information for the PGT-121LO6 antibody.

TABLE 1

Crystal structure and experimental analysis of an antibody highly related to the PGT-121LO6 antibody (i.e., PGT-122) indicate that PGT122 also utilizes amino acid residues other than the CDRs to bind antigen (along with the CDRs). For example, the antibody appears to have additional regions in the framework regions that contact the antigen (see, e.g., experimental differentiation for PGT121 and related antibodies: sok et al PLOS Pathogens,9, e1003754 (2013)). The high resolution structure of PGT122 bound to Env viral antigens has been determined (see, e.g., julien JP et al, science,342, 14777-14783 (2013) and panera, m. Et al, nature,514, 455-461 (2014)). The structure of PGT121 is described in Julie JP et al, PLOS Pathologens 9, E1003342 (2013) and Mouquet H et al, PNAS,109, E3268-E3277 (2012). The structure of PGT122 is described in Julie JP et al, PLOS Pathologens 9, e1003342 (2013), PDB ID 4JY 5; and the structure of PGT123 is described in Julie JP et al, PLOS Pathologens 9, e1003342 (2013). PGT122 and PGT123 antibodies are closely related to PGT121 antibodies, so PGT122/Env structure and knowledge of PGT121, PGT122 and PGT123 structure can be used to model the structure of PGT121 bound to Env very accurately and predict with high confidence the residues of PGT121 involved in binding to Env. Residues of the PGT121 LO6 antibody contacting gp120 antigen predicted based on similarity to PGT122 and PGT122/Env structures are provided below, with framework residues shown in bold (Kabat numbering):

VH (Kabat numbering):

33. 56, 58, 99, 100A, 100B, 100C, 100D, 100E, 100G, 100I, 100J, 100K, 100L; and

VL (Kabat numbering):

28、29、30、50、51、52、66、67、67A、67C、91、92、93、94、95、95A、95B。

PGT-121 LO6 antibodies have been shown to bind to a variety of different antigen variants (e.g., different virus strains), which may contact the antibody at unknown amino acid positions other than those listed above. Different virus strains have different Env (i.e. antigen) sequences and different glycosylation patterns, and even a single Env sequence may have heterogeneous glycosylation patterns, requiring extensive binding or neutralizing antibodies to recognize different Env proteins of different HIV-1 variants or even different glycosylation patterns on the same Env protein. For example, the epitope for PGT121 consists of the Env V3 loop, in particular the N-linked glycan at position N332. The V3 loop is the primary determinant of cell tropism and viral clade. In the 117 CCR5 tropic viruses of various clades, the presence of a potential N-linked glycosylation (PNG) motif in the viral DNA sequence encoding the N332 glycan was significantly associated with susceptibility to PGT121 neutralization in viruses of clades B, G, a, AC and AE. Of the 50 clade B Env sequences isolated from patients enrolled in a girlidard (Gilead) -sponsored clinical trial, 94% of the CCR5 tropic Env with the N332 PNG motif were readily neutralized by PGT121, compared to only 26% of non-CCR 5 tropic, N332 PNG-positive viruses susceptible (P < 0.0001). Thus, genetic determination of Env clade, tropism and presence of the N332 PNG motif are highly predictive of susceptibility to neutralization by PGT121 and can be used as markers to predict viral susceptibility to neutralization by PGT121 and its derivatives.

The present disclosure provides variants of PGT-121LO6 antibodies. In certain embodiments, these variants have substantially the same or increased binding affinity for gp120 as compared to the PGT-121LO6 antibody. Binding affinity can be determined using any assay known in the art, including ELISA, SPR, BLI, or flow cytometry. In certain embodiments, these variants have increased binding affinity for FcRn at pH 6.0 as compared to the PGT-121LO6 antibody. In some embodiments, these variants have increased HIV-1 neutralization relative to the PGT-121LO6 antibody. In certain embodiments, the variant has reduced immunogenicity as compared to the PGT-121LO6 antibody. In certain embodiments, the Env region in or around the following residues (HIV Env HXB2 numbering) is implicated in predicting binding of a variant of the disclosure to an Env protein: the V3 loop (324-328, 330) and the associated N332 glycan and a portion of the V1-loop (135-137) and the associated N137 glycan, residues 415-417. Paratope prediction for Env binding involves residues in direct contact with antigen in the following regions (Kabat numbering): CDRH1 (33), CDRH2 (50, 56, 58), CDRH3 (99, 100A, 100B, 100C, 100D, 100E, 100G, 100I, 100L), CDRL1 (28, 29, 30), CDRL2 (50, 51, 52), LFR3 (66, 67A, 67B, and 67C), and CDRL3 (93, 94, 95A, 95B).

PGT-121LO6 heavy chain variable domain sequence (with Kabat numbering) (SEQ ID NO: 126)

PGT-121LO6 light chain variable domain sequence (with Kabat numbering; note VL terminates at position 107 (V); i.e., G108 is not part of VL) (SEQ ID NO: 127)

An exemplary variant of the PGT-121LO6 antibody is the PGT-121.60 antibody, the relevant sequence information of which is provided in table 2 below.

TABLE 2

Exemplary anti-gp 120 antibody 1

Exemplary anti-gp 120 antibody 1 is related to the PGT-121.60 antibody. Table 3 provides the relevant amino acid sequences of an exemplary anti-gp 120 antibody 1 (PGT 121.60 human IgG1 FEARLS/human λ).

TABLE 3

The anti-gp 120 antibody can comprise the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3 of the exemplary anti-gp 120 antibody 1. In one embodiment, the CDRs are defined based on the Kabat definition. In another embodiment, the CDRs are defined based on the Chothia definition. In particular embodiments, chothia is defined from Discovery Studio using Chothia and Lesk, J Mol biol.196 (4): 901-17 (1987) and Morea et al, methods,20:267-279 (2000). In another specific embodiment, the Chothia definition is based on Chothia from the Abysis definition. In another embodiment, the CDRs are defined based on IMGT definitions. In another embodiment, the CDRs are defined based on the honeyger definition. In another embodiment, the CDRs are defined based on the contact definition. In some cases, an anti-gp 120 antibody of the disclosure binds to a polypeptide comprising or consisting of SEQ ID NO:21, and a protein consisting of the amino acid sequence shown in the figure 21. In some cases, an anti-gp 120 antibody of the present disclosure binds to a polypeptide comprising or consisting of SEQ ID NO: 38. In some cases, the anti-gp 120 antibodies of the disclosure bind to free HIV-1 virus. In some cases, the anti-gp 120 antibodies of the disclosure bind to HIV-1 infected cells. In some cases, the anti-gp 120 antibodies of the disclosure bind to free HIV-1 virus and HIV-1 infected cells. In some cases, an anti-gp 120 antibody of the present disclosure binds to at least two different HIV-1 strains (e.g., group M, group N, group O, or group P). In one embodiment, the anti-gp 120 antibodies of the disclosure bind pwito.c/2474 (accession No. JN944948 and NIH AIDS Reagent Program catalog No. 11739). In another embodiment, an anti-gp 120 antibody of the disclosure binds to pch058.C/2960 (accession No. JN944940 and NIH AIDS Reagent Program catalog No. 700010058).

In certain instances, the anti-gp 120 antibody comprises an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable heavy chain of exemplary anti-gp 120 antibody 1. In some embodiments, the anti-gp 120 antibody comprises an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain of exemplary anti-gp 120 antibody 1. In certain instances, the anti-gp 120 antibody comprises an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable light chain of exemplary anti-gp 120 antibody 1. In certain instances, the anti-gp 120 antibody comprises an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the light chain of exemplary anti-gp 120 antibody 1. In certain embodiments, the anti-gp 120 antibody comprises an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable heavy and variable light chains of exemplary anti-gp 120 antibody 1. In some embodiments, the anti-gp 120 antibody comprises an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain of exemplary anti-gp 120 antibody 1 and comprises an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the light chain 1 of the exemplary anti-gp 120 antibody. In some cases, an anti-gp 120 antibody of the present disclosure binds to a polypeptide comprising or consisting of SEQ ID NO:21, or a pharmaceutically acceptable salt thereof. In some cases, an anti-gp 120 antibody of the disclosure binds to a polypeptide comprising SEQ ID NO:38 or a protein consisting of the amino acid sequence shown in seq id no. In some cases, the anti-gp 120 antibodies of the present disclosure bind to free HIV-1 virus. In some cases, an anti-gp 120 antibody of the disclosure binds to an HIV-1 infected cell. In some cases, the anti-gp 120 antibodies of the disclosure bind to free HIV-1 virus and HIV-1 infected cells. In some cases, an anti-gp 120 antibody of the present disclosure binds to at least two different HIV-1 strains (e.g., group M, group N, group O, or group P). In one embodiment, the anti-gp 120 antibodies of the disclosure bind pwito.c/2474 (accession No. JN944948 and NIH AIDS Reagent Program catalog No. 11739). In another embodiment, an anti-gp 120 antibody of the disclosure binds pch058.C/2960 (accession No. JN944940 and NIH AIDS Reagent Program catalog No. 700010058).

In some embodiments, the variable heavy chain of exemplary anti-gp 120 antibody 1 is linked to a heavy chain constant region comprising a CH1 domain and a hinge region. In some embodiments, the variable heavy chain of exemplary anti-gp 120 antibody 1 is linked to a heavy chain constant region comprising a CH3 domain. In certain embodiments, the variable heavy chain of exemplary anti-gp 120 antibody 1 is linked to a heavy chain constant region comprising a CH1 domain, a hinge region, and a CH2 domain and a CH3 domain from IgG4 (e.g., igG 1). In certain embodiments, the variable heavy chain of exemplary anti-gp 120 antibody 1 is linked to a heavy chain constant region comprising a CH1 domain, a CH2 domain, and a CH3 domain from an IgG1 (e.g., a human IgG1, e.g., an IgG1m3 allotype), and an IgG3 hinge region (e.g., an "open" IgG3 hinge variant designated "IgG 3C" in WO 2017/096221 (see, e.g., fig. 2A of the PCT publication)). This IgG3 hinge variant is expected to exhibit improved Fab arm flexibility and 200A span ⁰ Distance (sufficient for trimeric internal interactions). In certain embodiments, such chimeric antibodies comprise one or more additional mutations in the heavy chain constant region that increase the stability of the chimeric antibody. In certain embodiments, the heavy chain constant region comprises a substitution that alters a property of the antibody (e.g., reduces Fc receptor binding, increases or decreases antibody glycosylation, reduces binding to C1q, increases half-life, decreases effector function).

In certain embodiments, the anti-gp 120 antibody is an IgG antibody. In one embodiment, the antibody is an IgG1. In another embodiment, the antibody is an IgG2. In some embodiments, the antibody has a chimeric heavy chain constant region (e.g., having the CH1, hinge, and CH2 regions of IgG4 and the CH3 region of IgG 1).

IgG antibodies exist in various allotypes and allotypes (isoallotypes). In particular embodiments, the antibodies of the disclosure comprise an IgG1 heavy chain having an allotype G1m1, nG1m2, G1m3, G1m17,1,2, G1m3,1, or G1m 17. Each of these allotypes or heteroallotypes is characterized by the following amino acid residues at designated positions (EU numbering) in the IgG1 heavy chain constant region (Fc):

G1m1:D356,L358；

nG1m1:E356,M358；

G1m3:R214,E356,M358,A431；

G1m17,1:K214,D356,L358,A431；

G1m17,1,2:K214,D356,L358,G431；

g1m3, 1; and

G1m17:K214,E356,M358,A431。

in particular embodiments, the VH of exemplary anti-gp 120 antibody 1 is linked, either directly or by insertion of an amino acid sequence (e.g., G-S linker), to the wild-type IgG1m3 sequence provided below (representative allotype-determining residues are in bold type)

In certain embodiments, the VH of exemplary anti-gp 120 antibody 1 (e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:7 or having 0 to 5 (i.e., 1,2, 3, 4, or 5) amino acid substitutions (e.g., conservative substitutions in SEQ ID NO: 7)) is identical to a VH having SEQ ID NO: the mutated IgG1m3 sequences of 1 to 10 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions in 77 (e.g., substitutions that reduce effector function and/or increase half-life) are linked directly or by intervening amino acid sequences (e.g., G-S linkers). Exemplary amino acid substitutions are described later in this disclosure.

In certain embodiments, the anti-gp 120 antibody is a human IgG 1/human kappa antibody. In some embodiments, an antibody of the present disclosure comprises a kappa light chain having an allotype selected from Km1,2, or Km 3. Each of these allotypes is characterized by the following amino acid residues at the designated positions (EU numbering) within the IgG1 light chain:

Km1:V153,L191；

km1,2, a153, l191; and

Km3:A153,V191。

in certain embodiments, the antibodies of the present disclosure comprise an IgG1 kappa light chain comprising one of the following amino acid sequences, wherein representative allotype-determining residues are indicated in bold:

Km1:

Km1,2:

or

Km3:

In certain embodiments, the anti-gp 120 antibody is a human IgG 1/human lambda antibody. Each human individual contains 7 to 11 different λ light chain genes encoding light chains selected from λ 1, λ 2, λ 3, λ 4, λ 5, λ 6 and λ 7. In particular embodiments, the antibodies of the present disclosure comprise a λ light chain selected from λ 1, λ 2, λ 3, λ 4, λ 5, λ 6, and λ 7. In particular embodiments, the antibodies described herein comprise a lambda light chain comprising one of the following amino acid sequences, wherein representative lambda determining residues are represented in bold:

λ1:

λ2:

λ3:

or

λ7:

In particular embodiments, the VL of exemplary anti-gp 120 antibody 1 (e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:8, or having from 0 to 5 (i.e., 1,2, 3, 4, or 5) amino acid substitutions (e.g., conservative substitutions in SEQ ID NO: 8)) is linked directly to a wild-type human λ 2 sequence (SEQ ID NO: 89) or via an intervening amino acid sequence (e.g., a G-S linker). In certain embodiments, the VL of exemplary anti-gp 120 antibody 1 is identical to a VL having SEQ ID NO:89 (i.e., 1,2, 3, 4, 5) of the substituted mutant human λ 2 sequences are linked directly or by insertion of an amino acid sequence (e.g., a G-S linker).

In a particular embodiment, the anti-gp 120 antibody is a human IgG1m 3/human λ 2 antibody.

For example, an antibody such as exemplary anti-gp 120 antibody 1 can be prepared by preparing and expressing a nucleic acid encoding an antibody amino acid sequence.

Exemplary anti-gp 120 antibody 2

Another exemplary anti-gp 120 antibody, exemplary anti-gp 120 antibody 2, has the same six CDRs as exemplary anti-gp 120 antibody 1. The antibody comprises a heavy chain variable region comprising SEQ ID NO:7 or a VH sequence consisting of it, and a VL sequence comprising or consisting of the amino acid sequence set forth in seq id no:

in some cases, the VL is linked to the human λ constant region directly or by intervening amino acid sequences (e.g., a G-S linker).

An exemplary anti-gp 120 antibody 2 comprises a heavy chain comprising SEQ ID NO:9 and a light chain comprising or consisting of the amino acid sequence set forth in seq id no:

in some cases, an anti-gp 120 antibody of the present disclosure binds to a polypeptide comprising or consisting of SEQ ID NO:21, and a protein consisting of the amino acid sequence shown in the figure 21. In some cases, an anti-gp 120 antibody of the disclosure binds to a polypeptide comprising or consisting of SEQ ID NO: 38. In some cases, the anti-gp 120 antibodies of the disclosure bind to free HIV-1 virus. In some cases, an anti-gp 120 antibody of the disclosure binds to an HIV-1 infected cell. In some cases, the anti-gp 120 antibodies of the disclosure bind to free HIV-1 virus and HIV-1 infected cells. In some cases, an anti-gp 120 antibody of the present disclosure binds to at least two different HIV-1 strains (e.g., group M, group N, group O, or group P). In one embodiment, an anti-gp 120 antibody of the disclosure binds pwito.c/2474 (accession No. JN944948 and NIH AIDS Reagent Program catalog No. 11739). In another embodiment, an anti-gp 120 antibody of the disclosure binds to pch058.C/2960 (accession No. JN944940 and NIH AIDS Reagent Program catalog No. 700010058).

In some embodiments, the variable heavy chain of exemplary anti-gp 120 antibody 2 (e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:7 or having 0 to 5 (i.e., 1, 2, 3, 4, or 5) amino acid substitutions (e.g., conservative substitutions in SEQ ID NO: 7)) is linked to a heavy chain constant region comprising a CH1 domain and a hinge region. In some embodiments, the variable heavy chain of exemplary anti-gp 120 antibody 2 (e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:7 or having 0 to 5 (i.e., 1, 2, 3, 4, or 5) amino acid substitutions (e.g., conservative substitutions in SEQ ID NO: 7)) is linked to a heavy chain constant region comprising a CH3 domain. In certain embodiments, the variable heavy chain of exemplary anti-gp 120 antibody 2 (e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:7 or having from 0 to 5 (i.e., 1, 2, 3, 4, or 5) amino acid substitutions (e.g., conservative substitutions in SEQ ID NO: 7)) is linked to a heavy chain constant region comprising a CH1 domain, a hinge region, and CH2 and CH3 domains from IgG4 (e.g., from IgG 1). In certain embodiments, the variable heavy chain of the exemplary anti-gp 120 antibody 1 is linked to a heavy chain constant region comprising a CH1 domain, a CH2 domain, and a CH3 domain from an IgG1 (e.g., a human IgG1, e.g., an IgG1m3 allotype), and an IgG3 hinge region (e.g., an "open" IgG3 hinge variant "IgG 3C-" described in WO 2017/096221). In certain embodiments, such chimeric antibodies comprise one or more additional mutations in the heavy chain constant region that increase the stability of the chimeric antibody. In certain embodiments, the heavy chain constant region comprises a substitution that alters a property of the antibody (e.g., reduces Fc receptor binding, increases or decreases antibody glycosylation, reduces binding to C1q, increases half-life, decreases effector function).

In certain embodiments, the anti-gp 120 antibody is an IgG antibody. In one embodiment, the antibody is an IgG1. In another embodiment, the antibody is an IgG2. In some embodiments, the antibody has a chimeric heavy chain constant region (e.g., having the CH1, hinge, and CH2 regions of IgG4 and the CH3 region of IgG 1).

In particular embodiments, the antibodies of the disclosure comprise an IgG1 heavy chain having an allotype G1m1, nG1m2, G1m3, G1m17,1,2, G1m3,1, or G1m 17.

In certain embodiments, the anti-gp 120 antibody is a human IgG 1/human kappa antibody. In some embodiments, an antibody of the present disclosure comprises a kappa light chain having an allotype selected from Km1,2, or Km 3. In certain embodiments, the anti-gp 120 antibody is a human IgG 1/human lambda antibody. In some embodiments, an antibody of the present disclosure comprises a light chain selected from λ 1, λ 2, λ 3, λ 4, λ 5, λ 6, and λ 7.

In a particular embodiment, the anti-gp 120 antibody is a human IgG1m 3/human λ 2 antibody.

In some embodiments, the VH of exemplary anti-gp 120 antibody 2 (e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:7, or having from 0 to 5 (i.e., 1,2, 3, 4, or 5) amino acid substitutions (e.g., conservative substitutions within SEQ ID NO: 7)) is linked to a wild-type IgG1m3 sequence (SEQ ID NO: 77) either directly or through an intervening amino acid sequence (e.g., a G-S linker). In certain embodiments, the VH of exemplary anti-gp 120 antibody 2 (e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:7, or having from 0 to 5 (i.e., 1,2, 3, 4, or 5) amino acid substitutions (e.g., conservative substitutions in SEQ ID NO: 7)) is identical to a VH having SEQ ID NO: 1 to 10 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions in 77 (e.g., substitutions that reduce effector function and/or increase half-life) are linked directly or by insertion of an amino acid sequence (e.g., a G-S linker). Exemplary amino acid substitutions are described later in this disclosure.

In some embodiments, the VL of exemplary anti-gp 120 antibody 2 (e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:81, or having from 0 to 5 (i.e., 1, 2, 3, 4, or 5) amino acid substitutions (e.g., a conservative substitution in SEQ ID NO: 81)) is linked directly to a wild-type human λ 2 sequence (SEQ ID NO: 89) or via an intervening amino acid sequence (e.g., a G-S linker). In certain embodiments, the VL of exemplary anti-gp 120 antibody 2 (e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:81, or having from 0 to 5 (i.e., 1, 2, 3, 4, or 5) amino acid substitutions (e.g., a conservative substitution in SEQ ID NO: 81)) is identical to a VL having SEQ ID NO:89 (i.e., 1, 2, 3, 4, 5) of the substituted mutant human λ 2 sequences are linked directly or by insertion of an amino acid sequence (e.g., a G-S linker).

Exemplary anti-gp 120 antibody 2 can be used as a monospecific antibody or a multispecific antibody (e.g., bispecific antibody). The disclosure encompasses whole antibodies or antigen-binding fragments (e.g., fab, F (ab) 2, fv, scFv, sc (Fv) 2, diabodies).

Antibodies such as exemplary anti-gp 120 antibody 2 can be prepared, for example, by making and expressing nucleic acids encoding the amino acid sequences of the antibodies.

Exemplary anti-gp 120 antibody 3

Another exemplary anti-gp 120 antibody, exemplary anti-gp 120 antibody 3, has the same six CDRs as exemplary anti-gp 120 antibody 1. The antibody comprises a heavy chain variable region comprising SEQ ID NO:7 or a VH sequence consisting of it, and a VL sequence comprising or consisting of the amino acid sequence set forth in seq id no:

in some cases, the VL is linked to the human λ constant region directly or through an intervening amino acid sequence (e.g., a G-S linker).

Exemplary anti-gp 120 antibody 3 comprises a heavy chain variable region comprising SEQ ID NO:9 and a light chain comprising or consisting of the amino acid sequence set forth in seq id no:

in some cases, an anti-gp 120 antibody of the disclosure binds to a polypeptide comprising or consisting of SEQ ID NO:21, and a protein consisting of the amino acid sequence shown in the sequence table 21. In some cases, an anti-gp 120 antibody of the disclosure binds to a polypeptide comprising or consisting of SEQ ID NO: 38. In some cases, the anti-gp 120 antibodies of the disclosure bind to free HIV-1 virus. In some cases, an anti-gp 120 antibody of the disclosure binds to an HIV-1 infected cell. In some cases, the anti-gp 120 antibodies of the present disclosure bind to free HIV-1 virus and HIV-1 infected cells. In some cases, an anti-gp 120 antibody of the present disclosure binds to at least two different HIV-1 strains (e.g., group M, group N, group O, or group P). In one embodiment, the anti-gp 120 antibodies of the disclosure bind pwito.c/2474 (accession No. JN944948 and NIH AIDS Reagent Program catalog No. 11739). In another embodiment, an anti-gp 120 antibody of the disclosure binds to pch058.C/2960 (accession No. JN944940 and NIH AIDS Reagent Program catalog No. 700010058).

In some embodiments, the variable heavy chain of exemplary anti-gp 120 antibody 3 is linked to a heavy chain constant region comprising a CH1 domain and a hinge region. In some embodiments, the variable heavy chain of exemplary anti-gp 120 antibody 3 is linked to a heavy chain constant region comprising a CH3 domain. In certain embodiments, the variable heavy chain of exemplary anti-gp 120 antibody 3 is linked to a heavy chain constant region comprising a CH1 domain, a hinge region, and a CH2 domain from IgG4 and a CH3 domain (e.g., from IgG 1). In certain embodiments, the variable heavy chain of exemplary anti-gp 120 antibody 1 is linked to a heavy chain constant region comprising a CH1 domain, a CH2 domain, and a CH3 domain from an IgG1 (e.g., a human IgG1, e.g., an IgG1m3 allotype), and an IgG3 hinge region (e.g., an "open" IgG3 hinge variant "IgG 3C-" described in WO 2017/096221). In certain embodiments, such chimeric antibodies comprise one or more additional mutations in the heavy chain constant region that increase the stability of the chimeric antibody. In certain embodiments, the heavy chain constant region comprises a substitution that alters a property of the antibody (e.g., reduces Fc receptor binding, increases or decreases antibody glycosylation, reduces binding to C1q, increases half-life, decreases effector function).

In certain embodiments, the anti-gp 120 antibody is an IgG antibody. In one embodiment, the antibody is an IgG1. In another embodiment, the antibody is an IgG2. In some embodiments, the antibody has a chimeric heavy chain constant region (e.g., having CH1, hinge, and CH2 regions of IgG4 and CH3 regions of IgG 1).

In particular embodiments, the antibodies of the disclosure comprise an IgG1 heavy chain having an allotype G1m1, nG1m2, G1m3, G1m17,1,2, G1m3,1, or G1m 17.

In certain embodiments, the anti-gp 120 antibody is a human IgG 1/human kappa antibody. In some embodiments, an antibody of the present disclosure comprises a kappa light chain having an allotype selected from Km1,2, or Km 3. In certain embodiments, the anti-gp 120 antibody is a human IgG 1/human lambda antibody. In some embodiments, an antibody of the present disclosure comprises a light chain selected from λ 1, λ 2, λ 3, λ 4, λ 5, λ 6, and λ 7.

In a particular embodiment, the anti-gp 120 antibody is a human IgG1m 3/human λ 2 antibody.

In some embodiments, the VH of exemplary anti-gp 120 antibody 3 (e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:7, or having from 0 to 5 (i.e., 1,2, 3, 4, or 5) amino acid substitutions (e.g., conservative substitutions within SEQ ID NO: 7)) is linked directly to a wild-type IgG1m3 sequence (SEQ ID NO: 77) or by insertion of an amino acid sequence (e.g., a G-S linker). In certain embodiments, the VH (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:7, or having 0 to 5 (i.e., 1,2, 3, 4, or 5) amino acid substitutions (e.g., conservative substitutions in SEQ ID NO: 7) amino acid sequence) of exemplary anti-gp 120 antibody 3 is identical to a VH having SEQ ID NO: 1 to 10 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions in 77 (e.g., to reduce effector function and/or extend half-life) are linked directly or by intervening amino acid sequences (e.g., G-S linkers). Exemplary amino acid substitutions are described later in this disclosure.

In some embodiments, the VL of exemplary anti-gp 120 antibody 3 (e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:82, or having from 0 to 5 (i.e., 1, 2, 3, 4, or 5) amino acid substitutions (e.g., conservative substitutions in SEQ ID NO: 82)) is linked directly to a wild-type human λ 2 sequence (SEQ ID NO: 89) or via an intervening amino acid sequence (e.g., a G-S linker). In certain embodiments, the VL of exemplary anti-gp 120 antibody 3 (e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:82, or having from 0 to 5 (i.e., 1, 2, 3, 4 or 5) amino acid substitutions (e.g., conservative substitutions in SEQ ID NO: 82)) is identical to a VL having SEQ ID NO:89 (i.e., 1, 2, 3, 4, 5) substituted mutant human λ 2 sequences are linked directly or via an intervening amino acid sequence (e.g., a G-S linker).

The exemplary anti-gp 120 antibody 3 can be used as a monospecific antibody or a multispecific antibody (e.g., bispecific antibody). The present disclosure encompasses whole antibodies or antigen-binding fragments (e.g., fab, F (ab) 2, fv, scFv, sc (Fv) 2, diabodies).

For example, an antibody such as exemplary anti-gp 120 antibody 3 can be prepared by preparing and expressing a nucleic acid encoding an antibody amino acid sequence.

Exemplary anti-gp 120 antibody 4

Another exemplary anti-gp 120 antibody, exemplary anti-gp 120 antibody 4, has the same six CDRs as exemplary anti-gp 120 antibody 1. The antibody comprises a heavy chain variable region comprising SEQ ID NO:7 or a VH sequence consisting of it, and a VL sequence comprising or consisting of the amino acid sequence set forth in seq id no:

in some cases, the VL is linked to the human λ constant region directly or through intervening amino acid sequences (e.g., a G-S linker).

Exemplary anti-gp 120 antibody 4 comprises a heavy chain variable region comprising SEQ ID NO:9 and a light chain comprising or consisting of the amino acid sequence set forth in seq id no:

in some cases, an anti-gp 120 antibody of the present disclosure binds to a polypeptide comprising or consisting of SEQ ID NO:21, and a protein consisting of the amino acid sequence shown in the figure 21. In some cases, an anti-gp 120 antibody of the present disclosure binds to a polypeptide comprising or consisting of SEQ ID NO:38, or a pharmaceutically acceptable salt thereof. In some cases, the anti-gp 120 antibodies of the present disclosure bind to free HIV-1 virus. In some cases, an anti-gp 120 antibody of the disclosure binds to an HIV-1 infected cell. In some cases, the anti-gp 120 antibodies of the present disclosure bind to free HIV-1 virus and HIV-1 infected cells. In some cases, the anti-gp 120 antibodies of the disclosure bind to at least two different HIV-1 strains (e.g., group M, group N, group O, or group P). In one embodiment, an anti-gp 120 antibody of the disclosure binds pwito.c/2474 (accession No. JN944948 and NIH AIDS Reagent Program catalog No. 11739). In another embodiment, an anti-gp 120 antibody of the disclosure binds to pch058.C/2960 (accession No. JN944940 and NIH AIDS Reagent Program catalog No. 700010058).

In some embodiments, the variable heavy chain of exemplary anti-gp 120 antibody 4 is linked to a heavy chain constant region comprising a CH1 domain and a hinge region. In some embodiments, the variable heavy chain of the exemplary anti-gp 120 antibody 4 is linked to a heavy chain constant region comprising a CH3 domain. In certain embodiments, the variable heavy chain of exemplary anti-gp 120 antibody 4 is linked to a heavy chain constant region comprising a CH1 domain, a hinge region, and CH2 and CH3 domains from IgG4 (e.g., from IgG 1). In certain embodiments, the variable heavy chain of exemplary anti-gp 120 antibody 1 is linked to a heavy chain constant region comprising a CH1 domain, a CH2 domain, and a CH3 domain from an IgG1 (e.g., a human IgG1, e.g., an IgG1m3 allotype), and an IgG3 hinge region (e.g., an "open" IgG3 hinge variant "IgG 3C-" described in WO 2017/096221). In certain embodiments, such chimeric antibodies comprise one or more additional mutations in the heavy chain constant region that increase the stability of the chimeric antibody. In certain embodiments, the heavy chain constant region comprises a substitution that alters a property of the antibody (e.g., reduces Fc receptor binding, increases or decreases antibody glycosylation, decreases binding to C1q, increases half-life, decreases effector function).

In certain embodiments, the anti-gp 120 antibody is an IgG antibody. In one embodiment, the antibody is an IgG1. In another embodiment, the antibody is an IgG2. In some embodiments, the antibody has a chimeric heavy chain constant region (e.g., having CH1, hinge, and CH2 regions of IgG4 and CH3 regions of IgG 1).

In particular embodiments, the antibodies of the disclosure comprise an IgG1 heavy chain having an allotype G1m1, nG1m2, G1m3, G1m17,1,2, G1m3,1, or G1m 17.

In certain embodiments, the anti-gp 120 antibody is a human IgG 1/human kappa antibody. In some embodiments, an antibody of the present disclosure comprises a kappa light chain having an allotype selected from Km1,2, or Km 3. In certain embodiments, the anti-gp 120 antibody is a human IgG 1/human lambda antibody. In some embodiments, an antibody of the present disclosure comprises a λ light chain selected from λ 1, λ 2, λ 3, λ 4, λ 5, λ 6, and λ 7.

In some embodiments, the VH of exemplary anti-gp 120 antibody 4 (e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:7, or having from 0 to 5 (i.e., 1,2, 3, 4, or 5) amino acid substitutions (e.g., conservative substitutions within SEQ ID NO: 7)) is linked directly to a wild-type IgG1m3 sequence (SEQ ID NO: 77) or by an intervening amino acid sequence (e.g., a G-S linker). In certain embodiments, the VH (e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID No. 7, or having from 0 to 5 (i.e., 1,2, 3, 4, or 5) amino acid substitutions (e.g., a conservative substitution in SEQ ID No. 7)) of exemplary anti-gp 120 antibody 4 is identical to a VH having SEQ ID NO: the mutated IgG1m3 sequences with 1 to 10 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions in 77 (e.g., to reduce effector function and/or increase half-life) are linked directly or via an intervening amino acid sequence (e.g., a G-S linker). Exemplary amino acid substitutions are described later in this disclosure.

In some embodiments, the VL of exemplary anti-gp 120 antibody 4 (e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:83, or having from 0 to 5 (i.e., 1, 2, 3, 4, or 5) amino acid substitutions (e.g., a conservative substitution in SEQ ID NO: 83)) is linked directly to a wild-type human λ 2 sequence (SEQ ID NO: 89) or by insertion of an amino acid sequence (e.g., a G-S linker). In certain embodiments, the VL of exemplary anti-gp 120 antibody 4 (e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:83, or having from 0 to 5 (i.e., 1, 2, 3, 4 or 5) amino acid substitutions (e.g., a conservative substitution in SEQ ID NO: 83)) is identical to the VL of SEQ ID NO:89 with 1 to 5 (i.e., 1, 2, 3, 4, 5) substitutions are linked directly or via an intervening amino acid sequence (e.g., a G-S linker).

In a particular embodiment, the anti-gp 120 antibody is a human IgG1m 3/human λ 2 antibody.

Exemplary anti-gp 120 antibody 4 can be used as a monospecific antibody or a multispecific antibody (e.g., bispecific antibody). Whole antibodies or antigen-binding fragments (e.g., fab, F (ab) 2, fv, scFv, sc (Fv) 2, diabodies) can be used.

For example, an antibody such as exemplary anti-gp 120 antibody 4 can be made by making and expressing a nucleic acid encoding the amino acid sequence of the antibody.

Exemplary anti-gp 120 antibody 5

Another exemplary anti-gp 120 antibody, exemplary anti-gp 120 antibody 5, has the same six CDRs as exemplary anti-gp 120 antibody 1. The antibody comprises a heavy chain variable region comprising SEQ ID NO:7 or a VH sequence consisting of it, and a VL sequence comprising or consisting of the amino acid sequence set forth in seq id no:

in some cases, the VL is linked to the human λ constant region directly or through an intervening amino acid sequence (e.g., a G-S linker).

An exemplary anti-gp 120 antibody 5 comprises a heavy chain variable region comprising SEQ ID NO:9 or a heavy chain consisting of the amino acid sequence shown below and a light chain comprising or consisting of the amino acid sequence shown below

In some cases, an anti-gp 120 antibody of the present disclosure binds to a polypeptide comprising or consisting of SEQ ID NO:21, and a protein consisting of the amino acid sequence shown in the figure 21. In some cases, an anti-gp 120 antibody of the disclosure binds to a polypeptide comprising or consisting of SEQ ID NO: 38. In some cases, the anti-gp 120 antibodies of the disclosure bind to free HIV-1 virus. In some cases, the anti-gp 120 antibodies of the disclosure bind to HIV-1 infected cells. In some cases, the anti-gp 120 antibodies of the disclosure bind to free HIV-1 virus and HIV-1 infected cells. In some cases, an anti-gp 120 antibody of the present disclosure binds to at least two different HIV-1 strains (e.g., group M, group N, group O, or group P). In one embodiment, an anti-gp 120 antibody of the disclosure binds pwito.c/2474 (accession No. JN944948 and NIH AIDS Reagent Program catalog No. 11739). In another embodiment, an anti-gp 120 antibody of the disclosure binds to pch058.C/2960 (accession No. JN944940 and NIH AIDS Reagent Program catalog No. 700010058).

In some embodiments, the variable heavy chain of exemplary anti-gp 120 antibody 5 is linked to a heavy chain constant region comprising a CH1 domain and a hinge region. In some embodiments, the variable heavy chain of exemplary anti-gp 120 antibody 5 is linked to a heavy chain constant region comprising a CH3 domain. In certain embodiments, the variable heavy chain of exemplary anti-gp 120 antibody 5 is linked to a heavy chain constant region comprising a CH1 domain, a hinge region, and CH2 and CH3 domains from IgG4 (e.g., from IgG 1). In certain embodiments, the variable heavy chain of the exemplary anti-gp 120 antibody 1 is linked to a heavy chain constant region comprising a CH1 domain, a CH2 domain, and a CH3 domain from IgG1 (e.g., human IgG1, e.g., igG1m3 allotype) and an IgG3 hinge region (e.g., the "open" IgG3 hinge variant "IgG 3C-" described in WO 2017/096221). In certain embodiments, such chimeric antibodies comprise one or more additional mutations in the heavy chain constant region that increase the stability of the chimeric antibody. In certain embodiments, the heavy chain constant region comprises a substitution that alters a property of the antibody (e.g., reduces Fc receptor binding, increases or decreases antibody glycosylation, decreases binding to C1q, increases half-life, decreases effector function).

In certain embodiments, the anti-gp 120 antibody is an IgG antibody. In one embodiment, the antibody is an IgG1. In another embodiment, the antibody is an IgG2. In some embodiments, the antibody has a chimeric heavy chain constant region (e.g., having CH1, hinge, and CH2 regions of IgG4 and CH3 regions of IgG 1).

In particular embodiments, the antibodies of the disclosure comprise an IgG1 heavy chain having an allotype G1m1, nG1m2, G1m3, G1m17,1,2, G1m3,1, or G1m 17.

In certain embodiments, the anti-gp 120 antibody is a human IgG 1/human kappa antibody. In some embodiments, an antibody of the present disclosure comprises a kappa light chain having an allotype selected from Km1,2, or Km 3. In certain embodiments, the anti-gp 120 antibody is a human IgG 1/human lambda antibody. In some embodiments, an antibody of the present disclosure comprises a λ light chain selected from λ 1, λ 2, λ 3, λ 4, λ 5, λ 6, and λ 7.

In some embodiments, the VH of exemplary anti-gp 120 antibody 5 (e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:7, or having from 0 to 5 (i.e., 1,2, 3, 4, or 5) amino acid substitutions (e.g., conservative substitutions within SEQ ID NO: 7)) is linked directly to a wild-type IgG1m3 sequence (SEQ ID NO: 77) or via an intervening amino acid sequence (e.g., a G-S linker). In certain embodiments, the VH (e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID No. 7, or having from 0 to 5 (i.e., 1,2, 3, 4, or 5) amino acid substitutions (e.g., conservative substitutions in SEQ ID No. 7)) of exemplary anti-gp 120 antibody 5 is identical to a VH having the amino acid sequence of SEQ ID NO: 1 to 10 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions in 77 (e.g., to reduce effector function and/or extend half-life) are linked directly or by insertion of an amino acid sequence (e.g., a G-S linker). Exemplary amino acid substitutions are described later in this disclosure.

In some embodiments, the VL of exemplary anti-gp 120 antibody 5 (e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:84, or having from 0 to 5 (i.e., 1, 2, 3, 4, or 5) amino acid substitutions (e.g., a conservative substitution in SEQ ID NO: 84)) is linked directly to a wild-type human λ 2 sequence (SEQ ID NO: 89) or by insertion of an amino acid sequence (e.g., a G-S linker). In certain embodiments, the VL of exemplary anti-gp 120 antibody 5 (e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:84, or having from 0 to 5 (i.e., 1, 2, 3, 4, or 5) amino acid substitutions (e.g., a conservative substitution in SEQ ID NO: 84)) is identical to the VL of SEQ ID NO:89 with 1 to 5 (i.e., 1, 2, 3, 4, 5) substitutions are linked directly or via an intervening amino acid sequence (e.g., a G-S linker).

In a particular embodiment, the anti-gp 120 antibody is a human IgG1m 3/human λ 2 antibody.

Exemplary anti-gp 120 antibody 5 can be used as a monospecific antibody or a multispecific antibody (e.g., bispecific antibody). The present disclosure encompasses whole antibodies or antigen-binding fragments (e.g., fab, F (ab) 2, fv, scFv, sc (Fv) 2, diabodies).

Antibodies such as exemplary anti-gp 120 antibody 5 can be prepared, for example, by preparing and expressing nucleic acids encoding the amino acid sequences of the antibodies.

CD3

Cluster of differentiation (CD 3) is a multimeric protein complex, which consists of four different polypeptide chains: epsilon lon (. Epsilon.), gamma (. Gamma.), delta (. Delta.), and zeta (. Zeta.), they assemble and function as three pairs of dimers (. Epsilon.gamma.,. Epsilon.delta., and. Zeta.). The CD3 protein has an N-terminal extracellular region, a transmembrane domain, and a cytoplasmic tail in which an Immunoreceptor Tyrosine Activation Motif (ITAM) is located. The extracellular domains of CD3 epsilon, gamma, and delta comprise immunoglobulin-like domains and are therefore considered to be part of the immunoglobulin superfamily. CD3/T cell co-receptor contributes to the activation of CD8 ⁺ T cell and CD4 ⁺ T cells.

The amino acid sequence of human CD3 epsilon can be found in unaprotkb-P07766 and is provided below (signal sequence underlined):

the amino acid sequence of human CD3 δ can be found in unaprotkb-P04234 and is provided below (signal sequence underlined):

antibodies that bind human CD3 are well known in the art (see, e.g., kuhn & Weiner, immunotherapy,8 (8): 889-906 (2016); WO 2015/104346). OKT3 (Muromab), an anti-CD 3 antibody directed against CD3 epsilon, has been approved clinically for use in humans to induce immunosuppression in solid organ transplants to prevent and treat rejection (Norman, therapeutic Drug Monitoring,17, 615-620 (1995)). Teplizumab, also known as hOKT 3. Gamma.1 (Ala-Ala) and MGA031, is a humanized IgG1 antibody developed by grafting the complementarity determining regions of OKT3 into the human IgG1 backbone. Introduction of two point mutations into the Fc portion thereof reduces FcR binding. Otelixizumab (chaclycd 3, TRX4, GSK 2136525) was derived from the rat antibody YTH12.5. The humanized IgG1 carries a single mutation in the γ 1Fc portion to avoid glycosylation and thus inhibit FcR binding. Visilizumab (Nuvion, huM 291) is a humanized IgG2 antibody that is made non-mitogenic by two point mutations in its Fc region. Foralumab (28F 11-AE; NI-0401) is a fully human anti-CD 3 mAb; the Fc portion of this human IgG1 was mutated so that the mAb was non-FcR binding in vitro and showed only a small amount of cytokine release in vivo while maintaining CD3/TCR modulation and T cell depletion.

Non-limiting examples of anti-CD 3 antibodies are also disclosed in US 2016/0333095 A1.

In certain embodiments, the anti-CD 3 antibodies of the present disclosure bind to human CD3. In some cases, an anti-CD 3 antibody of the present disclosure binds human CD3 epsilon. In other embodiments, the anti-CD 3 antibodies of the present disclosure bind to human CD3 δ.

Exemplary anti-CD 3 antibody 1

Relevant sequence information for exemplary anti-CD 3 antibody 1 is provided in table 4.

TABLE 4

The anti-CD 3 antibody can include heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 of exemplary anti-CD 3 antibody 1. In one embodiment, the CDRs are defined based on the Kabat definition. In another embodiment, the CDRs are defined based on the Chothia definition. In another embodiment, the CDRs are defined based on IMGT definitions. In another embodiment, the CDRs are defined based on the honeygger definition. In another embodiment, the CDRs are based on Chothia definitions from the Abysis definition. In another embodiment, the CDR based on Chothia/AbM CDR definition. In another embodiment, the CDRs are defined based on contact definitions. For example, these CDRs can be determined using the abbsis database (www. Biooil. Org. Uk/analysis/sequence _ input/key _ annotation. Cgi).

In certain instances, an anti-CD 3 antibody comprises an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable heavy chain of exemplary anti-CD 3 antibody 1. In some embodiments, the anti-CD 3 antibody comprises an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain of exemplary anti-CD 3 antibody 1. In some cases, an anti-CD 3 antibody comprises an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable light chain of exemplary anti-CD 3 antibody 1. In some cases, an anti-CD 3 antibody comprises an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the light chain of exemplary anti-CD 3 antibody 1. In certain embodiments, the anti-CD 3 antibody comprises an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable heavy and variable light chains of exemplary anti-CD 3 antibody 1. In some embodiments, the anti-CD 3 antibody comprises an amino acid sequence at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain of exemplary anti-CD 3 antibody 1 and comprises an amino acid sequence at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the light chain of exemplary anti-CD 3 antibody 1.

In some embodiments, the variable heavy chain of exemplary anti-CD 3 antibody 1 is linked to a heavy chain constant region comprising a CH1 domain and a hinge region. In some embodiments, the variable heavy chain of exemplary anti-CD 3 antibody 1 is linked to a heavy chain constant region comprising a CH3 domain. In certain embodiments, the variable heavy chain of exemplary anti-CD 3 antibody 1 is linked to a heavy chain constant region comprising a CH1 domain, a hinge region, and CH2 domain from IgG4 and a CH3 domain (e.g., from IgG 1). In certain embodiments, the variable heavy chain of exemplary anti-gp 120 antibody 1 is linked to a heavy chain constant region comprising a CH1 domain, a CH2 domain, and a CH3 domain from an IgG1 (e.g., a human IgG1, e.g., an IgG1m3 allotype), and an IgG3 hinge region (e.g., an "open" IgG3 hinge variant "IgG 3C-" described in WO 2017/096221). In certain embodiments, such chimeric antibodies comprise one or more additional mutations in the heavy chain constant region that increase the stability of the chimeric antibody. In certain embodiments, the heavy chain constant region comprises a substitution that alters a property of the antibody (e.g., reduces Fc receptor binding, increases or decreases antibody glycosylation, decreases binding to C1q, increases half-life, decreases effector function).

In certain embodiments, the anti-CD 3 antibody is an IgG antibody. In one embodiment, the antibody is an IgG1. In another embodiment, the antibody is an IgG2. In some embodiments, the antibody has a chimeric heavy chain constant region (e.g., having the CH1, hinge, and CH2 regions of IgG4 and the CH3 region of IgG 1).

In particular embodiments, the antibodies of the disclosure comprise an IgG1 heavy chain having an allotype G1m1, nG1m2, G1m3, G1m17,1,2, G1m3,1, or G1m 17.

In a specific embodiment, the VH of exemplary anti-CD 3 antibody 1 is linked to wild-type IgG1m3 Fc (SEQ ID NO: 77). In certain instances, the VH of exemplary anti-CD 3 antibody 1 is linked to a mutant IgG1m3 sequence having 1 to 10 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions in SEQ ID NO:77 (e.g., reducing effector function and/or increasing half-life). Exemplary amino acid substitutions are described below.

In a specific embodiment, the VL of exemplary anti-CD 3 antibody 1 is linked to a human λ 2 sequence (SEQ ID NO: 89). In some cases, the VL of anti-CD 3 antibody 1 is identical to the VL having SEQ ID NO:89 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions in a human λ 2 sequence. Such amino acid substitutions are described below.

In certain embodiments, the anti-CD 3 antibody is a human IgG 1/human λ antibody.

For example, an antibody such as exemplary anti-CD 3 antibody 1 can be prepared by preparing and expressing a nucleic acid encoding the amino acid sequence of the antibody.

CD89

CD89 (cluster of differentiation 89), also known as the Fc fragment of the IgA receptor (FCAR), is the transmembrane receptor Fc α RI. Fc α RI binds to the heavy chain constant region of immunoglobulin a (IgA) antibodies. Fc α RI is expressed on the cell surface of myeloid lineage cells (including neutrophils, monocytes, macrophages, and eosinophils).

The amino acid sequence of human CD89 from UniProtKB-P24071 is provided below:

antibodies that bind human CD89 are well known in the art (see, e.g., fishwild et al, nature Biotechnol.,14 (7): 845-851 (1996); duval et al, J.Virol.,82 (9): 4671-4674 (2008); US 2003/0082643). In some embodiments, the anti-CD 89 antibody is one of 14.1, 7.4, or 8.2 (also referred to as 14A8, 7F12, and 8D2, respectively). Any of these antibodies or variants thereof can be used in the multispecific antibodies disclosed herein.

In certain embodiments, an anti-CD 89 antibody of the present disclosure or a multispecific antibody disclosed herein binds to a polypeptide comprising SEQ ID NO:95 or a polypeptide consisting thereof.

Exemplary anti-CD 89 antibody 1

The following table provides relevant sequence information for exemplary anti-CD 89 antibody 1.

The anti-CD 89 antibody can include the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3 of exemplary anti-CD 89 antibody 1. In one embodiment, the CDRs are defined based on the Kabat definition. In another embodiment, the CDRs are defined based on the Chothia definition. In another embodiment, the CDRs are defined based on IMGT definitions. In another embodiment, the CDRs are defined based on the honeygger definition. In another embodiment, the CDRs are defined based on Chothia from the Abysis definition. In another embodiment, the CDRs are defined based on Chothia/AbM CDRs. In another embodiment, the CDRs are defined based on contact definitions. For example, these CDRs can be determined using the abbis database (www. Bio. Org. Uk/analysis/sequence _ input/key _ annotation.

In certain instances, the anti-CD 89 antibody comprises an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable heavy chain of exemplary anti-CD 89 antibody 1. In some embodiments, the anti-CD 89 antibody comprises an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain of exemplary anti-CD 89 antibody 1. In some cases, the anti-CD 89 antibody comprises an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the light chain of exemplary anti-CD 89 antibody 1. In certain instances, the anti-CD 89 antibody comprises an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an exemplary anti-CD 89 antibody 1 light chain. In certain embodiments, the anti-CD 89 antibody comprises an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the variable heavy and variable light chains of exemplary anti-CD 89 antibody 1. In some embodiments, the anti-CD 89 antibody comprises an amino acid sequence at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain of exemplary anti-CD 89 antibody 1 and comprises an amino acid sequence at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the light chain of exemplary anti-CD 89 antibody 1.

In some embodiments, the variable heavy chain of exemplary anti-CD 89 antibody 1 is linked to a heavy chain constant region comprising a CH1 domain and a hinge region. In some embodiments, the variable heavy chain of exemplary anti-CD 89 antibody 1 is linked to a heavy chain constant region comprising a CH3 domain. In certain embodiments, the variable heavy chain of exemplary anti-CD 89 antibody 1 is linked to a heavy chain constant region comprising a CH1 domain, a hinge region, and CH2 and CH3 domains from IgG4 (e.g., from IgG 1). In certain embodiments, the variable heavy chain of exemplary anti-CD 89 antibody 1 is linked to a heavy chain constant region comprising a CH1 domain, a CH2 domain, and a CH3 domain from an IgG1 (e.g., a human IgG1, e.g., an IgG1m3 allotype), and an IgG3 hinge region (e.g., an "open" IgG3 hinge variant "IgG 3C-" described in WO 2017/096221). In certain embodiments, such chimeric antibodies comprise one or more additional mutations in the heavy chain constant region that increase the stability of the chimeric antibody. In certain embodiments, the heavy chain constant region comprises a substitution that alters a property of the antibody (e.g., reduces Fc receptor binding, increases or decreases antibody glycosylation, decreases binding to C1q, increases half-life, decreases effector function).

In certain embodiments, the anti-CD 89 antibody is an IgG antibody. In one embodiment, the antibody is an IgG1. In another embodiment, the antibody is an IgG2. In some embodiments, the antibody has a chimeric heavy chain constant region (e.g., having the CH1, hinge, and CH2 regions of IgG4 and the CH3 region of IgG 1).

In particular embodiments, the antibodies of the disclosure comprise an IgG1 heavy chain having an allotype G1m1, nG1m2, G1m3, G1m17,1,2, G1m3,1, or G1m 17.

In a specific embodiment, the VH of exemplary anti-CD 89 antibody 1 is linked to a wild-type IgG1m3 Fc (SEQ ID NO: 77). In certain instances, the VH of exemplary anti-CD 89 antibody 1 is identical to the VH having SEQ ID NO: 1 to 10 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions in 77 (e.g., to reduce effector function and/or increase half-life) are linked to the mutant IgG1m3 sequence. Exemplary amino acid substitutions are described below.

In a specific embodiment, the VL of exemplary anti-CD 89 antibody 1 is linked to a human λ 2 sequence (SEQ ID NO: 89). In certain instances, the VL of exemplary anti-CD 89 antibody 1 is identical to the VL having SEQ ID NO:89 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions in a human λ 2 sequence. Such amino acid substitutions are described below.

In certain embodiments, the anti-CD 89 antibody is a human IgG 1/human λ antibody.

For example, an antibody such as exemplary anti-CD 89 antibody 1 can be prepared by preparing and expressing a nucleic acid encoding the amino acid sequence of the antibody.

Multispecific antibodies

In another aspect, the disclosure features a multispecific antibody. Multispecific antibodies are antibodies that bind two or more different epitopes (e.g., bispecific antibodies, trivalent antibodies, tetravalent antibodies). The anti-gp 120 and anti-CD 3 antibodies or anti-gp 120 and anti-CD 89 antibodies described above may be included as part of a multispecific antibody. The multispecific antibody may have a binding site for at least one other antigen or one other epitope that is not bound by the anti-gp 120 or anti-CD 3 (or anti-CD 89) antibody binding site of the multispecific antibody. The anti-gp 120/anti-CD 3 multispecific antibody or anti-gp 120/anti-CD 89 multispecific antibody may comprise a dimerization domain andthree or more (e.g., three, four, five, six) antigen binding sites. An exemplary dimerization domain comprises (or consists of) an Fc region. The anti-gp 120/anti-CD 3 multispecific antibody or anti-gp 120/anti-CD 89 multispecific antibody may comprise (or consist of) three to about eight (i.e., three, four, five, six, seven, eight) antigen binding sites. The multispecific antibody optionally comprises at least one polypeptide chain (e.g., two polypeptide chains, three polypeptide chains), wherein the polypeptide chain comprises three or more variable domains. For example, the polypeptide chain can comprise, e.g., VD1- (X1) _n -VD2-(X2) _n Fc or VD1- (X1) _n -VD2-(X2) _n -VD3-(X3) _n -Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, VD3 is a third variable domain, fc is a polypeptide chain of an Fc region, X1, X2 and X3 represent amino acid or peptide spacers, and n is 0 or 1. In certain instances, the variable domains may each be an scFv. Multispecific antibodies can be readily produced by recombinant expression of nucleic acids encoding the polypeptide chains of the antibody.

Bispecific antibodies

In one aspect, the multispecific antibody is a bispecific antibody. Bispecific antibodies are antibodies that have binding specificities for two different epitopes. Bispecific antibodies have two "arms". One arm of the bispecific antibody binds to one epitope and the other arm binds to the other epitope. In one embodiment, one arm of the bispecific antibody binds a first antigen and the other arm of the bispecific antibody binds a second antigen. In another embodiment, the two arms of the bispecific antibody bind two different epitopes of the same antigen.

In one aspect, the disclosure features bispecific antibodies that specifically bind gp120 and specifically bind a second antigen (e.g., a trigger molecule on a leukocyte, such as a T cell receptor molecule (e.g., CD 3), or an Fc receptor of IgG (Fc γ R), such as Fc γ RI (CD 64), fc γ RII (CD 32), fc γ RIII (CD 16), or CD 89), thereby focusing and localizing cellular defense mechanisms to infected cells.

In particular embodiments, one arm of the bispecific antibody specifically binds gp120, while the other arm specifically binds CD3 (e.g., human CD3 epsilon, human CD3 delta)). In another specific embodiment, one arm of the bispecific antibody specifically binds gp120 and the other arm specifically binds CD89 (e.g., human CD 89). In certain embodiments, the arms of the bispecific antibody that binds gp120 comprise the six CDRs of the exemplary anti-gp 120 antibody 1. In some cases, the CDRs are defined according to Kabat. In other embodiments, the CDRs are defined according to Chothia. In still other embodiments, the CDRs are defined according to the IMGT definition. In still other embodiments, the CDRs are defined according to the honeyger definition. In certain embodiments, the arm of a bispecific antibody that binds gp120 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH (SEQ ID NO: 7) of exemplary anti-gp 120 antibody 1. In certain embodiments, the arm of a bispecific antibody that binds gp120 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL (SEQ ID NO: 8) of exemplary anti-gp 120 antibody 1. In certain embodiments, the arm of a bispecific antibody that binds gp120 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH (SEQ ID NO: 7) and VL (SEQ ID NO: 8), respectively, of exemplary anti-gp 120 antibody 1. In certain embodiments, the arm of a bispecific antibody that binds gp120 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH (SEQ ID NO: 7) and VL (SEQ ID NO: 81), respectively, of exemplary anti-gp 120 antibody 2. In certain embodiments, the arm of a bispecific antibody that binds gp120 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH (SEQ ID NO: 7) and VL (SEQ ID NO: 82), respectively, of exemplary anti-gp 120 antibody 3. In certain embodiments, the arm of a bispecific antibody that binds gp120 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH (SEQ ID NO: 7) and VL (SEQ ID NO: 83), respectively, of exemplary anti-gp 120 antibody 4. In certain embodiments, the arm of a bispecific antibody that binds gp120 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH (SEQ ID NO: 7) and VL (SEQ ID NO: 84), respectively, of exemplary anti-gp 120 antibody 5. In certain embodiments, the arm of a bispecific antibody that binds gp120 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain of exemplary anti-gp 120 antibody 1 (SEQ ID NO: 9). In certain embodiments, the arm of a bispecific antibody that binds gp120 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the light chain of an exemplary anti-gp 120 antibody (SEQ ID NO: 10). In certain embodiments, the arm of a bispecific antibody that binds gp120 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the heavy chain (SEQ ID NO: 9) and light chain (SEQ ID NO: 10), respectively, of exemplary anti-gp 120 antibody 1. In certain instances, the arm of a bispecific antibody that binds gp120 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain (SEQ ID NO: 9) and light chain (SEQ ID NO: 40), respectively, of exemplary anti-gp 120 antibody 2. In certain instances, the arm of a bispecific antibody that binds gp120 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain (SEQ ID NO: 9) and light chain (SEQ ID NO: 78), respectively, of exemplary anti-gp 120 antibody 3. In certain instances, the arm of a bispecific antibody that binds gp120 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain (SEQ ID NO: 9) and light chain (SEQ ID NO: 79), respectively, of exemplary anti-gp 120 antibody 4. In certain instances, the arm of a bispecific antibody that binds gp120 comprises an amino acid sequence that is at least 75%, 80%, identical to 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain (SEQ ID NO: 9) and light chain (SEQ ID NO: 80), respectively, of exemplary anti-gp 120 antibody 5. In one embodiment, the arm of the bispecific antibody that binds gp120 comprises SEQ ID NO:7 and SEQ ID NO: 8. In another embodiment, the arm of the bispecific antibody that binds gp120 comprises SEQ ID NO:7 and SEQ ID NO:81, or a pharmaceutically acceptable salt thereof. In another embodiment, the arm of the bispecific antibody that binds gp120 comprises SEQ ID NO:7 and SEQ ID NO: 82. In yet another embodiment, the arm of the bispecific antibody that binds gp120 comprises SEQ ID NO:7 and SEQ ID NO:83 of the sequence listing. In a further embodiment, the arm of the bispecific antibody that binds gp120 comprises SEQ ID NO:7 and SEQ ID NO: 84. In a particular embodiment, the arm of the bispecific antibody that binds gp120 comprises SEQ ID NO:9 and SEQ ID NO: 10. In another specific embodiment, the arm of the bispecific antibody that binds gp120 comprises SEQ ID NO:9 and SEQ ID NO: 40. In yet another specific embodiment, the arm of the bispecific antibody that binds gp120 comprises SEQ ID NO:9 and SEQ ID NO:78, or a pharmaceutically acceptable salt thereof. In a further embodiment, the arm of the bispecific antibody that binds gp120 comprises SEQ ID NO:9 and SEQ ID NO: 79. In another embodiment, the arm of the bispecific antibody that binds gp120 comprises SEQ ID NO:9 and SEQ ID NO: 80. In some cases, the arm of a bispecific antibody that binds gp120 binds to a polypeptide comprising or consisting of SEQ ID NO:21, and a protein consisting of the amino acid sequence shown in the figure 21. In some cases, the arm of a bispecific antibody that binds gp120 binds to a polypeptide comprising or consisting of SEQ ID NO:38, or a pharmaceutically acceptable salt thereof. In some cases, the arms of the bispecific antibody that bind gp120 bind free HIV-1 virus. In some cases, arms of bispecific antibodies that bind gp120 bind to HIV-1 infected cells. In some cases, arms of bispecific antibodies that bind gp120 bind to free HIV-1 virus and HIV-1 infected cells. In some cases, the arms of a bispecific antibody that binds gp120 bind to at least two HIV-1 strains (e.g., group M, group N, group O, or group P). In one embodiment, the arms of the bispecific antibody that binds gp120 bind pwito. C/2474 (accession No. JN944948 and NIH AIDS Reagent Program catalog No. 11739). In another embodiment, the arms of the bispecific antibody that binds gp120 bind pch058.C/2960 (accession No. JN944940 and NIH AIDS Reagent Program catalog No. 700010058).

In certain embodiments, the arms of a bispecific antibody that binds human CD3 comprise the six CDRs of exemplary anti-human CD3 antibody 1. In some cases, the CDRs are according to the Kabat definition. In other embodiments, the CDRs are defined according to Chothia. In still other embodiments, the CDRs are defined according to the IMGT definition. In still other embodiments, the CDRs are defined according to the honeyger definition. In certain embodiments, the arm of a bispecific antibody that binds human CD3 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH (SEQ ID NO: 17) of exemplary anti-CD 3 antibody 1. In certain embodiments, the arm of a bispecific antibody that binds human CD3 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL (SEQ ID NO: 18) of exemplary anti-CD 3 antibody 1. In certain embodiments, the arm of a bispecific antibody that binds human CD3 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH (SEQ ID NO: 17) and VL (SEQ ID NO: 18), respectively, of exemplary anti-human CD3 antibody 1. In certain embodiments, the arm of a bispecific antibody that binds human CD3 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain of exemplary anti-human CD3 antibody 1 (SEQ ID NO: 19). In certain embodiments, the arm of a bispecific antibody that binds CD3 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the light chain of exemplary anti-human CD3 antibody 1 (SEQ ID NO: 20). In certain embodiments, the arm of a bispecific antibody that binds human CD3 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain (SEQ ID NO: 19) and light chain (SEQ ID NO: 20), respectively, of exemplary anti-human CD3 antibody 1. In a particular embodiment, the arm of the bispecific antibody that binds human CD3 comprises SEQ ID NO:17 and SEQ ID NO: 18. In another specific embodiment, the arm of the bispecific antibody that binds human CD3 comprises SEQ ID NO:19 and SEQ ID NO: 20. In certain embodiments, the arms of a bispecific antibody that binds human CD3 bind human CD3 epsilon.

In certain embodiments, the arm of a bispecific antibody that binds human CD89 comprises the six CDRs of exemplary anti-human CD89 antibody 1. In some cases, the CD is according to Kabat. In other embodiments, the CDRs are defined according to Chothia. In still other embodiments, the CDRs are defined according to the IMGT definition. In still other embodiments, the CDRs are defined according to the honeyger definition. In certain embodiments, the arm of a bispecific antibody that binds human CD89 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH (SEQ ID NO: 96) of exemplary anti-CD 89 antibody 1. In certain embodiments, the arm of a bispecific antibody that binds human CD89 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL of exemplary anti-CD 89 antibody 1 (SEQ ID NO: 101). In certain embodiments, the arm of a bispecific antibody that binds human CD89 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH (SEQ ID NO: 96) and VL (SEQ ID NO: 101), respectively, of exemplary anti-human CD89 antibody 1. In certain embodiments, the arm of a bispecific antibody that binds human CD89 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain of exemplary anti-human CD89 antibody 1 (SEQ ID NO: 97). In certain embodiments, the arm of a bispecific antibody that binds CD89 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the light chain of exemplary anti-human CD89 antibody 1 (SEQ ID NO: 102). In certain embodiments, the arm of a bispecific antibody that binds human CD89 comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain (SEQ ID NO: 97) and light chain (SEQ ID NO: 102), respectively, of exemplary anti-human CD89 antibody 1. In a particular embodiment, the arm of the bispecific antibody that binds human CD89 comprises SEQ ID NO:96 and SEQ ID NO:101, or a pharmaceutically acceptable salt thereof. In another specific embodiment, the arm of the bispecific antibody that binds human CD89 comprises SEQ ID NO:97 and SEQ ID NO: 102. In certain embodiments, the arm of a bispecific antibody that binds human CD89.

In certain embodiments, one arm of the bispecific antibody comprises an scFv that binds gp 120. In certain embodiments, one arm of the bispecific antibody comprises an scFv that binds human CD 3. In certain embodiments, one arm of the bispecific antibody comprises an scFv that binds human CD 89. In certain embodiments, a bispecific antibody may comprise a chimeric antibody or a humanized antibody. In certain embodiments, a bispecific antibody can comprise a F (ab') 2 fragment.

In one aspect, bispecific antibodies of the disclosure bind gp120 and human CD3 (e.g., CD3 epsilon, CD3 delta) and can effect killing of HIV-1 infected cells. In one instance, such bispecific antibody that binds gp120 comprises SEQ ID NO:1, VH-CDR1 of SEQ ID NO:2, VH-CDR2 of SEQ ID NO:3, VH-CDR3 of SEQ ID NO:4, VL-CDR1 of SEQ ID NO:5 and VL-CDR2 of SEQ ID NO:6 VL-CDR3. In one instance, such a bispecific antibody that binds human CD3 comprises SEQ ID NO:11, VH-CDR1 of SEQ ID NO:12, VH-CDR2 of SEQ ID NO:13, VH-CDR3 of SEQ ID NO:14, VL-CDR1 of SEQ ID NO:15 and VL-CDR2 of SEQ ID NO:16 VL-CDR3. In another instance, a bispecific antibody that binds gp120 and human CD3 comprises in its gp 120-binding arm: SEQ ID NO:1, VH-CDR1 of SEQ ID NO:2, VH-CDR2 of SEQ ID NO:3, VH-CDR3 of SEQ ID NO:4, VL-CDR1 of SEQ ID NO:5 and the VL-CDR2 of SEQ ID NO:6 VL-CDR3; and comprises on its human CD3 binding arm: the amino acid sequence of SEQ ID NO:11, VH-CDR1 of SEQ ID NO:12, VH-CDR2 of SEQ ID NO:13, VH-CDR3 of SEQ ID NO:14, VL-CDR1 of SEQ ID NO:15 and VL-CDR2 of SEQ ID NO:16, VL-CDR3.

In another instance, such a bispecific antibody comprises a heavy chain variable region comprising a heavy chain variable region having a sequence identical to SEQ ID NO:7 VH having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or at least 98% identical. In another instance, such a bispecific antibody comprises a substitution, insertion, and/or deletion in its gp 120-binding arm that is identical to SEQ ID NO:7 VH of the same amino acid sequence. In another instance, such a bispecific antibody comprises a substitution in its gp 120-binding arm that is identical to SEQ ID NO:7 VH of the same amino acid sequence. In another instance, such a bispecific antibody comprises a heavy chain variable region having a sequence identical to SEQ ID NO: 8. 81, 82, 83 or 84, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or at least 98% identical. In another instance, such a bispecific antibody comprises a heavy chain variable region having substitutions, insertions, and/or deletions in its gp 120-binding arm other than 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) with SEQ ID NO: 8. 81, 82, 83 or 84, or a pharmaceutically acceptable salt thereof. In another instance, such a bispecific antibody comprises a heavy chain variable region having a sequence identical to SEQ ID NO: 8. 81, 82, 83 or 84 of the same amino acid sequence. In another instance, such a bispecific antibody comprises a peptide having a sequence identical to SEQ ID NO:9, a heavy chain of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or at least 98% identical. In another instance, such a bispecific antibody comprises a substitution, insertion, and/or deletion in its gp 120-binding arm that is identical to SEQ ID NO:9 the same amino acid sequence. In another instance, such a bispecific antibody comprises a substitution in its gp 120-binding arm that is identical to SEQ ID NO:9 the same amino acid sequence. In another instance, such a bispecific antibody comprises a peptide having a sequence identical to SEQ ID NO: 10. 40, 78, 79 or 80, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or at least 98% identical. In another instance, such a bispecific antibody comprises a heavy chain variable region having from 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) substitutions, insertions, and/or deletions in its gp120 binding arm that is complementary to SEQ ID NO: 10. 40, 78, 79 or 80, or a pharmaceutically acceptable salt thereof. In another instance, such a bispecific antibody comprises a substitution in its gp 120-binding arm that is identical to SEQ ID NO: 10. 40, 78, 79 or 80, or a pharmaceutically acceptable salt thereof.

In another instance, such a bispecific antibody comprises on its human CD3 binding arm a heavy chain variable region having a sequence identical to SEQ ID NO:17 VH of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or at least 98% identical. In another instance, such a bispecific antibody comprises a substitution, insertion, and/or deletion on its human CD3 binding arm that is identical to SEQ ID NO:17 VH of the same amino acid sequence. In another instance, such a bispecific antibody comprises a heavy chain variable region having a sequence identical to SEQ ID NO:17 VH of the same amino acid sequence. In another instance, such a bispecific antibody comprises on its human CD3 binding arm a heavy chain variable region having a sequence identical to SEQ ID NO:18, VL of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or at least 98% identical. In another instance, such a bispecific antibody comprises on its human CD3 binding arm a sequence having an amino acid sequence identical to SEQ ID NO:18, VL of the same amino acid sequence. In another instance, such a bispecific antibody comprises a heavy chain variable region having a sequence identical to SEQ ID NO:18, and VL of the same amino acid sequence. In another instance, such a bispecific antibody comprises on its human CD3 binding arm a heavy chain variable region having a sequence identical to SEQ ID NO:19, a heavy chain of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or at least 98% identical. In another instance, such a bispecific antibody comprises in its human CD3 binding arm a substitution, insertion, and/or deletion that has a sequence identical to SEQ ID NO:19 the same amino acid sequence. In another instance, such a bispecific antibody comprises a heavy chain variable region having a sequence identical to SEQ ID NO:19 the same amino acid sequence. In another instance, such a bispecific antibody comprises on its human CD3 binding arm a heavy chain variable region having a sequence identical to SEQ ID NO:20, a light chain of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or at least 98% identical. In another instance, such a bispecific antibody comprises on its human CD3 binding arm a sequence having an amino acid sequence identical to SEQ ID NO:20, and a light chain of the same amino acid sequence. In another instance, such a bispecific antibody comprises a sequence having a substitution in its human CD3 binding arm that is identical to SEQ ID NO:20, and a light chain of the same amino acid sequence.

In one aspect, the bispecific antibodies of the present disclosure bind gp120 and human CD89 and can effect killing of HIV-1 infected cells. CD89 is an IgA receptor that is expressed predominantly on neutrophils, and thus this bispecific antibody enhances recruitment of neutrophils to kill HIV-infected cells. In one instance, such bispecific antibodies that bind gp120 comprise SEQ ID NO:1, VH-CDR1 of SEQ ID NO:2, VH-CDR2 of SEQ ID NO:3, VH-CDR3 of SEQ ID NO:4, VL-CDR1 of SEQ ID NO:5 and the VL-CDR2 of SEQ ID NO:6 VL-CDR3. In one instance, such a bispecific antibody that binds human CD89 comprises SEQ ID NO:98, VH-CDR1 of SEQ ID NO:99, VH-CDR2 of SEQ ID NO:100, VH-CDR3 of SEQ ID NO:103, VL-CDR1 of SEQ ID NO:104 and the VL-CDR2 of SEQ ID NO:105, VL-CDR3. In another instance, a bispecific antibody that binds gp120 and human CD89 comprises on its gp 120-binding arm the amino acid sequence of SEQ ID NO:1, VH-CDR1 of SEQ ID NO:2, VH-CDR2 of SEQ ID NO:3, VH-CDR3 of SEQ ID NO:4, VL-CDR1 of SEQ ID NO:5 and VL-CDR2 of SEQ ID NO:6 VL-CDR3; and comprises on its human CD89 binding arm: the amino acid sequence of SEQ ID NO:98, VH-CDR1 of SEQ ID NO:99, VH-CDR2 of SEQ ID NO:100, VH-CDR3 of SEQ ID NO:103, VL-CDR1 of SEQ ID NO:104 and the VL-CDR2 of SEQ ID NO:105 VL-CDR3.

In another instance, such a bispecific antibody comprises a heavy chain variable region having a sequence identical to SEQ ID NO:7 VH of an amino acid sequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or at least 98% identical. In another instance, such a bispecific antibody comprises, in its gp120 binding arm, a sequence having an amino acid sequence identical to SEQ ID NO:7 VH of the same amino acid sequence. In another instance, such a bispecific antibody comprises a heavy chain variable region having a sequence identical to SEQ ID NO:7 VH of the same amino acid sequence. In another instance, such a bispecific antibody comprises a peptide having a sequence identical to SEQ ID NO: 8. 81, 82, 83 or 84 has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or at least 98% identical to the VL. In another instance, such a bispecific antibody comprises in its gp120 binding arm an amino acid sequence having an amino acid sequence identical to SEQ ID NO: 8. 81, 82, 83 or 84 of the same amino acid sequence. In another instance, such a bispecific antibody comprises a heavy chain variable region having a sequence identical to SEQ ID NO: 8. 81, 82, 83 or 84 of the same amino acid sequence. In another instance, such a bispecific antibody comprises a heavy chain variable region having a sequence identical to SEQ ID NO:9, a heavy chain of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or at least 98% identical. In another instance, such a bispecific antibody comprises in its gp120 binding arm a substitution, insertion and/or deletion with SEQ ID NO:9 the same amino acid sequence. In another instance, such a bispecific antibody comprises a substitution in its gp 120-binding arm that has a sequence identical to SEQ ID NO:9 the same amino acid sequence. In another instance, such a bispecific antibody comprises a peptide having a sequence identical to SEQ ID NO: 10. 40, 78, 79 or 80, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or at least 98% identical. In another instance, such a bispecific antibody comprises in its gp120 binding arm an amino acid sequence having a sequence identical to SEQ ID NO: 10. 40, 78, 79 or 80, or a pharmaceutically acceptable salt thereof. In another instance, such a bispecific antibody comprises a substitution in its gp 120-binding arm that has a sequence identical to SEQ ID NO: 10. 40, 78, 79 or 80, or a pharmaceutically acceptable salt thereof.

In another instance, such a bispecific antibody comprises on its human CD89 binding arm a polypeptide having an amino acid sequence identical to SEQ ID NO:96 VH having an amino acid sequence at least 90%,91%,92%,93%,94%,95%, 96%,97% or at least 98% identical. In another instance, such a bispecific antibody comprises on its human CD89 binding arm a sequence having an amino acid sequence identical to SEQ ID NO:96 VH of the same amino acid sequence. In another instance, such a bispecific antibody comprises a heavy chain variable region having a sequence identical to SEQ ID NO:96 VH of the same amino acid sequence. In another instance, such a bispecific antibody comprises on its human CD89 binding arm a polypeptide having an amino acid sequence identical to SEQ ID NO:101 has an amino acid sequence that is at least 90%,91%,92%,93%,94%,95%, 96%,97%, or at least 98% identical. In another instance, such a bispecific antibody comprises, in its human CD89 binding arm, a substitution, insertion, and/or deletion that has a sequence identical to SEQ ID NO:101, VL of the same amino acid sequence. In another instance, such a bispecific antibody comprises a heavy chain variable region having a sequence identical to SEQ ID NO:101, VL of the same amino acid sequence. In another instance, such a bispecific antibody comprises on its human CD89 binding arm a polypeptide having an amino acid sequence identical to SEQ ID NO:97, at least 90%,91%,92%,93%,94%,95%96%,97% or at least 98% identical to the heavy chain of the amino acid sequence. In another example, such a bispecific antibody comprises on its human CD89 binding arm a sequence having an amino acid sequence identical to SEQ ID NO:97 of the same amino acid sequence. In another instance, such a bispecific antibody comprises a heavy chain variable region having a sequence identical to SEQ ID NO:97 of the same amino acid sequence. In another instance, such a bispecific antibody comprises on its human CD89 binding arm a polypeptide having an amino acid sequence identical to SEQ ID NO:102 have an amino acid sequence that is at least 90%,91%,92%,93%,94%,95%, 96%,97%, or at least 98% identical. In another instance, such a bispecific antibody comprises on its human CD89 binding arm a sequence having an amino acid sequence identical to SEQ ID NO:102, and a light chain of the same amino acid sequence. In another instance, such a bispecific antibody comprises a heavy chain variable region having a sequence identical to SEQ ID NO:102, and a light chain of the same amino acid sequence.

In certain embodiments, the bispecific antibody has an Fc domain from a human IgG1 antibody with 0-10 amino acid substitutions therein. The Fc domain comprises one "branch" (hinge-CH 2-CH 3) from one component of the bispecific antibody (e.g., gp 120-binding portion) and another "branch" from a second component of the bispecific antibody (e.g., CD 3-or CD 89-binding portion). The permutation may be in one or two "branches". In certain embodiments, the Fc domain has one or more (1, 2, 3, 4, or 5) of the following mutations (EU numbering) in one or both "branches": N297A or N297Q, L234F, L235E, D265A or P331S.

Bispecific antibodies that bind gp120 and CD3 or bind gp120 and CD89 as disclosed herein can be prepared by chemically linking two different monoclonal antibodies or by fusing two hybridoma cell lines to produce a hybrid hybridoma. Other bispecific antibody technology platforms that can be used include, for example, K lambda antiAntibodies, SMIP, DNL, covx bodies, peptide antibodies, chain exchange engineered domain antibodies (SEEDbodes), dAbs, diabodies, affinibois, fynomers, kunitz Domains, tandAbs, nanobodies, albu-dabs, DARTs, DVD-IG, scFv-Igs, SVD-Igs, dAb-Igs, knobs-in-Holes, biTe platform, A platform,Andnon-limiting examples of bispecific formats that can be used to prepare the bispecific Antibodies disclosed herein are described in Del Bano et al, antibodies,5:1 (2016); garber et al, nature Reviews Drug Discovery,13:799-801 (2014).

In one embodiment, a bispecific antibody molecule of the present disclosure comprises a single antibody having two arms comprising different antigen-binding regions, one arm specific for a first antigen, such as gp120, and a second arm specific for a second antigen, such as human CD3 or human CD 89. In another embodiment, a bispecific antibody molecule of the present disclosure comprises a single antibody having one antigen-binding region or arm with specificity for a first antigen, e.g., gp120, and a second antigen-binding region or arm with specificity for a second antigen, e.g., human CD3 or human CD 89. In yet another embodiment, a bispecific antibody molecule of the present disclosure comprises a single chain antibody having a first specificity for a first antigen, such as gp120, and a second specificity for a second antigen, such as human CD3 or human CD89, e.g., via two scfvs linked in series by an additional peptide linker. In a further embodiment, bispecific antibody molecules of the present disclosure include double Variable Domain antibodies (DVD-Ig), in which each light and heavy chain comprises two Variable domains in tandem connected by a short peptide (Wu et al, generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig) ^TM ) Molecule, antibody Engineering, springer Berlin Heidelberg (2010)). In some casesIn embodiments, the bispecific antibody is a chemically linked bispecific (Fab') 2 fragment. In other embodiments, the bispecific antibody comprises Tandab (i.e., a fusion of two single chain diabodies, thereby producing a tetravalent bispecific antibody with two binding sites for each target antigen). In certain embodiments, the bispecific antibody is a flexible body (flexobody), which is a combination of a scFv and a diabody, resulting in a multivalent molecule. In yet another embodiment, the bispecific antibody comprises a "docking and locking" molecule based on a "dimerization and docking domain" in Protein Kinase (Protein Kinase) a, which when applied to Fab, can produce a trivalent bispecific binding Protein consisting of two identical Fab fragments linked to different Fab fragments. In another instance, the bispecific antibody of the present disclosure comprises a "Scorpion molecule" (which comprises, e.g., two scfvs fused to the two ends of a human Fab arm. In yet another embodiment, a bispecific antibody of the present disclosure comprises a diabody.

Exemplary classes of bispecific antibodies include, but are not limited to, igG-like molecules with complementary CH3 domains to promote heterodimerization; an IgG fusion molecule in which a full-length IgG antibody is fused to an additional Fab fragment or portion of a Fab fragment; an Fc fusion molecule in which a single chain Fv molecule or a stable diabody is fused to a heavy chain constant domain, fc region, or portion thereof; a Fab fusion molecule in which different Fab fragments are fused together; a recombinant IgG-like dual targeting molecule, wherein the molecule is flanked by at least two Fab fragments or portions of Fab fragments of different antibodies; scFv-based and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies), wherein different single chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule.

Examples of Fab fusion bispecific antibodies include, but are not limited to, F (ab) 2 (Metarex/AMGEN), dual-Action or Bis-Fab (Genentech), dock-and-Lock (DNL) (ImmunoMedics), bivalent bispecific (Biotecnol) and Fab-Fv (UCB-Celltech).

Examples of scFv-based, diabody-based, and domain antibodies include, but are not limited to, bispecific T cell adaptors (BITE) (Micromet), tandem diabodies (Tandab) (affected), parental and retargeting techniques (DART) (macrogenetics), single chain diabodies (Academic), TCR-like antibodies (AIT, receptorLogics), human serum albumin scFv fusions (Merrimack), and comboyy (Epigen Biotech), double targeting nanobodies (Ablynx), and double targeting heavy chain-only domain antibodies.

Duobodies

Bispecific antibodies of the disclosure can be those that bind gp120 and a second antigen (e.g., human CD3, e.g., human CD3 epsilon or human CD3 delta; human CD 89)Is a bispecific IgG1 antibody comprising a K409R mutation in the CH3 region of the constant region of one heavy chain and a mutation selected from the group consisting of F405L, F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W and F405Y in the CH3 region of the constant region of the other heavy chain of the bispecific antibody. In a particular embodiment of the process of the present invention,is a bispecific IgG1 antibody comprising a K409R mutation in the CH3 region of the constant region of one heavy chain and a F405L mutation in the CH3 region of the constant region of the other heavy chain of the bispecific antibody.

In certain embodiments, the first antigen-binding domain that binds gp120 as described above comprises a human IgG1 heavy chain constant region comprising a mutation selected from the group consisting of F405L, F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, and F405Y, and the second antigen-binding domain that binds human CD3 (or human CD 89) as described above comprises a human IgG1 heavy chain constant region comprising a K409R mutation.

In one embodiment, the first antigen-binding domain that binds gp120 as described above comprises a human IgG1 heavy chain constant region comprising the F405L mutation, and the second antigen-binding domain that binds human CD3 as described above comprises a human IgG1 heavy chain constant region comprising the K409R mutation.

In other embodiments, the first antigen-binding domain that binds gp120 as described above comprises a human IgG1 heavy chain constant region comprising a K409R mutation, and the second antigen-binding domain that binds human CD3 (or human CD 89) as described above comprises a human IgG1 heavy chain constant region comprising a mutation selected from the group consisting of F405L, F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, and F405Y.

In one embodiment, the first antigen-binding domain that binds gp120 as described above comprises a human IgG1 heavy chain constant region comprising a K409R mutation, and the second antigen-binding domain that binds human CD3 (or human CD 89) as described above comprises a human IgG1 heavy chain constant region comprising a F405L mutation.

The CDRs of (a) and the VH and VL may be any of the anti-gp 120, anti-CD 3 or anti-CD 89 amino acid sequences described in detail above.

In one embodiment of the process of the present invention,the gp 120-binding arm of (a) comprises SEQ ID NO:1, VH-CDR1 of SEQ ID NO:2, VH-CDR2 of SEQ ID NO:3, VH-CDR3 of SEQ ID NO:4, VL-CDR1 of SEQ ID NO:5 and the VL-CDR2 of SEQ ID NO:6 VL-CDR3. In one embodiment of the process of the present invention,the CD 3-binding arm of (a) comprises SEQ ID NO:11, VH-CDR1 of SEQ ID NO:12, VH-CDR2 of SEQ ID NO:13, VH-CDR3 of SEQ ID NO:14, VL-CDR1 of SEQ ID NO:15 and VL-CDR2 of SEQ ID NO:16 VL-CDR3. In another instance, binding gp120 and human CD3 Comprising in its gp120 binding arm:SEQ ID NO:1, VH-CDR1 of SEQ ID NO:2, VH-CDR2 of SEQ ID NO:3, VH-CDR3 of SEQ ID NO:4, VL-CDR1 of SEQ ID NO:5 and the VL-CDR2 of SEQ ID NO:6 VL-CDR3; and comprises on its human CD3 binding arm: the amino acid sequence of SEQ ID NO:11, VH-CDR1 of SEQ ID NO:12, VH-CDR2 of SEQ ID NO:13, VH-CDR3 of SEQ ID NO:14, VL-CDR1 of SEQ ID NO:15 and VL-CDR2 of SEQ ID NO:16, VL-CDR3.

In one embodiment of the process of the present invention,the gp120 binding arm of (a) comprises SEQ ID NO:7 and the VH of SEQ ID NO:8 VL. In one embodiment of the process of the present invention,the gp120 binding arm of (a) comprises SEQ ID NO:7 and the VH of SEQ ID NO: VL of 81. In one embodiment of the process of the present invention,the gp120 binding arm of (a) comprises SEQ ID NO:7 and the VH of SEQ ID NO:82 VL. In one embodiment of the process of the present invention,the gp120 binding arm of (a) comprises SEQ ID NO:7 and the VH of SEQ ID NO:83 VL of seq id no. In one embodiment of the process of the present invention,the gp120 binding arm of (a) comprises SEQ ID NO:7 and the VH of SEQ ID NO: VL of 84. In one embodiment of the process of the present invention,the CD 3-binding arm of (a) comprises SEQ ID NO:17 and SEQ ID NO:18 VL of. In one embodiment of the process of the present invention,the CD89 binding arm of (a) comprises SEQ ID NO: VH of 96 and SEQ ID NO:101 VL. In another case, binding of gp120 and human CD3 Comprising in its gp120 binding arm: SEQ ID NO:7 and the VH of SEQ ID NO:8, and comprises in its human CD3 binding arm: SEQ ID NO:17 and SEQ ID NO:18 VL of. In another instance, binding gp120 and human CD89Comprising in its gp120 binding arm: the amino acid sequence of SEQ ID NO:7 and the VH of SEQ ID NO:8, and comprises in its human CD89 binding arm: SEQ ID NO:96 and the VH of SEQ ID NO:101 VL. In one instance, binding of gp120 and human CD3Comprising in its gp120 binding arm: SEQ ID NO:7 and the VH of SEQ ID NO:81, and comprises in its human CD3 binding arm: SEQ ID NO:17 and SEQ ID NO:18 VL. In one instance, binding gp120 and human CD89Comprising in its gp120 binding arm: the amino acid sequence of SEQ ID NO:7 and the VH of SEQ ID NO:81, and comprises in its human CD3 binding arm: the amino acid sequence of SEQ ID NO: VH of 96 and SEQ ID NO:101 VL. In one instance, binding gp120 and human CD3Comprising in its gp120 binding arm: SEQ ID NO:7 and the VH of SEQ ID NO:82, and comprises in its human CD3 binding arm: the amino acid sequence of SEQ ID NO:17 and SEQ ID NO:18 VL of. In one instance, binding gp120 and human CD89Comprising in its gp120 binding arm: SEQ ID NO:7 and the VH of SEQ ID NO:82, and comprises in its human CD3 binding arm: SEQ ID NO: VH of 96 and SEQ ID NO:101 VL. In one instance, binding gp120 and human CD3 Comprising in its gp120 binding arm: SEQ ID NO:7 and the VH of SEQ ID NO:83, and comprises in its human CD3 binding arm: SEQ ID NO:17 and SEQ ID NO:18 VL of. In one instance, binding gp120 and human CD89Comprising in its gp120 binding arm: SEQ ID NO:7 and the VH of SEQ ID NO:83, and comprises in its human CD89 binding arm: the amino acid sequence of SEQ ID NO: VH of 96 and SEQ ID NO:101 VL. In one instance, binding of gp120 and human CD3Comprising in its gp120 binding arm: SEQ ID NO:7 and the VH of SEQ ID NO:84, and comprises in its human CD3 binding arm: SEQ ID NO:17 and SEQ ID NO:18 VL of. In one instance, binding gp120 and human CD89Comprising in its gp120 binding arm: SEQ ID NO:7 and the VH of SEQ ID NO:84, and comprises in its human CD89 binding arm: SEQ ID NO: VH of 96 and SEQ ID NO:101 VL. In another embodiment, binding of gp120 and human CD3Comprises the amino acid sequence of SEQ ID NO:9 and the first heavy chain of SEQ ID NO:10, a first light chain; and SEQ ID NO:19 and the second heavy chain of SEQ ID NO:20, a second light chain. In another embodiment, binding gp120 and human CD89Comprises the amino acid sequence of SEQ ID NO:9 and the first heavy chain of SEQ ID NO: 10; and SEQ ID NO:97 and the second heavy chain of SEQ ID NO:102, a second light chain. In another embodiment, binding of gp120 and human CD3 Comprises the amino acid sequence of SEQ ID NO:9 th ofA heavy chain and SEQ ID NO: 40; and SEQ ID NO:19 and the second heavy chain of SEQ ID NO:20, a second light chain. In another embodiment, binding gp120 and human CD89Comprises the amino acid sequence of SEQ ID NO:9 and the first heavy chain of SEQ ID NO: 40; and SEQ ID NO:97 and the second heavy chain of SEQ ID NO:102, a second light chain. In another embodiment, binding gp120 and human CD3Comprises the amino acid sequence of SEQ ID NO:9 and the first heavy chain of SEQ ID NO:78 with a first light chain; and SEQ ID NO:19 and the second heavy chain of SEQ ID NO:20, a second light chain. In another embodiment, binding gp120 and human CD89Comprises SEQ ID NO:9 and the first heavy chain of SEQ ID NO:78 with a first light chain; and SEQ ID NO:97 and the second heavy chain of SEQ ID NO:102, a second light chain. In another embodiment, binding of gp120 and human CD3Comprises the amino acid sequence of SEQ ID NO:9 and the first heavy chain of SEQ ID NO:79 of a first light chain; and SEQ ID NO:19 and the second heavy chain of SEQ ID NO:20, a second light chain. In another embodiment, binding gp120 and human CD89Comprises the amino acid sequence of SEQ ID NO:9 and the first heavy chain of SEQ ID NO:79 of a first light chain; and SEQ ID NO:97 and the second heavy chain of SEQ ID NO:102, a second light chain. In another embodiment, binding gp120 and human CD3 Comprises the amino acid sequence of SEQ ID NO:9 and the first heavy chain of SEQ ID NO: 80; and SEQ ID NO:19 and the second heavy chain of SEQ ID NO:20, a second light chain. In addition toIn one embodiment, binding gp120 and human CD89Comprises SEQ ID NO:9 and the first heavy chain of SEQ ID NO: 80; and SEQ ID NO:97 and the second heavy chain of SEQ ID NO:102, a second light chain. In one embodiment, anti-gp 120 x CD3Or anti-gp 120 x CD89Comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94% or 95%, 96%, 97%, 98%, 99% or 100% identical to one of the amino acid sequences set forth below, or identical to one of the amino acid sequences provided below except for 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid substitutions:

in one embodiment, anti-gp 120 x CD3Or anti-gp 120 x CD89Comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94% or 95%, 96%, 97%, 98%, 99% or 100% identical to one of the amino acid sequences set forth below, or identical (except for 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid substitutions) to one of the amino acid sequences provided below:

In one embodiment of the process of the present invention,comprises the heavy chain constant region of SEQ ID NO:64, andcomprises the amino acid sequence of SEQ ID NO: 74. In a further embodiment of the process of the present invention,comprises the amino acid sequence of SEQ ID NO:65, andcomprises the amino acid sequence of SEQ ID NO:75 is an amino acid represented byAnd (4) sequencing. In a further embodiment of the process of the present invention,comprises the amino acid sequence of SEQ ID NO:62, andcomprises the heavy chain constant region of SEQ ID NO:72, or a pharmaceutically acceptable salt thereof. In yet another embodiment of the present invention,comprises the amino acid sequence of SEQ ID NO:63, andcomprises the heavy chain constant region of SEQ ID NO:73, or a pharmaceutically acceptable salt thereof.

In one embodiment of the process of the present invention,the heavy chain of the gp 120-binding arm of (a) has the amino acid sequence of SEQ ID NO:9, andthe light chain of the gp120 binding arm of (a) has the amino acid sequence of SEQ ID NO:10, or a pharmaceutically acceptable salt thereof. In a further embodiment of the process of the present invention,the heavy chain of the gp 120-binding arm of (a) has the amino acid sequence of SEQ ID NO:9, andthe light chain of the gp120 binding arm of (a) has the amino acid sequence of SEQ ID NO:40, or a pharmaceutically acceptable salt thereof. In a further embodiment of the process of the present invention, The heavy chain of the gp 120-binding arm of (a) has the amino acid sequence of SEQ ID NO:9, andand is provided withThe light chain of the gp120 binding arm of (a) has the amino acid sequence of SEQ ID NO:78, or a pharmaceutically acceptable salt thereof. In a further embodiment of the process of the present invention,the heavy chain of the gp 120-binding arm of (a) has the amino acid sequence of SEQ ID NO:9, andthe light chain of the gp120 binding arm of (a) has the amino acid sequence of SEQ ID NO:79, or a pharmaceutically acceptable salt thereof. In yet another embodiment of the present invention,the heavy chain of the gp 120-binding arm of (a) has the amino acid sequence of SEQ ID NO:9, andthe light chain of the gp120 binding arm of (a) has the amino acid sequence of SEQ ID NO:80, or a pharmaceutically acceptable salt thereof.

In one embodiment, gp 120X CD3The heavy chain of the CD3 binding arm of (a) comprises SEQ ID NO:19 or consists thereof, andthe light chain of the CD3 binding arm of (a) comprises SEQ ID NO:20 or consists thereof.

In one embodiment, gp 120X CD89The heavy chain of the CD89 binding arm of (a) comprises SEQ ID NO:97 or consists thereof, andCD3 binding arm of (1)Comprises SEQ ID NO:102 or consists thereof.

In one embodiment, gp 120X CD3Comprising two arms, the first arm comprising a heavy chain and a light chain that bind gp120, wherein The heavy chain of the gp120 binding arm of (a) comprises a heavy chain identical to SEQ ID NO:9, and the light chain of the gp120 binding arm comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identical to the amino acid sequence set forth in SEQ ID NO: 10. 40, 78, 79 or 80, is at least 80%, 81%, 82%, an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identical, and the second arm comprises a heavy chain and a light chain that bind CD3, whereinThe heavy chain of the CD3 binding arm of (a) comprises a heavy chain identical to SEQ ID NO:19, and the light chain of the CD3 binding arm comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identical to the amino acid sequence set forth in SEQ ID NO:20, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identical to the amino acid sequence set forth in seq id no.

In one embodiment, gp 120X CD89Comprising two arms, the first arm comprising a heavy chain and a light chain that bind gp120, whereinThe heavy chain of the gp 120-binding arm of (a) comprises a heavy chain identical to SEQ ID NO:9, and the light chain of the gp120 binding arm comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identical to the amino acid sequence set forth in SEQ ID NO: 10. 40, 78, 79 or 80, is at least 80%, 81%, 82%, an amino acid sequence that is at least 80%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identical, and the second arm comprises heavy and light chains that bind CD89, whereinThe heavy chain of the CD89 binding arm of (a) comprises a heavy chain identical to SEQ ID NO:97, an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identical to the amino acid sequence set forth in SEQ ID NO:102, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identical thereto.

In a specific embodiment, gp 120X CD3Comprising two arms, the first arm comprising a heavy chain and a light chain that bind gp120, whereinThe heavy chain of the gp 120-binding arm of (a) comprises SEQ ID NO:9 and the light chain of the gp120 binding arm comprises the amino acid sequence set forth in SEQ ID NO:10 and the second arm comprises a heavy chain and a light chain that bind CD3, whereinThe heavy chain of the CD3 binding arm of (a) comprises SEQ ID NO:19, and the light chain of the CD3 binding arm comprises the amino acid sequence set forth in SEQ ID NO:20, or a pharmaceutically acceptable salt thereof. In certain embodiments, can beFrom 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions are made in any component (e.g., CH1, hinge, CH2, CH 3) of one or both heavy chain constant regions of (a). In certain instances, amino acid substitutions alter (e.g., decrease) effector function and/or increase half-life relative to an unaltered polypeptide. In certain embodiments, these substitutions may be conservative amino acid substitutions.

In a specific embodiment, gp 120X CD89Comprising two arms, the first arm comprising a heavy chain and a light chain that bind gp120, whereinThe heavy chain of the gp 120-binding arm of (a) comprises SEQ ID NO:9, and the light chain of the gp120 binding arm comprises the amino acid sequence set forth in SEQ ID NO:10 and the second arm comprises a heavy chain and a light chain that bind CD89, wherein The heavy chain of the CD89 binding arm of (a) comprises SEQ ID NO:97, and the light chain of the CD89 binding arm comprises the amino acid sequence set forth in SEQ ID NO:102, or a pharmaceutically acceptable salt thereof. In certain embodiments, can beFrom 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions are made in any component (e.g., CH1, hinge, CH2, CH 3) of one or both heavy chain constant regions of (a). In certain instances, amino acid substitutions alter (e.g., decrease) effector function and/or increase half-life relative to the unaltered polypeptide. In some instancesIn embodiments, these substitutions may be conservative amino acid substitutions.

In another specific embodiment, gp 120X CD3Comprising two arms, the first arm comprising a heavy chain and a light chain that bind gp120, whereinThe heavy chain of the gp 120-binding arm of (a) comprises SEQ ID NO:9, and the light chain of the gp120 binding arm comprises the amino acid sequence set forth in SEQ ID NO:40 and the second arm comprises a heavy chain and a light chain that bind CD3, whereinThe heavy chain of the CD3 binding arm of (a) comprises SEQ ID NO:19, and the light chain of the CD3 binding arm comprises the amino acid sequence set forth in SEQ ID NO:20, or a pharmaceutically acceptable salt thereof. In certain embodiments, can be From 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions are made in any component (e.g., CH1, hinge, CH2, CH 3) of one or both heavy chain constant regions of (a). In certain instances, amino acid substitutions alter (e.g., decrease) effector function and/or increase half-life relative to an unaltered polypeptide. In certain embodiments, these substitutions may be conservative amino acid substitutions.

In another specific embodiment, gp 120X CD89Comprising two arms, the first arm comprising a heavy chain and a light chain that bind gp120, whereinThe heavy chain of the gp120 binding arm of (a) comprises SEQ ID NO:9, and the light chain of the gp120 binding arm comprises the amino acid sequence set forth in SEQ ID NO:40 is an amino acid represented by formula (I)A sequence and a second arm comprising a heavy chain and a light chain that bind CD89, whereinThe heavy chain of the CD89 binding arm of (a) comprises SEQ ID NO:97, and the light chain of the CD89 binding arm comprises the amino acid sequence set forth in SEQ ID NO:102, or a pharmaceutically acceptable salt thereof. In certain embodiments, can beFrom 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions are made in any component (e.g., CH1, hinge, CH2, CH 3) of one or both heavy chain constant regions of (a). In certain instances, amino acid substitutions alter (e.g., decrease) effector function and/or increase half-life relative to the unaltered polypeptide. In certain embodiments, these substitutions may be conservative amino acid substitutions.

In another specific embodiment, gp 120X CD3Comprising two arms, the first arm comprising a heavy chain and a light chain that bind gp120, whereinThe heavy chain of the gp 120-binding arm of (a) comprises SEQ ID NO:9, and the light chain of the gp120 binding arm comprises the amino acid sequence set forth in SEQ ID NO:78 and a second arm comprising a heavy chain and a light chain that bind CD3, whereinThe CD3 binding arm of (a) comprises SEQ ID NO:19, and the light chain of the CD3 binding arm comprises the amino acid sequence set forth in SEQ ID NO:20, or a pharmaceutically acceptable salt thereof. In certain embodiments, can beFrom 1 to 10 (i.e., 1, 2, 3, etc.) of any component (e.g., CH1, hinge, CH2, CH 3) of one or both heavy chain constant regions of (A),4. 5, 6, 7, 8, 9, or 10) amino acid substitutions. In certain instances, amino acid substitutions alter (e.g., decrease) effector function and/or increase half-life relative to the unaltered polypeptide. In certain embodiments, these substitutions may be conservative amino acid substitutions.

In another specific embodiment, gp 120X CD89Comprising two arms, the first arm comprising a heavy chain and a light chain that bind gp120, whereinThe heavy chain of the gp 120-binding arm of (a) comprises SEQ ID NO:9, and the light chain of the gp120 binding arm comprises the amino acid sequence set forth in SEQ ID NO:78 and the second arm comprises a heavy chain and a light chain that bind CD89, wherein The heavy chain of the CD89 binding arm of (a) comprises SEQ ID NO:97, and the light chain of the CD89 binding arm comprises the amino acid sequence set forth in SEQ ID NO:102, or a pharmaceutically acceptable salt thereof. In some embodiments, can be inFrom 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions are made in any component (e.g., CH1, hinge, CH2, CH 3) of one or both heavy chain constant regions of (a). In certain instances, amino acid substitutions alter (e.g., decrease) effector function and/or increase half-life relative to an unaltered polypeptide. In certain embodiments, these substitutions may be conservative amino acid substitutions.

In another specific embodiment, gp 120X CD3Comprising two arms, the first arm comprising a heavy chain and a light chain that bind gp120, whereinThe heavy chain of the gp 120-binding arm of (a) comprises SEQ ID NO:9, and the light chain of the gp120 binding arm comprises the amino acid sequence set forth in SEQ ID NO:79 and the second arm comprises a heavy chain and a light chain that bind CD3, whereinThe heavy chain of the CD3 binding arm of (a) comprises SEQ ID NO:19, and the light chain of the CD3 binding arm comprises the amino acid sequence set forth in SEQ ID NO:20, or a pharmaceutically acceptable salt thereof. In some embodiments, can be in From 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions are made in any component (e.g., CH1, hinge, CH2, CH 3) of one or both heavy chain constant regions of (a). In certain instances, amino acid substitutions alter (e.g., decrease) effector function and/or increase half-life relative to the unaltered polypeptide. In certain embodiments, these substitutions may be conservative amino acid substitutions.

In another specific embodiment, gp 120X CD89Comprising two arms, the first arm comprising a heavy chain and a light chain that bind gp120, whereinThe heavy chain of the gp 120-binding arm of (a) comprises SEQ ID NO:9, and the light chain of the gp120 binding arm comprises the amino acid sequence set forth in SEQ ID NO:79 and the second arm comprises a heavy chain and a light chain that bind CD89, whereinThe heavy chain of the CD89 binding arm of (a) comprises SEQ ID NO:97, and the light chain of the CD89 binding arm comprises the amino acid sequence set forth in SEQ ID NO:102, or a pharmaceutically acceptable salt thereof. In some embodiments, can be inFrom 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions are made in any component (e.g., CH1, hinge, CH2, CH 3) of one or both heavy chain constant regions of (a). In some cases, the amino acid substitution alters (e.g., reduces) effector function and/or increases half-life relative to an unaltered polypeptide. In certain embodiments, these substitutions may be conservative amino acid substitutions.

In another specific embodiment, gp 120X CD3Comprising two arms, the first arm comprising a heavy chain and a light chain that bind gp120, whereinThe heavy chain of the gp 120-binding arm of (a) comprises SEQ ID NO:9, and the light chain of the gp120 binding arm comprises the amino acid sequence set forth in SEQ ID NO:80 and the second arm comprises a heavy chain and a light chain that bind CD3, whereinThe heavy chain of the CD3 binding arm of (a) comprises SEQ ID NO:19, and the light chain of the CD3 binding arm comprises the amino acid sequence set forth in SEQ ID NO:20, or a pharmaceutically acceptable salt thereof. In certain embodiments, can beFrom 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions are made in any component (e.g., CH1, hinge, CH2, CH 3) of one or both heavy chain constant regions of (a). In certain instances, amino acid substitutions alter (e.g., decrease) effector function and/or increase half-life relative to an unaltered polypeptide. In certain embodiments, these substitutions may be conservative amino acid substitutions.

In another specific embodiment, gp 120X CD89Comprising two arms, the first arm comprising a heavy chain and a light chain that bind gp120, whereinThe heavy chain of the gp120 binding arm of (a) comprises SEQ ID NO:9, and the light chain of the gp120 binding arm comprises the amino acid sequence set forth in SEQ ID NO:80 and the second arm comprises a heavy chain and a light chain that bind CD89, wherein The CD89 binding arm of (a) comprises SEQ ID NO:97, and the light chain of the CD89 binding arm comprises the amino acid sequence set forth in SEQ ID NO:102, or a pharmaceutically acceptable salt thereof. In certain embodiments, can beFrom 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions are made in any component (e.g., CH1, hinge, CH2, CH 3) of one or both heavy chain constant regions of (a). In certain instances, amino acid substitutions alter (e.g., decrease) effector function and/or increase half-life relative to an unaltered polypeptide. In certain embodiments, these substitutions may be conservative amino acid substitutions.

In particular embodiments, the antibody is defucosylated. In some embodiments, the antibody comprises one or more tags. In certain embodiments, the one or more labels comprise an avidin label.

Fc modification

In certain embodiments, an antibody of the present disclosure (e.g.,) One or more amino acid sequence modifications are included in the heavy chain constant region (Fc). In certain embodiments, an antibody of the present disclosure (e.g.,) One or more amino acid sequence modifications are included in the heavy chain constant region (Fc). In particular embodiments, these modifications increase the stability or improve binding affinity of the modified antibody compared to the PGT-121LO6 antibody. In particular embodiments, these modifications increase the stability or improve binding affinity of the modified antibody compared to the PGT-121.60 antibody. In particular embodiments, certain of these modifications increase the half-life of the antibody. In certain embodiments, certain of these modifications reduce antibody effector function. In other embodiments, certain of these modifications reduce antibody effector function and increase the half-life of the antibody.

In certain embodiments, the one or more modifications are selected from the following Fc amino acid substitutions (EU numbering) or combinations thereof: L234F; L235E; G236A; S239D; F243L; D265E; D265A; S267E; H268F; R292P; N297Q; N297A; S298A; S324T; I332E; S239D; A330L; L234F; L235E; P331S; F243L; Y300L; V305I; P396L; S298A; E333A; K334A; E345R; L235V; F243L; R292P; Y300L; P396L; M428L; E430G; N434S; G236A, S267E, H268F, S324T, and I332E; G236A, S239D and I332E; S239D, A330L and I332E; L234F, L235E, and P331S; F243L, R292P, Y300L, V305I and P396L; G236A, H268F, S324T and I332E; S239D, H268F, S324T and I332E; S298A, E333A, and K334A; L235V, F243L, R292P, Y300L, and P396L; S239D and I332E; S239D, S298A, and I332E; G236A, S239D, I332E, M428L and N434S; G236A, S239D, a330L, I332E, M428L and N434S; S239D, I332E, G236A, and a330L; M428L and N4343S; M428L, N434S; G236A, S239D, a330L, and I332E; and G236A and I332E. In certain embodiments, the one or more modifications are selected from: N297A, D265A, L234F, L235E, N297Q and P331S. In certain embodiments, the one or more modifications are N297A or D265A. In certain embodiments, the one or more modifications are L234F and L235E. In certain embodiments, the one or more modifications are L234F, L234E and D265A. In certain embodiments, the one or more modifications are L234F, L234E, and N297Q. In certain embodiments, the one or more modifications are L234F, L235E, and P331S. In certain embodiments, the one or more modifications are D265A and N297Q. In certain embodiments, the one or more modifications are L234F, L235E, D265A, N297Q and P331S.

Mutations that reduce Fc receptor binding include, for example, N297A; N297Q; D265A; L234F/L235E; L234F/L235E/N297Q; L234F/L235E/P331S; D265A/N297Q; and L234F/L235E/D265A/N297Q/P331S (all EU numbering). In certain embodiments, an antibody disclosed herein (e.g., duobodies) comprises L234F and L235E mutations. In certain embodiments, an antibody disclosed herein (e.g., duobodies) comprises L234F, L235E, and D265A mutations. In certain embodiments, an antibody disclosed herein (e.g., duobodies) comprises L234F, L235E, and D265A mutations. In certain embodiments, an antibody disclosed herein (e.g., duobodes) comprises an N297A or N297Q mutation. In certain embodiments, an antibody disclosed herein (e.g., duobodies) comprises an N297A or N297Q mutation, as well as L234F, L235E, and D265A mutations. In certain embodiments, one, two, three, four, or more amino acid substitutions are introduced into the Fc region to alter the effector function of the antibody. For example, these substitutions are at a position selected from the group consisting of amino acid residues 234, 235, 236, 237, 265, 297, 318, 320 and 322 (according to EU numbering). These positions may be substituted with different amino acid residues to give the antibody an altered (e.g., reduced) affinity for an effector ligand (e.g., an Fc receptor or the C1 component of complement), but retain the antigen binding ability of the parent antibody. In certain embodiments, an antibody disclosed herein (e.g., duobodies) comprises E233P, L234V, L235A, and G236A mutations (EU numbering). In some embodiments, the antibody comprises a327G, a330S, and P331S mutations (EU numbering). In some embodiments, the antibody comprises a K322A mutation (EU numbering). In some embodiments, the antibody comprises E318A, K320A, and K322A (EU numbering) mutations. In certain embodiments, the antibody comprises an L235E (EU numbering) mutation.

Mutations that increase the half-life of an antibody are known in the art. In one embodiment, an antibody described herein (e.g.,) The constant region of (a) comprises a methionine to tyrosine substitution at position 252 (EU numbering), a serine to threonine substitution at position 254 (EU numbering), and a threonine to glutamic acid substitution at position 256 (EU numbering). See, for example, U.S. patent No. 7,658,921. This type of mutant, referred to as the "YTE mutant", exhibits a four-fold increased half-life relative to the wild-type form of the same antibody (Dall' Acqua et al, J Biol Chem,281, 23514-24 (2006); robbie et al, antimicrob Agents Chemotherap.,57 (12): 6147-6153 (2013)). In certain embodiments, the antibody comprises an IgG constant domain comprising amino acid substitutions of one, two, three, or more amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436 (EU numbering). In other embodiments, the antibodies described herein (e.g.,) Including M428L and N4343S substitutions (EU numbering). In other embodiments, the antibodies described herein (e.g.,) Contains the T250Q and M428L (EU numbering) mutations. In other embodiments, the antibodies described herein (e.g., ) Comprising H433K and N434F (EU numbering) mutations.

In particular embodiments, an antibody (e.g.,) Comprising two or more, three or more, four or more, five or more, six or less, five or less, four or less, three or less, two or less, or one modified Fc amino acid residue. In certain embodiments, the antibody comprises L234F, L235E, D264A mutations collectively referred to as "FEA". In certain embodiments, the antibody comprises L234F, collectively referred to as "FEALL235E, D264A and F405L mutations. In certain embodiments, the antibody comprises L234F, L235E, D264A, and a mutation selected from the group consisting of F405L, F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, and F405Y. In certain embodiments, the antibody comprises L234F, L235E, D264A, and K409R mutations collectively referred to as "FEAR. In certain embodiments, FEAL and FEAR comprise a bispecific antibody described herein (e.g.,) In (1). In certain embodiments, the antibody comprises M428L and N434S mutations, collectively referred to as LS. In certain embodiments, the antibody comprises L234F, L235E, D264A, F405L, M428L, and N434S mutations, collectively referred to as "FEALLS. In certain embodiments, the antibody comprises L234F, L235E, D264A, M428L, and N434S mutations and one additional mutation selected from F405L, F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, and F405Y. In certain embodiments, the antibody comprises the L234F, L235E, D264A, K409R, M428L and N434S mutations collectively referred to as "FEARLS". In certain embodiments, FEALLS and FEARLS comprise a bispecific antibody described herein (e.g., ) In (1). In certain embodiments, the antibody comprises S239D, I332E, G236A, a330L ("DEAL"). In certain embodiments, the antibody comprises the S239D, I332E, G236A, a330L, M428L, and N434S mutations ("DEALLS"). FEA mutations reduce or eliminate effector function, while DEAL mutations increase or enhance effector function by enhancing Fc binding to activated Fc γ R. The LS mutation increases the pharmacokinetic half-life of the antibody.

By reducing or eliminating effector function, the CD 3X gp120 multispecific/bispecific antibody (i) ensures that T cells (which include non-HIV infected T cells) bound by the bispecific molecule are not killed by innate effector cells (e.g., NK cells, macrophages); and (ii) also ensures that T cells are not activated in the absence of target cells by not binding or reducing binding to Fc γ Rs on innate effector cells. Activation of T cells in the absence of target cells leads to a cytokine response and is not tolerated. Binding of bispecific molecules to Fc γ Rs on innate effector cells results in clustering of CD3 molecules on T cells, leading to antigen-independent T cell activation.

Conjugated antibodies

Any of the antibodies disclosed herein can be conjugated antibodies that bind to a variety of molecules, including macromolecular species such as polymers (e.g., polyethylene glycol (PEG), polyethyleneimine modified with PEG (PEI-PEG), polyglutamic acid (PGA) (N- (2-hydroxypropyl) methacrylamide (HPMA) copolymer), hyaluronic acid, radioactive species (e.g., a polymer such as polyethylene glycol (PEG), polyethylene imine modified with PEG (PEI-PEG), and poly (N- (2-hydroxypropyl) methacrylamide (HPMA)) ⁹⁰ Y、 ¹³¹ I、 ¹²⁵ I、 ³⁵ S、 ³ H、 ¹²¹ In、 ⁹⁹ Tc), fluorescent substances (e.g., fluorescein and rhodamine), luminescent substances (e.g., luminol), haptens, enzymes (e.g., glucose oxidase), metal chelates, biotin, avidin, and drugs.

Such conjugated antibodies can be prepared by chemical modification of an antibody or lower molecular weight version thereof as described herein. Methods of modifying antibodies are well known in the art (e.g., US 5,057,313 and US 5,156,840).

Nucleic acids

The disclosure also features polynucleotides comprising nucleotide sequences encoding polynucleotide antibodies described herein that bind gp120 and human CD3 antigen or bind gp120 and CD89 antigen, vectors comprising such polynucleotides, and host cells (e.g., mammalian cells, yeast, e.coli) comprising such polynucleotides or expression vectors. Provided herein are polynucleotides comprising nucleotide sequences encoding any of the antibodies provided herein, as well as vectors comprising such polynucleotide sequences, e.g., expression vectors for efficient expression in a host cell, e.g., a mammalian cell.

In one aspect, the disclosure provides a polynucleotide comprising a nucleotide sequence encoding the VH, VL, or VH and VL of an antibody that binds gp120 (e.g., exemplary anti-gp 120 antibody 2; exemplary anti-gp 120 antibody 3; exemplary anti-gp 120 antibody 4; exemplary anti-gp 120 antibody 5).

In another aspect, the disclosure provides a polynucleotide comprising a nucleotide sequence encoding an antibody that binds gp120 and a human CD3 polypeptide and comprises an amino acid sequence described herein.

In another aspect, the disclosure provides a polynucleotide comprising a nucleotide sequence encoding an antibody that binds gp120 and a human CD89 polypeptide and comprises an amino acid sequence described herein.

In another aspect, the disclosure provides a polynucleotide or nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to the invention. In some embodiments, the nucleic acid molecule encodes an antibody light chain (or fragment thereof) or both of the present application. In other embodiments, the nucleic acid is DNA, cDNA, or mRNA. In some other embodiments, the nucleic acid molecule is codon optimized to enhance expression in the host cell.

In another aspect, provided herein is a polynucleotide comprising a nucleotide sequence encoding a CDR, light chain or heavy chain of an antibody described herein. The polynucleotide may comprise a nucleotide sequence encoding a light chain or light chain variable domain comprising the VL CDRs of the antibodies described herein (see, e.g., the tables above). The polynucleotide may comprise a nucleotide sequence encoding a heavy chain or a heavy chain variable domain comprising the VH CDRs of the antibodies described herein (see, e.g., the tables above). In one embodiment, the polynucleotides described herein encode a polypeptide having a sequence comprising SEQ ID NOs: 4. 5 and 6 or SEQ ID NO: 14. 15 and 16, or a variable light chain of the VL-CDRs of the amino acid sequences set forth in seq id no. In another embodiment, the polynucleotides described herein encode a polypeptide having a sequence comprising SEQ ID NOs: 1. 2 and 3 or SEQ ID NOs: 11. 12 and 13, or a variable heavy chain or heavy chain of a VH-CDR of an amino acid sequence set forth in seq id no. In one embodiment, the polynucleotide described herein encodes a polypeptide comprising SEQ ID NO: 8. 18 or 101, or a VL domain of an amino acid sequence set forth in seq id No. 18 or 101. In another embodiment, the polynucleotide described herein encodes a polypeptide comprising SEQ ID NO: 7. 17 or 96. In yet another embodiment, the polynucleotide described herein encodes a polypeptide comprising SEQ ID NO: 10. 20 or 102, or a light chain of an amino acid sequence as set forth in seq id no. In another embodiment, the polynucleotide described herein encodes a polypeptide comprising SEQ ID NO: 9. 19 or 97, or a pharmaceutically acceptable salt thereof.

The disclosure also includes polynucleotides encoding anti-gp 120 and anti-CD 3 (or anti-CD 89) antibodies optimized, for example, by codon optimization, substitution of heterologous signal sequences, and elimination of mRNA instability elements. Methods of producing optimized nucleic acids can be achieved by employing, for example, U.S. Pat. Nos. 5,965,726;6,174,666;6,291,664;6,414,132; and 6,794,498.

Vectors and host cells

The disclosure also includes vectors comprising the nucleic acids disclosed herein. The vector may be of any type, e.g., a recombinant vector, such as an expression vector. Vectors include, but are not limited to, plasmids, cosmids, bacterial Artificial Chromosomes (BACs) and Yeast Artificial Chromosomes (YACs), as well as vectors derived from bacteriophages or plant or animal (including human) viruses. The vector may contain an origin of replication recognized by the proposed host cell, and in the case of an expression vector, a promoter and other regulatory regions recognized by the host cell. In particular embodiments, the vector comprises a polynucleotide encoding an antibody of the present disclosure operably linked to a promoter and optionally additional regulatory elements. Certain vectors are capable of autonomous replication in a host into which they are introduced (e.g., vectors having a bacterial origin of replication may replicate in bacteria). Other vectors may be integrated into the genome of a host upon introduction into the host, and thereby replicated together with the host genome. Vectors include, but are not limited to, those suitable for recombinant production of the antibodies disclosed herein.

The choice of vector will depend on the recombination procedure to be followed and the host to be used. The vector may be introduced into the host cell by calcium phosphate transfection, viral infection, DEAE-dextran mediated transfection, lipofectamine transfection or electroporation, among others. Vectors may replicate autonomously or may replicate together with the chromosome(s) into which they have been integrated. In certain embodiments, the vector comprises one or more selectable markers. The choice of marker may depend on the host cell chosen. These include, but are not limited to, kanamycin, neomycin, puromycin, hygromycin, bleomycin, thymidine kinase gene from herpes simplex virus (HSV-TK), and dihydrofolate reductase gene from mice (dhfr). The present disclosure also encompasses vectors comprising one or more nucleic acid molecules encoding the antibodies described herein operably linked to one or more nucleic acid molecules encoding proteins or peptides useful for isolating the antibodies. These proteins or peptides include, but are not limited to, glutathione-S-transferase, maltose binding protein, metal binding polyhistidine, green fluorescent protein, luciferase, and beta-galactosidase.

In a particular embodiment, the vector used is pcDNA ^TM 3.1+(ThermoFisher，MA)。

The present disclosure also provides host cells comprising a nucleic acid or vector described herein. Any of a variety of host cells may be used. In one embodiment, the host cell is a prokaryotic cell, such as E.coli. In another embodiment, the host cell is a eukaryotic cell, e.g., a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a COS cell, a BHK cell, an NSO cell, or a bowden melanoma cell. Examples of human host cells are inter alia HeLa, 911, AT1080, a549, 293 and HEK293T cells.

The term "nucleic acid molecule" refers to a polymeric form of nucleotides and includes RNA, cDNA, sense and antisense strands of genomic DNA, as well as synthetic forms and mixed polymers of the foregoing. As used herein, the term "nucleic acid molecule" is interchangeable with the term "polynucleotide". In some embodiments, a nucleotide refers to a ribonucleotide, a deoxynucleotide, or a modified form of either type of nucleotide, as well as combinations thereof. The term also includes, but is not limited to, single-stranded and double-stranded forms of DNA. In addition, a polynucleotide such as a cDNA or mRNA can include either or both naturally occurring nucleotides and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages. As is readily understood by those skilled in the art, nucleic acid molecules may be chemically or biochemically modified or may comprise non-natural or derivatized nucleotide bases. Such modifications include, for example, labels, methylation, substitution of one or more naturally occurring nucleotides with an analog, internucleotide modifications such as without an electrical connection (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.), charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.), pendant moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylating agents, and modified linkages (e.g., α anomeric nucleic acids, etc.). The above terms are also intended to include any topological conformation, including single-stranded, double-stranded, partially duplex, triplex, hairpin, circular, and padlock conformations. Unless otherwise indicated, reference to a nucleic acid sequence includes its complement. Thus, reference to a nucleic acid molecule having a particular sequence is understood to include the complementary strand thereof and the complementary sequence thereof. The term also includes codon optimized nucleic acids.

The term "operably linked" refers to two or more nucleic acid sequence elements that are typically physically linked and in a functional relationship with each other. For example, a promoter is operably linked to a coding sequence if it is capable of promoting or regulating the transcription or expression of the coding sequence, in which case the coding sequence is understood to be "under the control" of the promoter.

As used herein, "substitution" means the replacement of one or more amino acids or nucleotides with a different amino acid or nucleotide, respectively.

An "isolated" nucleic acid is a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. "isolated nucleic acid encoding an antibody or fragment thereof" refers to one or more nucleic acid molecules encoding the heavy and light chains of an antibody (or fragment thereof), including such nucleic acid molecules in a single vector or separate vectors, as well as such nucleic acid molecules present at one or more locations in a host cell.

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors which are self-replicating nucleic acid structures, as well as vectors which are incorporated into the genome of a host cell into which they are introduced. Some vectors are suitable for delivery of the nucleic acid molecules or polynucleotides of the present application. Certain vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as expression vectors.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom (regardless of passage number). Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Progeny of the mutants having the same function or biological activity as screened or selected in the originally transformed cell are included herein.

The term "variant" of a polynucleotide as used herein is a polynucleotide that differs from the polynucleotide specifically disclosed herein, typically in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be produced synthetically, e.g., by modifying one or more polynucleotide sequences of the invention and assessing one or more biological activities of the encoded polypeptide as described herein and/or using any of a variety of techniques well known in the art.

The term "variant" of a polypeptide as used herein is a polypeptide that differs from the polypeptide specifically disclosed herein, typically in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring, or may be synthetically produced, for example, by modifying one or more of the above-described polypeptide sequences of the invention and assessing one or more biological activities of the polypeptide as described herein and/or using any of a variety of techniques well known in the art. In one embodiment, the antibody or antigen binding fragment thereof comprises variant 1, variant 2, variant 3, and/or variant 4. In some embodiments, the antibody or antigen-binding fragment thereof comprises variant 1. In some embodiments, the antibody or antigen-binding fragment thereof comprises variant 2. In some embodiments, the antibody or antigen-binding fragment thereof comprises variant 3. In some embodiments, the antibody or antigen-binding fragment thereof comprises variant 4.

The term "variant" may also refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations. In one embodiment, the molecule is an antibody. For example, a somatic variant may include all relevant naturally occurring antibodies that are part of or derived from the same B cell lineage. Engineered variants may include all single mutations or combined mutations made to an antibody.

Method for producing antibody

Monospecific antibodies that bind gp120 and bispecific antibodies that bind gp120 and human CD3 (e.g., human CD3 epsilon or human CD3 delta) can be generated by any method known in the art for antibody synthesis, e.g., by chemical synthesis or by recombinant expression techniques.

Methods for making monospecific antibodies are well known in the art. Methods for making bispecific antibodies are described, for example, in U.S. Pat. nos. 5,731,168;5,807,706;5,821,333; and U.S. application publication Nos. 2003/020734 and 2002/0155537. Bispecific tetravalent antibodies and methods for their preparation are described, for example, in WO 02/096948 and WO 00/44788, the disclosures of both of which are incorporated herein by reference in their entirety. In addition, other publications relevant to the preparation of bispecific antibodies include WO 91/00360, WO 92/08802, WO92/05793, and WO 93/17715; tutt et al, j.immunol.147:60-69 (1991); U.S. Pat. Nos. 4,474,893;4,714,681;4,925,648;5,573,920;5,601,819 and 9,212,230; and Kostelny et al, j.immunol.148:1547-1553 (1992).

In one embodiment, the bispecific antibody of the present disclosure isDuobodiesCan be prepared by, for example, those described in International publication Nos. WO 2008/119353, WO 2011/131746, WO 2011/147986 and WO 2013/060867, labrijn AF et al PNAS,110 (13): 5145-5150 (2013), gramer et al mAbs,5 (6): 962-973 (2013), and Labrijn et al Nature Protocols,9 (10): 2450-2463 (2014)Preparation of technical platform (Genmab A/S). This technique can be used to bind half of a first monospecific antibody comprising two heavy chains and two light chains to half of a second monospecific antibody comprising two heavy chains and two light chains. The resulting heterodimer comprises one heavy chain and one light chain from the first antibody paired with one heavy chain and one light chain from the second antibody. When two monospecific antibodies recognize different epitopes on different antigens, the resulting heterodimer is a bispecific antibody.

ForPlatform, each monospecific antibody includes a heavy chain constant region with a single point mutation in the CH3 domain. These point mutations allow for stronger interactions between the CH3 domains in the resulting bispecific antibody than between the CH3 domains in any monospecific antibody without the mutation. A single point mutation in each monospecific antibody may be at residue 366, 368, 370, 399, 405, 407 or 409 (EU numbering) in the CH3 domain of the heavy chain constant region (see WO 2011/131746). Furthermore, a single point mutation is located at a different residue in one monospecific antibody relative to another monospecific antibody. For example, one monospecific antibody may comprise the mutation F405L (EU numbering; phenylalanine to leucine mutation at residue 405), or one of the mutations F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, and F405Y, while another monospecific antibody may comprise the mutation K409R (EU numbering; lysine to arginine mutation at residue 409). The heavy chain constant region of a monospecific antibody may be of the IgG1, igG2, igG3 or IgG4 isotype (e.g., human IgG1 isotype) and is produced by Bispecific antibodies produced by the technology can be modified to alter (e.g., reduce) Fc-mediated effector function and/or improve half-life. Preparation methodThe method comprises the following steps: (i) Separately expressing two parental IgG1 comprising a single matched point mutation in the CH3 domain, i.e., K409R and F405L (or F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, and F405Y mutations) (EU numbering); (ii) Mixing the parental IgG1 in vitro under permissive redox conditions to allow half-molecule recombination; (iii) removing the reducing agent to reoxidise the interchain disulfide bonds; and (iv) analysis of exchange efficiency and final product using chromatography-based or Mass Spectrometry (MS) -based methods (see Labrijn et al, nature Protocols,9 (10): 2450-2463 (2014)).

Another exemplary method for making bispecific antibodies is by knob-and-hole structured antibodies (knobs-int-holes) technology (Ridgway et al, protein Eng.,9, 617-621 (1996); WO 2006/028936). The mismatch problem of Ig heavy chains is reduced in this technique by mutating selected amino acids forming the interface of the CH3 domain in IgG, which is a major drawback in the preparation of bispecific antibodies. At the position in the CH3 domain where the two heavy chains interact directly, an amino acid with a small side chain (hole) is introduced into the sequence of one heavy chain, while an amino acid with a large side chain (knob) is introduced into the corresponding interaction residue position of the other heavy chain. In some cases, the antibodies of the present disclosure have an immunoglobulin chain in which the CH3 domain is modified by mutating selected amino acids that interact at the interface between the two polypeptides, thereby preferentially forming a bispecific antibody. Bispecific antibodies may consist of immunoglobulin chains of the same subclass or of different subclasses. In one instance, bispecific antibodies that bind gp120 and CD3 contain a T366W (EU numbering) mutation in the "knob chain" and a T366S, L368A, Y407V (EU numbering) mutation in the "hole chain". In certain embodiments, additional interchain disulfide bridges are introduced between CH3 domains, for example, by introducing a Y349C mutation into the "knob chain" and an E356C mutation or S354C mutation into the "hole chain". In certain embodiments, the R409D, K370E mutation is introduced in the "knob chain" and the D399K, E357K mutation is introduced in the "hole chain". In other embodiments, the Y349C, T366W mutation is introduced into one strand, and the E356C, T366S, L368A, Y407V mutation is introduced into the corresponding strand. In some embodiments, the Y349C, T366W mutations are introduced in one chain and the S354C, T366S, L368A, Y407V mutations are introduced in the corresponding chain. In some embodiments, the Y349C, T366W mutation is introduced into one chain, while the S354C, T366S, L368A, Y407V mutation is introduced into the corresponding chain. In yet other embodiments, the Y349C, T366W mutations are introduced into one chain and the S354C, T366S, L368A, Y407V mutations are introduced into the corresponding chain (all EU numbering).

Another exemplary method of making bispecific antibodies is by using bispecific T cell adaptorsA platform. BiTE is made by genetically fusing a first scFv (e.g., a scFv that binds gp 120) to a second scFv (e.g., a scFv that binds human CD 3) via a flexible peptide linker (e.g., GGGGS (SEQ ID NO: 76)). See, e.g., staerz et al, nature,314:628-631 (1985); mack et al, PNAS,92:7021-7025 (1995); huehls et al, immunol. Cell biol.,93:290-296 (2015).

Another exemplary method of making bispecific antibodies is by using a Dual Affinity Retargeting (DART) platform. This technique is based on a diabody version of Holliger et al (PNAS, 90, 6444-6448 (1993)) and further improves stability and optimal pairing of VH and VL chains (Johnson et al, J mol. Biol., 399.

Yet another exemplary method for making bispecific antibodies is through the use of a trifunctional hybrid antibody platformThe platform adopts two different same speciesType (i) full-length antibody (mouse IgG2a and rat IgG2 b). This technique relies on species-preferential heavy/light chain pairing. See Lindhofer et al, J immunol, 155:219-225 (1995).

A further exemplary method of making a bispecific antibody is by usingA platform. This technology is based on a diabody concept, but is designed to contain a single polypeptide chain VH1-VL2-VH2-VL1 with a short linker to prevent intra-chain pairing. The head-to-tail dimerization of this single chain results in the formation of tetravalent homodimers (Kipriyanov et al, J mol. Biol.,293, 41-56 (1999)).

Yet another method for making bispecific antibodies is CrossMab technology. CrossMab is a chimeric antibody composed of half of two full-length antibodies. For proper strand pairing, it combines two techniques: (i) A knob and mortar structure which facilitates correct pairing between the two heavy chains; and (ii) exchange between the heavy and light chains of one of the two fabs to introduce an asymmetry that avoids light chain mispairing. See, ridgway et al, protein eng, 9:617-621 (1996); schaefer et al, PNAS,108:11187-11192 (2011). CrossMab can combine two or more antigen binding domains to target two or more targets or to introduce a bivalent to one target, e.g., 2:1 format.

Multispecific antibodies of the present disclosure may be produced in bacteria or eukaryotic cells. Antibodies can also be produced in eukaryotic cells, such as transformed cell lines (e.g., CHO, 293E, 293T, COS, NIH3T 3). In addition, antibodies (e.g., scFv) can be expressed in yeast cells such as Pichia pastoris (see, e.g., powers et al, J Immunol methods.251:123-35 (2001)), hansenula, or Saccharomyces. In one embodiment, the bispecific antibody described herein is produced in a CHO cell line. To produce the antibody of interest, a polynucleotide encoding the antibody is constructed, introduced into an expression vector, and then expressed in a suitable host cell. Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture the host cells and recover the antibodies.

If the antibody is expressed in a bacterial cell (e.g., E.coli), the expression vector should have characteristics that allow the vector to be amplified in the bacterial cell. In addition, when Escherichia coli such as JM109, DH 5. Alpha., HB101 or XL1-Blue is used as a host, the vector must have a promoter which can be expressed efficiently in Escherichia coli, such as lacZ promoter (Ward et al, 341, 544-546 (1989)), araB promoter (Better et al, science,240, 1041-1043 (1988)), or T7 promoter. Examples of such vectors include, for example, M13 series vectors, pUC series vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), "QIAexpress System" (QIAGEN), pEGFP and pET (when the expression vector is used, the host is preferably BL21 expressing T7 RNA polymerase). The expression vector may comprise a signal sequence for secretion of the antibody. For production in the periplasm of E.coli, the pelB signal sequence (Lei et al, J.Bacteriol.,169, 4379 (1987)) can be used as a signal sequence for antibody secretion. For bacterial expression, the expression vector can be introduced into bacterial cells using calcium chloride methods or electroporation.

If the antibody is expressed in animal cells such as CHO, COS and NIH3T3 cells, the expression vector includes promoters required for expression in these cells, such as the SV40 promoter (Mullgan et al, nature,277, 108 (1979)), MMLV-LTR promoter, EF 1. Alpha. Promoter (Mizushima et al, nucleic Acids Res.,18 (1990)), or CMV promoter. In addition to the nucleic acid sequence encoding the immunoglobulin or domain thereof, the recombinant expression vector may carry additional sequences, such as sequences that regulate replication of the vector in a host cell (e.g., an origin of replication) and a selectable marker gene. The selectable marker gene facilitates selection of the host cell into which the vector has been introduced (see, e.g., U.S. Pat. nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically a selectable marker gene confers resistance to a drug such as G418, hygromycin or methotrexate to the host cell into which the vector has been introduced. Examples of vectors with selectable markers include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

In one embodiment, the antibody is produced in a mammalian cell. Exemplary mammalian host cells for expression of antibodies include Chinese Hamster Ovary (CHO) cells (including DHFR-CHO cells, as described in Urlaub and Chasin (1980) proc.natl.acad.sci.usa 77, 4216-4220, used with DHFR selectable markers, e.g., as described in Kaufman and Sharp (1982) mol.biol.159: 601-621), human embryonic kidney 293 cells (e.g., 293, 293e, 293t), COS cells, NIH3T3 cells, lymphocyte cell lines, e.g., NS0 myeloma and SP2 cells, and cells from transgenic animals (e.g., transgenic mammals). For example, the cell is a mammary epithelial cell.

In an exemplary system for antibody expression, recombinant expression vectors encoding the antibody heavy and light chains of bispecific antibodies of the disclosure are introduced into dhfr by calcium phosphate-mediated transfection ^- In CHO cells. In a specific embodiment, the dhfr-CHO cell is a cell of the DG44 cell line, such as DG44i (see, e.g., derouaz et al, biochem Biophys Res Commun.,340 (4): 1069-77 (2006)). In recombinant expression vectors, the antibody heavy and light chain genes are each operably linked to enhancer/promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus, etc., such as CMV enhancer/AdMLP promoter regulatory elements or SV40 enhancer/AdMLP promoter regulatory elements) to drive high levels of transcription of the genes. The recombinant expression vector also carries the DHFR gene, which allows the use of methotrexate selection/amplification to select CHO cells transfected with the vector. The selected transformant host cells are cultured to allow expression of the heavy and light chains of the antibody, and the antibody is recovered from the culture medium.

Multispecific antibodies can also be produced by transgenic animals. For example, U.S. Pat. No.5,849,992 describes a method of expressing antibodies in the mammary gland of transgenic mammals. A transgene comprising a milk-specific promoter and nucleic acid encoding the antibody of interest and a signal sequence for secretion is constructed. The milk produced by the female of such a transgenic mammal includes the antibody of interest secreted therein. The antibody may be purified from milk or, for some applications, used directly. Also provided are animals comprising one or more of the nucleic acids described herein.

Multispecific antibodies of the present disclosure can be isolated from the interior or exterior (e.g., culture medium) of a host cell and purified as substantially pure and homogeneous antibodies. The method of separation and purification generally used for antibody purification may be used for separation and purification of antibodies, and is not limited to any particular method. The antibody can be isolated and purified by appropriately selecting and combining, for example, column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization. Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography and adsorption chromatography (stratgies for Protein Purification and chromatography: A Laboratory Course Manual. Ed Daniel R. Marshak et al, cold Spring Harbor Laboratory Press, 1996). The chromatographic analysis can be performed using liquid chromatography such as HPLC and FPLC. Columns for affinity chromatography include protein a columns and protein G columns. Examples of chromatography columns using protein A columns include Hyper D, POROS and Sepharose FF (GE Healthcare Biosciences). The present disclosure also includes antibodies that are highly purified using these purification methods.

Pharmaceutical composition

The disclosure also includes pharmaceutical compositions comprising an antibody described herein or a polynucleotide encoding an antibody described herein and a pharmaceutically acceptable diluent, carrier, or excipient. In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an antibody or polynucleotide.

In light of this disclosure, those skilled in the art are aware of various pharmaceutically acceptable diluents, carriers, and excipients, as well as techniques for preparing and using pharmaceutical compositions. Illustrative pharmaceutical compositions and pharmaceutically acceptable diluents, carriers and excipients are also described in Remington: the Science and Practice of Pharmacy 20th Ed. (Lippincott, williams & Wilkins 2003). In particular embodiments, each carrier, diluent or excipient is "acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical composition and not injurious to the subject. Typically, the pharmaceutically acceptable carrier is an aqueous pH buffered solution. Some examples of materials that may be used as pharmaceutically acceptable carriers, diluents or excipients include: sterile water; buffers, for example, phosphate buffered saline; sugars such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; radix astragali powder; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol; a phosphate buffer solution; and other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, for example, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, mold release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

The methods of formulation and delivery of the pharmaceutical compositions are generally tailored to the site and disease being treated. Exemplary formulations include, but are not limited to, those suitable for parenteral administration, such as intravenous, intraarterial, intramuscular, or subcutaneous administration, including formulations encapsulated in micelles, liposomes, or drug release capsules (with the active agent incorporated into a biocompatible coating designed for sustained release); an ingestible formulation; formulations for topical use, such as creams, ointments and gels; and other formulations such as inhalants, aerosols and sprays.

Method of treatment

The present disclosure provides methods for treating or preventing HIV infection or a related disease or disorder in a subject in need thereof (e.g., a human subject), comprising providing to a subject in need thereof an effective amount of an antibody or a polynucleotide encoding an antibody described herein. As used herein, the term "effective amount" in the context of administering a therapy to a subject refers to the amount of the therapy that achieves the desired prophylactic or therapeutic effect. Multi-coreThe nucleotide may be present in a vector, such as a viral vector. In a particular embodiment, the associated disease or condition is caused by HIV infection. In a particular embodiment, it is acquired immunodeficiency syndrome (AIDS). In certain embodiments, the subject is a virally-inhibited HIV-infected mammal, while in other embodiments, the subject is an untreated HIV-infected mammal. In certain embodiments, the viral load of the untreated subject is at 10 ³ To 10 ⁵ Between copies/ml, and in certain embodiments, virologically suppressed subjects have a viral load < 50 copies/ml. In particular embodiments, the subject is a mammal, e.g., a human. In certain embodiments, the subject is diagnosed with, or is considered at risk for developing, an HIV (such as HIV-1 or HIV-2) infection or associated disease or condition (such as AIDS). Subjects at risk for an HIV-associated disease or disorder include patients who have been in contact with or otherwise exposed to HIV from an infected person. Administration of a prophylactic agent can be carried out prior to the onset of symptoms characteristic of an HIV-associated disease or condition, such that the disease or condition is prevented or, optionally, its development is delayed.

Also provided are methods for preventing or inhibiting HIV viral titer, viral replication, viral propagation, or an increase in the amount of HIV viral DNA, HIV proviral DNA, or HIV viral protein in a subject (e.g., a human subject). In one embodiment, the method comprises providing to a subject in need thereof an antibody described herein or a polynucleotide encoding the antibody effective to prevent an increase in HIV titer, viral replication, or HIV protein amount of one or more HIV strains or isolates in the subject. In certain embodiments, the method further comprises measuring the amount of HIV viral or proviral DNA or protein at one or more time points before and after the subject is provided with the antibodies of the present disclosure. Methods and biomarkers for determining the amount of HIV viral or proviral DNA or protein in a subject are known in the art and are available and are described, for example, in silicano, j.d. et al, curr Opin HIV AIDS,5 (6): 491-7 (2010) and Rouzioux, c. et al, curr Opin HIV AIDS,8 (3): 170-5 (2013).

As described hereinCD4 isolatable from patients with combined antiretroviral therapy (cART) inhibition ⁺ The T cells reactivate latent HIV ex vivo and thus can actually increase the titer of the HIV virus. This is thatBecause it can activate latently infected cells to express gp120 and then potentially be targeted for clearance. Binding to CD3 induces a partial T cell activation phenotype while latently infected CD4 ⁺ Activation of T cells then results in viral expression. Thus, also featured are methods of reversing HIV latency in a subject in need thereof. The method comprises administering gp120X CD3 as described herein to a human subject in need thereofIn certain embodiments, the method further comprises, e.g., in providing the subject with a composition of the present disclosureThe amount of HIV RNA, viral or proviral DNA or protein is measured at one or more time points before and after.

In certain aspects, the antibodies of the present disclosure may be used, for example, in methods of inhibiting certain viruses, such as HIV isolates described herein, prophylactically inhibiting or preventing infection of certain viruses, such as HIV isolates described herein, detecting certain viruses in a sample, such as HIV isolates described herein, inhibiting certain viruses, such as HIV isolates described herein, diagnosing certain viruses, such as HIV isolates described herein.

For in vivo treatment of a mammalian subject, e.g., a human, the subject can administer or provide a pharmaceutical composition comprising a multispecific antibody described herein. When used for in vivo therapy, the antibodies described herein are typically administered or provided to a patient in a therapeutically effective amount (i.e., to eliminate or reduce the viral load and/or viral pool of the patient). The antibody is administered or provided to a mammalian subject, e.g., a human, according to known methods, such as, but not limited to, intravenous administration (e.g., bolus injection or by continuous infusion over a period of time), by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes. The antibody may be administered parenterally or intravenously at the site of the target cell where possible. In one embodiment, the antibody is administered to the subject by an intravenous route. In another embodiment, the antibody is administered to the subject by a subcutaneous route. In particular embodiments, the pharmaceutical compositions of the present disclosure are administered to a subject systemically, parenterally, or topically.

In certain embodiments, the present disclosure provides a method for treating HIV infection comprising administering to a patient in need thereof a therapeutically effective amount of an antibody disclosed herein.

Combination therapy

In certain embodiments, a method is provided for treating or preventing HIV infection in a human having or at risk of having the infection, the method comprising administering to the human a therapeutically effective amount of an antibody disclosed herein, or a pharmaceutical composition thereof, in combination with a therapeutically effective amount of one or more (e.g., one, two, three, one or two, or one to three) other therapeutic agents. In one embodiment, a method is provided for treating an HIV infection in a human having or at risk of having the infection, comprising administering to the human a therapeutically effective amount of an antibody disclosed herein, or a pharmaceutically acceptable salt thereof, in combination with an effective amount of one or more (e.g., one, two, three, one or two, or one to three) other therapeutic agents.

In one embodiment, a pharmaceutical composition is provided comprising an antibody disclosed herein, or a pharmaceutical composition thereof, in combination with one or more (e.g., one, two, three, one or two, or one to three) additional therapeutic agents, and a pharmaceutically acceptable carrier, diluent, or excipient.

In certain embodiments, the methods provided by the present disclosure for treating HIV infection comprise administering to a patient in need thereof a therapeutically effective amount of an antibody disclosed herein, or a pharmaceutical composition thereof, in combination with a therapeutically effective amount of one or more other therapeutic agents useful for treating HIV infection.

In certain embodiments, the antibodies disclosed herein or pharmaceutical compositions thereof are combined with one, two, three, four, or more additional therapeutic agents. In certain embodiments, the antibodies disclosed herein or pharmaceutical compositions thereof are combined with two additional therapeutic agents. In other embodiments, the antibodies disclosed herein or pharmaceutical compositions thereof are combined with three additional therapeutic agents. In further embodiments, the antibodies disclosed herein or pharmaceutical compositions thereof are combined with four additional therapeutic agents. The one, two, three, four or more additional therapeutic agents may be different therapeutic agents selected from the same therapeutic agent class, and/or they may be selected from different therapeutic agent classes.

In certain embodiments, the antibodies disclosed herein are administered with one or more additional therapeutic agents. Co-administration of an antibody disclosed herein with one or more additional therapeutic agents generally refers to the simultaneous or sequential administration of a compound disclosed herein and one or more additional therapeutic agents such that a therapeutically effective amount of both the antibody disclosed herein and the one or more additional therapeutic agents are present in the patient. When administered sequentially, the combination may be administered two or more times.

Co-administration includes administering a unit dose of an antibody disclosed herein before or after administration of a unit dose of one or more other therapeutic agents. For example, an antibody disclosed herein can be administered within seconds, minutes, or hours of administration of one or more additional therapeutic agents. In some embodiments, a unit dose of an antibody disclosed herein is administered first, followed by a unit dose of one or more additional therapeutic agents within seconds or minutes. Alternatively, a unit dose of one or more additional therapeutic agents is administered first, followed by administration of a unit dose of an antibody disclosed herein within seconds or minutes. In other embodiments, a unit dose of an antibody disclosed herein is administered first, followed by administration of a unit dose of one or more additional therapeutic agents several hours (e.g., 1-12 hours) later. In still other embodiments, a unit dose of one or more additional therapeutic agents is administered first, followed by administration of a unit dose of an antibody disclosed herein after several hours (e.g., 1-12 hours).

In certain embodiments, the antibodies disclosed herein are combined with one or more additional therapeutic agents in a single dosage form for simultaneous administration to a patient, e.g., as a solid dosage form for oral administration.

In certain embodiments, the antibodies of the present disclosure are formulated as liquids, which may optionally comprise additional therapeutic agents for the treatment of HIV. In certain embodiments, the liquid may comprise another active ingredient for the treatment of HIV, such as HIV protease inhibitors, HIV non-nucleoside or non-nucleotide reverse transcriptase inhibitors, HIV nucleoside or nucleotide reverse transcriptase inhibitors, HIV integrase inhibitors, HIV non-catalytic site (or allosteric) integrase inhibitors, pharmacokinetic enhancers, and combinations thereof.

In certain embodiments, such formulations are suitable for once-a-day administration. In the above embodiments, the additional therapeutic agent may be an anti-HIV agent. In some cases, it is possible to use, additional therapeutic agents may be HIV protease inhibitors, HIV non-nucleoside or non-nucleotide reverse transcriptase inhibitors, HIV nucleoside or nucleotide reverse transcriptase inhibitors, HIV integrase inhibitors, HIV non-catalytic site (or allosteric) integrase inhibitors, HIV entry inhibitors, HIV maturation inhibitors, latency reversal agents, compounds targeting the HIV capsid, immune-based therapies, phosphatidylinositol 3-kinase (PI 3K) inhibitors, HIV antibodies, bispecific antibodies and "antibody-like" therapeutic proteins, HIV p17 matrix protein inhibitors, IL-13 antagonists, peptidyl-prolyl cis-trans isomerase A modulators, protein disulfide isomerase inhibitors, complement C5a receptor antagonists, DNA methyltransferase inhibitors, HIV Vif gene modulators, vif dimerization antagonists, HIV-1 viral infection factor inhibitors, TAT protein inhibitors, HIV-1Nef modulators, hck tyrosine kinase modulators, mixed kinase lineage 3 (MLK-3) inhibitors, HIV-1 splicing inhibitors, rev protein inhibitors, integrin antagonists, nucleoprotein inhibitors, HIV factor modulators, cycM 1-containing modulators, cycM 1 domain modulators, HIV-tyrosine kinase inhibitors, HIV-dependent cyclin domain inhibitors, cDNA kinase inhibitors, cDNA 1-dependent cyclin inhibitors, cDNA kinase inhibitors, cDNA 1-inducible protein kinase inhibitors, cDNA-inducible protein inhibitors, cDNA 9 inhibitors, cDNA-inducible protein kinase inhibitors, cDNA-inducible protein inhibitors, cDNA-converting inhibitors, cDNA-like, G6PD and NADH-oxidase inhibitors, pharmacokinetic enhancers, HIV gene therapy, HIV vaccines, and combinations thereof.

In some embodiments, the additional therapeutic agent is selected from the group consisting of combination drugs for HIV, other drugs for HIV, HIV protease inhibitors, HIV reverse transcriptase inhibitors, HIV integrase inhibitors, HIV non-catalytic site (or allosteric) integrase inhibitors, HIV entry (fusion) inhibitors, HIV maturation inhibitors, latency reversal agents, capsid inhibitors, immune-based therapies, PI3K inhibitors, HIV antibodies and bispecific antibodies, and "antibody-like" therapeutic proteins, and combinations thereof.

Examples of combination drugs that can be used with the antibodies of the present disclosure include(efavirenz, tenofovir disoproxil fumarate and emtricitabine);(rilpivirine, tenofovir disoproxil fumarate, and emtricitabine);(rilpivirine, tenofovir disoproxil fumarate and emtricitabineShore);(elvitegravir, coltstat, tenofovir disoproxil fumarate, and emtricitabine);(tenofovir disoproxil fumarate and emtricitabine; TDF + FTC);(tenofovir alafenamide and emtricitabine);(tenofovir alafenamide, emtricitabine, and rilpivirine);(tenofovir alafenamide, emtricitabine, cosmostat and elvitegravir); darunavir, tenofovir alafenamide hemifumarate, emtricitabine, and costatate; efavirenz, lamivudine and tenofovir disoproxil fumarate; lamivudine and tenofovir disoproxil fumarate; tenofovir and lamivudine; tenofovir alafenamide and emtricitabine; tenofovir alafenamide hemifumarate and emtricitabine; tenofovir alafenamide hemifumarate, emtricitabine and rilpivirine; tenofovir alafenamide hemifumarate, emtricitabine, cosotal and elvitegravir; (zidovudine and lamivudine; AZT +3 TC);(abacavir sulfate and lamivudine; ABC +3 TC);(lopinavir and ritonavir);(dolutegravir, abacavir and lamivudine);(abacavir sulfate, zidovudine and lamivudine; ABC + AZT +3 TC); atazanavir and comparastat; atazanavir sulfate and comparastat; atazanavir sulfate and ritonavir; darunavir and costal; dolutegravir and rilpivirine; dolutegravir and rilpivirine hydrochloride; dolutegravir, abacavir sulfate and lamivudine; lamivudine, nevirapine, and zidovudine; rosiglivir and lamivudine; dolavine, lamivudine and tenofovir disoproxil fumarate; dolavine, lamivudine and tenofovir disoproxil; dolutegravir + lamivudine, lamivudine + abacavir + zidovudine, lamivudine + abacavir, lamivudine + tenofovir fumarate, lamivudine + zidovudine + nevirapine, lopinavir + ritonavir + abacavir + lamivudine, lopinavir + ritonavir + zidovudine + lamivudine, tenofovir + lamivudine, and tenofovir fumarate + ritonavir + rillitabine + rilpivirine hydrochloride, lopinavir, ritonavir, zidovudine, and lamivudine; vacc-4x and romidepsin; and APH-0812.

Examples of other drugs that may be used in combination with the antibodies of the present disclosure for the treatment of HIV include: acetyl-morphinan, aprevir, banLec, deferiprone, gamimone, maytansinoid, naltrexone, prolactin, REP 9, RPI-MN, VSSP, H1viral, SB-728-T, 1, 5-dicaffeoylquinic acid, rHIV7-shl-TAR-CCR5RZ, AAV-eCD4-Ig gene therapy, mazF gene therapy, blockAide, ABX-464, AG-1105, APH-0812, BIT-225, CYT-107, HGTV-43, HPH-1023H-116, HS-635, IMO-3100, IND-02, MK-1376, MK-8507, MK-8591, NOV-205, PA-1050040 (PA-PGN-007, SCY-635, SB-9200, SCB-90452, TEV-90110, TEV 719-90112, TEV-040, VIMUV-576, and VIPGR-576.

Examples of HIV protease inhibitors that can be combined with the antibodies of the present disclosure include amprenavir, atazanavir, brecanavir, darnaravir, fosamprenavir calcium, indinavir sulfate, lopinavir, nelfinavir mesylate, ritonavir, saquinavir mesylate, tipranavir, DG-17, TMB-657 (PPL-100), T-169, BL-008, and TMC-310911.

Examples of HIV nucleoside or non-nucleotide reverse transcriptase inhibitors that may be combined with the antibodies of the present disclosure include dapivirine, delavirdine mesylate, dolavine, efavirenz, etravirine, lentinan, nevirapine, rilpivirine, ACC-007, AIC-292, KM-023, and VM-1500.

Examples of HIV nucleoside or nucleotide reverse transcriptase inhibitors that may be combined with an antibody of the present disclosure include adefovir, adefovir dipivoxil, alfvudine, emtricitabine, tenofovir alafenamide fumarate, tenofovir alafenamide hemifumarate, tenofovir disoproxil fumarate, tenofovir disoproxil hemifumarate,and(didanosine, ddl), abacavir sulfate, alovudine, aplidine, censvudine, didanosine, elvitein, filtinivir, fosalvudine tidoxil, CMX-57, dapivirine, dolavine, etravirine, OCR-5753, tenofovir disoproxil orotate, fosalvudine tidoxil, lamivudine, phosphazene, stavudine, zalcitabine, zidovudine, GS-9131, GS-9148, and KP-1461.

Examples of HIV integrase inhibitors that may be combined with the antibodies of the present disclosure include elvitegravir, curcumin derivatives, sebacic acid derivatives, 3, 5-dicaffeoylquinic acid, aurintricarboxylic acid, derivatives of aurintricarboxylic acid, caffeic acid phenethyl ester, derivatives of caffeic acid phenethyl ester, tyrphostin (tyrphostin), tyrphostin derivatives, quercetin derivatives, raltegravir, dolutegravir, JTK-351, bictegravir, AVX-15567, cabotegravir (long acting injection), diketimine 4-1 derivatives, integrase LEDGF inhibitors, ledggins, M-522, M-532, NSC-310217, NSC-371056, NSC-48240, NSC-642710, NSC-699171, NSC-699172, stilbene C-699173, NSC-699174, disulfonic acid, and cacivir 169.

Examples of HIV non-catalytic sites or allosteric integrase inhibitors (NCINIs) that can be combined with the antibodies of the present disclosure include CX-05045, CX-05168, and CX-14442.

Examples of HIV entry (fusion) inhibitors that may be combined with the antibodies of the present disclosure include cericiviroc, CCR5 inhibitors, gp41 inhibitors, CD4 attachment inhibitors, gp120 inhibitors, and CXCR4 inhibitors.

Examples of CCR5 inhibitors that may be combined with the antibodies of the present disclosure include apviravir, vicrivaroc, maraviroc, cenicriviroc, PRO-140, adaptavir (RAP-101), nifeviroc (TD-0232), anti-GP 120/CD4 or CCR5 bispecific antibodies, B-07, MB-66, polypeptide C25P, TD-0680, and vMIP (Haimipouu).

Examples of gp41 inhibitors that can bind to the antibodies of the present disclosure include abacavir peptide, enfuvirtide, BMS-986197, enfuvirtide biologics, enfuvirtide biosimiders, HIV-1 fusion inhibitors (P26-Bapc), ITV-1, ITV-2, ITV-3, ITV-4, PIE-12 trimer, and Cifuvirtide.

Examples of CD4 attachment inhibitors that can be combined with the antibodies of the present disclosure include ibalizumab and CADA analogs.

Examples of gp120 inhibitors that can bind to the antibodies of the present disclosure include Radha-108 (receptor) 3B3-PE38, banLec, bentonite-based nanomedicines, fostemavir tromethamine, IQP-0831, and BMS-663068

Examples of CXCR4 inhibitors that can be combined with the antibodies of the present disclosure include plexafor, ALT-1188, N15 peptide, and vMIP (Haimipu).

Examples of HIV maturation inhibitors that can be combined with the antibodies of the present disclosure include BMS-955176 and GSK-2838232.

Examples of latency reversing agents that can bind to the antibodies of the present disclosure include Histone Deacetylase (HDAC) inhibitors, proteasome inhibitors such as velcade, protein Kinase C (PKC) activators, smyd2 inhibitors, BET-bromodomain 4 (BRD 4) inhibitors, ionomycin, PMA, SAHA (mercaptohydroxamic acid or sulfinyl, aniline and hydroxamic acid), NIZ-985, IL-15, JQ1, disulfiram, amphotericin B, and ubiquitin inhibitors (e.g., lagrazole analogs and GSK-343). Examples of HDAC inhibitors include romidepsin, vorinostat and panobinostat. Examples of PKC activators include indomethacin, prostratin, eugenol B, and DAG lactone.

Examples of capsid inhibitors that may be combined with the antibodies of the present disclosure include capsid polymerization inhibitors or capsid disrupting compounds, HIV nucleocapsid p7 (NCp 7) inhibitors such as azodicarbonamide, HIV p24 capsid protein inhibitors, AVI-621, AVI-101, AVI-201, AVI-301, and AVI-CAN1-15 series.

Examples of immune-based therapies that can be combined with the antibodies of the present disclosure include toll-like receptor modulators, such as TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13; programmed cell death protein 1 (PD-1) modulators; programmed death ligand 1 (PD-L1) modulators; an IL-15 agonist; dermaVir; interleukin 7; plaquenil (hydroxychloroquine); proleukin (aldesleukin, IL-2); interferon alpha; interferon alpha-2 b; interferon alpha-n 3; pegylated interferon alfa; an interferon gamma; a hydroxyurea; mycophenolic acid (MPA) and its ester derivative Mycophenolate Mofetil (MMF); ribavirin; ritatolimumod, polymeric Polyethyleneimine (PEI); gepon; ritatolimud; IL-12; WF-10; VGV-1; MOR-22; BMS-936559; CYT-107, interleukin 15/Fc fusion protein, norferon, pegylated interferon alpha-2 a, pegylated interferon alpha-2 b, recombinant interleukin 15, RPI-MN, GS-9620, and IR-103.

Examples of PI3K inhibitors that may be combined with the antibodies of the present disclosure include idarasib (idelalisib), abelisib (aleglisib), buparlisib (buparlisib), CAI orotic acid, copanlisib, duvelisib, gedatolisib, neratinib, panculisib, periplosine, pictilisib, pilalaisib, puquitinib mesylate, rigosetib sodium, sonolisib, taselisib, AMG-319, AZD-8186, BAY-1082439, CLR-1401, CLR-457, CUDC-907, DS-7423, SAR-245042, GSK-2126458, GSK-2269577, GSK-2636771, INC-36093, INN-3024, MLN-3417, SAR-1727667, TGR-17280, TGRG-849, TGRG-04017, TGRG-310, TGS-04017, TGS-849, TGS-310, VSF-103, TGS-310, ZSR-103, and ZSR-103.

Examples of integrin alpha-4/beta-7 antagonists that can be combined with the antibodies of the present disclosure include PTG-100, TRK-170, albugumab (abrilumab), epratuzumab (etrolizumab), carprosettemethyl (carotegrast methyl), and vedolizumab (vedolizumab).

Examples of HIV antibodies, bispecific antibodies, and "antibody-like" therapeutic proteins that can be combined with the antibodies of the present disclosure includeFab derivatives, bNAbs (broadly neutralizing HIV-1 antibody), BMS-936559, TMB-360 and antibodies targeting HIV GP120 or GP41, HIV-targeting antibody recruiting molecules, anti-CD 63 monoclonal antibodies, anti-GB virus C antibodies, anti-GP 120/CD4, CCR5 bispecific antibodies, anti-nef single domain antibodies, anti-Rev antibodies, camel-derived anti-CD 18 antibodies, camel-derived anti-ICAM-1 antibodies, DCVax-001, GP140 targeting antibodies, GP 41-based HIV therapeutic antibodies, human recombinant mAb (PGT-121), ebalizumab, immuglo, MB-66. Examples of those that target HIV in this manner include bavimab, UB-421, C2F5, C2G12, C4E10, C2F5+ C2G12+ C4E10, 3BNC-117, PGT145, PGT121, PGDM1400, MDX010 (ipilimumab), VRC01, A32, 7B2, 10E8, VRC-07-523, VRC-HIVMAB080-00-AB, MGD-014, and VRC07. Other example packages of antibodies targeting HIV Including PGT122, PGT123, PGT124, 10-1074, PGT133, PGT134, PG16, PG9, PGT151, etc.

Examples of pharmacokinetic enhancers that may be combined with the antibodies of the present disclosure include cobicistat and ritonavir.

Examples of other therapeutic agents that may be combined with the antibodies of the present disclosure include compounds disclosed in WO 2004/096286 (Gilead Sciences), WO 2006/015261 (Gilead Sciences), WO 2006/110157 (Gilead Sciences), WO 2012/003497 (Gilead Sciences), WO 2012/003498 (Gilead Sciences), WO 2012/145728 (Gilead Sciences), WO 2013/006006006484 (Gilead Sciences), WO 2013/159064 (Gilead Sciences), WO 2014/100323 (Gilead Sciences), 201us 3/0165489 (University of Pennsylvania), US/1378 (Japan tobacaco), US/0221380 (Japan tobacaco), WO 2009/WO inehringe (ingenhingenhingenhinger), WO 062mange 201wo 201022 (WO 201wo 200022), WO 201wo 062/WO 2014, WO 43006436 (WO 2014/WO 2014) and WO 2014/1303 (WO 2014) and WO 0226 (WO 2014).

Examples of HIV vaccines that can be combined with the antibodies of the disclosure include peptide vaccines, recombinant subunit protein vaccines, live vector vaccines, DNA vaccines, CD 4-derived peptide vaccines, vaccine combinations, rgp120 (AIDSVAX), ALVAC HIV (vCP 1521)/AIDSVAX B/E (GP 120) (RV 144), monomeric GP120 HIV-1 subtype C vaccines, remune, ITV-1, contre Vir, ad5-ENVA-48, DCVax-001 (CDX-2401), vacc-4x, vacc-C5, VAC-3S, multiclade DNA recombinant adenovirus 5 (rAd 5), pennvax-G, pennvax-GP, HIV-Trimix-mRNA vaccines, HIV-LAMP-vax, ad35-GRIN, NAcGM 3/VSSP-51, poly-ImICLC adjuvants, tatmone vaccines, tatmuXe vaccines, and HIV-V-I vaccines GTU-MultiHIV (FIT-06), GP140[ Delta ] V2.TV1+ MF-59, rVSVIN HIV-1 Gag vaccine, seV-Gag vaccine, AT-20, DNK-4, ad35-GRIN/ENV, TBC-M4, HIVAX-2, NYVAC-HIV-PT1, NYVAC-HIV-PT4, DNA-HIV-PT123, rAAV1-PG9DP, GOVX-B11, GOVX-B21, TVI-HIV-1, VX-1, VVX-4, and HIV-4 Ad-4 (Ad 4-ENV clade C + Ad 4-mGag), EN41-UGR7C, EN41-FPA2, preVaxtat, AE-H, MYM-V101, combiHIVvac, ADVAX, MYM-V201, MVA-CMDR, DNA-Ad5 Gag/pol/nef/nev (HVTN 5), MVGTG-17401, ETV-01, CDX-1401, rcAD26.MOS1.HIV-ENV, ad26.Mod. HIV vaccine, and, AGS-004, AVX-101, AVX-201, PEP-6409, SAV-001, thV-01, TL-01, TUTI-16, VGX-3300, IHV-001, and virus-like particle vaccines such as pseudovirion vaccines, combivicvac, LFn-p 24B/C fusion vaccines, GTU-based DNA vaccines, HIV gag/pol/nef/Env DNA vaccines, anti-TAT HIV vaccines, binding polypeptide vaccines, dendritic cell vaccines, gag-based DNA vaccines, GI-2010, GP41 HIV-1 vaccines, HIV vaccines (PIKA adjuvants), ii-key/MHC class II epitope hybrid peptide vaccines, ITV-2, ITV-3, ITV-4, evolutionary LIPO-5, clade Env vaccines, MVA vaccines, pevax-GP, pp71 deficient HIV vector, HIV vaccine (SCBcMV), recombinant peptide vaccine (HIV infection), NCI 160, HIV-120, HIV RNgt-703, HIV-5, HIV-HBr vaccine, HIV-4-HBr vaccine, HIV-HBr virus variant vaccine, HIV-HBr virus-B vaccine, HIV-5, HIV-HBc vaccine, HIV-7, HIV-HBc vaccine, HIV-4 and HIV vaccine variants thereof.

Therapeutic agents for birth control (contraceptives) that can be combined with the antibodies of the present disclosure include cyproterone acetate, desogestrel, dienogest, drospirenone, estradiol valerate, ethinyl estradiol, norethindrone, etonogestrel, levofolinic acid, levonorgestrel, ethinyl estrenol, medroxyprogesterone acetate, mestranol, mifepristone, misoprostol acetate, nomegestrol acetate, norelgestromin, norethindrone, norgestimate, oxymetafene, segastersone acetate, ulipristal acetate, and any combination thereof.

In certain embodiments, the antibodies disclosed herein, or pharmaceutically acceptable salts thereof, are conjugated with one, two, three, four, or more members selected from the group consisting of(efavirenz, tenofovir fumarate and emtricitabine);(rilpivirine, tenofovir disoproxil fumarate, and emtricitabine);(elvitegravir, coltstat, tenofovir disoproxil fumarate, and emtricitabine);(tenofovir disoproxil fumarate, emtricitabine; TDF + FTC);(tenofovir alafenamide and emtricitabine);(tenofovir alafenamide, emtricitabine, and rilpivirine);(tenofovir alafenamide, emtricitabine, cosotal and elvitegravir); adefovir dipivoxil; adefovir dipivoxil; can be compared with the sitagliptin; emtricitabine; tenofovir; tenofovir disoproxil fumarate; tenofovir disoproxil fumarate; tenofovir alafenamide; tenofovir alafenamide hemifumarate; (dolutegravir, abacavir and lamivudine); dolutegravir, abacavir sulfate and lamivudine; leegievir; rosiglivir and lamivudine; maravir oc; enfuvirtide;(lopinavir and ritonavir);(zidovudine and lamivudine; AZT +3 TC);(abacavir sulfate and lamivudine; ABC +3 TC);(abacavir sulfate, zidovudine and lamivudine; ABC + AZT +3 TC); rilpivirine; rilpivirine hydrochloride; atazanavir sulfate and comparastat; atazanavir and comparastat; darunavir and cobicistat; atazanavir; atazanavir sulfate; dolutegravir; elvitegravir; ritonavir; atazanavir sulfate and ritonavir; darunavir; lamivudine; prolactin; fosamprenavir; fosamprenavir calcium efavirenz; etravirine; nelfinavir; nelfinavir mesylate; an interferon; didanosine; stavudine; indinavir; indinavir sulfate; tenofovir and lamivudine; zidovudine; nevirapine; saquinavir; saquinavir mesylate; aldesleukin; zalcitabine; tipranavir; apravir; delavirdine; delavirdine mesylate; radha-108 (receptor); lamivudine and tenofovir disoproxil fumarate; efavirenz, lamivudine and tenofovir disoproxil fumarate; phosphazenes; lamivudine, nevirapine, and zidovudine; abacavir; and abacavir sulfate.

In a specific embodiment, the antibodies disclosed herein or pharmaceutical compositions thereof are combined with an HIV nucleoside or nucleotide inhibitor of reverse transcriptase and an HIV non-nucleoside inhibitor of reverse transcriptase. In another specific embodiment, the antibodies disclosed herein or pharmaceutical compositions thereof are combined with an HIV nucleoside or nucleotide inhibitor of reverse transcriptase and an HIV protease inhibiting compound. In additional embodiments, the antibodies disclosed herein or pharmaceutical compositions thereof are combined with an HIV nucleoside or nucleotide inhibitor of reverse transcriptase, an HIV non-nucleoside inhibitor of reverse transcriptase, and a pharmacokinetic enhancer. In certain embodiments, the antibodies disclosed herein or pharmaceutical compositions thereof are combined with at least one HIV nucleoside inhibitor of reverse transcriptase, an integrase inhibitor, and a pharmacokinetic enhancer. In another embodiment, the antibodies disclosed herein or pharmaceutical compositions thereof are combined with HIV nucleoside or nucleotide inhibitors of two reverse transcriptases.

In particular embodiments, the antibodies disclosed herein or pharmaceutical compositions thereof are combined with abacavir sulfate, tenofovir disoproxil, tenofovir fumarate, tenofovir alafenamide hemifumarate, tenofovir alafenamide, or tenofovir alafenamide hemifumarate.

In particular embodiments, the antibodies disclosed herein or pharmaceutical compositions thereof are combined with tenofovir, tenofovir disoproxil fumarate, tenofovir alafenamide, or tenofovir alafenamide hemifumarate.

In a particular embodiment, the antibody or pharmaceutical composition thereof disclosed herein is combined with a first additional therapeutic agent selected from abacavir sulfate, tenofovir disoproxil fumarate, tenofovir alafenamide, and tenofovir alafenamide hemifumarate, and a second additional therapeutic agent selected from emtricitabine and lamivudine.

In a particular embodiment, the antibody disclosed herein or a pharmaceutical composition thereof is combined with a first additional therapeutic agent selected from the group consisting of tenofovir, tenofovir disoproxil, tenofovir alafenamide, and tenofovir alafenamide hemifumarate, and a second additional therapeutic agent, wherein the second additional therapeutic agent is emtricitabine.

In another specific embodiment, the antibody disclosed herein or a pharmaceutical composition thereof is combined with a first additional therapeutic agent (contraceptive) of cyproterone acetate, desogestrel, dienogest, drospirenone, estradiol valerate, ethinyl estradiol, norethindrone, etonogestrel, levofolinic acid, levonorgestrel, ethinyl estrenol acetate, medroxyprogesterone acetate, mestranol, mifepristone, misoprostol acetate, nomegestrol, norelgestromin, norethindrone, norgestimate, oxymetafil, segetssone acetate, ulipristal acetate, and any combination thereof.

Reagent kit

The present disclosure also encompasses kits comprising one or more of the antibodies described herein or conjugates thereof. In one instance, provided herein is a pharmaceutical package or kit comprising one or more containers filled with one or more ingredients of a pharmaceutical composition described herein, e.g., one or more antibodies provided herein. In some cases, a kit comprises a pharmaceutical composition described herein. In one embodiment, a kit is provided comprising an antibody disclosed herein or a pharmaceutical composition thereof in combination with one or more (e.g., one, two, three, one or two, or one to three) additional therapeutic agents (e.g., those described above). Optionally, associated with such containers may be a notice in a form prescribed by an institution for regulating the production, use, or sale of a pharmaceutical or biological product, the notice reflecting approval by the institution for manufacture, use, or sale for human administration.

Examples

The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. To the extent that specific materials are mentioned, this is for illustrative purposes only and is not intended to limit the invention. Those skilled in the art can develop equivalent means or reactants without the inventive faculty and without departing from the scope of the invention.

Example 1 Virus-neutralizing Activity

PGT121 is a highly neutralizing antibody with broad coverage of HIV subtype B isolates (IC 50 of 0.03. Mu.g/ml, breadth of 80%). The effectiveness (measured as IC50 or IC 95) and breadth (percentage of neutralized isolates of the tested groups) of neutralization of PGT121 and its variants were examined using two different published test methods:

i) Analysis based on CEM-NKr-CCR5-LucR reporter cell line (Li et al 2005.J Vir 79 (16): 10108-10125) suitable for screening antibodies against pseudotyped and replication competent HIV isolates; and

ii) Monogram HIV PhenoSense neutralization assay (Monogram Biosciences) using a luciferase reporter virus pseudotyped with the HIV Env variant of interest (Richman et al 2003.Pnas 100 (7): 4144-4149).

In a reporter cell line based CEM-NKr-CCR5-LucR neutralization assay (a multicycle virus replication assay) (Spenlehauer et al 2001.Virology, doi.

For the five viruses tested, neutralization potency of several variant PGT-121 antibodies was observed to be comparable to that of PGT-121 (also referred to herein as PGT-121LO 6), indicating that the modifications present in these antibodies had little effect on determinants of antigen recognition and binding compared to PGT-121 (for CEM-NKr-CCR5-Luc cells, table 5 below). Other variants (e.g., PGT121.60 and PGT 121.61) showed 2-3 fold improvement in neutralizing potency over this limited group of viruses compared to PGT121.

Table 5 pgt121 and selected variants were directed against the neutralizing activity of HIV-1 strains BaL, HT593, US657, US712 and US727 as observed using cem. Nkr. Ccr5.Lucr based assays. Data are presented as the average of 2-3 replicates

In the Monogram neutralization assay, the Env (gp 160) coding region was amplified from plasma viral RNA isolated from HIV + ART proviral blood patients and cloned into an expression vector so that the viral quasispecies profile present in the patient plasma samples was maintained. The expression vector is then used to generate a population of HIV-1 pseudoviruses expressing patient-derived Env proteins. Two sets of clade B clinical isolates were generated for Monogram neutralization analysis: group 1 (Monogram clinical isolate group) contained 63 isolates from the Monogram library pool and included 33 or more CCR 5-tropic viruses, 15 or more CXCR 4-tropic (X4) viruses, and 15 or more Dual Mixed (DM) tropic viruses; group 2 (Gilead clinical isolate group) contained 142 subtype B viruses isolated from pre-ART baseline plasma samples from HIV patients not receiving ART treatment participating in the clinical trial and included 113 CCR5 tropic (R5) viruses, 28 dual or mixed tropic (DM) viruses and one CXCR4 tropic (X4) virus. Given that HIV-1 Env exhibits significant diversity among patient isolates, between clades, and within clades, the neutralizing activity of PGT121 and variants was also profiled against viruses representing non-B clades using a group of viruses from the Monogram pool set. The Monogram HIV PhenoSense neutralization assay was used to profile large sets of patient isolates, enabling more rigorous profiling of both the breadth and potency of PGT121 and the variants generated. The results are shown in tables 6 to 9. The results indicate that variants of PGT121 (e.g., PGT 121.60) show enhanced neutralizing activity against the selected virus.

Table 6 neutralizing activity (IC) of PGT121 and PGT121.60 against subtype B viruses ₅₀ )

Table 7 neutralizing potency (IC) of PGT121 and PGT121.60 against subtype B virus ₅₀ )

Table 8 neutralizing potency and coverage of pgt121 and selected variants against 92 subtype B viruses

Table 9 neutralizing activity of PGT121 and PGT121.60 against multiclade virus

These experiments demonstrate an unexpected improvement in X4 tropic HIV neutralization of Fc-enhanced PGT121. In addition to CD4, HIV can utilize two co-receptors to enter T cells-CXCR 4 or CCR5. Co-receptor binding is mediated by Env, which is the target of broadly neutralizing antibodies described herein. Thus, different HIV strains with different sequences preferentially use CXCR4 (referred to as X4-tropism), CCR5 (referred to as R5-tropism), or both (referred to as X4/R5 or dual tropism). A viral pool exhibiting both R5 and X4 tropisms (referred to as Dual-Mixed or DM) may comprise a mixture of R5, X4 and/or Dual tropism strains. PGT121 generally showed low sensitivity (low potency and breadth) to X4 isolates, while preferentially neutralizing R5 tropic viruses. The addition of the Fc mutation DEAL + LS (PGT 121.56) to PGT121 specifically enhanced its neutralizing activity against DM and X4 tropic viruses (median IC50 enhancement 2-fold, and up to about 20-fold for at least one isolate). Although some PGT121 Fab variants (e.g., PGT121.13 and PGT 121.22) showed reduced neutralizing potency against R5 DM and X4 virus, several engineered PGT121 Fab variants with the DEAL + LS Fc mutation, including PGT121.56 with WT Fab, were more potent in neutralizing DM and X4 virus compared to R5 virus (P < 0.0001) (data not shown). This is highly unexpected because HIV neutralization is believed to be mediated only by the Fab domain (and not the Fc domain). In the R5 isolate, a 2 to 3 fold increase in neutralization was observed in about 46% of the isolates tested. DEAL + LS mutations are present in certain antibodies and fragments thereof of the present disclosure. Introduction of additional modifications of PGT121.56 further improved the neutralizing activity of the selected variants (e.g., PGT 121.60).

Coverage was calculated as the percentage of virus neutralized with IC95<15 μ g/ml. Potency was determined by calculating the median IC95 value in viruses with an IC95<15 μ g/ml. When tested against a panel of two HIV-1 isolates including a total of 89 clade B isolates, the antibodies of the present disclosure showed no loss of neutralizing activity compared to PGT121 (data not shown). Some antibodies were nearly as potent as PGT121, with a slightly increased extent of neutralization. Neutralization profiling was also used as an alternative assessment of the ability of antibodies to recognize and bind diverse Env antigens from a wide range of HIV-1 clinical isolates relative to PGT-121. Data from profiling of various antibodies showed that antibodies with reduced neutralizing potency also exhibited reduced ADCC activity (data not shown), indicating a positive correlation between neutralizing activity and ADCC activity of the antibody, and supporting the use of neutralizing breadth as an alternative to ADCC breadth assessment.

Example 2 immunogenicity Studies

Three methods were used to assess immunogenicity and to guide engineering to eliminate immunogenic motifs in PGT 121. Computer modeling prediction tools were used to identify sites of potential immunogenicity risk in PGT121 antibodies and also to direct engineering efforts to improve processability (e.g., removal of glycosylation sites, improvement of low pH to maintain stability) while preventing the introduction of new T cell epitopes. Based on this analysis, modifications of the framework regions to reduce immunogenicity are performed in the antibodies of the present disclosure, which have a low risk of affecting functional activity. In addition, to further identify potential immunogenic motifs within the variable domains of an antibody of the present disclosure, an ex vivo human T cell activation assay, epiScreen, was used ^TM (Antitope, ltd., cambridge, UK). CD4+ T cell responses induced in 50 healthy donors (representing multiple HLA haplotypes) in response to antibody-derived overlapping 15 amino acid peptides and KLH (keyhole limpet hemocyanin, positive control) were assessed using H-thymidine incorporation assays to measure T cell proliferation. This assay enables the localization of specific T cell epitopes in the primary antibody sequence to guide antibody engineering. It also provides a ranking of the relative immunogenicity of the T cell epitopes of the test antibodies (data not shown).

To assess the clinical immunogenicity risk of selected antibody variants, epiScreen was used ^TM Time course T cell analysis (antope, ltd., cambridge, UK) measures T cell activation induced by intact antibodies. As described (Baker and Jones 2007.Curr. Opin. Drug Discov. Devel.10: 219-227) was performed. Thus, the assay takes into account not only T cell epitope content, but also processing of native IgG. Unlike computer simulations and peptide scanning assays, whole molecule ex vivo T cell activation assays can assess the relative clinical risk of a given antibody, and in some cases can be used to predict clinical immunogenicity rates as described (Baker and Jones 2007curr. Opin. Drug discov.devel.10.

Many clinical phase antibodies were run in this assay, and antibodies that showed little or no clinical immunogenicity in this assay had scores near or below 10%, while antibodies that showed high clinical immunogenicity (such as alemtuzumab and infliximab) had scores in the range of 25% to 40% (Baker and Jones 2007.Curr. Opin. Drug discov. Devel.10. PGT121.42, PGT121.60, PGT121.61 and PGT121.65 showed reduced donor response rates compared to PGT121 WT (i.e. PGT-121LO 6), supporting a reduced risk of clinical immunogenicity for these variants (data not shown).

Example 3 fcrn binding

Neonatal Fc receptors (FcRn) are Fc receptors that have been shown to play a major role in regulating the pharmacokinetics of IgG molecules in human and preclinical species. After endocytosis, fcRn binds with high affinity to the Fc portion of IgG at acidic pH (< 6.5). FcRn-bound IgG circulates back to the extracellular space where at physiological pH, igG binding affinity decreases and IgG is released back into circulation. Free IgG, which is not rescued by the FcRn pathway, is degraded in lysosomes as endogenous amino acids. The relative binding affinity characteristics of IgG to FcRn at pH 6.0/7.0 have been a relevant factor for the determination of IgG half-life grading in vivo and are a design feature for pharmacokinetic optimization.

The binding of the antibody to FcRn of various antibody variants at different pH was determined. 96-well Maxisorp plates were coated with 100ul of 5. Mu.g/ml FcRn. The plates were incubated overnight at 4 ℃ and then blocked with 4% skim milk for 2 hours at room temperature after 3 washes with 0.05% Tween 20 wash buffer. Plates were incubated with 3-fold serial dilutions of primary antibody for 1 hour at room temperature. The plate was then washed 3 times and 100 μ L of either Fab-anti-human Fab-HRP or goat-anti-human IgG-HRP secondary antibody diluted in 4% skim milk was added. The plate was then incubated at room temperature for 50 minutes, washed 3 times, and 100 μ Ι _ of fresh TMB substrate was added. The plate was developed for 3 minutes on a gently shaken platform. The plate was quenched with 100. Mu.L of 1M HCl, shaken briefly, and read on a Spectrummax M5 plate reader under A450.

Antibodies of the present disclosure comprising LS mutations in the IgG Fc portion that interacts with FcRn show significant improvement in FcRn binding at pH 6.0, while having less impact on binding at neutral pH 7.0, relative to PGT-121, as represented by the pH 7.0/6.0 ratio for human FcRn. The improved binding is due to the presence of the LS mutation and is predicted to provide an extended half-life in humans relative to PGT-121. The data are shown in table 10.

Table 10 human FcRn binding data for pgt121 and variants

This data shows a significant improvement in PGTs 121.60 and 61 over PGT121.56 or PGT 121.42. PGT121.56 is WT Fab with DEAL + LS Fc. This indicates that Fab mutations in PGTs 121.60 and 61 improved FcRn binding. PGT121.64 and PGT121.65 did not show this improvement, indicating that Fab modifications in both variants can actually reduce FcRn binding.

Example 4 in vivo assay

PGT121 and several antibodies from the present application were analyzed to characterize their basic pharmacokinetic profile, ensuring enhanced Fab/Fc modification present in the antibodies of the present disclosure without significantly interfering with the intrinsic pharmacokinetic properties of PGT121. In vivo treatment of PGT121 and several other antibodies of the present disclosure was characterized after a single Intravenous (IV) 1.0mg/kg dose in two male naive cynomolgus monkeys (n = 2). Serum samples were collected from monkeys and analyzed using bioanalytical methods (described herein) to determine serum concentration-time curves and mean serum pharmacokinetic parameters by non-compartmental pharmacokinetic analysis (NCA).

In a separate study, the intrinsic pharmacokinetic behavior of PGT121, PGT121 LS and a new batch of PGT121.42 and PGT121.60 was characterized after a single intravenous 10.0mg/kg dose in three male naive cynomolgus monkeys (n = 3). Serum samples were collected and analyzed using bioanalytical methods (described herein) to determine serum concentration-time curves and mean serum pharmacokinetic parameters by non-compartmental pharmacokinetic analysis (NCA). The mean serum pharmacokinetic parameters for PGT121, PGT121.42, PGT121.43, PGT121.60 and PGT121.61 were determined from non-compartmental pharmacokinetic analysis of the concentration-time profile and are depicted in table 11. All antibodies of the present disclosure tested in vivo have comparable or improved pharmacokinetics (as defined herein) relative to PGT121.

TABLE 11 pharmacokinetic parameters of PGT121 and variants in primary cynomolgus monkeys (n = 2) after IV administration (1 mg/kg)

Mean serum pharmacokinetic parameters for PGT121, PGT121 LS, PGT121.42 and PGT121.60 were determined from non-compartmental pharmacokinetic analysis of the concentration-time profiles and are depicted in table 12.

TABLE 12 pharmacokinetic parameters of PGT121, PGT121 LS, PGT121.42, and PGT121.60 in naive cynomolgus monkeys (n = 3) after IV administration (10 mg/kg)

All antibodies of the present disclosure tested in vivo have comparable or improved pharmacokinetics (as defined herein) relative to PGT121. PGT121.60 showed enhanced efficacy against the test virus compared to PGT121 (data not shown). PGT121 variants (e.g. PGT121.60, PGT121.64 and PGT 121.65) showed increased potency in all tested virus isolates (data not shown). This indicates that the modifications made (possibly to antigen contact residues outside the CDRs) improve neutralization efficacy. PGT121.60 showed enhanced neutralizing activity against viruses representing subtypes B and non-B compared to PGT121 (data not shown).

Example 5 evaluation of gp120 X CD3 Ability of Duobody to kill HIV-infected cells

Exemplary gp 120X CD3Killing activity of (provided below) The heavy chain sequence of the gp120 portion of (a),the light chain of the gp120 portion of (a) has the amino acid sequence of SEQ ID NO: 10; are as followsThe heavy chain sequence of the CD3 portion of (1),the light chain of the CD3 portion of (a) has the sequence shown in SEQ ID NO: 20) and monospecific PGT121.60 (SEQ ID NO:41 and 10) were evaluated against 22 major HIV-1 isolates or clones. Each virus was evaluated with an average of 4 healthy PBMC donors (see table 13). Exemplary gp 120X CD3(mean. + -. SD; 70%. + -. 11%) the proportion of infected cells killed (Emax) was significantly higher compared to PGT121.60 (mean. + -. SD; 56%. + -. 16%; paired T test, P = 0.001). Exemplary gp 120X CD3The efficacy of (D) is also significantly higher than that of PGT121.60 and that of PGT121.601.034. Mu.g/mL. + -. 1.408. Mu.g/mL, an EC of 0.129. Mu.g/mL. + -. 0.074. Mu.g/mL was obtained ₅₀ Values (paired T-test, P = 0.007). Exemplary gp 120X CD3 in comparison to PGT121.60EC of (1) ₅₀ The average fold change of (a) is 21 fold.

TABLE 13 exemplary gp 120X CD3And a summary of killing activity of PGT-121.60

In summary, exemplary gp 120X CD3A significantly greater proportion of HIV-infected cells were killed at a much lower concentration than PGT 121.60.

The heavy chain sequence of the gp120 portion of (a):

the heavy chain sequence of the CD3 portion of (a):

Method

Infected cell killing by PBMC effector cells

CD4 infection with primary resting HIV ⁺ T cells as target cells and autologous PBMC as effector cells for exemplary gp 120X CD3Dependent and PGT-121.60 dependent killing of HIV infected CD 4T cells was studied in vitro. Infection of primary CD4 by spinoction at 1200 Xg with 50-100ng p24/million cells ⁺ T cells for 2 hours, and at 37 degrees C in 30U/mL IL-2 (Roche catalog No. 11011456001) RPMI medium (supplement 10% FBS and 1% penicillin/streptomycin) cultured for 5 days. Transfected (transfected) CD4 after 5 days of standing to allow expression from the head antigen ⁺ T cell cultures were washed 3 times to remove free virus at 2x 10 ⁵ Cells/well were seeded in 96-well plates and mixed with 7 concentrations of exemplary gp 120X CD3 in a 10-fold serial dilution in the presence of human serum IgG (final concentration 5 mg/mL)Or PGT-121.60 for 1 hour. When CD4 ⁺ When the T cell target is conditioned, effector cells are prepared. Cryopreserved autologous PBMC were thawed and membrane stained with PKH67 and at 4x 10 according to manufacturer's instructions ⁵ Cells/well were added to conditioned target cells to produce an E: T ratio of 2. Effector cells were co-cultured with conditioned target cells at a final volume of 200 μ Ι _, per well for 24-48 hours.

Killing of HIV-infected target cells was determined by flow cytometry. At the end of the co-cultivation period, cells were washed 2 times with PBS, stained with 100 μ L Live/Dead Aqua (1/1000 dilution in PBS) for 10 minutes until the stain was inactivated by addition of 100 μ L FACS buffer (PBS +2% FBS). The cells were then washed with FACS buffer and incubated with anti-CD 4-PE/Cy7mAb (diluted 1/50 in FACS buffer) for 20 min, then washed 3 times with FACS buffer and fixed and permeabilized with 100. Mu.L of Cytofix/Cytoperm for 10 min. Cells were then washed once with PermWash and incubated with anti-p 24-PE mAb in FACS buffer +10% PermWash for 25 minutes. Finally, cells were washed 3 times with FACS buffer, resuspended in 120 μ L FACS buffer, and flow cytometric data were obtained on LSR Fortessa or X20 FACS (BD Biosciences, san Jose, CA) and analyzed using FlowJo software (TreeStar).

Data analysis

Killing infected primary CD4 by PBMC ⁺ T cells, HIV-infected target cells were counted by flow cytometry using the following gating strategy: lymphocytes were selected based on forward and side scatter, and Live lymphocytes were selected by negative staining of Live/Dead Aqua. CD4 representative of the vaccination was then selected ⁺ PKH67 negative viable lymphocytes of T cells and identifying HIV-infected cells as p24 Gag +, CD4 ^{Is low in} (due to HIV-mediated down-regulation of CD 4) cells. Percentage of HIV infection by HIV (p 24 Gag) ⁺ ,CD4 ^{Is low in} Positive) vaccination (PKH 67 negative) CD4 ⁺ The percentage of T cells is expressed.

Exemplary gp 120X CD3The percentage of HIV-infected target cells in treated or PGT-121.60 treated wells was compared to the average percentage of HIV-infected target cells in untreated wells (treated with human serum IgG only, n =2-10/96 well plates). The percent killing of HIV-infected target cells was calculated using the following formula:

100- ((% HIV-infected target cells in treated wells/% HIV-infected target cells in untreated wells) × 100).

Exemplary gp 120X CD3Or the maximum fraction of infected cells killed by PGT-121.60 (Emax) and the concentration giving half-maximal killing (EC) ₅₀ ) Obtained from dose-response curves (equation 1) fitted by three-parameter nonlinear regression using GraphPad Prism (La Jolla, CA) software.

Equation 1:

where Y =% killing, X = antibody concentration, bottom = reaction in the absence of antibody, and top = maximal reaction.

The apparent Emax <40% dose-response curve, EC50 values reported as > 100. Mu.g/mL and Emax absolute value < 0% assigned as 0%.

Example 6 evaluation of anti-gp 120 antibody variants

The aspartic acid at position 59 of the PGT-121.60 light chain variable domain isomerized to IsoAsp under accelerated stress conditions (25 ℃,40 ℃). Asp59 forms part of the binding motif for HIV Env N332 and is critical for gp120 binding to PGT-121.60, resulting in loss of antibody dependent cell mediated cytotoxicity (ADCC). Lyophilization of a pharmaceutical product can mitigate this chemical propensity.

PGT-121.60 is selected asOne of the arms of the molecule. Although lyophilization is a successful commercial strategy, it is desirable to eliminate the isomerization tendency from PGT-121.60 to simplify development and production, improve batch consistency and achieveLiquid formulations in the form. The engineering goal is to remove the aspartate isomerization site while maintaining biological activity and without reintroducing T cell epitopes. Characterization was continued from the first four engineered variants (variant 1, variant 2, variant 3, and variant 4), including binding and potency testing using PGT-121.60ADCC reporter cell-based assays.

All four variants have the same heavy chain amino acid sequence provided below:

variant 1 has the sequence of SEQ ID NO:40, or a light chain sequence as set forth in seq id no. Variant 2 has the amino acid sequence of SEQ ID NO:78 to seq id no. Variant 3 has the amino acid sequence of SEQ ID NO:79, or a pharmaceutically acceptable salt thereof. Variant 4 has the sequence of SEQ ID NO:80, or a light chain sequence as set forth in seq id no.

Binding study

The binding of the various variant antibodies described above to gp120 HIV ENV protein was determined. 384 well Maxisorp plates were coated with 25. Mu.l of 5. Mu.g/ml gp120. The plates were incubated overnight at 40 ℃. The plates were washed 4 times with PBS 0.05% Tween20 washing buffer and blocked with 75. Mu.l PBS 5% BSA shaking at 600rpm for 1 hour at room temperature. Plates were incubated with three-fold serial dilutions of primary antibody for 1 hour at room temperature with shaking at 600 rpm. The plates were then washed 4 times with PBS 0.05% Tween20 and 25. Mu.l of goat anti-human IgG (H + L) HRP secondary antibody was diluted in PBS 1% BSA and incubated for 40 minutes at room temperature with shaking at 600 rpm. The plates were washed 4 times with PBS 0.05%Tweenn 20 and 25. Mu.l of fresh TMB substrate was added. The plate was developed for 90 seconds with shaking at 600rpm and then quenched with 25. Mu.l of 1M HCl. Plates were read on a Spectramax m5 plate reader under a 450.

Variant antibodies of the present disclosure comprising a mutation at the aspartate isomerization site of the framework insertion loop have improved and reduced binding to gp120 relative to PGT-121.60. Sequence activity relationship analysis showed a clear and strong preference for maintaining aspartate at residue 59. Glycine is tolerated at residue 59, while glutamine, glutamic acid and asparagine negatively affect the ability of the antibody to bind gp120. At residue 60, a bulky side group for the introduced amino acid is tolerated. The data are shown in Table 14 below.

Table 14 gp120 binding data for pgt121.60 and variants

Molecule	WITO EC50(nM)
		PGT121.60(DS)	0.36
Variant 2 (DS->DF)	0.15
		Variant 4 (DS->GS)	0.84
Variant 5 (DS->GF)	0.91
		Variant 6 (DS->ES)	21
Variant 7 (DS->NS)	59
		Variant 1 (DS->DY)	0.05
Variant 3 (DS->DT)	0.20
		Variant 8 (DS->QS)	1.37
Variant 9 (DS->EY)	19
		Variant 10 (DS->ET)	18

( In the above table, the letters next to the molecules correspond to the amino acids at positions 59 and 60 (Kabat numbering) of the light chain. The mutated residues are bolded. )

The data show that the improved gp120 binding of variants 2, 1 and 3 relative to PGT121.60 and other variants indicates that aspartate isomerization site mutations can affect binding to gp 120. These variants were tested with DEAL + LS Fc (i.e., fc with mutations at S239D, I332E, G236A, a330L, M428L, and N434S ("DEALLS").

Study of efficacy

PGT-121.60ADCC assay was chosen because it included a target cell line expressing HT593, and HT593 was known to be sensitive to isoAsp propensity (liability) in PGT-121.60. The ability of PGT-121.60 to induce nuclear factor of activated T cells (NFAT) -mediated luciferase expression was measured as a surrogate measure of ADCC activity based on an ADCC reporter assay. In this assay, jurkat cells expressing Fc γ RIIIa receptor (V158) and NFAT-regulated luciferase were incubated with gp 120-expressing Human Embryonic Kidney (HEK) cells in the presence of increased concentrations of PGT-121.60 reference standards, controls and samples. PGT-121.60 binds gp120 on the surface of HEK cells, effectively crosslinking Fc γ RIIIa on Jurkat cells and activating luciferase gene expression by NFAT. Relative Luminescence Units (RLU) were plotted against PGT-121.60 concentration and the efficacy of the controls and samples relative to a reference standard was determined using a parallel line assay procedure. As a first Reference Standard (RS), PGT-121.60 assigned a Relative Potency (RP) value of 100%.

Samples of PGT-121.60 variants (i.e., variant 1, variant 2, variant 3, and variant 4) were diluted to an intermediate concentration of 0.5mg/mL, and the protein concentration (maximum absorbance at 280 nm-absorbance at 320 nm) was confirmed by uv spectrophotometry. The intermediate concentration was then diluted to an initial concentration range of 0.6ng/mL to 4.96. Mu.g/mL. The samples were then tested according to the ADCC assay described above (control, 25 ℃,4 weeks) except that the heat stressed samples were quantified against their respective controls (designated 100% RP) instead of the PGT-121.60 reference standard.

Samples of pgt.121.60 variants (variant 1, variant 2, variant 3 and variant 4) were subjected to heat stress by incubation at 25 ℃ for 4 weeks. They were then tested against their corresponding untreated controls (stored at 2-8 ℃) in a pgt.121.60adcc reporter cell based assay. The results are shown in Table 15.

TABLE 15 ADCC Activity of Heat-stressed PGT-121.60 and IsoAsp variants

^a. PGT-121.60 was tested only once and the results were consistent with historical data.

The results show that while the activity of heat treated PGT-121.60 is reduced (RP of 65%), the ADCC activity of variant 2, variant 3, variant 4 and variant 1 (96%, 104%, 95% and 100%, respectively) is not affected by treatment at 25 ℃ for 4 weeks.

Example 7 evaluation of HIV neutralizing Activity

The antibodies were examined for their potency (measured as IC50 or IC 95) and breadth (% of neutralized isolates in the tested groups) of HIV-1 neutralization (as IC50 or IC 95) in (i) a CEM-NKr-CCR5-LucR reporter cell line-based assay and ii) a Monogram HIV PhenoSenses neutralization assay (Monogram Biosciences) as described in example 1.

In a neutralization assay based on the CEM-NKr-CCR5-LucR reporter cell line, antibodies were examined against a panel of 40 clade B replication competent HIV-1 isolates and clones.

The neutralizing potency of variants 1-4 was similar to that of PGT-121.60 (Student's paired T-test P value of each variant compared to PGT-121.60 ≧ 0.372). IC of variants compared to PGT-121.60 ₅₀ The geometric mean fold changes in values ranged from 1.035 for variant 1 to 1.539 for variant 2, indicating that mutations that abrogate the propensity for isoaspartic acid in PGT-121.60 did not significantly affect the HIV neutralizing activity of PGT-121.60. For 1 of the 40 viruses tested (virus 8339), the neutralizing activity of variants 1-4 was completely abolished. The results are shown in Table 16.

Table 16 neutralizing activity using an assay based on cem. Nkr. Ccr5. Lucr. Data represent the average of 2 replicates.

^a. na means "not applicable"

Geometric mean of calculation, exclusion of resistant viruses (IC 50) >100ug/mL)

In the Monogram HIV phenostrain neutralization assay, HIV neutralization activity of variants 1-3 was evaluated against a panel of Gilead clinical isolates (n =142 viruses) in a bispecific format with FEAR/FEAL Fc. When evaluated in the FEAR/FEAL bispecific format, variants 1-3 decreased in potency by a factor of 4.1 to 5.3 compared to PGT-121.60DEAL + LS (Table 17). Combined with the results of cem. Nkr. Ccr5.Lucr based assays (where variants were evaluated in DEAL + LS format), the results indicate bispecificForms, rather than mutations that eliminate the isoaspartic acid propensity in PGT-121.60, impair HIV neutralizing activity 4 to 5-fold.

TABLE 17 neutralization activity by neutralization assay using Monogram HIV PhenoSense. Data are representative of a single experiment.

^a. na means "not applicable"

Calculated geometric mean excludes resistant viruses (IC 50)>100ug/mL)

Example 8

The linkage of CD3+ T cells to target cells expressing the antigen of interest via CD 3-bispecific antibodies results in T cell activation. In the absence of antigen-positive target cells, no T cell activation is generally seen for CD3 bispecific antibodies. This dependence of T cell activation on the presence of antigen-positive target cells can prevent global T cell activation and limit adverse events associated with global T cell activation, such as cytokine release syndrome.

To evaluate gp120X CD3Ability to activate T cells only in the presence of both antigens, we isolated primary PBMCs from non-HIV infected donors (n = 2) and HIV infected donors (n = 2) with gp120X CD3Variant 1, gp120X CD3Incubation of variant 2 or PGT-121.60 with CEM-NKr-CCR5-LucR CD 4T cells (i) infected with gp120x CD3Killing-sensitive HIV-1 virus (7552), (ii) infection with gp120x CD3(ii) killing-resistant HIV-1 virus (THRO), or (iii) no infection. PBMC and CEM-NKr-CCR5-LucR CD 4T cells were co-cultured at a 1. Effector cells from non-HIV-infected (n = 2) and HIV-infected (n = 2) donors were evaluated. The results are shown in tables 18 to 23.

Both gp120X CD3 Duobody variant 1 (tables 18 and 19) and gp120X CD3 Duobody variant 2 (tables 20 and 21) induced dose-dependent upregulation of the T cell activation markers CD69 and CD25 on primary CD 4T cells and CD 8T. A slight upregulation of PD-1 was also observed. No up-regulation of Ki67 was observed. T cell responses from non-HIV-infected and HIV-infected donors were similar. Upregulation of the activation marker was dependent on gp120X CD3 Duobody binding to both antigens, as upregulation of the activation marker was only observed in the presence of CEM-NKr-CCR5-LucR CD 4T cells infected with HIV-1 virus (7552) sensitive to gp120X CD3 Duobody killing. No upregulation of activation markers was observed when effector cells were co-cultured with CEM-NKr-CCR5-LucR CD 4T cells infected with gp120X CD3 Duobody killing resistant HIV virus (THRO) or uninfected CEM-NKr-CCR5-LucR CD 4T cells. Whether or not CEM-NKr-CCR5-LucR CD 4T cells are infected with gp120x CD3 Killing sensitive viruses, PGT-121.60 that did not bind to T cells did not induce upregulation of T cell activation markers (tables 22 and 23).

Table 18 gp120 x CD3 Duobody variant 1CD4T cell activation

Table 19 gp120 x CD3 Duobody variant 1CD8T cell activation.

Table 20 gp120 x CD3 Duobody variant 2CD4T cell activation.

Table 21 gp120 x CD3 Duobody variant 2CD8T cell activation.

TABLE 22 PGT-121.60CD4T cell activation.

Table 23.

Method

gp 120X CD3 Duobody-induced upregulation of T cell activation markers by co-culture of 1x 10 at 37 ℃ ⁴ Human primary T cells isolated by Ficol paque from leukopak obtained from non-HIV infected (n = 2) and HIV infected donors (n = 2) were separated from 1x 10 ⁵ One CEM-NKr-CCR5-LucR CD 4T cell, which was infected with gp120 x CD3, was evaluated for 24 hours(iii) a killing-sensitive HIV-1 virus (7552), (ii) infection with gp120 x CD3(ii) killing-resistant HIV-1 virus (THRO), or (iii) no infection. Wells were then washed 3 times with FACS buffer, stained with Live/Dead Amcyan (Thermo Fisher, cat. No. l34966) as per the manufacturer's instructions, washed 3 times with FACS buffer and incubated with the following antibodies diluted in FACS buffer for 20 min at room temperature: anti-CD 4-BV711 (BD Biosciences catalog number 56028); anti-CD 8-APC/Cy7 (BD Biosciences, cat No. 560179); anti-CD 25-PE/Cy7 (BD Biosciences, cat. No. 557741); anti-CD 69-PerCP/Cy5.5 (BioLegend, cat. No. 310926); anti-PD-1-BV 605 (BioLegend, cat. No. 329924); anti-Ki 67-AF700 (BD biosciences. Catalog No. 561277). The cells were then washed 3 times with FACS buffer, fixed and permeabilized for 10 minutes with 100 μ Ι _ Cytofix/Cytoperm, washed once with PermWash, resuspended in 120 μ Ι _ FACS buffer and flow cytometric data were obtained on lsrfortessa or X20 FACS (BD Biosciences, san Jose, CA) and using FlowJo software (TreeSt) ar) for analysis.

The maximal fraction of CD4 and CD 8T cells expressing each activation marker (Emax) and the concentration that yields 50% Emax (EC 50) were obtained from dose response curves (equation 1) fitted by three-parameter non-linear regression using GraphPad Prism (La Jolla, CA) software.

Equation 1:

where Y =% activation, X = antibody concentration, bottom = response in the absence of antibody, and top = maximal response.

For dose-response curves with an Emax of T cell activation above baseline (no antibody control wells) <5%, EC50 values were reported as >100 μ g/mL (maximum concentration tested).

Example 9 killing of hiv-infected CEM-NKr-CCR5-LucR CD 4T cells using PBMC effector cells.

Killing activity was assessed against 4 major HIV-1 isolates or molecular clones. Each virus was evaluated using PBMC effector cells from 2 healthy donors and the results are shown in table 24. gp 120X CD3The proportion of killed infected cells (Emax) (median 76%) for (exemplary variant 1 and variant 2) was greater than the ratio of PGT-121.60 (median 18%, mann-Whitney, P)<0.0001 And PGT-121 (median 0%, mann-Whitney, P)<0.0001 ) is significantly higher.

In addition to killing significantly more infected cells (Emax) than PGT-121.60 and PGT-121, gp 120X CD3 Duobodies (exemplary variants 1 and 2; median EC50,0.042 ug/mL) were more potent in killing infected cells than PGT-121 (median EC50, >100ug/mL; mann-Whitney, P < 0.0001) and tended to be more efficient than PGT-121.60 (median EC50, >100ug/mL; mann-Whitney, P = 0.1157).

The difference in killing efficacy (Emax) of infected cells between PGT-121.60 (median 18%) and PGT-121 (median 0%) was statistically significant (Mann Whitney, P = 0.018). Furthermore, PGT-121.60 tends to be more effective than PGT-121 (P = 0.128). The results indicate that the CD3 bispecific antibody shows improved killing of HIV infected cells compared to PGT-121.60 (Effector enhanced IgG1 mAb) or PGT-121 (IgG 1 mAb).

Table 24 killing of hiv-infected CEM-NKr-CCR5-LucR CD 4T cells.

Method

CEM-NKr-CCR5-LucR CD 4T cells were infected with HIV-1 isolates 92US657, 1489, 8398 and 7552 in R10+1 (RMPI plus 10% FBS,1% penicillin/streptomycin, 1% HEPES) medium containing 20. Mu.g/mL DEAE dextran and incubated at 37 ℃ for 4 hours. Four hours after inoculation, CEM-NKr-CCR5-LucR CD 4T cells were diluted 3X with R10+1 and cultured for 48-72 hours to allow for de novo expression of HIV Env. Infected CEM-NKr-CCR5-LucR CD 4T cells were washed 3 times to remove free virus, 2x 10 ⁴ Individual cells/well were seeded in white 96-well plates and mixed with 7 concentrations of gp 120X CD3Or 10-fold serial dilutions of PGT-121.60 incubated for 1 hour in the presence of human serum IgG (final concentration 5 mg/mL) followed by 2x 10 ⁵ PBMC/well were added to conditioned CEM-NKr-CCR5-LucR CD 4T cells and incubated for 48 hours at 37 ℃ in a final volume of 100. Mu.L. By adding 100. Mu.L/well ONE-Glo ^TM Luciferase reagents determine killing of HIV-infected CEM-NKr-CCR5-LucR CD 4T cells and measure Relative Luminescence Units (RLU) in a luminometer according to the manufacturer's instructions.

Assay of gp120 x CD3 from RLU of gp120 x CD3 Duobody or PGT-121.60 treated wellsAnd PGT-121.60 CEM-NKr-activated protein for HIV infectionKilling of CCR5-LucR CD 4T cells and comparison with RLU of HIV-infected CEM-NKr-CCR5-LucR CD 4T cells in untreated wells (treated with human serum IgG only, n =2-10 per 96 well plate). The percentage of killing of HIV-infected CEM-NKr-CCR5-LucR CD 4T cells was calculated using the following formula:

100- ((RLU of HIV-infected target cells in treated wells/RLU of HIV-infected target cells in untreated wells) 100).

The maximum fraction of killed infected cells (Emax) and the concentration that produced 50% killing (IC) were obtained from the dose-response curve (equation 1) fitted by three-parameter non-linear regression using GraphPad Prism (La Jolla, CA) software ₅₀ )。

Equation 1:

where Y =% killing, X = antibody concentration, bottom = reaction in the absence of antibody, and top = maximal reaction.

For dose response curves with apparent Emax <10% killing of infected cells, IC50 values are reported as >100 μ g/mL, and Emax assignments of 0% with absolute values < 0%.

Example 10 killing of hiv-infected primary CD 4T cells using tonsil-derived monocytes

The major reservoir of latent HIV-infected cells in cART-inhibited, HIV-infected subjects is located in the lymph nodes. The ability of the antibodies to utilize effector cells present in lymphoid tissues was examined in an in vitro killing assay using monocytes isolated from HIV-1 seronegative tonsils as effector cells and HIV-1 infected primary CD 4T cells as target cells. Since autologous PBMCs from tonsil donors were not available, allogeneic primary CD 4T cells were used as a source of target cells and PBMC effector cells.

Tables 25 and 26 show the results of tonsil-derived monocytes (TDMC) from a single donor, peripheral Blood Mononuclear Cells (PBMC) from two donors, and target cells infected with two HIV-1 viruses (CHO 58 and 92US 727). TDMC and PBMC mediate strong killing of HIV-infected CD 4T cells by exemplary gp120 x CD3 Duobody and Duobody consisting of variant 1 or variant 2, with Emax and IC50 concentrations ranging from 66% -82.6% and 0.013-0.053 μ g/mL for TDMC, and Emax and IC50 concentrations ranging from 62% -84% and 0.001-0.024ug/mL for PBMC, respectively. In contrast, only PBMC, but not TDMC, were able to mediate killing of HIV-infected CD 4T cells using PGT121.60 (or the negative control, duobody, palivizumab x CD 3).

TABLE 25 maximum percentage of HIV-infected cells killed using tonsil-derived and peripheral blood-derived monocyte effector cells.

TABLE 26 efficacy of killing HIV-infected cells using tonsil-derived and peripheral blood-derived mononuclear effector cells (IC 50).

Method

CD4 infection with primary resting HIV ⁺ T cells as target cells and monocytes derived from peripheral blood and monocytes derived from tonsil as effector cells against gp 120X CD3 in vitroAnd PGT-121.60 dependent killing of HIV infected CD4T cells was studied. Non-diseased tonsils were obtained from healthy, consented donors who received a tonsillectomy. Tonsils were transported to the laboratory in DMEM medium containing antibiotics and treated within 8 hours of tonsillectomy. To isolate tonsil-derived mononuclear cells (TDMC), fat and quartered tissue were first removed. Cutting the tonsil into 2mm with scalpel ³ And dispersed through a 100 μm nylon cell filter (Falcon). After washing with DMEM plus 1% FBS, TDMC was recovered from the cell suspension by Ficol paque, cryopreserved in 90% DMSO,10% FBS, and stored in liquid nitrogen.

Primary CD4 ⁺ T cells were infected at 1200 Xg with 50-100ng p24/million cell spinoction for 2 hours and cultured for 5 days in RPMI medium (supplemented with 10% FBS and 1% penicillin/streptomycin) with 30U/mL IL-2 (Roche catalog No. 11011456001) at 37 ℃. After standing for 5 days to allow expression from the head antigen, transfected CD4 was ⁺ T cell cultures were washed 3 times to remove free virus at 2x 10 ⁵ Cells/well were seeded in 96-well plates and mixed with 7 concentrations of gp 120X CD3 in 10-fold serial dilutions in the presence of human serum IgG (final concentration 5 mg/mL)Or PGT-121.60 for 1 hour. When CD4 ⁺ When the target T cell is conditioned, effector cells are prepared. Cryopreserved PBMCs and TDMC were thawed and membrane stained with PKH67 according to the manufacturer's instructions and at 4X 10 ⁵ Cells/well were added to conditioned target cells to generate 2: 1E: and (3) a T ratio. Effector cells were co-cultured with conditioned target cells for 48 hours at a final volume of 200 μ Ι _, per well.

Killing of HIV-infected target cells was determined by flow cytometry. At the end of the co-culture period, cells were washed 2X with PBS, stained with 100 μ L Live/Dead Aqua (1/1000 dilution in PBS) for 10 min until the dye was inactivated by the addition of 100 μ L FACS buffer (PBS +2% FBS). The cells were then washed with FACS buffer and incubated with anti-CD 4-PE/Cy7mAb (diluted 1/50 in FACS buffer) for 20 minutes, then washed 3 times with FACS buffer and fixed and permeabilized with 100. Mu.L of Cytofix/Cytoperm for 10 minutes. Cells were then washed once with PermWash and incubated with anti-p 24-PE mAb in FACS buffer +10% PermWash for 25 minutes. Finally, cells were washed 3 times with FACS buffer, resuspended in 120 μ Ι _ FACS buffer, and flow cytometric analysis data were obtained on LSR Fortessa or X20 FACS (BD Biosciences, san Jose, CA) and analyzed using FlowJo software (TreeStar).

In primary infection with PBMC and TDMC CD4 ⁺ In T cell killing, HIV-infected target cells were counted by flow cytometry using the following gating strategy: lymphocytes are selected based on forward and side scatter, anViable lymphocytes were selected by Live/Dead Aqua negative staining. CD4 representative of the vaccination was then selected ⁺ PKH67 negative live lymphocytes of T cells and identified HIV-infected cells as p24 Gag +, CD4 low (due to HIV-mediated down regulation of CD 4) cells. Percentage of HIV infection was expressed as vaccination against HIV infection (PKH 67 negative) CD4 ⁺ T cells (p 24 Gag) ⁺ ,CD4 ^{Is low in} Positive) percent.

The percentage of HIV-infected target cells in PGT-121.60 treated wells was compared to the average percentage of HIV-infected target cells in untreated wells (treated with human serum IgG only, n =2-10 per 96 well plate). The percent killing of HIV-infected target cells was calculated using the following formula:

100- ((% HIV-infected target cells in treated wells/% HIV-infected target cells in untreated wells) × 100).

The maximum fraction of infected cells killed by PGT-121.60 (Emax) and the concentration giving 50% killing (IC) were obtained from the dose-response curve fitted by three-parameter nonlinear regression (equation 1) using GraphPad Prism (La Jolla, CA) software ₅₀ )。

Equation 1:

where Y =% killing, X = antibody concentration, bottom = reaction in the absence of antibody, and top = maximal reaction.

For dose-response curves with apparent Emax of infected cells killing <40%, IC50 values are reported as >10 μ g/mL, and Emax assignments for absolute values < 0% are 0%.

Example 11 HIV-infected CD4 Killing of T cells by T cell subsets

To investigate which T cell subsets are capable of mediating gp120x CD3 Duobody-dependent killing of HIV-infected cells, we assessed the ability of isolated T cell subsets, i.e. memory CD 8T cells, naive CD 4T cells, memory CD 4T cells, effector memory CD 4T cells and γ Δ T cells, to mediate killing of HIV-infected CD 4T cells.

The results shown in table 27 show that,all T cell subsets examined (i.e., memory CD 8T cells, naive CD 4T cells, memory CD 4T cells, effector memory CD 4T cells, and γ Δ T cells) can utilize exemplary gp120x CD3gp120 x CD3Variant 1 and gp120x CD3Variant 2 mediates the Duobody-dependent killing of potent (EC 50; 0.006-0.14. Mu.g/mL), potent (Emax; 27% -68%) T cells clonally infected with the HIV-1 WITO infectious molecule. In contrast, the CD8 and CD 4T cell subsets were unable to mediate efficient killing of HIV-infected T cells using PGT-121.60. The results also show that γ Δ T cells are able to mediate efficient killing of HIV infected T cells by PGT-121.60 with Emax comparable to that obtained by gp120x CD3 duobes (48% vs 43% -68%), although less potent than gp120x CD3 duobes (EC 50 ug/mL vs 0.02ug/mL-0.14 ug/mL). The ability of PGT-121.60 to mediate killing of antibody-dependent HIV-infected T cells by γ Δ T cells is in contrast to the disclosure that γ Δ T cells express Fc γ R CD16 and are capable of mediating antibody-dependent cellular cytotoxicity (Tokuyama et al, 2008 &Freedman, 2008).

TABLE 27 Emax (%) and potency (IC 50) of killing by HIV-infected cells according to various T cell subsets.

Method

Gp 120X CD3 passage through T cell subsets Using infected cell killing by PBMC effector cells as described in example 5And killing of PGT-121.60-dependent HIV-infected CD 4T cells in vitroWith the following modifications: in contrast to using whole PBMCs as effector cells, T cell subsets isolated using the cell isolation kit detailed in table 21 were separated according to the manufacturer's protocol at 2: an effector-target ratio of 1 was used as effector cells. If Emax is less than or equal to 10%, the EC50 value is reported as>10ug/mL (maximum concentration tested).

Table 28 t cell subset isolation kit

Example 11 human platelet binding

To assess binding to human platelets, a flow cytometry-based platelet binding assay was performed and compared to PGT-121.60. Platelet-rich plasma (PRP) samples were prepared from whole blood of 3 human healthy donors and treated with the test article at a concentration of 1000. Mu.g/ml or 250. Mu.g/ml. An RSV fusion protein targeting the monoclonal antibody palivizumab (Pali) and its derivative Duobody (Pali x CD3 was used as a non-anti-HIV Env control antibody.

The results shown in table 29 indicate that in samples from 3 donors 1000 μ g/ml of PGT-121.60 bound MFI to human platelets increased 40-100 fold against background staining, while the MFI of gp 120X CD3 Duobody variant increased 15-50 fold in the same samples. When the test article is tested at 250 μ g/ml in the binding assay, platelet staining MFI is reduced by 4-7 fold compared to PGT-121.60 and 1-3 fold for the variant. MFI of non-anti-HIV Env control antibodies Pali and Pali X CD3 were at background levels at both tested concentrations. The variant showed lower human platelet binding activity compared to PGT-121.60. The mean bound MFI of the Duobody variants was 35-47% of PGT-121.60 at a concentration of 1000. Mu.g/ml (Table 30).

TABLE 29 fold increase in human platelets binding MFI against stained background.

Table 30 comparison of binding of gp120X CD3 Duobody variant and PGT-121.60 to human platelets

Method

Platelet Rich Plasma (PRP) samples were prepared from human whole blood samples. Briefly, whole blood samples were centrifuged at 170Xg for 15 minutes at room temperature without braking. After centrifugation, PRP was collected from the top layer of each sample, which was then washed in modified HT (mHT) buffer (10mM HEPES,137mM NaCl,2.8mM KCl,1mM MgCl) ₂ 、12mM NaHCO ₃ 、0.4mM Na ₂ HPO ₄ 0.35% BSA,5.5mM glucose, pH 7.4). Diluted test antibody (50 μ Ι _) was added to an equal volume of PRP sample and incubated for 45 minutes at room temperature. At the end of incubation, an equal volume of BD FACS staining buffer (phosphate buffered saline containing 2% fetal bovine serum) was added and the assay plates were centrifuged at 2000xg for 5 minutes at room temperature. The supernatant was aspirated, the washed PRP samples were resuspended in mHT buffer and stained with PE anti-CD 61, FITC anti-CD 41 and APC anti-human IgG secondary antibodies for 30 min at 4 ℃. After staining, PRP samples were washed with and resuspended in 125 μ L BD FACS staining buffer and using BD LSRFortessa ^TM Cell analyzers (BD Biosciences, san Jose, CA) and FlowJo software (TreeStar, ashland, OR) were analyzed by flow cytometry.

Platelet populations were defined as PE anti-CD 41 and FITC anti-CD 61 double positive FACS events. Mean Fluorescence Intensity (MFI) values of APC anti-human IgG of platelet populations were quantified. The fold increase in MFI for each test article relative to the staining background (determined by APC-only anti-human IgG secondary antibody) was then calculated. To compare the platelet binding activity of gp 120X CD3 Duobody variants with PGT-121.60, the MFI percentage of each Duobody variant relative to PGT-121.60 at 1000. Mu.g/ml in each sample was calculated.

Other embodiments

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims (41)

1. A multispecific antibody that binds human immunodeficiency virus-1 (HIV-1) envelope (Env) glycoprotein gp120 (gp 120) and a second human antigen, wherein the antibody comprises:

(a) A first antigen-binding domain comprising a first heavy chain variable domain (VH) and a first light chain variable domain (VL), wherein the first antigen-binding domain binds gp120, and wherein:

Said first VH consists of the amino acid sequence of SEQ ID NO. 7 and said first VL consists of the amino acid sequence of SEQ ID NO. 81;

said first VH consists of the amino acid sequence of SEQ ID NO. 7 and said first VL consists of the amino acid sequence of SEQ ID NO. 82;

said first VH consists of the amino acid sequence of SEQ ID NO. 7 and said first VL consists of the amino acid sequence of SEQ ID NO. 83; or

Said first VH consists of the amino acid sequence of SEQ ID NO. 7 and said first VL consists of the amino acid sequence of SEQ ID NO. 84; and

(b) A second antigen-binding domain that binds to human CD3 or CD 89.

2. The antibody of claim 1, wherein the first antigen binding domain comprises a heavy chain having an amino acid sequence at least 95% identical to SEQ ID No. 9.

3. The antibody of claim 1, wherein the first antigen binding domain comprises a light chain having an amino acid sequence at least 95% identical to SEQ ID NO. 10.

4. The antibody of any one of claims 1-3, wherein the multispecific antibody is a bispecific antibody.

5. The antibody of any one of claims 1 to 3, wherein the antibody is a kappa-lambda body, an amphiphilic and re-targeting molecule (DART), "knob-hole" structured antibody, a chain exchange engineered domain antibody (SEEDbody), a bispecific T cell engager (BiTe), a CrossMab, an Fcab, a diabody, or a DuoBody.

6. The antibody of claim 5, wherein the diabody is a tandem diabody (TandAb).

7. The antibody of any one of claims 1-3, wherein the first antigen binding domain is attached to a peptide selected from the group consisting of human IgG, either directly or by insertion of an amino acid sequence ₁ Human IgG ₂ Human IgG ₃ Human IgG ₄ Human IgA ₁ And human IgA ₂ And wherein the second antigen-binding domain is fused, directly or by insertion of an amino acid sequence, to a first heavy chain constant region selected from human IgG ₁ Human IgG ₂ Human IgG ₃ Human IgG ₄ Human IgA ₁ And human IgA ₂ The second heavy chain constant region fusion of (3).

8. The antibody of claim 7, wherein the first heavy molecule isThe chain constant region is human IgG ₁ And wherein the second heavy chain constant region is human IgG ₁ 。

9. The antibody of claim 7, wherein the first antigen-binding domain is fused, directly or by way of an intervening amino acid sequence, to a first light chain constant region that is a human lambda constant region, and wherein the second antigen-binding domain is fused, directly or by way of an intervening amino acid sequence, to a second light chain constant region that is a human lambda constant region.

10. The antibody of claim 7, wherein

(a) The first heavy chain constant region comprises a F405L amino acid mutation; and

(b) The second heavy chain constant region comprises a K409R amino acid mutation.

11. The antibody of claim 7, wherein

(a) The first heavy chain constant region comprises a K409R amino acid mutation; and

(b) The second heavy chain constant region comprises a F405L amino acid mutation.

12. The antibody of claim 7, wherein the effector functions of the first heavy chain constant region and the second heavy chain constant region are reduced or eliminated.

13. The antibody of claim 7, wherein the first heavy chain constant region comprises a human IgG comprising an N297A mutation or an N297Q mutation ₁ The heavy chain constant region and/or the second heavy chain constant region comprises a human IgG comprising an N297A mutation or an N297Q mutation ₁ A heavy chain constant region.

14. The antibody of any one of claims 1-3, further comprising a cytotoxic agent, a radioisotope, a therapeutic agent, an antiviral agent, or a detectable label.

15. A composition comprising (i) a nucleic acid molecule encoding a first light chain variable region or a first light chain of a first antigen-binding fragment of the antibody of any one of claims 1-14, (ii) a nucleic acid molecule encoding a first heavy chain variable region or a first heavy chain of a first antigen-binding fragment of the antibody of any one of claims 1-14, (iii) a nucleic acid molecule encoding a first light chain variable region or a first light chain of a second antigen-binding fragment of the antibody of any one of claims 1-14, and (iv) a nucleic acid molecule encoding a second heavy chain variable region or a second heavy chain of a second antigen-binding fragment of the antibody of any one of claims 1-14.

16. A host cell comprising (i) a nucleic acid molecule encoding a first light chain variable region or a first light chain of a first antigen-binding fragment of the antibody of any one of claims 1-14, (ii) a nucleic acid molecule encoding a first heavy chain variable region or a first heavy chain of a first antigen-binding fragment of the antibody of any one of claims 1-14, and/or (iii) a nucleic acid molecule encoding a first light chain variable region or a first light chain of a second antigen-binding fragment of the antibody of any one of claims 1-14, and (iv) a nucleic acid molecule encoding a second heavy chain variable region or a second heavy chain of a second antigen-binding fragment of the antibody of any one of claims 1-14.

17. The host cell of claim 16, which is selected from the group consisting of E.coli, pseudomonas, bacillus, streptomyces, yeast, CHO, YB/20, NS0, PER-C6, HEK-293T, NIH-3T3, heLa, BHK, hep G2, SP2/0, R1.1, B-W, L-M, COS 1, COS 7, BSC1, BSC40, BMT10 cells, plant cells, insect cells, and human cells in tissue culture.

18. A method of producing an antibody that binds gp120 and a second human antigen, wherein the second human antigen is human CD3 or human CD89, comprising culturing the host cell of claim 16 under conditions such that the nucleic acid molecule is expressed and the antibody is produced.

19. Use of an antibody according to any one of claims 1 to 14 in the preparation of a test agent for detecting gp120 and a second human antigen in a sample, wherein the second human antigen is human CD3 or human CD89, said detecting comprising contacting the sample with the antibody.

20. A pharmaceutical composition comprising the antibody of any one of claims 1-14 and a pharmaceutically acceptable excipient.

21. A kit comprising the antibody of any one of claims 1-14, and a) a detection reagent, b) gp120 and/or a second human antigen selected from human CD3 and human CD89, c) a notice reflecting approval for use or sale for human administration, or d) a combination thereof.

22. Use of the antibody of any one of claims 1-14 or the pharmaceutical composition of claim 20 in the manufacture of a medicament for treating or preventing human immunodeficiency virus infection in a human subject in need thereof.

23. An antibody that binds gp120, wherein said antibody comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein said VH consists of the amino acid sequence of SEQ ID NO:7 and said VL consists of the amino acid sequence of SEQ ID NO: 81; the VH consists of the amino acid sequence of SEQ ID NO. 7 and the VL consists of the amino acid sequence of SEQ ID NO. 82; the VH consists of the amino acid sequence of SEQ ID NO. 7 and the VL consists of the amino acid sequence of SEQ ID NO. 83; or said VH consists of the amino acid sequence of SEQ ID NO. 7 and said first consists of the amino acid sequence of SEQ ID NO. 84.

24. The antibody of claim 23, wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO 9.

25. The antibody of claim 23, wherein the antibody comprises a light chain comprising an amino acid sequence set forth in any one of SEQ ID NOs 40, 78, 79, or 80.

26. The antibody of claim 23, wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO 9 and a light chain comprising the amino acid sequence set forth in any one of SEQ ID NO 40, 78, 79, or 80.

27. The antibody of any one of claims 23-26, further comprising a cytotoxic agent, a radioisotope, a therapeutic agent, an antiviral agent, or a detectable label.

28. A pharmaceutical composition comprising the antibody of any one of claims 23-27 and a pharmaceutically acceptable carrier.

29. One or more nucleic acids encoding the antibody of any one of claims 23-26.

30. One or more vectors comprising one or more nucleic acids of claim 29.

31. A host cell comprising one or more vectors of claim 30.

32. A method of producing an anti-gp 120 antibody, the method comprising culturing the host cell of claim 31 under conditions such that the one or more nucleic acids are expressed and the antibody is produced.

33. Use of the antibody of any one of claims 23-27 or the pharmaceutical composition of claim 28 in the manufacture of a medicament for treating or preventing human immunodeficiency virus infection in a human subject in need thereof.

34. An antibody fragment that binds gp120, wherein said antibody fragment comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein said VH consists of the amino acid sequence of SEQ ID NO:7 and said VL consists of the amino acid sequence of SEQ ID NO: 81; the VH consists of the amino acid sequence of SEQ ID NO. 7 and the VL consists of the amino acid sequence of SEQ ID NO. 82; the VH consists of the amino acid sequence of SEQ ID NO. 7 and the VL consists of the amino acid sequence of SEQ ID NO. 83; or said VH consists of the amino acid sequence of SEQ ID NO. 7 and said first consists of the amino acid sequence of SEQ ID NO. 84.

35. The antibody fragment of claim 34, which is a Fab, F (ab) 2, fv, scFv, sc (Fv) 2, or diabody.

36. The antibody fragment of any one of claims 34-35, further comprising a cytotoxic agent, a radioisotope, a therapeutic agent, an antiviral agent, or a detectable label.

37. A pharmaceutical composition comprising the antibody fragment of any one of claims 34-36 and a pharmaceutically acceptable carrier.

38. One or more nucleic acids encoding the antibody fragment of any one of claims 34-36.

39. One or more vectors comprising one or more nucleic acids of claim 38.

40. A host cell comprising one or more vectors of claim 39.

41. A method of producing an anti-gp 120 antibody fragment, said method comprising culturing the host cell of claim 40 under conditions such that said one or more nucleic acids are expressed and said antibody fragment is produced.