WO2013163427A1

WO2013163427A1 - Antibodies to treat hiv-1 infection

Info

Publication number: WO2013163427A1
Application number: PCT/US2013/038214
Authority: WO
Inventors: Gary J. Nabel; Zhi-Yong Yang; Amarendra PEGU; Xiaoti GUO; Lan Wu
Original assignee: The United States Of America, As Represented By The Secretary, Department Of Health & Human Services
Priority date: 2012-04-25
Filing date: 2013-04-25
Publication date: 2013-10-31

Abstract

Bispecific antibodies are disclosed that include a first antigen binding domain that specifically binds to an HIV-1 envelope protein (such as gp120 or gp41), and a second antigen binding domain that is T cell activating and HIV-1 -activating, such as an scFv that specifically binds to CD3. In some embodiments, the bispecific antibody includes a scFv that specifically binds to an HIV-1 envelope protein (such as gp120 or gp41) and a scFv that that is T cell activating and HIV-1 -activating, such as an scFv that specifically binds to CD3. In additional embodiments, the bispecific antibody includes a Fab that specifically binds to an HIV-1 envelope protein (such as gp120 or gp41) and a scFv that that is T cell activating and HIV-1 -activating, such as an scFv that specifically binds to CD3. Multispecific antibodies are also disclosed that include these bispecific antibodies. Methods of using these bispecific antibodies, multispecific antibodies and nucleic acids encoding these antibodies are also disclosed.

Description

ANTIBODIES TO TREAT HIV-1 INFECTION

RELATED APPLICATIONS

This claims the benefit of U.S. Provisional Application No. 61/638,437, filed April 25, 2012, which is incorporated by reference herein in its entirety.

FIELD OF THE DISCLOSURE

This application relates to multispecific antibodies that specifically bind an human

immunodeficiency virus (HIV)-l envelope protein and CD3, and their use to treat an HIV-1 infection.

BACKGROUND

Highly Active Antiretroviral Therapy (HAART) has been effective in reducing the viral burden and ameliorating the effects of HIV-1 infection in infected individuals. However, a latent reservoir of HIV infected cells evades this treatment, allowing the virus to persist in the individual despite therapy and requiring continued treatment. The lifelong dependence on anti-retro viral drugs along with the emergence of resistant strains over time shows the limited practicality of HAART for long term treatment of HIV-1 infection and underscores the importance of elimination of the latent reservoir of HIV-1 infected cells that persists in an infected individual. Therefore, there is a need for therapeutic agents for treatment of HIV-1 infected individuals, as well as agents that are capable of reducing the latent reservoir of HIV infected cells.

SUMMARY

Compositions and methods are disclosed for treatment of HIV-1 infection in a subject. In some embodiments, the compositions and methods can be used to reduce the latent reservoir of HIV-1 infected cells in a subject.

In several embodiments, bispecific antibodies are disclosed that include a first antigen binding domain that specifically binds to an HIV-1 envelope protein (such as gpl20 or gp41) and is neutralizing, and a second antigen binding domain that specifically binds to CD3 and is T cell activating and HIV-1 activating. In some such embodiments, binding to CD3 on the surface of a T cell by the second antigen binding domain triggers cellular signals resulting in activation of the T cell and, if present, activation of the latent HIV-1 reservoir in the T cell. Activation of the latent HIV-1 reservoir results in replication of the HIV-1 virus in the T cell, and expression of HIV-1 envelope proteins (such as gpl20 and gp41) on the surface of the T cell. The exposed HIV-1 envelope proteins are targeted by the first binding domain of the bispecific antibody, which is HIV-1 neutralizing. The first and second binding domains of the bispecific antibodies are capable of functioning synergistically to target the latent reservoir of HIV-1 virus in CD3 expressing T cells, in such an embodiment.

In some embodiments, an isolated bispecific monoclonal antibody is disclosed that includes a first antigen binding domain and a second antigen binding domain. The first antigen binding domain can be a Fab or a scFv, specifically binds to gpl20 or gp41, and is neutralizing. The Fab and/or the scFv of the first antigen binding domain includes a heavy chain variable region including a heavy chain complementarity determining region (H-CDR)l, an H-CDR2, an H-CDR3, and a light chain variable region including a light chain complementarity determining region (L-CDR)l, an L-CDR2, and an L- CDR3. The second antigen binding domain can also be a Fab or a scFv. The Fab and/or the scFv of the second antigen binding domain includes a heavy chain variable region including a HCDRl, HCDR2, and a HCD3 and a light chain variable region including a LCDR1, a LCDR2, and a LCDR3. The second antigen binding domain specifically binds CD3, activates T cells, and activates expression of proviral HIV-1 DNA in T cells. In some specific non-limiting examples, the first antigen binding domain includes the heavy chain variable region and the light chain variable region from a VRCOl-like antibody.

In some embodiments, each antigen binding domain of the bispecific antibody is an scFv. In other embodiments, the first antigen binding domain is a Fab and the second antigen binding domain is a scFv. In some embodiments, the bispecific antibody includes a scFv or Fab from an HIV-1 neutralizing antibody (such as, but not limited to, VRCOl, VRC07, 10E8, or a variant thereof), and an scFv from a T cell activating anti-CD3 antibody (such as, but not limited to, TR66). Also disclosed herein are compositions including these bispecific antibodies, nucleic acids encoding these bispecific antibodies, expression vectors including the nucleic acids, and isolated host cells that express the nucleic acids.

In one non-limiting example, the first antigen binding domain of the isolated bispecific antibody is a Fab comprising the heavy chain variable region of the first antigen binding domain, a heavy chain constant 1(CH1) region, the light chain variable region of the first antigen binding domain, and a light chain constant (CL) region, and the second antigen binding domain of the isolated bispecific antibody is a scFv. In some such embodiments, the isolated bispecific antibody includes a first polypeptide and a second polypeptide, wherein the first polypeptide includes, N-terminal to C-terminal, the heavy chain variable region of the Fab and the CHI region, and the second polypeptide includes, N-terminal to C- terminal, the light chain variable region of the Fab, the CL region, and the scFv. In some such embodiments, the first antigen binding domain specifically binds gpl20, and the first and second polypeptides include the amino acid sequences set forth as SEQ ID NO: 1516 and SEQ ID NO: 1508, respectively; SEQ ID NO: 1749 and SEQ ID NO: 1508, respectively; SEQ ID NO: 1516 and SEQ ID NO: 1510, respectively; SEQ ID NO: 1749 and SEQ ID NO: 1510, respectively; SEQ ID NO: 1516 and SEQ ID NO: 1751, respectively; or SEQ ID NO: 1749 and SEQ ID NO: 1751, respectively. The additional embodiments, the first antigen binding domain specifically binds gp41, and the first and second polypeptides include the amino acid sequences set forth as SEQ ID NO: 1757 and SEQ ID NO: 1488, respectively; SEQ ID NO: 1496 and SEQ ID NO: 1488, respectively; SEQ ID NO: 1757 and SEQ ID NO: 1490, respectively; or SEQ ID NO: 1496 and SEQ ID NO: 1490, respectively.

In additional embodiments, trispecific antibodies are provided that include the first and second antigen binding domains of any one of the disclosed bispecific antibodies, and further include a third antigen binding domain that specifically binds to CD28, CD40L, CD137, or CD137L. The provided antibodies can be used for a variety of purposes, for example, for treating a subject with an HIV-1 infection, such as, but not limited to, a subject with acquired immunodeficiency syndrome (AIDS). The methods include administering a therapeutically effective amount of a provided antibody that specifically binds to an HIV envelope protein (such as gpl20 or gp41) and CD3 to the subject. In several embodiments, HAART is also administered to the subject. In some embodiments, the disclosed methods are useful to reduce or eliminate the latent reservoir of HIV-1 infected cells in a subject. The provided compositions and methods have multiple features enabling reduction or elimination of the reservoir of HIV-1 -infected cells in a subject and which distinguish them from previously described antibodies.

The foregoing and other features and advantages of this disclosure will become more apparent from the following detailed description of a several embodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a series of schematic diagrams depicting scFv, bispecific scFv₂, bispecific Fab-scFv

(bibody), and trispecific Fab-scFv-scFv (tribody) antibody formats. The heavy chain variable region (VH), light chain variable region (VL), heavy chain constant region 1(CH1), and light chain constant region (CL) and peptide linkers (linker) are indicated.

FIGs. 2A-2E are a series of graphs and a digital image illustrating purification and

characterization of the VRC2455 bispecific single chain antibody, which includes an scFv including heavy and light chain variable domains from the VRCOl monoclonal antibody linked to an scFv including heavy and light chain variable domains from the CD3 specific monoclonal antibody TR66. (A) VRCOl based bispecific single chain antibodies were expressed by transient transfection of 293F cells and the bispecific single chain antibody was purified from the culture supernatant (light grey trace) using Ni-affinity column, followed by size exclusion column chromatography. The molecular weight markers (black trace) were also run to obtain the correct monomeric fraction. (B) The purified monomeric VRCOl based bispecific single chain antibody fraction was run on a SDS-PAGE gel to characterize the purity and size of the protein. FIG. 2C is a set of flow cytometry graphs illustrating that the VRC2455 bispecific single chain antibody binds to peripheral blood mononuclear cells (PBMCs) in vitro. Normal human donor PBMCs were first incubated in the presence or absence of VRC2455 bispecific single chain antibody, then stained with anti-human (h)CD3 along with either a VRCOl cognate antigen RSC3 (left) or anti-His tag antibody (VRC2455 was engineered with a histidine-tag) (right). The successful binding was shown as resurfaced stabilized core (RSC)3 positive or His tag positive within CD3+ populations. (D) Human PBMCs were incubated with the indicated bispecific single chain antibody and bound bispecific single chain antibody was detected on CD3+ T cells by a phcyerythrin (PE)-conjugated anti- HisTag antibody. (E) Human PBMCs were incubated with VRC2455 bispecific single chain antibody and binding to HIV envelope protein by the bound VRC2455 bispecific single chain antibody on CD3+ T cells was detected by using a fluorescein isothiocyanate (FITC)-conjugated RSC3. FIGs. 3A-3B are a set of graphs illustrating the ability of bispecific single chain antibodies to activate T cells in vitro. Normal human donor PBMCs (effector; E) were co-cultured with CEM-NKr- CCR5 cells that are chronically infected with HIV-1 (HIV+ target) or uninfected CEM-NKr-CCR5 cells (HIV- target; T) at an E:T ratio of 10: 1, in the presence of Brefeldin A and 1 μg/ml of three different bispecific single chain antibodies - VRC2455, VRC2678 and VRC2442. The VRC2678 bispecific single chain antibody includes an scFv including heavy and light chain variable domains from the VRCOl monoclonal antibody joined to an scFv including heavy and light chain variable domains from the rhesus macaque CD3 specific monoclonal antibody C207. The control VRC2442 bispecific single chain antibody includes an scFv including heavy and light chain variable domains from the human 5B8 monoclonal antibody joined to a scFv including heavy and light chain variable domains from the human CD3 specific monoclonal antibody TR66. The percentage of CD4+ (A) or CD8+ (B) T cells expressing interferon (IFN)-y was measured by intracellular cytokine staining after 20-24 hours of co-culture.

FIG. 4 is a set of graphs showing the in vitro cytotoxic activity of VRC2455 bispecific single chain antibody using HIV-1 infected cell lines as target cells and human PBMCs as effector cells. The indicated HIV-1 infected cell lines were incubated with human PBMCs from normal donors in the presence of 10-fold serial dilutions of VRC2455 (VRCOl scFv - anti-hCD3 scFv (TR66) or VRCOl IgGl for 20-24 hours. The percentage of lysed cells was determined by flow cytometry using a live/dead cell stain to measure amount of cell lysis for each protein.

FIG. 5 is a graph illustrating the plasma viremia levels of SHIV in two rhesus macaques infected with SHIV-BaL before and during anti-retro viral treatment (ART).

FIG. 6 is a graph illustrating the proviral DNA level in PBMCs harvested from the two rhesus macaques (animal Nos. A8V091 and A8V102) infected with SHIV-BaL before and during treatment with the bispecific single chain antibodies (which was administered after HAART was completed). A8V102 was treated with the VRC2678 bispecific single chain antibody (VRCOl scFv - C207 scFv) and A8V091 was treated with the control antibody VRC2442 (5B8 scFv - C207 scFv).

FIG. 7 is a graph illustrating that the VRC2678 bispecific single chain antibody binds to monkey PBMCs in vivo. PBMCs were isolated at different time points from rhesus macaques (animal Nos. A8V091 and A8V102) infected with SHIV-BaL and treated with either VRC2678 or VRC2442 bispecific single chain antibody. The PBMCs were double-stained with the VRCOl cognate antigen RSC3 and anti-His tag antibody (VRC2678 has a histidine-tag). As shown in FIG. 8, a dramatic increase of the RSC3⁺HisTag⁺ cell population was detected in A8V102 at only six hours following the first dose of VRC2678, which then dropped to the baseline level afterwards.

FIG. 8 is a graph illustrating binding of anti-gpl20 and anti-CD3 -bispecific antibodies to RSC3 (gpl20 antigen). Binding was detected using horse radish peroxidase (HRP) labeled anti-Fab conjugate. The ELISA plate was coated with 200 ng/well RSC3, followed by incubation with serial diluted VRC07 Fab, VRC07 G54W Fab - TR66 scFv bispecific antibody, or VRC07 G54W Fab - C207 scFv bispecific antibody and detected using an HRP-labeled anti-Fab conjugate. The VRC07 G54W Fab - bispecific antibodies were constructed by co-transfection into 293F cells of cDNA encoding the VRC07 G54W heavy chain variable and CHI regions with a C-terminal HisTag (VRC3125) and VRCOl light chain linked to anti-CD3 scFv (VRC3122 for anti-hCD3 (TR66)) and VRC3123 for anti-rhesus (rh)CD3 (C207)).

FIG. 9 is a graph illustrating binding of a gp41- and CD3-bispecific antibody to an MPER peptide (gp41 antigen). The enzyme linked immunosorbent assay (ELISA) plate was coated with 100 ng/well MPER peptide (SEQ ID NO: 1539), followed by incubation with serial diluted 10E8 antibody or 10E8 variant Fab - C207 scFv bispecific antibody, and detected using an HRP-labeled anti-Fab conjugate. The 10E8 - anti-RhCD3 bispecific antibody was constructed by co-transfection into 293F cells of cDNA encoding a 10E8 variant heavy chain variable and CHI regions with C-terminal HisTag (VRC 3115) and a 10E8 variant light chain fused to C207 scFv (VRC3113).

FIGs. 10A and 10B are a set of flow cytometry graphs illustrating that a bispecific antibody including VRC07 G54W Fab linked to anti-hCD3 scFv (described in FIG. 8), or anti-rhCD3 scFv (similarly described in FIG. 8), binds to PBMCs in vitro. Version 1 (vl) has a shorter linker (GGGGS)₂ (SEQ ID NO: 1764), while version 2 (v2) has a longer linker (GGGGS)₃ (SEQ ID NO: 1765), between the light chain of the VRC07 Fab and the anti-CD3 scFv. VRC07 Fab fragment was used as a negative control. HIV-1 infected human PBMCs were incubated with the indicated bispecific antibodies and bound bispecific antibody was detected on CD4+ or CD8+ T cells by a FITC-conjugated anti-Fab antibody. Binding to HIV envelope protein by the bound bispecific Abs was detected by using an APC- conjugated RSC3 (VRC07 antigen).

FIGs. 11A-11B are a set of graphs illustrating the in vitro cytotoxic activity of the indicated bispecific antibodies. An HIV-1 infected cell line was labeled with a fluorescent membrane dye (PKH26) and incubated with human PBMCs from normal donors at an E:T ratio of 10: 1 in the presence of 10-fold serial dilutions of indicated bispecific antibodies for 20-24 hours. The percentage of dead cells was determined by flow cytometry using a live/dead cell stain to measure amount of cell lysis for each protein. The VRC07 G54W Fab - anti-CD3 scFv bispecific antibodies were constructed by co- transfection into 293F cells of cDNA encoding VRC07 (G54W) heavy chain variable and CHI regions with C-terminal HisTag (VRC3125) and VRCOl light chain fused to anti-CD3 scFv (VRC3121 for anti- hCD3, and VRC3123 for anti-RhCD3 in FIG. 11A; VRC3121 for v2 and VRC3122 for vl in FIG. 11B). 5B8 Fab-CD3 bispecific antibodies were used for the negative control, and were constructed by co- transfection into 293F cells of cDNA encoding humanized 5B8 heavy chain variable and CHI regions with C-terminal HisTag (VRC3120) and humanized 5B8 light chain linked to anti-CD3 scFv (VRC3116 for anti-hCD3 and VRC3118 for anti-RhCD3 in FIG. 11A).

FIGs. 12A-12C show a graph and coomassie stained gel illustrating purification of bispecific antibodies that bind the CD4 binding site (CD4bs) of HIV envelope (Env) and the CD3 ε chain. (A) Molecular characterization of the bispecific antibody. The chromatogram of a representative bispecific antibody simVRC07 Fab -anti-rhCD3 run through a size exclusion column shows the correct molecular size for the bispecific antibody (left). The simVRC07 Fab -anti-rhCD3 antibody includes a simianized version of VRC07 G54W and a ScFv including the heavy and light variable regions of the rhesus macaques C207 antibody. The heavy and light chain fragments of indicated Fab and bispecific antibodies can be observed when they were run on a reducing SDS-PAGE gel (right). (B) VRC07 Fab and its bispecific derivatives bind to HIV Env in solution. The antibodies at increasing five fold concentrations were allowed to bind to a resurfaced HIV Env fragment containing the CD4bs that was coated on ELISA plates and the bound antibodies were detected using an HRP-conjugated anti-Fab probe. (C) Bispecific antibodies bind to CD3 and HIV-1 Env on the cell surface. Human or monkey T cells and HIV-1 infected CEM cells were incubated with bispecific antibodies of the indicated specificities and bound antibodies were detected by a FITC-conjugated anti-Fab probe.

FIGs. 13A-13B show a series of graphs illustrating activation of T cells by a bispecific antibody targeting the CD4bs of HIV Env and CD3. (A) CD4⁺ and (B) CD8⁺ T cells are specifically activated by the bispecific antibody. Enriched human T cells were cocultured with either uninfected or HIV-infected CEM cells (indicated by - or + at the top of each column) in the presence of the indicated bispecific proteins (0.5 μg/ml) and Brefeldin A overnight. The T cells were then stained with antibodies against interferon (IFN)-y, tumor necrosis factor (TNF)-OC and CD69 and the percentage of T cells expressing each molecule was measured by flow cytometry.

FIGs. 14A-14D are a series of graphs illustrating that activation and targeted lysis of chronic and latent HIV-infected cells by VRC07 Fab-anti-hCD3 scFv bispecific antibody. (A) Induction of HIV in latent cell lines. Latent cell lines (ACH2, Jl.l and OM10) and a chronic cell line (CEM-IIIb) were cultured in the absence or presence of TNF-a for 14-16 hours and the expression of HIV Env on the cell surface using an allophycocyanin (APC) -conjugated 2G12 antibody (which specifically binds HIV-1 gpl20) was measured by flow cytometry. The increase in the expression of HIV Env indicates the inducible expression in the latent cell lines compared to the constitutive expression in the chronic cell line. (B) Targeted lysis of HIV-infected cell lines by VRC07 Fab - anti-hCD3 scFv bispecific antibody. The indicated chronic and latent HIV-infected cell lines were cocultured with enriched human T cells in the presence of increasing concentrations of VRC07 Fab - anti-hCD3 scFv or a control antibody for 14- 16 hours and percent lysis of the infected cell line was measured by flow cytometry after staining with a live/dead cell marker. (C, D) Reduction in the number of latently infected primary CD4⁺ T cells.

Resting CD4⁺ T cells were enriched from PBMCs and infected with HIV-1 BaL after culture in the presence of CCL19 for 3 days. These CD4⁺ T cells were then co-cultured with allogeneic CD8⁺ T cells in the presence of VRC07 Fab-anti-hCD3 scFv or control bispecific antibody for 14-16 hours. The expression of HIV Env on the cell surface of CD4⁺ T cells was then measured by flow staining with a fluorescently labeled 2G12 antibody. Representative data from one donor is shown in (C) and data from three independent donors showing a statistically significant reduction in HIV Env⁺ CD4 T cells in the presence of the bispecific antibodies was plotted in (D).

FIGs. 15A-15C show a series of graphs illustrating T cell depletion and cytokine release in peripheral blood induced by a VRC07 G54W Fab - anti-rhCD3 scFv bispecific antibody. SHIV-BaL infected rhesus macaques were infused with either VRC07 G54W Fab -anti-rhCD3 scFv (Treatment, n=5) or 5B8 Fab -anti-hCD3 (Control, n=4). Peripheral blood was collected at 0, 1 and 24 hours post- infusion at each dosing (arrows). (A) A dramatic decline of the CD3⁺ T cell population was detected in the treatment group at 1 hour post infusion, then rebounded to original levels by 24 hr. The sharp decline is less prominent after dose 5. (B, C) Cytokines released in plasma were quantified by Luminex multiplex cytokine assay. Spikes of TNF-a (B) and macrophage inflammatory protein (ΜΙΡ)-Ιβ (C) were detected in VRC07 G54W Fab-anti-rhCD3 scFv treated animals (right) at 1 hour post-infusion during the first 4 doses, less remarkable after day 14. Values represent means ± SEs.

FIGs. 16A-16B show a series of graphs illustrating reduced HIV-1 proviral DNA in PBMCs treated with VRC07 G54W Fab - anti-rhCD3 scFv. Relative proviral DNA level in PBMCs was quantified by real-time PCR as SIV gag copies per 10⁶ cells. (A) Proviral DNA was reduced by 4-fold in animals treated with VRC07 G54W Fab - anti-rhCD3 scFv (right, n=5) while remained unchanged in the control group (left, n=4). The times of infusion (in days) are indicated by arrows. Values represent means ± SEs. (B) Reduction in proviral DNA compared to the pre-treatment level is shown. A significantly greater reduction is observed at day 20 in the VRC07 G54W Fab - anti-rhCD3 scFv treated group compared with controls (P = 0.002, Student's t test).

FIGs. 17A-17B show a schematic diagram and a graph illustrating bispecific antibody treatment of SHIV infected Indian rhesus macaques. (A) Schematic of bispecific antibody treatment study. (B) Plasma viral loads in rhesus macaques challenged with SHIV-BaL and treated with ART drugs and bispecific antibody.

FIGs. 18A-18C show a series of graphs illustrating concentrations of bispecific antibody and cytokines in plasma following infusion. (A) Decay of plasma concentration of VRC07 G54W Fab-anti- rhCD3 scFv after infusion was measured by ELISA and data is shown as means ± SEs from 5 animals. (B, C) Cytokines released in plasma were quantified by LUMINEX® multiplex cytokine assay. Spikes of interleukin (IL)-10 and IFN-γ are detected in VRC07 G54W Fab-anti-rhCD3 scFv treated animals (Treatment) at 1 hour post-infusion. Values represent means ± SEs.

FIGs. 19A-19B show a set of graphs illustrating binding of VRC07 Fab-anti-CD3 scFv bispecific antibodies to human T cells (A) and to rhesus T cells (B). Human or Rhesus T cells were incubated with the indicated bispecific antibodies and bound antibodies were detected by an anti-Fab probe using flow cytometry.

FIG. 20 shows a graph illustrating that binding of VRC07 Fab-anti-CD3 scFv bispecific antibodies to RSC3 detected by ELISA.

FIGs. 21 A and 21B show a set of graphs illustrating binding of 10E8 Fab-anti-CD3 scFv bispecific antibodies to human T cells (A) and to rhesus T cells (B). 10E8 antibody specifically binds to the membrane proximal extracellular region of gp41. Human or Rhesus T cells were incubated with the indicated bispecific antibodies and bound antibodies were detected by an anti-Fab probe using flow cytometry.

FIG. 22 shows a graph illustrating that binding of 10E8 Fab-anti-CD3 scFv bispecific antibodies to 3AGJ (which includes the MPER peptide and specifically binds to 10E8) detected by ELISA. SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and one or three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the

complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file in the form of the file named "Sequence.txt" (~ 1.7MB), which was created on April 25, 2013, and is incorporated by reference herein. A summary of the sequences included in the accompanying sequence listing is provided in section VII, below. DETAILED DESCRIPTION

/. Introduction

Persistence of latently infected cells even after prolonged periods of HAART in HIV-1 infected patients remains a major hurdle towards finding a cure for HIV. Latency leads to the integration of the viral genome into the host chromosome generally without any active transcription of viral genes. Since the HAART drugs target viral proteins involved in the viral replication cycle, they are unable to eliminate these quiescent cells that harbor proviral DNA.

Activation and elimination of the latently infected cells in HIV-1 infection has become a major focus of HIV research. However, use of agents known to stimulate T cell activation (such as anti-CD3 antibodies or IL-2) to purge the latent HIV-1 reservoir in patients on HAART therapy led to deleterious effects on the immune system, and also failed to eliminate the latently infected cells (Chun et al, Nature 401, 874-875, 1999; Abadie et al, Astron Astrophys 539, 2012; Prins et al, AIDS 13, 2405-2410, 1999; Fraser et al, AIDS 14, 659-669, 2000; van Praag et al, Journal of clinical immunology 21, 218-226, 2001 ; Kulkosky et al, The Journal of infectious diseases 186, 1403-1411, 2002). Currently, no approach has substantially reduced or eliminated the latent infection or prevented viral rebound after termination of therapy. For example, attempts to use bispecific constructs that bind to an HIV antigen and to CD3 were ineffective for reducing the latent HIV-1 reservoir (Traunecker et al, The EMBO journal 10, 3655-3659, 1991 ; Chamow et al, J Immunol 153, 4268-4280, 1994; Okada et al, Immunology letters 38, 195-199, 1993; Yin et al, Microbiology and immunology 45, 101-108, 2001).

HIV-1 antigens normally are not present on the surface of latently infected cells. Thus, it is unexpected that a bispecific T cell engager (BiTE) that targets an HIV-1 antigen and CD3 could be used to reduce the latent reservoir of HIV-1 infected cells, because HIV-1 antigens are not available for binding on the surface of latently infected T cells. The prior unsuccessful attempts to use bispecific antibody constructs that bind to an HIV antigen and to CD3 for reducing the latent HIV-1 reservoir support this view.

A class of multispecific immunotherapeutic antibodies is disclosed herein that can be used to treat HIV. In some embodiments, these multispecific antibodies can activate latent viral gene expression and direct T lymphocytes to lyse these latently infected cells in vitro and/or in vivo. In some

embodiments, these these multispecific antibodies include a first antigen binding domain that specifically binds to an HIV-1 antigen and is neutralizing, and a second antigen binding domain that specifically binds to CD3, activates T cells, and activates HIV-1 in T cells. Through the CD3 interaction, the disclosed multispecific antibodies activate CD4 and CD 8 T cells and, importantly, stimulate pro viral HIV-1 gene expression in latently infected T cell lines and primary T cells. This mode of action is fundamentally different from the therapeutic mechanism of tumor targeting BiTEs, which target a constitutively expressed tumor associated antigen. In several embodiments, bispecific antibodies falling within this novel class of multispecific immunotherapeutic proteins dramatically reduced the latent reservoir of infected cells in a primate model of HIV-1 infection.

//. Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular

Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). Terms describing protein structure and structural elements of proteins can be found in Creighton, Proteins, Structures and Molecular Properties, W.H. Freeman & Co., New York, 1993 (ISBN 0-717-7030) which is incorporated by reference herein in its entirety.

As used herein, the term "comprises" means "includes." Thus, "comprising an antigen" means "including an antigen" without excluding other elements.

It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described below. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various embodiments of this disclosure, the following explanations of terms are provided:

Administration: The introduction of a composition into a subject by a chosen route.

Administration can be local or systemic. For example, if the chosen route is intravenous, the composition is administered by introducing the composition into a vein of the subject. In some embodiments a disclosed multispecific antibody, or one or more nucleic acid molecules encoding the antibody, is administered to a subject.

Agent: Any substance or any combination of substances that is useful for achieving an end or result; for example, a substance or combination of substances useful for inhibiting or treating HIV infection in a subject. Agents include proteins, antibodies, nucleic acid molecules, compounds, small molecules, organic compounds, inorganic compounds, or other molecules of interest. An agent can include a therapeutic agent (such as an anti-retro viral agent), a diagnostic agent or a pharmaceutical agent. In some embodiments, the agent is a polypeptide agent (such as a disclosed antibody), or an antiviral agent. The skilled artisan will understand that particular agents may be useful to achieve more than one result.

Amino acid substitution: The replacement of one amino acid in peptide with a different amino acid.

Antibody: A polypeptide ligand typically including light chain and heavy chain

immunoglobulin variable regions that specifically binds an epitope of an antigen. Immunoglobulin molecules are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (V_H) region and the variable light (V_L) region. Together, the V_H region and the V_L region are responsible for binding the antigen recognized by the antibody. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes.

Antibodies include intact immunoglobulins and the variants and portions of antibodies known in the art, such as single -domain antibodies (e.g. VH domain antibodies), Fab fragments, Fab' fragments, F(ab)'₂ fragments, single chain Fv proteins ("scFv"), disulfide stabilized Fv proteins ("dsFv"), diabodies, linear antibodies, small modular immunopharmaceuticals (SMIP), binding-domain immunoglobulin fusion proteins, camelid antibodies, as well as multispecific antibody fragments such as a bispecific, trispecific, or multispecific antibodies (e.g., diabodies, a triabodies, a tetrabodies), minibodies, a chelating recombinant antibodies, tribodies, bibodies, intrabodies, nanobodies, and combinations thereof.

Fab fragments include a first polypeptide including the heavy chain variable region and the first constant domain (CHI) of the heavy chain, and a second polypeptide including the light chain variable region and the light chain constant region (LC). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. F(ab')₂ antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. A scFv is a fusion protein in which a light chain variable region and a heavy chain variable region are bound by a linker (see, e.g., Ahmad et al., Clin. Dev. Immunol., 2012,

doi: 10.1155/2012/980250; Marbry, IDrugs, 13:543-549, 2010). The intramolecular orientation of the V_H-domain and the V_L-domain in a scFv, is typically not decisive for scFvs. Thus, scFvs with both possible arrangements (V_H-domain-linker domain- V_L-domain; V_L-domain-linker domain- V_H-domain) may be used.

An antibody variant is an antibody polypeptide sequence that contains at least one amino acid substitution, deletion, or insertion in the variable region of the reference antibody variable region domains. Variants may be substantially homologous or substantially identical to the unmodified antibody.

Antibodies also include genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3^rd Ed., W. H. Freeman & Co., New York, 1997.

Typically, a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda (λ) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variable region, (the regions are also known as "domains"). In combination, the heavy and the light chain variable regions specifically bind the antigen. References to "V_H" or "VH" refer to the heavy chain variable region, and references to "V_L" or "VL" refer to the light chain variable region.

Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs." The extent of the framework region and CDRs can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. ("Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991; "Kabat" numbering scheme), Al-Lazikani et al., (JMB 273,927-948, 1997; "Chothia" numbering scheme), and Lefranc et al. ("EVICT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev. Comp. Immunol., 27:55-77, 2003; "EVICT" numbering scheme). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species, such as humans. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDRl, CDR2, and CDR3, numbered sequentially starting from the N- terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a V_H CDR3 (or H-CDR3) is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a V_L CDRl (or L-CDRl) is the CDRl from the variable domain of the light chain of the antibody in which it is found. An antibody that binds gpl20, for example, will have a specific V_H region and the V_L region sequence, and thus specific CDR sequences. Antibodies with different specificities (i.e. different combining sites for different antigens) have different CDRs. Only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs).

A "monoclonal antibody" or a "mAb" is an antibody produced by a single clone of

B -lymphocytes or by a cell into which nucleic acid encoding the light and heavy chains of a single antibody have been transfected. mAbs are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. mAbs include humanized mAbs.

A "chimeric antibody" has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species, such as a murine antibody that specifically binds gpl20 or that specifically binds CD3.

A "human" antibody (also called a "fully human" antibody) is an antibody that includes human framework regions and all of the CDRs from a human antibody. In one example, the framework and the CDRs are from the same originating human heavy and/or light chain amino acid sequence. However, frameworks from one human antibody can be engineered to include CDRs from a different human antibody.

A "humanized" antibody is an antibody including a human framework region and one or more CDRs from a non-human (for example a mouse, rat, or synthetic) antibody. The non-human antibody providing the CDRs is termed a "donor," and the human antibody providing the framework is termed an "acceptor." In one embodiment, all the CDRs are from the donor antibody in a humanized antibody. Constant regions need not be present, but if they are, they are typically substantially identical to human antibody constant regions, i.e., at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized antibody, except possibly the CDRs, are substantially identical to corresponding parts of natural human antibody sequences. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Humanized antibodies can be constructed by means of genetic engineering (see for example, U.S. Patent No. 5,585,089).

Antibody Immunogenicity: A property of an antibody, whereby the antibody generates an immune response when administered to a subject, such as a human subject. In several embodiments, a disclosed antibody is not immunogenic or has low immunogenicity, for example, a disclosed antibody is not significantly more immunogenic compared to a standard control, or a reference antibody. Methods of determining the immunogenicity of an antibody are known to the person of ordinary skill in the art (see, e.g., Krieckaert et al , Current Opin Rheumatol., 24:306-311, 2012; Stas and Lasters, IDrugs, 12: 169- 173, 2009). In one non-limiting example, immunogenicity can be determined by assaying plasma or serum from a test subject using an ELISA against the antibody of interest.

Antibody self-reactivity or autoreactivity: A property of an antibody, whereby the antibody reacts with self -epitopes, that is epitopes of proteins and/or lipids that are produced by the subject. An antibody that does not have self -reactivity does not substantially bind to epitopes or lipids present on the membrane of a cell from a subject. Methods of determining if an antibody reacts with self epitopes are known to the person of ordinary skill in the art and described herein (for example, in Examples 1 and 8). In one example, antibody self reactivity is evaluated using an anti-cardiolipin assay or an anti-nuclear antigen (ANA) assay. The anti-ANA assay can include an anti-ANA LUMINEX® assay or an ANA cell-staining assay, for example. In several embodiments, a disclosed antibody is not self-reactive (or autoreactive), or is minimally self-reactive. In one non-limiting example, a disclosed antibody is not significantly more self -reactive compared to the VRCOl antibody, for example as measured using an anti-ANA LUMINEX® assay or an ANA cell-staining assay. In another non-limiting example, a disclosed antibody noes not have self reactivity above background levels, for example, as measured using an anti-ANA LUMINEX® assay or an ANA cell-staining assay.

Antigen: A polypeptide that can stimulate the production of antibodies or a T cell response in an animal, including polypeptides that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the disclosed antigens. "Epitope" or "antigenic determinant" refers to the region of an antigen to which B and/or T cells respond. In one embodiment, T cells respond to the epitope, when the epitope is presented in conjunction with an MHC molecule. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and nuclear magnetic resonance.

Immunogenic polypeptides and immunogenic peptides are non-limiting examples of antigens. In some examples, antigens include polypeptides derived from a pathogen of interest, such as a virus. An antigen that can stimulate the production of antibodies or a T cell response in a subject to a polypeptide expressed by a virus is a viral antigen. An "HIV antigen" can stimulate the production of antibodies or a T cell response in a subject to a polypeptide expressed by HIV. In some embodiments, an HIV antigen is a polypeptide expressed by HIV, such as HIV ENV, or a fragment thereof, such as gpl20.

A "target epitope" is a specific epitope on an antigen that specifically binds an antibody of interest, such as a monoclonal antibody. In some examples, a target epitope includes the amino acid residues that contact the antibody of interest, such that the target epitope can be selected by the amino acid residues determined to be in contact with the antibody of interest. Antigen binding domain: A region of a polypeptide or polypeptide complex that specifically binds to an antigen. The antigen binding domain includes the heavy and light chain CDRs of a monoclonal antibody. Non-limiting examples of antigen binding domains include antibodies and functional fragments thereof, such as a Fab and a scFv. A single polypeptide can contain more than one antigen binding domain.Anti-retroviral agent: An agent that specifically inhibits a retrovirus from replicating or infecting cells. Non-limiting examples of antiretroviral drugs include entry inhibitors (e.g. , enfuvirtide), CCR5 receptor antagonists (e.g. , aplaviroc, vicriviroc, maraviroc), reverse transcriptase inhibitors (e.g. , lamivudine, zidovudine, abacavir, tenofovir, emtricitabine, efavirenz), protease inhibitors (e.g. , lopivar, ritonavir, raltegravir, darunavir, atazanavir), maturation inhibitors (e.g. , alpha interferon, bevirimat and vivecon).

Anti-retroviral therapy (ART): A therapeutic treatment for HIV infection involving administration of at least one anti-retroviral agents (e.g. , one, two, three or four anti-retroviral agents) to an HIV infected individual during a course of treatment. Non-limiting examples of antiretroviral agents include entry inhibitors (e.g. , enfuvirtide), CCR5 receptor antagonists (e.g. , aplaviroc, vicriviroc, maraviroc), reverse transcriptase inhibitors (e.g. , lamivudine, zidovudine, abacavir, tenofovir, emtricitabine, efavirenz), protease inhibitors (e.g. , lopivar, ritonavir, raltegravir, darunavir, atazanavir), maturation inhibitors (e.g. , alpha interferon, bevirimat and vivecon). One example of an ART regimen includes treatment with a combination of tenofovir, emtricitabine and efavirenz. In some examples, ART includes HAART.

Bispecific antibody: A recombinant molecule composed of two different antigen binding domains that consequently binds to two different antigenic epitopes. Bispecific antibodies include chemically or genetically linked molecules of two antigen-binding domains. The antigen binding domains can be linked using a linker. The antigen binding domains can be monoclonal antibodies, antigen-binding fragments (e.g. , Fab, scFv), or combinations thereof. A bispecific antibody can include one or more constant domains, but does not necessarily include a constant domain. An example of a bispecific antibody is a bispecific single chain antibody including a scFv that specifically binds to gpl20 joined (via a peptide linker) to a scFv that specifically binds CD3. Another example is a bispecific antibody including a Fab that specifically binds to gpl20 joined to a scFv that specifically binds to CD3. Production of bispecific Fab-scFv ("bibody") molecules are described, for example, in Schoonjans et al. (J. Immunol. 165:7050-57, 2000) and Willems et al. (J Chromatogr B Analyt Technol Biomed Life Sci. 786: 161-76, 2003). For bibodies, a scFv molecule is fused to one of the VL-CL (L) or VH-CH1 chains, e.g. , to produce a bibody one scFv is fused to the C-term of a Fab chain.

Bispecific single chain antibody: A bispecific antibody composed of a single chain of amino acids that includes a first antigen binding domain that specifically binds a first antigen and a second antigen binding domain that specifically binds a second antigen, wherein the first and second antigen are different. In several embodiments, the first antigen binding domain is a scFv and the second antigen binding domain is a scFv. In some embodiments, the first and second antigen binding domains (e.g. , a first and second scFv) are joined via a peptide linker. In the event that a linker is used, the linker is preferably of a length and sequence sufficient to ensure that each of the first and second antigen binding domains can, independently from one another, retain their differential binding specificities. Bispecific single chain antibodies can be encoded by a single nucleic acid molecule. Examples of bispecific single chain antibodies, as well as methods of constructing such antibodies are known in the art (see, e.g. , U.S. Pat. Nos. 8,076,459, 8,017,748, 8,007,796, 7,919,089, 7,820,166, 7,635,472, 7,575,923, 7,435,549, 7,332,168, 7,323,440, 7,235,641, 7,229,760, 7,112,324, 6,723,538, incorporated by reference herein). Additional examples of bispecific single chain antibodies can be found in PCT application No. WO 99/54440; Mack, . Immunol. , 158:3965-3970, 1997; Mack, PNAS, 92:7021-7025, 1995; Kufer, Cancer Immunol. Immunother. , 45:193-197, 1997; Loffler, Blood, 95:2098-2103, 2000; and Bruhl, . Immunol , 166:2420-2426, 2001.

CD3: Cluster of differentiation 3, which is a T cell co-receptor: the CD3 protein complex including at least four polypeptide chains, which are non-covalently associated with the T cell receptors on the surface of T cells. The four polypeptide chains include two CD3-epsilon chains, a CD3-delta chain and a CD3-gamma chain. CD3 is present on both helper T cells and cytotoxic T cells.

CD4: Cluster of differentiation factor 4 polypeptide; a T cell surface protein that mediates interaction with the MHC class II molecule. CD4 also serves as the primary receptor site for HIV on T cells during HIV-1 infection, and binds gpl20.

CD4 binding site (CD4BS) antibodies: Antibodies that bind to or substantially overlap the CD4 binding surface of a gp 120 polypeptide. The antibodies interfere with or prevent CD4 from binding to a gpl20 polypeptide.

Conservative variants: "Conservative" amino acid substitutions that do not substantially affect or decrease the affinity of a protein, such as an antibody that specifically binds gpl20 or an antibody that specifically binds CD3, for their respective target antigen. For example, a antibody that specifically binds gpl20 can include at most about 1, at most about 2, at most about 5, and most about 10, or at most about 15 conservative substitutions and specifically bind the gpl20 polypeptide with a similar affinity. The term conservative variantalso includes the use of a substituted amino acid in place of an

unsubstituted parent amino acid, provided that antibody specifically binds the target antigen. Non- conservative substitutions are those that reduce an activity or binding to the target antigen, such as gpl20 or CD3.

Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). Contacting: Placement in direct physical association; includes both in solid and liquid form, which can take place either in vivo or in vitro. Contacting includes contact between one molecule and another molecule, for example the amino acid on the surface of one polypeptide, such as an antigen, that contacts another polypeptide, such as an antibody. Contacting can also include contacting a cell for example by placing an antibody in direct physical association with a cell.

Control: A reference standard. In some embodiments, the control is a negative control, such as sample obtained from a healthy patient not infected with HIV or not administered a particular treatment. In other embodiments, the control is a positive control, such as a tissue sample obtained from a patient diagnosed with HIV infection. In still other embodiments, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of HIV patients with known prognosis or outcome, or group of samples that represent baseline or normal values).

A difference between a test sample and a control can be an increase or conversely a decrease. The difference can be a qualitative difference or a quantitative difference, for example a statistically significant difference. In some examples, a difference is an increase or decrease, relative to a control, of at least about 5%, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, or at least about 500%.

Cross complementation: Formation of an antibody including heavy and light chain variable domains using a heavy chain variable domain of an antibody that specifically binds an epitope of an antigen of interest from first antibody and a light chain variable domain of an antibody that specifically binds the same epitope from a second antibody, wherein the antibody that is formed from the heavy chain variable domain and the light chain variable domain retains its ability to bind the epitope and wherein the first and the second antibodies are different antibodies. Thus, in cross complementation, the light chain variable domains and the heavy chain variable domains that form an antibody are from different sources, but the chimeric antibody that is formed still binds the epitope. In one embodiment, the antigen is gpl20. In one embodiment an antibody that specifically binds to gpl20 includes a heavy chain cross- complemented with a light chain, wherein the heavy chain includes the heavy chain variable domain of VRC07 and the light chain includes the light chain variable domain of VRC01.

Degenerate variant: In the context of the present disclosure, a "degenerate variant" refers to a polynucleotide encoding a polypeptide (such as an antibody that binds gpl20) that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences encoding a polypeptide are included as long as the amino acid sequence of the polypeptide encoded by the nucleotide sequence is unchanged.

Effector molecule: The portion of a chimeric molecule that is intended to have a desired effect on a cell to which the chimeric molecule is targeted. Effector molecule is also known as an effector moiety (EM), therapeutic agent, or diagnostic agent, or similar terms. Therapeutic agents include such compounds as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, lipids, carbohydrates, or recombinant viruses. Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking with single or duplex DNA, and triplex forming oligonucleotides. Alternatively, the molecule linked to a targeting moiety, such as an anti-gpl20 antibody, may be an encapsulation system, such as a liposome or micelle that contains a therapeutic composition such as a drug, a nucleic acid (such as an antisense nucleic acid), or another therapeutic moiety that can be shielded from direct exposure to the circulatory system. Means of preparing liposomes attached to antibodies are well known to those of skill in the art (see, for example, U.S. Patent No. 4,957,735; and Connor et al , Pharm. Ther. 28:341-365, 1985). Diagnostic agents or moieties include radioisotopes and other detectable labels.

Detectable labels useful for such purposes are also well known in the art, and include radioactive isotopes such as ³⁵S, ⁿC, ¹³N, ¹⁵0, ¹⁸F, ¹⁹F, ^99mTc, ¹³¹1, ³H, ¹⁴C, ¹⁵N, ⁹⁰Y, ⁹⁹Tc, ^mIn and ¹²⁵I, fluorophores, chemiluminescent agents, and enzymes.

Epitope: An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, i.e. that elicit a specific immune response. An antibody specifically binds a particular antigenic epitope on a polypeptide. In some embodiments a disclosed antibody specifically binds to an epitope on the surface of gpl20 from HIV, and/or specifically binds to CD3.

Expressed: Translation of a nucleic acid into a protein. Proteins may be expressed and remain intracellular, become a component of the cell surface membrane, or be secreted into the extracellular matrix or medium.

Expression Control Sequences: Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e.,

ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term "control sequences" is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.

A promoter is a minimal sequence sufficient to direct transcription. Also included are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters are included (see for example, Bitter et al., Methods in Enzymology 153:516-544, 1987). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used. In one embodiment, when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (such as metallothionein promoter) or from mammalian viruses (such as the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences. A polynucleotide can be inserted into an expression vector that contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells.

Fc polypeptide: The polypeptide including the constant region of an antibody excluding the first constant region immunoglobulin domain. Fc region generally refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM. An Fc region may also include part or all of the flexible hinge N-terminal to these domains. For IgA and IgM, an Fc region may or may not include the tailpiece, and may or may not be bound by the J chain. For IgG, the Fc region includes immunoglobulin domains Cgamma2 and Cgamma3 (Cy2 and Cy3) and the lower part of the hinge between Cgammal (Cyl) and Cy2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat. For IgA, the Fc region includes immunoglobulin domains Calpha2 and Calpha3 (Ca2 and Ca3) and the lower part of the hinge between Calphal (Cal) and Ca2.

Framework region: Amino acid sequences interposed between CDRs. Framework regions include variable light and variable heavy framework regions. The framework regions serve to hold the CDRs in an appropriate orientation for antigen binding.

gp41: A HIV envelope protein that contains a transmembrane domain and remains in a trimeric configuration; it interacts with gpl20 in a non-covalent manner. The amino acid sequence of an example of gp41 is set forth in GENBANK® Accession No. CAD20975 (as available on October 16, 2009) which is incorporated by reference herein. It is understood that the sequence of gp41 can vary from that given in GENBANK® Accession No. CAD20975. gp41 contains a transmembrane domain and typically remains in a trimeric configuration; it interacts with gpl20 in a non-covalent manner.

gpl20: A HIV envelope protein that interacts with gp41 in a non-covalent manner. gpl20 contains most of the external, surface-exposed, domains of the HIV envelope glycoprotein complex, and it is gpl20 which binds both to cellular CD4 receptors and to cellular chemokine receptors (such as CCR5).

The mature gpl20 wildtype polypeptides have about 500 amino acids in the primary sequence. gpl20 is heavily N-glycosylated giving rise to an apparent molecular weight of 120 kD. The polypeptide is comprised of five conserved regions (C1-C5) and five regions of high variability (VI -V5). Exemplary sequence of wt gpl20 polypeptides are shown on GENBANK®, for example accession numbers

AAB05604 and AAD12142 (as available on October 16, 2009), incorporated by reference herein. It is understood that there are numerous variation in the sequence of gpl20 from what is given in GENBANK®, for example accession numbers AAB05604 and AAD12142, and that these variants are skill recognized in the art as gpl20.

The numbering used in gpl20 polypeptides disclosed herein is relative to the HXB2 numbering scheme as set forth in Numbering Positions in HIV Relative to HXB2CG Bette Korber et al, Human Retroviruses and AIDS 1998: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Korber B, Kuiken CL, Foley B, Hahn B, McCutchan F, Mellors JW, and Sodroski J, Eds. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM which is incorporated by reference herein in its entirety.

Highly active anti- retroviral therapy (HAART): A therapeutic treatment for HIV infection involving administration of multiple anti-retroviral agents {e.g. , two, three or four anti-retroviral agents) to an HIV infected individual during a course of treatment. Non-limiting examples of antiretroviral agents for use with HAART include entry inhibitors {e.g., enfuvirtide), CCR5 receptor antagonists {e.g., aplaviroc, vicriviroc, maraviroc), reverse transcriptase inhibitors {e.g., laniivudine, zidovudine, abacavir, tenofovir, emtricitabine, efavirenz), protease inhibitors {e.g., lopivar, ritonavir, raltegravir, darunavir, atazanavir), maturation inhibitors {e.g. , alpha interferon, bevirimat and vivecon). The person of ordinary skill in the art is familiar with anti-retroviral agents and dosages thereof for use with HAART. One example of a HAART regimen includes treatment with a combination of tenofovir, emtricitabine and efavirenz.

Host cells: Cells in which a vector can be propagated and its DNA expressed. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used.

Human Immunodeficiency Virus (HIV): A retrovirus that causes immunosuppression in humans (HIV disease), and leads to a disease complex known as the acquired immunodeficiency syndrome (AIDS). HIV disease refers to a well-recognized constellation of signs and symptoms

(including the development of opportunistic infections) in persons who are infected by an HIV virus.

Laboratory findings associated with this disease include a progressive decline in T cells. HIV includes, for example, HIV type 1 and type 2 (HIV-1 and HIV-2).

Treatment of HIV-1 with HAART has been effective in reducing the viral burden and ameliorating the effects of HIV-1 infection in infected individuals. However, a latent reservoir of HIV infected cells evades this treatment, allowing the virus to persist in the individual despite therapy and requiring continued treatment. The latent reservoir of cells includes cells infected with the HIV-1 virus, wherein the virus is not replicating (see, e.g. , Rong and Perelson, J. Theor. Biol., 260:308-331, 2009).

This latent reservoir is thought to persist at least in a subpopulation of resting CD4⁺ T cells. Since the HAART drugs target viral proteins involved in the viral replication cycle, they are unable to eliminate these quiescent cells that harbor pro viral HIV-1 DNA.

HIV-1 activation: Activation of HIV-1 replication, for example activation of HIV-1 replication in the latent reservoir of HIV infected cells. An HIV-1 activating agent (such as a small molecule or antibody) is an agent capable of inducing HIV-1 activation, for example, activation of HIV-1 replication in the latent reservoir of HIV infected cells. Methods of detecting and/or measuring HIV-1 activation are known to the skilled artisan and further described herein. HIV-1 activation can include activation of expression of HIV-1 proviral DNA in HIV-1 infected T cells.

HIV Envelope protein (Env): The HIV envelope protein is initially synthesized as a longer precursor protein of 845-870 amino acids in size, designated gpl60. gpl60 forms a homotrimer and undergoes glycosylation within the Golgi apparatus. In vivo, it is then cleaved by a cellular protease into gpl20 and gp41.

Immune complex: The binding of antibody to a soluble antigen forms an immune complex. The formation of an immune complex can be detected through conventional methods known to the skilled artisan, for instance immunohistochemistry, immunoprecipitation, flow cytometry,

immunofluorescence microscopy, ELISA, immunoblotting (for example, Western blot), magnetic resonance imaging, CT scans, X-ray and affinity chromatography. Immunological binding properties of selected antibodies may be quantified using methods well known in the art.

Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response"). In one embodiment, an immune response is a T cell response, such as a CD4⁺ response or a CD8⁺ response. In another embodiment, the response is a B cell response, and results in the production of specific antibodies.

Immunologically reactive conditions: Includes reference to conditions which allow an antibody raised against a particular epitope to bind to that epitope to a detectably greater degree than, and/or to the substantial exclusion of, binding to substantially all other epitopes. Immunologically reactive conditions are dependent upon the format of the antibody binding reaction and typically are those utilized in immunoassay protocols or those conditions encountered in vivo. See Harlow & Lane, supra, for a description of immunoassay formats and conditions. The immunologically reactive conditions employed in the methods are "physiological conditions" which include reference to conditions (such as temperature, osmolarity, pH) that are typical inside a living mammal or a mammalian cell.

While it is recognized that some organs are subject to extreme conditions, the intra-organismal and intracellular environment normally lies around pH 7 (i.e., from pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), contains water as the predominant solvent, and exists at a temperature above 0°C and below 50°C. Osmolarity is within the range that is supportive of cell viability and proliferation.

Inhibiting or treating a disease: Inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as acquired immunodeficiency syndrome (AIDS). "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. The term "ameliorating," with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the viral load, an improvement in the overall health or well- being of the subject, or by other parameters well known in the art that are specific to the particular disease. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology. In one embodiment, a disclosed antibody is administered to a subject to treat HIV-1 infection in the subject, for example, by reducing the latent reservoir of HIV-1 infected cells in the subject.

Isolated: An "isolated" biological component (such as a cell, for example a B cell, a nucleic acid, peptide, protein or antibody) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, such as, other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins which have been "isolated" thus include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids. An isolated biological component does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its natural environment within a cell. In one embodiment, a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation. In some embodiments, the biological component is at least 95%, 96%, 97%, 98% or 99% pure. In some embodiments an antibody, such as an antibody specific for gpl20 can be isolated. The antibodies disclosed herein can be purified by any of the means known in the art. See for example Guide to Protein Purification, ed. Deutscher, Meth. Enzymol. 185, Academic Press, San Diego, 1990; and Scopes, Protein Purification: Principles and Practice, Springer Verlag, New York, 1982.

Substantial purification denotes purification from other proteins or cellular components. A substantially purified protein is at least 60%, 70%, 80%, 90%, 95% or 98% pure. Thus, in one specific, non-limiting example, a substantially purified protein is 90% free of other proteins or cellular components.

K_d: The dissociation constant for a given interaction, such as a polypeptide -ligand interaction or an antibody-antigen interaction. For example, for the bimolecular interaction of an antibody or a functional fragment thereof and an antigen, it is the concentration of the individual components of the bimolecular interaction divided by the concentration of the complex.

Label: A detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes. In some examples, a disclosed antibody as labeled.

Linker: A bi-functional molecule that can be used to link two molecules into one contiguous molecule, for example, to link a first antigen binding domain to a second antigen binding domain in a multispecific antibody. In some cases, a linker is a peptide within an antigen binding fragment (such as an Fv fragment) which serves to indirectly bond the variable heavy chain to the variable light chain. In some examples, the linker is a peptide linker, such as a (GGGGS)i (SEQ ID NO: 1763), (GGGGS)₂ (SEQ ID NO: 1764), or a (GGGGS)₃ (SEQ ID NO: 1765) linker.

In several embodiments, the terms "conjugating," "joining," "bonding" or "linking" refer to making two polypeptides into one contiguous polypeptide molecule, to covalently attaching a radionuclide or other effector molecule to a polypeptide, such as an antibody that specifically binds gpl20, or an antigen binding fragment thereof. In some embodiments, the terms include reference to joining a ligand, such as an antibody moiety, to an effector molecule. The linkage can be either by chemical or recombinant means. "Chemical means" refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule.

Membrane-proximal external region (MPER) of gp41: A region that is immediately N- terminal of the transmembrane region of gp41. The MPER is highly hydrophobic (50% of residues are hydrophobic) and is highly conserved across many HIV clades (Zwick, M.B., et al, J Virol, 75 (22): p. 10892-905, 2001). The sequence of the MPER from an exemplary HIV-1 strain is included herein as SEQ ID NO: 1539.

Multispecific Antibody: An antibody that includes at least two antigen binding domains, each of which specifically binds a different antigen. Multispecific antibodies include bispecific antibodies and trispecific antibodies.

Neutralizing antibody: An antibody which reduces the infectious titer of an infectious agent by binding to a specific antigen on the infectious agent. In some examples the infectious agent is a virus. In some examples, an antibody that is specific for gpl20 neutralizes the infectious titer of HIV. A "broadly neutralizing antibody" is an antibody that binds to and inhibits the function of related antigens, such as antigens that share at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity antigenic surface of antigen. With regard to an antigen from a pathogen, such as a virus, the antibody can bind to and inhibit the function of an antigen from more than one class and/or subclass of the pathogen. For example, with regard to a human immunodeficiency virus, the antibody can bind to and inhibit the function of an antigen, such as gpl20 from more than one clade. In one embodiment, broadly neutralizing antibodies to HIV are distinct from other antibodies to HIV in that they neutralize a high percentage of the many types of HIV in circulation.

Nucleic acid: A polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non- naturally occurring analogs thereof. Thus, the term includes nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs, such as, for example and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide -nucleic acids (PNAs), and the like. Such

polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "oligonucleotide" typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e. , A, T, G, C), this also includes an RNA sequence (i.e. , A, U, G, C) in which "U" replaces "T."

"Nucleotide" includes, but is not limited to, a monomer that includes a base linked to a sugar, such as a pyrimidine, purine or synthetic analogs thereof, or a base linked to an amino acid, as in a peptide nucleic acid (PNA). A nucleotide is one monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide.

Conventional notation is used herein to describe nucleotide sequences: the left-hand end of a single-stranded nucleotide sequence is the 5'-end; the left-hand direction of a double-stranded nucleotide sequence is referred to as the 5'-direction. The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the "coding strand;" sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5' to the 5'-end of the RNA transcript are referred to as "upstream sequences;" sequences on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the coding RNA transcript are referred to as "downstream sequences."

"cDNA" refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form.

"Encoding" refers to the inherent property of specific sequences of nucleotides in a

polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (for example, rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and non-coding strand, used as the template for transcription, of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns. In some examples, a nucleic acid encodes a disclosed antibody.

"Recombinant nucleic acid" refers to a nucleic acid having nucleotide sequences that are not naturally joined together. This includes nucleic acid vectors including an amplified or assembled nucleic acid which can be used to transform a suitable host cell. A host cell that includes the recombinant nucleic acid is referred to as a "recombinant host cell." The gene is then expressed in the recombinant host cell to produce, such as a "recombinant polypeptide." A recombinant nucleic acid may serve a non- coding function (such as a promoter, origin of replication, ribosome-binding site, etc.) as well.

Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter, such as the CMV promoter, is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.

Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein- coding regions, in the same reading frame.

Polypeptide: Any chain of amino acids, regardless of length or post-translational modification

(such as glycosylation or phosphorylation). "Polypeptide" applies to amino acid polymers including naturally occurring amino acid polymers and non-naturally occurring amino acid polymer as well as in which one or more amino acid residue is a non-natural amino acid, for example an artificial chemical mimetic of a corresponding naturally occurring amino acid. A "residue" refers to an amino acid or amino acid mimetic incorporated in a polypeptide by an amide bond or amide bond mimetic. A polypeptide has an amino terminal (N-terminal) end and a carboxy terminal (C-terminal) end. Reference to a first region of a polypeptide that is "C-terminal" to a second region of a polypeptide indicates that the first region is closer to the C-terminal end of the polypeptide than the second region. Reference to a first region of a polypeptide that is "N-terminal" to a second region of a polypeptide indicates that the first region is closer to the N-terminal end of the polypeptide than the second region. "Polypeptide" is used interchangeably with peptide or protein, and is used interchangeably herein to refer to a polymer of amino acid residues.

Amino acids in a peptide, polypeptide or protein generally are chemically bound together via amide linkages (CONH). Additionally, amino acids may be bound together by other chemical bonds. For example, linkages for amino acids or amino acid analogs can include CH₂NH-, -CH₂S-, -CH₂-CH₂ -, -CH=CH- (cis and trans), -COCH₂ -, -CH(OH)CH₂-, and -CHH₂SO- (These and others can be found in Spatola, in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci pp. 463-468, 1980;

Hudson, et al, Int J Pept Prot Res 14: 177-185, 1979; Spatola et al. Life Sci 38: 1243-1249, 1986; Harm . Chem. Soc Perkin Trans. 1307-314, 1982; Almquist et al. J. Med. Chem. 23: 1392-1398, 1980; Jennings- White et al. Tetrahedron Lett 23:2533, 1982; Holladay et al. Tetrahedron. Lett 24:4401-4404, 1983; and Hruby Life Sci 31: 189-199, 1982.

Peptide modifications: Polypeptides, such as multispecific antibodies that specifically bind to gpl20, gp41, and/or CD3, include synthetic embodiments of peptides described herein. In addition, analogs (non-peptide organic molecules), derivatives (chemically functionalized peptide molecules obtained starting with the disclosed peptide sequences) and variants (homologs) of these proteins can be utilized in the methods described herein. Each polypeptide is comprised of a sequence of amino acids, which may be either L- and/or D- amino acids, naturally occurring and otherwise.

Peptides may be modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified peptides, and optionally having other desirable properties. For example, carboxylic acid groups of the protein, whether carboxyl-terminal or side chain, may be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified to form a Ci-Ci₆ ester, or converted to an amide of formula NRiR₂ wherein R and R₂ are each independently H or Ci-Ci₆ alkyl, or combined to form a heterocyclic ring, such as a 5- or 6- membered ring. Amino groups of the peptide, whether amino-terminal or side chain, may be in the form of a pharmaceutically-acceptable acid addition salt, such as HC1, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or may be modified to Ci-Ci₆ alkyl or dialkyl amino or further converted to an amide.

Hydroxyl groups of the peptide side chains may be converted to C₁-C₁₆ alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl and phenolic rings of the peptide side chains may be substituted with one or more halogen atoms, such as fluorine, chlorine, bromine or iodine, or with C₁-C₁₆ alkyl, C₁-C₁₆ alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids. Methylene groups of the peptide side chains can be extended to homologous C₂-C₄ alkylenes. Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups. Those skilled in the art will also recognize methods for introducing cyclic structures into the peptides to select and provide conformational constraints to the structure that result in enhanced stability.

Pharmaceutical agent: A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject or a cell. In some examples a pharmaceutical agent includes one or more of the disclosed antibodies.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington 's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 15th Edition, 1975, describes compositions and formulations suitable for pharmaceutical delivery of the antibodies herein disclosed.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (such as powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Sample (or biological sample): A biological specimen containing genomic DNA, RNA

(including mRNA), protein, or combinations thereof, obtained from a subject. Examples include, but are not limited to, peripheral blood, tissue, cells, urine, saliva, tissue biopsy, fine needle aspirate, surgical specimen, and autopsy material. In one example, a sample includes a HCC tissue biopsy.

Sequence identity: The similarity between amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981;

Needleman and Wunsch, /. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., . Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.

Homologs and variants of a V_L or a V_H of an antibody that specifically binds a polypeptide are typically characterized by possession of at least about 75%, for example at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full length alignment with the amino acid sequence of interest. Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.

Specifically bind: When referring to an antibody, refers to a binding reaction which determines the presence of a target protein, peptide, or polysaccharide in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated conditions, an antibody binds preferentially to a particular target protein, peptide or polysaccharide (such as an antigen present on the surface of a pathogen, for example gpl20, gp41, or CD3) and do not bind in a significant amount to other proteins or polysaccharides present in the sample or subject. As used herein, reference to an antigen specific monoclonal antibody (such as a CD3 specific monoclonal antibody) refers to a monoclonal antibody that specifically binds to the indicated antigen (such as a monoclonal antibody that specifically binds to CD3). Specific binding can be determined by methods known in the art. With reference to an antibody antigen complex, specific binding of the antigen and antibody has a K_D of less than about 10^"6 Molar, such as less than about 10^"6 Molar, 10^"7 Molar, 10^"8 Molar, 10^"9, or even less than about 10^"10 Molar.

Subject: Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals. Therapeutic agent: Used in a generic sense, it includes treating agents, prophylactic agents, and replacement agents. A therapeutic agent is used to ameliorate a specific set of conditions in a subject with a disease or a disorder.

Therapeutically effective amount: A quantity of a specific substance, such as a disclosed antibody, sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to inhibit HIV replication, reduce the latent reservoir of HIV-1 infected cells, or treat AIDS. In several embodiments, a therapeutically effective amount is the amount necessary to reduce a sign or symptom of AIDS, and/or to decrease viral titer in a subject. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations that has been shown to achieve a desired in vitro effect.

T Cell: A white blood cell critical to the immune response. T cells include, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ T lymphocyte is an immune cell that carries a marker on its surface known as "cluster of differentiation 4" (CD4). These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses. CD8⁺ T cells carry the "cluster of differentiation 8" (CD8) marker. In one embodiment, a CD8 T cell is a cytotoxic T lymphocyte. In another embodiment, a CD8 cell is a suppressor T cell. T cell activation can be induced by a number of factors, including binding of cognate antigen to the T cell receptor on the surface of T cells. Activation of a T cell leads to immune response, such as T cell proliferation and differentiation (see, e.g., Smith-Garvin et al , Annu. Rev. Immunol., 27:591-619, 2009). A molecule (such as an antibody) that is T cell activating is a molecule that specifically binds to a T cell (e.g., specifically binds to an epitope on the surface of the T cell), leading to activation of the T cell. For example, clustering of CD3 on T cells, e.g. by immobilized anti-CD3 antibodies, leads to T cell activation similar to the engagement of the T cell receptor but independent of its clone-typical specificity (see PCT Publication No. WO 99/54440 or Hoffman (1985) J. Immunol. 135:5-8). Thus, in some embodiments, activation of a cytotoxic T cell may occur via binding of CD3 as effector antigen on the surface of the cytotoxic T cell by an antibody provided herein. For example, certain antibodies (such as the TR66 antibody) that specifically bind to CD3 on the surface of a T cell are known to activate the T cell and can be included in the provided antibodies.

Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art.

Virus: Microscopic infectious organism that reproduces inside living cells. A virus consists essentially of a core of a single nucleic acid surrounded by a protein coat, and has the ability to replicate only inside a living cell. "Viral replication" is the production of additional virus by the occurrence of at least one viral life cycle. A virus may subvert the host cells' normal functions, causing the cell to behave in a manner determined by the virus. For example, a viral infection may result in a cell producing a cytokine, or responding to a cytokine, when the uninfected cell does not normally do so. "Retroviruses" are RNA viruses wherein the viral genome is RNA. When a host cell is infected with a retrovirus, the genomic RNA is reverse transcribed into a DNA intermediate which is integrated very efficiently into the chromosomal DNA of infected cells. The integrated DNA intermediate is referred to as a provirus. The term "lentivirus" is used in its conventional sense to describe a genus of viruses containing reverse transcriptase. The lentiviruses include the "immunodeficiency viruses" which include HIV-1, HIV-2, SIV and FIV.

VRCOl-like antibody, heavy chain or light chain: Exemplary VRC-01 antibodies, and methods for identifying and producing these antibodies, are disclosed, for example, in International (PCT) App. Nos. PCT/US2010/050295 and PCT/US2012/030465, and Kwong et al., Immunity, 37:412- 425, 2012, each of which is incorporated herein by reference in its entirety. Generally, these antibodies bind to the CD4 binding surface of gpl20 in substantially the same orientation as VRCOl, and are broadly neutralizing. VRCOl-like antibody heavy chain or light chain that can complement with a corresponding heavy chain or light chain from VRCOl, as described defined herein. VRCOl-like antibodies, and methods for identifying and producing these antibodies, are also disclosed herein.

VRCOl, VRC07, and their variants provided herein are examples of VRCOl-like antibodies.

///. Isolated Antibodies

Isolated multispecific antibodies, including bispecific and trispecific antibodies, are disclosed herein.

In several embodiments, the isolated multispecific antibodies includes a first antigen binding domain that specifically binds an HIV-1 epitope (e.g. , an epitope on gpl20 or gp41) and is neutralizing, and a second antigen binding domain that specifically binds an epitope present on the surface of a T cell (e.g., an epitope on CD3), and is T cell activating and HIV-1 activating. In some examples the isolated multispecific antibody is a trispecific antibody and further includes a third antigen binding domain that specifically binds to a costimulatory molecule on the surface of a T cell, and is T cell activating (for example, the third antigen binding domain can specifically bind to one of CD28, CD40L, CD 137 or CD137L.

In some embodiments, the disclosed multispecific antibody is a bispecific antibody including a first antigen binding domain and a second antigen binding domain. In several such embodiments, the first and second antigen binding domains are a Fab or a scFv. For example, the first antigen binding domain is an scFv, the second antigen binding domain is an scFv or both the first and second antigen binding domains are scFvs. In other embodiments, the first antigen binding domain is an Fab and the second antigen binding domain is an scFv, or the first antigen binding domain is a scFv and the second antigen binding domain is a Fab, or both the first and second antigen binding domains are Fabs. In some embodiments including a trispecific antibody, the third antigen binding domain can also be a Fab or an scFv. The person of skill in the art will appreciate that other antigen binding formats are available.

The bispecific antibodies can be generated in a variety of formats, including, for example, scFv- scFv (e.g., BiTE), Fab-scFv (e.g., bibody), Fab - scFv2 (e.g., tribody), and crossmab formats. In several embodiments, the multispecific antibody is a bibody, wherein the first antigen binding domain is a Fab that specifically binds to an HIV-1 epitope and is neutralizing, and the second antigen binding domain is a scFv that specifically binds to CD3 and is T cell activating and HIV-1 activating. The N-terminus of the scFv can be linked to the C-terminus of the light chain or the Fab or the CHI region of the heavy chain of the Fab. Construction and production of tribodies is well known in the art, and is described, for example, in Schoonjans et al. (J Immunol. 165:7050-57, 2000, which is incorporated by reference herein in its entirety).

In additional embodiments, the multispecific antibody is a crossmab antibody, including a full IgG with a first arm (antigen binding domain) that specifically binds to an HIV antigen, and a second arm (antigen binding domain) that specifically binds to CD3. Construction and production of crossmab antibodies is well known in the art, and is described, for example, in Schaefer et al. (Proc. Natl. Acad. Sci. U.S.A., 108: 11187-11192, 2011, which is incorporated by reference herein in its entirety). In several embodiments, the multispecific antibody is a tribody, wherein the first antigen binding domain is a Fab that specifically binds to an HIV-1 epitope and is neutralizing, and the second antigen binding domain is a scFv that specifically binds to CD3 and is T cell activating and HIV-1 activating, and the third antigen binding domain specifically binds to CD28, CD40L, CD 137 or CD137L, and is T cell activating.

Construction and production of tribodies is well known in the art, and is described, for example, in Schoonjans et al. (J Immunol. 165:7050-57, 2000, which is incorporated by reference herein in its entirety).

In several embodiments, the first antigen binding domain specifically binds to an HIV-1 envelope protein (such as gpl20 or gp41) and is neutralizing and the second antigen binding domain specifically binds to CD3, activates T cells and is HIV-1 activating (for example, activates expression of HIV-1 proviral DNA in HIV-1 infected T cells). In some embodiments, binding to CD3 on the surface of a T cell by the second antigen binding domain of the bispecific antibody triggers cellular signals resulting in activation of the T cell and, if present, activation of the latent HIV-1 reservoir in the T cell. Activation of the latent HIV-1 reservoir results in replication of the HIV-1 virus in the T cell, and expression of HIV-1 envelope proteins (such as gpl20 and gp41) on the surface of the T cell. The exposed HIV-1 envelope proteins are targeted by the first binding domain of the disclosed bispecific antibody, which is HIV-1 neutralizing. Thus, the first and second binding domains of the bispecific antibodies function synergistically to target the latent reservoir of HIV-1 virus in CD3 expressing T cells.

In some embodiments, an isolated bispecific monoclonal antibody is disclosed that includes a first antigen binding domain and a second antigen binding domain. The first antigen binding domain can be a Fab or a scFv, specifically binds to gpl20 or gp41, and is neutralizing. The Fab and/or the scFv of the first antigen binding domain includes a heavy chain variable region including a heavy chain complementarity determining region (H-CDR)l, an H-CDR2, an H-CDR3, and a light chain variable region including a light chain complementarity determining region (L-CDR)l, an L-CDR2, and an L- CDR3. The second antigen binding domain can also be a Fab or a scFv. The Fab and/or the scFv of the second antigen binding domain includes a heavy chain variable region including a HCDR1, HCDR2, and a HCD3 and a light chain variable region including a LCDR1, a LCDR2, and a LCDR3. The second antigen binding domain specifically binds CD3, activates T cells, and activates expression of proviral HIV-1 DNA in T cells. In some specific non-limiting examples, the first antigen binding domain includes the heavy chain variable region and the light chain variable region from a VRCOl-like antibody.

The antibody can be fully human, or can be humanized. Also disclosed are compositions including the bispecific antibodies and a pharmaceutically acceptable carrier. Nucleic acids encoding these antibodies, expression vectors including such nucleic acids, and isolated host cells that express the nucleic acids are also provided.

Compositions including the antibodies can be used for research, diagnostic and therapeutic purposes. For example, the bispecific antibody can be used to diagnose or treat a subject having an HIV- 1 infection and/or AIDS.

In several embodiments, reference to particular amino acid substitutions in the heavy or light chains of the disclosed antibodies is made according to the Kabat or IMGT numbering schemes. For example, the amino acid substitution G54H in the heavy chain variable domain of VRC07 referenced herein refers to the Kabat numbering scheme. The skilled artisan will understand reference to these (and other) substitutions using different antibody numbering schemes. For example, using the IMGT numbering scheme, the VRC07 G54H substitution would be referred to as VRC07 G55H; in both cases, this substation refers to substitution of the glycine residue at position 55 of SEQ ID NO: 1461.

Additionally, the discussion of monoclonal antibodies below refers to isolated monoclonal antibodies that include heavy and light chain variable domains including at least one complementarity determining region (CDR), such as a CDRl, CDR2 and CDR3. The person of ordinary skill in the art will understand that various CDR numbering schemes (such as the Kabat, Chothia or IMGT numbering schemes) can be used to determine CDR positions, and that the CDs included in the heavy and light chain variable regions of the antigen binding domains include CDR positions as defined by a single (and not multiple) CDR numbering scheme. For example, the antigen binding domains can include a HCDR1, HCDR2, HCD3, LCDR1, LCDR2, and LCDR3 defined by the Kabat or IMGT numbering scheme, but not an HCD1, HCD2 and HCD3 defined by the Kabat numbering scheme and a LCDR1, LCDR2 and LCDR3 defined by the IMGT numbering scheme. The person of skill in the art will readily understand use of various CDR numbering schemes when referencing particular amino acids of the antibodies disclosed herein.

A. First antigen binding domain - HIV specific

1. gpl20 specific antigen binding domains

In several embodiments, the isolated multispecific antibody includes a first antigen binding domain that specifically binds to gpl20, such as a VRCOl-like monoclonal antibody, or a functional fragment thereof {e.g., an scFv or a Fab), for example, as described herein. In some embodiments, the first antigen binding domain specifically binds to the CD4 binding site on gpl20. The HIV-1 specific antigen binding domain is neutralizing. In some embodiments, the first antigen binding domain includes the heavy and light chain CDRs of a VRCOl-like monoclonal antibody, such as VRCOl, VRC07, or a variant thereof. VRCOl-like monoclonal antibodies include, but are not limited to VRCOl, VRC07, and variants thereof, such as those described in PCT Application Nos. PCT/US 10/50295 (filed September 24, 2010) and

PCT/US2012/030465 (filed March 23, 2012), and U.S. Provisional App. Nos. 61/568,520 (field

December 8, 2011) and 61/613,431 (Filed March 20, 2012), each of which is incorporated by reference herein in its entirety. Several VRCOl-like antibodies are available, including those disclosed herein, as well as:

VRCOl-like antibodies, heavy chains and light chains disclosed in Scheid et al, "Sequence and structural convergence of broad and potent HIV antibodies that mimic CD4 binding," Science,

333(6049): 1633-1637, 2011, incorporated by reference herein. These include the heavy and light chains of the 3BNC117, 3BNC60, 12A12, 12A21, NIH4546, 8ANC131, 8ANC134, 1B2530, 1NC9 antibodies (corresponding Accession Nos. shown in Table 1, below).

VRCOl-like antibodies, heavy chains and light chains disclosed in Wu et al , "Focused evolution of HIV-1 neutralizing antibodies revealed by structures and deep sequencing," Science, 333(6049): 1593- 1602, 2011, incorporated herein by reference. These certain VRCOl-like antibodies, heavy chains and light chains include the heavy and light chains of the VRC-PG04 and VRC-PG04b antibodies

(GENBANK® Accession Nos. JN159464 to JN159467, respectively), VRC-CH30, VRC-CH31, and VRC-CH32 antibodies (GENBANK® Accession Nos. JN159434 to JN159439, respectively), and VRC- CH33 and VRC-CH34 antibodies (GENBANK® Accession Nos. JN159470 to 159473, respectively). These certain VRCOl-like antibodies, heavy chains and light chains also include 24 heavy chains from donor 74, 2008 (GENBANK® Accession Nos. JN159440 to JN159463), two heavy chains from donor 45, 2008 (GENBANK® Accession Nos. JN159474 and JN159475) and two light chains from donor 45, 2001 (GENBANK® Accession Nos. JN159468 and JN159469).

VRCOl-like antibodies, heavy chains and light chains disclosed in Diskin et al , "Increasing the potency and breadth of an HIV antibody by using structure -based rational design," Science,

334(6060): 1289-93, 2011, incorporated by reference herein and U.S. Pat. App. Pub No. 2012/0288502 Al, incorporated by reference herein. These include the heavy and light chains of the NIH4546 antibody with a G54W amino acid substitution (Kabat numbering) in the heavy chain variable domain.

All the Accession Nos. discussed in this definition of "VRCOl-like antibody, heavy chain or light chain," are incorporated by reference as available on April 10, 2013, examples of such Accession Numbers are shown in Table 1.

Table 1. VRCOl-like antibody heavy and light chains

NIH4546 EMBL Acc. No. HE584543 EMBL Acc. No. HE584544

8ANC131 EMBL Acc. No. HE584540 EMBL Acc. No. HE584550

8ANC134 EMBL Acc. No. HE584551 EMBL Acc. No. HE584552

1B2530 EMBL Acc. No. HE584545 EMBL Acc. No. HE584546

1NC9 EMBL Acc. No. HE584547 EMBL Acc. No. HE584548

VRC07 is highly related to VRCOl, and includes a four amino acid insertion in the heavy chain CDR3. The heavy chains of VRCOl-like antibodies (such as VRCOl and VRC07) can be complemented with the light chains of the same or a different VRCOl-like antibody and still retain binding for gpl20. Thus, any of the VRCOl-like heavy chains referred to herein can be complemented with any of the

VRCOl-like light chains to form a functional antigen binding domain that specifically binds to gpl20 and is neutralizing. In some embodiments, the antibody includes an antigen binding domain including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes a H-CDR1, an H-CDR2 and an H-CDR3 from a VRCOl-like antibody, and wherein the light chain variable domain includes a L-CDR1, an L-CDR2 and an L-CDR3 from a VRCOl-like antibody, and wherein the antigen binding domain specifically binds gpl20 and is neutralizing.

(i) VRCOl HC, VRC07 Heavy Chain (HC), and variants thereof

In some embodiments, the first antigen binding domain includes a heavy chain variable region including the CDR1 , CDR2, and/or CDR3 according to IMGT or Kabat of one of SEQ ID NOs: 1 , 3, 27, or 760-1459 (VRCOl HC and variants thereof), wherein the antibody including the antigen binding domain specifically binds to gpl20 and is neutralizing. In some embodiments, the first antigen binding domain includes a heavy chain variable region including amino acids 26-33 (CDR1), 51-58 (CDR2), and/or 97-110 (CDR3) of one of SEQ ID NOs: 1, 3, 27, or 760-1459 (IMGT CDRs). In some embodiments, the first antigen binding domain includes a heavy chain variable region including amino acids 26-33 (CDR1), 51-58 (CDR2), and 97-110 (CDR3) of one of SEQ ID NOs: 1, 3, 27, or 760-1459 (IMGT CDRs). In some embodiments, the first antigen binding domain includes a heavy chain variable region including amino acids 31-35 (CDR1), 50-66 (CDR2), and/or 99-110 (CDR3) of one of SEQ ID NOs: 1, 3, 27, or 760-1459 (Kabat CDRs). In some embodiments, the first antigen binding domain includes a heavy chain variable region including amino acids 31-35 (CDR1), 50-66 (CDR2), and 99-110 (CDR3) of one of SEQ ID NOs: 1, 3, 27, or 760-1459 (Kabat CDRs). In specific examples, the first antigen binding domain includes a heavy chain variable region including one of SEQ ID NOs: 1, 3, 27, or 760-1459.

In some embodiments, the first antigen binding domain includes a heavy chain variable region including the CDR1 , CDR2, and/or CDR3 according to IMGT or Kabat of one of SEQ ID NOs: 1460- 1463, 1478-1483, or 1724-1727 (VRC07 HC and variants thereof). In some embodiments, the first antigen binding domain includes a heavy chain variable region including amino acids 26-33 (CDR1), 51- 58 (CDR2), and/or 97-114 (CDR3) of one of SEQ ID NOs: 1460-1463, 1478-1483, or 1724-1727 (IMGT CDRs). In some embodiments, the first antigen binding domain includes a heavy chain variable region including amino acids 26-33 (CDRl), 51-58 (CDR2), and 97-114 (CDR3) of one of SEQ ID NOs: 1460-1463, 1478-1483, or 1724-1727 (IMGT CDRs). In some embodiments, the first antigen binding domain includes a heavy chain variable region including amino acids 31-35 (CDRl), 50-66 (CDR2), and/or 99-114 (CDR3) of one of SEQ ID NOs: 1460-1463, 1478-1483, or 1724-1727 (Kabat CDRs). In some embodiments, the first antigen binding domain includes a heavy chain variable region including amino acids 31-35 (CDRl), 50-66 (CDR2), and 99-114 (CDR3) of one of SEQ ID NOs: 1460- 1463, 1478-1483, or 1724-1727 (Kabat CDRs). In specific examples, the first antigen binding domain includes a heavy chain variable region including one of SEQ ID NOs: 1460-1463, 1478-1483, or 1724- 1727.

(ii) VRCOl Light Chain (LC) and VRC07 LC and variants thereof

As discussed above, VRCOl-like heavy chains can be complemented with VRCOl-like light chains to form a functional antigen binding domain that specifically binds to gpl20 and is neutralizing. Thus, any one of the heavy chains of the VRCOl, VRC07, or variants thereof discussed in the previous section can be complemented with any one of the light chains of the VRCOl, VRC07 or variants thereof, discussed below to form a functional antigen binding domain that specifically binds to gpl20 and is neutralizing.

In some embodiments, the first antigen binding domain includes a light chain variable region including the CDRl, CDR2, and/or CDR3 according to IMGT or Kabat of SEQ ID NOs: 2, 4, 28, 1464, 1645, 1466, 1484, or 1728-1748 (VRCOl LC and VRC07 LC and variants thereof), wherein the antibody including the antigen binding domain specifically binds to gpl20 and is neutralizing. In some embodiments, the first antigen binding domain includes a light chain variable region including amino acids 27-30 (CDRl), 48-50 (CDR2), and/or 87-91 (CDR3) of one of SEQ ID NOs: 2, 4, 28, 1464, 1645, 1466, 1484, 1737-1740, or 1748 (IMGT CDRs). In some embodiments, the first antigen binding domain includes a light chain variable region including amino acids 27-30 (CDRl), 48-50 (CDR2), and 87-91 (CDR3) of one of SEQ ID NOs: 2, 4, 28, 1464, 1645, 1466, 1484, 1737-1740, or 1748 (IMGT CDRs). In some embodiments, the first antigen binding domain includes a light chain variable region including amino acids 24-32 (CDRl), 48-54 (CDR2), and/or 87-91 (CDR3) of one of SEQ ID NOs: 2, 4, 28, 1464, 1645, 1466, 1484, 1737-1740, or 1748 (Kabat CDRs). In some embodiments, the first antigen binding domain includes a light chain variable region including amino acids 24-32 (CDRl), 48-54 (CDR2), and 87-91 (CDR3) of one of SEQ ID NOs: 2, 4, 28, 1464, 1645, 1466, 1737-1740, or 1748 (Kabat CDRs). In some embodiments, the first antigen binding domain includes a light chain variable region including amino acids 25-28 (CDRl), 46-48 (CDR2), and/or 85-89 (CDR3) of one of SEQ ID NOs 1728-1736, or 1741-1747 (IMGT CDRs). In some embodiments, the first antigen binding domain includes a light chain variable region including amino acids 25-28 (CDRl), 46-48 (CDR2), and 85-89 (CDR3) of one of SEQ ID NOs 1728-1736, or 1741-1747 (IMGT CDRs). In some embodiments, the first antigen binding domain includes a light chain variable region including amino acids 22-30 (CDRl), 46-52 (CDR2), and 85-89 (CDR3) of one of SEQ ID NOs 1728-1736, or 1741-1747 (Kabat CDRs). In some embodiments, the first antigen binding domain includes a light chain variable region including amino acids 22-30 (CDRl), 46-52 (CDR2), and/or 85-89 (CDR3) of one of SEQ ID NOs 1728-1736, or 1741-1747 (Kabat CDRs). In specific examples, the first antigen binding domain includes a light chain variable region including one of SEQ ID NOs: 2, 4, and 28, 1464, 1645, 1466, 1484, or 1728-1748.

2. gp41 specific antigen binding domains

In several embodiments, the antibody includes an antigen binding domain that specifically binds to gp41. For example the first antigen binding domain can include the heavy and light chain CDRs from the 10E8 antibody or a variant thereof, such as antibodies described in U.S. Prov. App. No. 61/556,660, filed November 7, 2011 (the disclosure of which is incorporated by reference herein), or a functional fragment thereof {e.g. , an scFv or Fab), for example, as described herein. In several such embodiments, the gp41 antigen binding domain is neutralizing.

Any one of the 10E8 or variant 10E8 heavy chains provided herein can be complemented with any one of the 10E8 or variant 10E8 light chains to form a functional antigen binding domain that specifically binds to gp41 and is neutralizing. In some embodiments, the bispecific antibody includes an antigen binding domain including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes a H-CDR1, an H-CDR2 and an H-CDR3 from a 10E8 or 10E8 variant antibody, and wherein the light chain variable domain includes a L-CDR1, an L-CDR2 and an L-CDR3 from a 10E8 or 10E8 variant antibody, and wherein the antigen binding domain specifically binds gp41 and is neutralizing.

( i) 10E8 HC and variants thereof

In some embodiments, the first antigen binding domain includes a heavy chain variable region including the CDRl , CDR2, and/or CDR3 according to IMGT or Kabat of one of SEQ ID NOs: 1485, 1517, 1519, 1525, 1660-1663, 1667-1677, 1701-1706, or 1714-1718 (10E8 HC and variants thereof), wherein the antibody including the antigen binding domain specifically binds to gp41 and is neutralizing. In some embodiments, the first antigen binding domain includes a heavy chain variable region including amino acids 26-33 (CDRl), 51-60 (CDR2), and/or 99-120 (CDR3) of one of SEQ ID NOs: 1485, 1517, 1519, 1525, 1660-1663, 1667-1677, 1701-1706, or 1714-1718 (IMGT CDRs). In some embodiments, the first antigen binding domain includes a heavy chain variable region including amino acids 26-33 (CDRl), 51-60 (CDR2), and 99-120 (CDR3) of one of SEQ ID NOs: 1485, 1517, 1519, 1525, 1660- 1663, 1667-1677, 1701-1706, or 1714-1718 (IMGT CDRs). In some embodiments, the first antigen binding domain includes a heavy chain variable region including amino acids 26-33 (CDRl), 51-60 (CDR2), and/or 101-120 of one of SEQ ID NOs: 1485, 1517, 1519, 1525, 1660-1663, 1667-1677, 1701- 1706, or 1714-1718 (Kabat CDRs). In some embodiments, the first antigen binding domain includes a heavy chain variable region including amino acids 26-33 (CDRl), 51-60 (CDR2), and 101-120 of one of SEQ ID NOs: 1485, 1517, 1519, 1525, 1660-1663, 1667-1677, 1701-1706, or 1714-1718 (Kabat CDRs). In specific examples, the first antigen binding domain includes a heavy chain variable region including any one of SEQ ID NOs: 1485, 1517, 1519, 1525, 1660-1663, 1667-1677, 1701-1706, or 1714-1718.

( ii) 10E8 LC and variants thereof

As discussed above, any one of the 10E8 or variant 10E8 heavy chains provided herein can be complemented with any one of the 10E8 or variant 10E8 light chains to form a functional antigen binding domain that specifically binds to gp41 and is neutralizing. Thus, any one of the heavy chains of the 10E8 or variant 10E8 heavy chains discussed in the previous section can be complemented with any one of the 10E8 or variant 10E8 light chains discussed below to form a functional antigen binding domain that specifically binds to gp41 and is neutralizing.

Thus, in some embodiments, the antigen binding domain includes a light chain variable region including the CDR1, CDR2, and/or CDR3 according to IMGT or Kabat of one of SEQ ID NOs: 1486, 1518, 1520, 1526, 1664-1666, 1678-1700, 1711-1713, or 1719-1723 (10E8 LC and variants thereof), wherein the antibody including the antigen binding domain specifically binds to gp41 and is neutralizing. In some embodiments, the first antigen binding domain includes a light chain variable region including amino acids 26-33 (CDR1), 51-60 (CDR2), and/or 99-120 (CDR3) of one of SEQ ID NOs: 1486, 1518, 1520, 1526, 1664-1666, 1678-1700, 1711-1713, or 1719-1723 (IMGT CDRs). In some embodiments, the first antigen binding domain includes a light chain variable region including amino acids 26-33 (CDR1), 51-60 (CDR2), and 99-120 (CDR3) of one of SEQ ID NOs: 1486, 1518, 1520, 1526, 1664- 1666, 1678-1700, 1711-1713, or 1719-1723 (IMGT CDRs). In some embodiments, the first antigen binding domain includes a light chain variable region including amino acids 26-33 (CDR1), 51-60 (CDR2), and/or 101-120 of one of SEQ ID NOs: 1486, 1518, 1520, 1526, 1664-1666, 1678-1700, 1711- 1713, or 1719-1723 (Kabat CDRs). In some embodiments, the first antigen binding domain includes a light chain variable region including amino acids 26-33 (CDR1), 51-60 (CDR2), and 101-120 of one of SEQ ID NOs: 1486, 1518, 1520, 1526, 1664-1666, 1678-1700, 1711-1713, or 1719-1723 (Kabat CDRs). In specific examples, the first antigen binding domain includes a light chain variable region including one of SEQ ID NOs: 1486, 1518, 1520, 1526, 1664-1666, 1678-1700, 1711-1713, or 1719-1723.

B. Second antigen binding domain - CD3 specific

In several embodiments, the second antigen binding domain of the isolated multispecific antibody specifically binds to CD3 and is T cell activating and HIV activating. The person of ordinary skill in the art will appreciate that any of the above HIV-1 specific antigen binding domains can be combined with any of the CD3-specfic and T cell activating binding domains disclosed below in the disclosed multispecific antibody.

( i) CD3 specific antigen binding domains

In one non-limiting example, the second antigen binding domain specifically binds to CD3, for example, the multispecific antibody can include a CD3-specific monoclonal antibody, or an antigen binding fragment thereof (e.g. , an scFv or a Fab). In several embodiments, binding of the antibody to CD3 on the surface of a T cell activates the T cell. In additional embodiments, binding of the antibody to CD3 on the surface of a T cell activates HIV-1 replication in the T cell (i.e., is HIV-1 activating).

Examples of antigen binding domains that specifically bind CD3, including monoclonal antibodies and functional fragments thereof (such as an scFv) that specifically bind to CD3 are known in the art (see, e.g. , U.S. Pat. Nos. 8,076,459, 8,017,748, 8,007,796, 7,919,089, 7,820,166, 7,635,472, 7,575,923, 7,435,549, 7,332,168, 7,323,440, 7,235,641, 7,229,760, 7,112,324, 6,723,538, incorporated by reference herein.

Numerous examples of monoclonal antibodies that specifically bind CD3 are known in the art and commercially available. See, e.g. , CD3 antibody clones available from Abeam, such as SP7, OKT3, PS1, B355.1, MEM-57, EP4426, UCHT1, UCH-T1, MEM-92, SPV-T3b, B-B l l, F7.2.38, CA-3, LT3, BB12, CLB-T3/4.E). Other examples of CD3 specific monoclonal antibodies include the X35-3, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, Fl 11-409, CLB-T3.4.2, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3, OKT3D, M-T301, SMC2, TR66, WT31 or F101.01 monoclonal antibodies (for example, as described in Tunnacliffe, Int. Immunol. , 1:546- 550, 1989, incorporated by reference herein). In some embodiments, the second antigen binding domain includes one or more of the heavy and/or light chain CDRs of one of the above described monoclonal antibodies that specifically bind CD3. For example, in some embodiments, the second antigen binding domain includes a heavy chain variable domain including a CDRl, CDR2 and/or CDR3 of the heavy chain variable domain of one of the above described monoclonal antibodies that specifically bind CD3. In some embodiments, the second antigen binding domain includes a heavy chain variable domain including the CDRl, CDR2 and CDR3 of the heavy chain variable domain of one of the above described monoclonal antibodies that specifically bind CD3. In some embodiments, the second antigen binding domain includes a light chain variable domain including the CDRl, CDR2 and/or CDR3 of the light chain variable domain of one of the above described monoclonal antibodies that specifically bind CD3. In some embodiments, the second antigen binding domain includes a light chain variable domain including the CDRl, CDR2 and CDR3 of the light chain variable domain of one of the above described monoclonal antibodies that specifically bind CD3. In some embodiments, the second antigen binding domain includes a heavy chain variable domain and a light chain variable domain, including the heavy chain CDRl , CDR2 and CDR3, and light chain CDRl , CDR2 and CDR3, respectively, of one of the above described monoclonal antibodies that specifically bind CD3.

Thus, in some embodiments, the second antigen binding domain includes one or more of the heavy and/or light chain CDRs of the TR66 monoclonal antibody, which specifically binds to CD3 (the TR66 antibody is described in, e.g., Traunecker, EMBO J., 10:3655-3659, 1991, and is commercially available, e.g., from Enzo Life Sciences, Corp). The sequence of the heavy chain variable domain of the TR66 antibody is provided as residues 240-360 of SEQ ID NO: 1510. The sequence of the light chain variable domain of the TR66 antibody is provides as residues 375-482 of SEQ ID NO: 1510. The heavy chain Kabat CDRs of the TR66 antibody are provided as amino acids 265- 274 (CDRl), 289 - 305 (CDR2), and 338-347 (CDR3) of SEQ ID NO: 1510. Further, the light chain Kabat CDRs of the TR66 antibody are provided as amino acids 400 - 409 (CDRl), 425-431 (CDR2), and 464-472 (CDR3) of one of SEQ ID NO: 1510. SEQ ID NO: 1510 is provided below:

MGWS C I I LFLVATATGVHSE IVLTQSPGTL SL SPGETAI I SCRT SQYGSLAWYQQRPGQAPRLVIYSGST RAAGIPDRFS GSRWGPDYNLT I SNLE SGDFGVYYCQQYEFFGQGTKVQVD IKRTVAAPSVF I FPPS DEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDS TYSL S S TLTL SKADYEKHKVYACE VTHQGL S SPVTKSFNRGECGGGGS GGGGSQ IQLVQS GAEVAKPGASVKVS CKAS GYTFTRYTMHWVRQRP GQGLEWI GYINPSRGYTNYNQKFKDRATLTTDKS TS TAYMEL S S LT SEDTAVYYCARYYDDHYCLDYWGQ GTTVTVS SVEGGSGGS GGSGGS GGVDDI QLTQSPS S LSASVGDRVT I TCRAS S SVSYMNWYQQKSGTAPK RWIYDT SKVASGVPYRFS GS GS GT SYTLT I S S LQPEDAATYYCQQWS SNPLTFGGGTKVE IK (SEQ ID NO: 1510)

In some embodiments, the second antigen binding domain includes a heavy chain variable region including amino acids 265- 274 (CDRl), 289 - 305 (CDR2), and/or 338-347 (CDR3) of SEQ ID NO: 1510. In some embodiments, the second antigen binding domain includes a heavy chain variable region including amino acids 265- 274 (CDRl), 289 - 305 (CDR2), and 338-347 (CDR3) of SEQ ID NO: 1510. In some embodiments, the second antigen binding domain includes a light chain variable region including amino acids 400 - 409 (CDRl), 425-431 (CDR2), and/or 464-472 (CDR3) of SEQ ID NO: 1510. In some embodiments, the second antigen binding domain includes a light chain variable region including amino 400 - 409 (CDRl), 425-431 (CDR2), and 464-472 (CDR3) of SEQ ID NO: 1510. In some embodiments, the second antigen binding domain includes a heavy chain variable region including amino acids 265- 274 (CDRl), 289 - 305 (CDR2), and/or 338-347 (CDR3) of SEQ ID NO: 1510 and a light chain variable region including amino acids 400 - 409 (CDRl), 425-431 (CDR2), and/or 464-472 (CDR3) of SEQ ID NO: 1510. In some embodiments, the second antigen binding domain includes a heavy chain variable region including amino acids 265- 274 (CDRl), 289 - 305 (CDR2), and 338-347 (CDR3) of SEQ ID NO: 1510 and a light chain variable region including amino acids 400 - 409 (CDRl), 425-431 (CDR2), and 464-472 (CDR3) of SEQ ID NO: 1510.

In some embodiments, the second antigen binding domain includes a heavy chain variable region including the amino acid sequence set forth as amino acids 240-360 of SEQ ID NO: 1510. In additional embodiments, the second antigen binding domain includes a light chain variable region including the amino acid sequence set forth as amino acids 375-482 of SEQ ID NO: 1510. In further embodiments, the second antigen binding domain includes a heavy chain variable region including the amino acid sequence set forth as amino acids 240-360 of SEQ ID NO: 1510, and a light chain variable region including the amino acid sequence set forth as amino acids 375-482 of SEQ ID NO: 1510.

In several embodiments, the second antigen binding domain includes a heavy chain variable domain and a light chain variable domain having the heavy chain CDRs and light chain CDRs, respectively, of one of the above-described monoclonal antibodies that specifically bind CD3, wherein the heavy chain variable domain and the light chain variable domain include human framework regions C. Third antigen binding domain - CD28, CD40L, CD137 or CD137L specific

In some examples the isolated multispecific antibody optionally includes a third antigen binding domain that specifically binds to a costimulatory molecule on the surface of a T cell, and is T cell activating. For example, the third antigen binding domain can specifically bind to one of CD28, CD40L, CD 137 or CD137L. The person of ordinary skill in the art will appreciate that any combination of the above first and second antigen binding domains can be combined with any of the CD28, CD40L, CD 137 or CD137L specific and T cell activating antigen binding domains disclosed below in the disclosed multispecific antibody.

Examples of antigen binding domains that specifically bind to CD28, CD40L, CD 137 or CD137L, including monoclonal antibodies and functional fragments thereof (such as an scFv) that specifically bind to CD28, CD40L, CD 137 or CD137L are known in the art and are commercially available.

For instance, examples of monoclonal antibodies that specifically bind to CD28 are known in the art and are commercially available (see, e.g. , U.S. Pat. App. Pub. No. 2010/0168400 and Bjorndahl et al., Eur. J. Immunol., 19:881-887, 1989; the description of each of which concerning CD28 antibodies is incorporated herein by reference). Examples of monoclonal antibodies that specifically bind to CD40L are also known in the art and are commercially available (see, e.g., U.S. Pat. App. Pub. Nos.

2011/0027276, 20100092483; PCT App. No. PCT/US2008/003735; Elgueta et al , Immunobiol Rev. , 229: 152-172, 2009; the description of each of which concerning CD40L antibodies is incorporated herein by reference). Further, examples of monoclonal antibodies that specifically bind to CD 137 or CD137L are known in the art and are commercially available (see, e.g. , International (PCT) Nos.

PCT/US2005/005405, PCT/US2009/059518, PCT/US2010/034295; Mittler et al, Immunol. Res., 29: 197-208, 2004; the description of each of which concerning CD137 antibodies is incorporated herein by reference).

In some embodiments, the antibody includes a third antigen binding domain including one or more of the heavy and/or light chain CDRs (e.g., Kabat or IMGT CDRs) of one of the above described monoclonal antibodies that specifically bind CD3.

For example, in some embodiments, the third antigen binding domain includes a heavy chain variable domain including a CDRl, CDR2 and/or CDR3 of the heavy chain variable domain of the above disclosed monoclonal antibody that specifically binds CD28. In some embodiments, the third antigen binding domain includes a heavy chain variable domain including the CDRl, CDR2 and CDR3 of the heavy chain variable domain of the above disclosed monoclonal antibodies that specifically bind CD28. In some embodiments, the third antigen binding domain includes a light chain variable domain including the CDRl, CDR2 and/or CDR3 of the light chain variable domain of the above disclosed monoclonal antibodies that specifically bind CD28. In some embodiments, the third antigen binding domain includes a light chain variable domain including the CDRl, CDR2 and CDR3 of the light chain variable domain of the above disclosed monoclonal antibodies that specifically bind CD28. In some embodiments, the third antigen binding domain includes a heavy chain variable domain and a light chain variable domain, including the heavy chain CDRl, CDR2 and CDR3, and light chain CDRl, CDR2 and CDR3, respectively, of one of the above disclosed monoclonal antibodies that specifically bind CD28.

For example, in some embodiments, the third antigen binding domain includes a heavy chain variable domain including a CDRl, CDR2 and/or CDR3 of the heavy chain variable domain of the above disclosed monoclonal antibodies that specifically bind CD 137. In some embodiments, the third antigen binding domain includes a heavy chain variable domain including the CDRl, CDR2 and CDR3 of the heavy chain variable domain of the above disclosed monoclonal antibodies that specifically bind CD 137. In some embodiments, the third antigen binding domain includes a light chain variable domain including the CDRl, CDR2 and/or CDR3 of the light chain variable domain of the above disclosed monoclonal antibodies that specifically bind CD137. In some embodiments, the third antigen binding domain includes a light chain variable domain including the CDRl, CDR2 and CDR3 of the light chain variable domain of the above disclosed monoclonal antibodies that specifically bind CD 137. In some embodiments, the third antigen binding domain includes a heavy chain variable domain and a light chain variable domain, including the heavy chain CDRl, CDR2 and CDR3, and light chain CDRl, CDR2 and CDR3, respectively, of the above disclosed monoclonal antibodies that specifically bind CD137.

For example, in some embodiments, the third antigen binding domain includes a heavy chain variable domain including a CDRl, CDR2 and/or CDR3 of the heavy chain variable domain of the above disclosed monoclonal antibodies that specifically bind CD40L. In some embodiments, the third antigen binding domain includes a heavy chain variable domain including the CDRl, CDR2 and CDR3 of the heavy chain variable domain of the above disclosed monoclonal antibodies that specifically bind CD40L. In some embodiments, the third antigen binding domain includes a light chain variable domain including the CDRl, CDR2 and/or CDR3 of the light chain variable domain of the above disclosed monoclonal antibodies that specifically bind CD40L. In some embodiments, the third antigen binding domain includes a light chain variable domain including the CDRl, CDR2 and CDR3 of the light chain variable domain of the above disclosed monoclonal antibodies that specifically bind CD40L. In some embodiments, the third antigen binding domain includes a heavy chain variable domain and a light chain variable domain, including the heavy chain CDRl, CDR2 and CDR3, and light chain CDRl, CDR2 and CDR3, respectively, of the above disclosed monoclonal antibodies that specifically bind CD40L.

For example, in some embodiments, the third antigen binding domain includes a heavy chain variable domain including a CDRl, CDR2 and/or CDR3 of the heavy chain variable domain of the above disclosed monoclonal antibodies that specifically bind CD137L. In some embodiments, the third antigen binding domain includes a heavy chain variable domain including the CDRl, CDR2 and CDR3 of the heavy chain variable domain of the above disclosed monoclonal antibodies that specifically bind CD137L. In some embodiments, the third antigen binding domain includes a light chain variable domain including the CDRl, CDR2 and/or CDR3 of the light chain variable domain of the above disclosed monoclonal antibodies that specifically bind CD137L. In some embodiments, the third antigen binding domain includes a light chain variable domain including the CDRl, CDR2 and CDR3 of the light chain variable domain of the above disclosed monoclonal antibodies that specifically bind CD137L. In some embodiments, the third antigen binding domain includes a heavy chain variable domain and a light chain variable domain, including the heavy chain CDR1, CDR2 and CDR3, and light chain CDR1, CDR2 and CDR3, respectively, of the above disclosed monoclonal antibodies that specifically bind CD137L.

In several embodiments, the third antigen binding domain includes a heavy chain variable domain and a light chain variable domain having the heavy chain CDRs and light chain CDRs, respectively, of one of the above-disclosed monoclonal antibodies that specifically binds to CD28, CD40L, CD 137 or CD137L, and wherein the heavy chain variable domain and the light chain variable domain include a human framework region. Thus, in some non-limiting examples, the third antigen binding domain is fully human.

In several embodiments, the third antigen binding domain includes a heavy chain variable domain and a light chain variable domain from one of the above -described monoclonal antibodies that specifically binds to CD28, CD40L, CD 137 or CD137L.

D. Exemplary Bispecific Antibodies

In several embodiments, the bispecific antibody is a bibody, including a Fab that specifically binds to an HIV-1 antigen, and a scFv that specifically binds to CD3. Construction and production of bispecific Fab-scFv antibodies is well known in the art, and is described, for example, in Schoonjans et al. (J Immunol. 165:7050-57, 2000) and Willems et al. (J Chromatogr B Analyt Technol Biomed Life Sci. 786: 161-76, 2003).

In some embodiments, the first antigen binding domain of the bispecific antibody, which binds to an HIV-1 epitope, is a Fab, and the second antigen binding domain, which binds to a T cell epitope, is a scFv. The heavy chain variable region of the first antigen binding domain is linked to a heavy chain constant 1 (CHI) region and the light chain variable region of the first antigen binding domain is linked to a light chain constant (CL) region. The N-terminus of the scFv of the second antigen binding domain is linked to the C-terminus of the CHI region of the first antigen binding domain, or the N-terminus of the scFv of the second antigen binding domain is linked to the C-terminus of the CL region of the first antigen binding domain. The linkage is typically by way of a peptide linker, for example, a (GGGGS)i (SEQ ID NO: 1763), (GGGGS)₂ (SEQ ID NO: 1764), or (GGGGS)₃ (SEQ ID NO: 1765) linker.

In some embodiments, the first antigen binding domain of the bispecific antibody specifically binds to gpl20 and is a Fab, and the second antigen binding domain specifically binds to CD3and is a scFv. The Fab of the first antigen binding domain includes a heavy chain variable region linked to a CHI region and a light chain variable region linked to a CL region. The N-terminus of the scFv of the second antigen binding domain is linked to the C-terminus of the CL region of the first antigen binding domain. In several embodiments, the bispecific antibody includes a first polypeptide including the heavy chain variable region linked to the CHI region of the first antigen binding domain, and a second polypeptide including the light chain variable region and CL region of the first antigen binding domain linked to the scFv of the second antigen binding domain. In specific examples, the amino acid sequences of the first and second polypeptides include one of: (a) SEQ ID NO: 1516 (VRC3125; VRC07H G54W Fab-HRV3c-His), and SEQ ID NO: 1508 (VRC3121 ; VRC01L-(GGGGS)₃-hCD3), respectively;

(b) SEQ ID NO: 1749 (VRC 3487; VRC07H G54H Fab-HRV3c-His), and SEQ ID NO: 1508 (VRC3121 ; VRC01L-(GGGGS)₃-hCD3), respectively;

(c) SEQ ID NO: 1516 (VRC3125; VRC07H G54W Fab-HRV3c-His), and SEQ ID NO: 1510

(VRC3122; VRC01L-(GGGGS)₂-hCD3), respectively;

(d) SEQ ID NO: 1749 (VRC 3487; VRC07H G54H Fab-HRV3c-His), and SEQ ID NO: 1510 (VRC3122; VRC01L-(GGGGS)₂-hCD3), respectively;

(e) SEQ ID NO: 1516 (VRC3125; VRC07H G54W Fab-HRV3c-His), and SEQ ID NO: 1751 (VRC 3485; VRC01L El/I2del V3S-(GGGGS)₂-hCD3), respectively;

(f) SEQ ID NO: 1749 (VRC 3487; VRC07H G54H Fab-HRV3c-His), and SEQ ID NO: 1751 (VRC 3485; VRC01L El/I2del V3S-(GGGGS)₂-hCD3), respectively.

In additional embodiments, the first antigen binding domain of the bispecific antibody specifically binds to gp41 and is a Fab, and the second antigen binding domain specifically binds to CD3and is a scFv. The Fab of the first antigen binding domain includes a heavy chain variable region linked to a CHI region and a light chain variable region linked to a CL region. The N-terminus of the scFv of the second antigen binding domain is linked to the C-terminus of the CL region of the first antigen binding domain. In several embodiments, the bispecific antibody includes a first polypeptide including the heavy chain variable region linked to the CHI region of the first antigen binding domain, and a second polypeptide including the light chain variable region and CL region of the first antigen binding domain linked to the scFv of the second antigen binding domain. In specific examples, the amino acid sequences of the first and second polypeptides include one of: (a) SEQ ID NO: 1757 (VRC 3490; 10E8HC6 Fab-HRV3c-His), and SEQ ID NO: 1488 (VRC3111 ; 10E8gL03-(GGGGS)₃-hCD3), respectively;

(b) SEQ ID NO: 1496 (VRC3115; 10E8gH03_4 Fab-HRV3c-His), and SEQ ID NO: 1488

(VRC3111 ; 10E8gL03-(GGGGS)₃-hCD3), respectively;

(c) SEQ ID NO: 1757 (VRC 3490; 10E8HC6 Fab-HRV3c-His), and SEQ ID NO: 1490

(VRC3112; 10E8gL03-(GGGGS)₂-hCD3), respectively; or

(d) SEQ ID NO: 1496 (VRC3115; 10E8gH03_4 Fab-HRV3c-His), and SEQ ID NO: 1490 (VRC3112; 10E8gL03-(GGGGS)₂-hCD3), respectively.

In additional embodiments, the bispecific antibody is a scFv-scFv, including a first scFv that specifically binds to an HIV-1 antigen, and a second scFv that specifically binds to CD3. Construction and production of bispecific scFv-scFv antibodies is well known in the art, and is described, for example, in Schoonjans et al. (J Immunol. 165:7050-57, 2000) and Willems et al. (J Chromatogr B Analyt Technol Biomed Life Sci. 786: 161-76, 2003). In one example, the scFv-scFv bispecific antibody includes the amino acid sequence set forth as SEQ ID NO: 1475 (VRC2455; VRCOl scFv + anti-hCD3 TR66 scFv). E. Additional description of the disclosed antibodies

The first antigen binding domain that specifically binds to an HIV-1 epitope can neutralize HIV- 1. In several embodiments, the first antigen binding domain is a broadly neutralizing antigen binding domain. In some embodiments, the first antigen binding domain can neutralize at least 80% (such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% or at least 99%) of circulating HIV-1 isolates with an IC50 of less than 50 μg/ml. In some embodiments, the first antigen binding domain can neutralize at least 80% (such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% or at least 99%) of the HIV-1 isolates listed in FIGs. 16-24 of

International (PCT) application No. PCT/US2010/050295 (incorporated by reference herein) with an IC50 of less than 50 μg/ml. The person of ordinary skill in the art if familiar with methods of measuring neutralization breadth and potency, for example such methods include the single-round HIV-1 Env- pseudoviruses infection of TZM-bl cells (see, e.g., Li et al, J Virol 79, 10108-10125, 2005, incorporated by reference herein; see also, PCT Pub. No. WO2011/038290, incorporated by reference herein).

Methods to assay for neutralization activity also include, but are not limited to, a single -cycle infection assay as described in Martin et al. (2003) Nature Biotechnology 21:71-76. In this assay, the level of viral activity is measured via a selectable marker whose activity is reflective of the amount of viable virus in the sample, and the IC50 is determined. In other assays, acute infection can be monitored in the PMl cell line or in primary cells (normal PBMC). In this assay, the level of viral activity can be monitored by determining the p24 concentrations using ELISA. See, for example, Martin et al. (2003) Nature

Biotechnology 21:71-76.

In some examples, the first antigen binding domain can specifically bind to the HIV-1 epitope (e.g., to gpl20 or gp41) with an affinity of at least about 1.0 x 10^~8 M, at least about 2.0 x 10^~8 M, at least about 3.0 x 10^"8 M, at least about 4.0 x 10^"8 M, at least about 5.0 x 10^"8 M or at least about 1.0 x 10^"9 M. In some examples, the second antigen binding domain can specifically bind to CD3 with an affinity of at least about 1.0 x 10^"8 M, at least about 2.0 x 10^"8 M, at least about 3.0 x 10^"8 M, at least about 4.0 x 10^"8 M, at least about 5.0 x 10^"8 M or at least about 1.0 x 10^"9 M. In some examples, the third antigen binding domain can specifically bind to CD28, CD40L, CD137 or CD137L with an affinity of at least about 1.0 x 10^"8 M, at least about 2.0 x 10^"8 M, at least about 3.0 x 10^"8 M, at least about 4.0 x 10^"8 M, at least about 5.0 x 10^"8 M or at least about 1.0 x 10^"9 M.

In embodiments where the antibody includes a full constant domain, the antibody can be, for example, an IgM or an IgG antibody, such as IgGior an IgG₂. The class of an antibody that specifically binds, e.g. , gpl20, gp41 or CD3, can be switched with another. In one aspect, a nucleic acid molecule encoding V_L or V_H is isolated using methods well-known in the art, such that it does not include any nucleic acid sequences encoding the constant region of the light or heavy chain, respectively. The nucleic acid molecule encoding V_L or V_H is then operatively linked to a nucleic acid sequence encoding a C_L or C_H from a different class of immunoglobulin molecule. This can be achieved using a vector or nucleic acid molecule that includes a C_L or C_H chain, as known in the art. For example, an antibody that specifically binds gpl20 that was originally IgM may be class switched to an IgG. Class switching can be used to convert one IgG subclass to another, such as from IgGi to IgG₂.

An antigen binding domain included in the disclosed antibodies can be a functional fragment

(antigen binding fragment), such as a scFv which includes a heavy chain and light chain variable region and is capable of binding the epitopic determinant on a HIV-1 envelope protein (such as gpl20 or gp41) or CD3. The antibody fragments retain the ability to selectively bind with the antigen and can be included in a multispecific antibody. These fragments include, but are not limited to:

(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;

(2) Fab', the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;

(3) (Fab')₂, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')₂ is a dimer of two Fab' fragments held together by two disulfide bonds;

(4) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and

(5) Single chain antibody (such as scFv), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. A scFv is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker (see, e.g., Ahmad et al., Clin. Dev. Immunol., 2012,

doi: 10.1155/2012/980250; Marbry, IDrugs, 13:543-549, 2010). The intramolecular orientation of the V_H-domain and the V_L-domain in a scFv, is not decisive for the provided antibodies (e.g. , for the provided multispecific antibodies). Thus, scFvs with both possible arrangements (V_H-domain-linker domain-V_L-domain; V_L-domain-linker domain- V_H-domain) may be used.

(6) A dimer of a single chain antibody (scFV₂), defined as a dimer of an scFV. This has also been termed a "miniantibody."

Methods of making these fragments and combining them into a multispecific format are known in the art (see for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988; Schoonjans et al., J Immunol. 165:7050-57, 2000; and Willems et al., J Chromatogr B Analyt Technol Biomed Life Sci. 786: 161-76, 2003).

In a further group of embodiments, the multispecific antibody includes an Fv antibody, which is typically about 25 kDa and contains a complete antigen-binding site with three CDRs per each heavy chain and each light chain. To produce these antibodies, the V_H and the V_L can be expressed from two individual nucleic acid constructs in a host cell. If the V_H and the V_L are expressed non-contiguously, the chains of the Fv antibody are typically held together by noncovalent interactions. However, these chains tend to dissociate upon dilution, so methods have been developed to crosslink the chains through glutaraldehyde, intermolecular disulfides, or a peptide linker. Thus, in one example, the Fv can be a disulfide stabilized Fv (dsFv), wherein the heavy chain variable region and the light chain variable region are chemically linked by disulfide bonds.

In an additional example, the multispecific antibody includes a scFv including V_H and V_L chains joined by a peptide linker. In several embodiments, the peptide linker does not include any

polymerization activity. In some embodiments, the peptide linker includes an amino acid sequence having four glycine residues followed by a serine, or polymers thereof. Also included are peptide linkers that include fewer amino acid residues, for example a peptide linker with fewer than five amino acids can include four, three, two or one amino acid. Thus, in some embodiments, a peptide linker having only one amino acid, e.g. , Glycine, is included. The characteristics of the peptide linker, which include the absence of the promotion of secondary structures are known in the art, e.g. , in Dall'Acqua et al., Biochem., 37:9266-9273, 1988, Cheadle et al., Mol Immunol., 29: 21-30, 1992 and Raag and Whitlow, FASEB, 9:73-80, 1995, and further described herein.

In several embodiments, the scFvs are prepared by constructing a structural gene including DNA sequences encoding the V_H and V_L domains joined by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFvs and multispecific antibodies including scFvs are known in the art (see Whitlow et al., Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991 ; Bird et al., Science 242:423, 1988; U.S. Patent No. 4,946,778; Pack et al., Bio/Technology 11 : 1271, 1993; and Sandhu, supra) and further described herein. Dimers of a single chain antibody (scFV₂), are also contemplated.

Camelid heavy chain antibodies exist as homodimers of a single heavy chain, dimerized via their constant regions (U.S. Patent Nos. 5,840,526 and 6,838,254; and U.S. Patent Application Publication No. 2003-0088074). The variable domains of these camelid heavy chain antibodies, referred to as V_HH domains, retain the ability, when isolated as fragments of the V_H chain, to bind antigen with high specificity (Hamers-Casterman et al. Nature 363:446-448, 1993; Gahroudi et al. FEBS Lett. 414:521- 526, 1997). The HCDRs disclosed herein can be included in a camelid antibody.

Antigen binding single V_H domains, called domain antibodies (dAb), have also been identified from a library of murine V_H genes amplified from genomic DNA of immunized mice (Ward et al. Nature 347:544-546, 1989). Human single immunoglobulin variable domain polypeptides capable of binding antigen with high affinity have also been described (see, for example, PCT Publication Nos. WO

2005/035572 and WO 2003/002609). The CDRs disclosed herein can also be included in a dAb.

Antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')₂. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly (see U.S. Patent No. 4,036,945 and U.S. Patent No. 4,331,647, and references contained therein; Nisonhoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73: 119, 1959; Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press, 1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

One of skill will realize that conservative variants of the antibodies can be produced. Such conservative variants employed in antibody fragments, such as dsFv fragments or in scFv fragments, will retain critical amino acid residues necessary for correct folding and stabilizing between the V_H and the V_L regions, and will retain the charge characteristics of the residues in order to preserve the low pi and low toxicity of the molecules. Amino acid substitutions (such as at most one, at most two, at most three, at most four, or at most five amino acid substitutions) can be made in the V_H and the V_L regions to increase yield. Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art (see above). Thus, one of skill in the art can readily review the sequences shown above, identify a conservative substitution, and produce the conservative variant using well-known molecular techniques.

In some embodiments, the multispecific antibody is produced by crosslinking two or more antibodies, antigen binding fragments (such as scFvs) of the same type or of different types. Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (such as disuccininiidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, 111. Exemplary bispecific antibodies are disclosed in the examples section.

Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH₂) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule. Alternatively, the multispecific antibody is derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford, IL. The linker can be any molecule used to join the antibody to the effector molecule. The linker is capable of forming covalent bonds to both the antibody and to the effector molecule. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the multispecific antibody and the effector molecule are polypeptides, the linkers may be joined to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids. In fact, similar methods can be used to form a multispecific antibody.

The multispecific antibodies disclosed herein that specifically bind an HIV-1 envelope protein

(such as gpl20 or gp41) and CD3 can be derivatized or linked to another molecule (such as another peptide or protein). In general, the antibodies or portion thereof is derivatized such that the binding to the HIV-1 envelope protein (such as gpl20 or gp41) and CD3 is not affected adversely by the derivatization or labeling. For example, the antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (for example, to form a multispecific antibody), a detection agent, a pharmaceutical agent, and/or a protein or peptide that can mediate associate of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).

Effector molecules, such as therapeutic, diagnostic, or detection moieties can be linked to the multispecific antibody that specifically binds an HIV-1 envelope protein (such as gpl20 or gp41) and CD3, using any number of means known to those of skill in the art. Both covalent and noncovalent attachment means may be used. The procedure for attaching an effector molecule to an antibody varies according to the chemical structure of the effector. In some circumstances, it is desirable to free the effector molecule from the multispecific antibody when the immunoconjugate has reached its target site. Therefore, in these circumstances, immunoconjugates will include linkages that are cleavable in the vicinity of the target site. Cleavage of the linker to release the effector molecule from the antibody may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site. In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules) drugs, toxins, and other agents, one skilled in the art will be able to determine a suitable method for attaching a given agent to an antibody or other polypeptide.

A multispecific antibody that specifically binds an HIV-1 envelope protein (such as gpl20 or gp41) and CD3 can be labeled with a detectable moiety. Useful detection agents include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l- napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like. Bioluminescent markers are also of use, such as luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP). A multispecific antibody can also be labeled with enzymes that are useful for detection, such as horseradish peroxidase, β- galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. A detectable enzyme can be detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is visually detectable. A multispecific antibody may also be labeled with biotin, and detected through indirect measurement of avidin or streptavidin binding. It should be noted that the avidin itself can be labeled with an enzyme or a fluorescent label.

A multispecific antibody may be labeled with a magnetic agent, such as gadolinium. A multispecific antibody can also be labeled with lanthanides (such as europium and dysprosium), and manganese. Paramagnetic particles such as superparamagnetic iron oxide are also of use as labels. A multispecific antibody may also be labeled with a predetermined polypeptide epitopes recognized by a secondary reporter (such as leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

A multispecific antibody can also be labeled with a radiolabeled amino acid. The radiolabel may be used for both diagnostic and therapeutic purposes. For instance, the radiolabel may be used to detect IGF-II by x-ray, emission spectra, or other diagnostic techniques. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionuclides: ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ^mIn, ¹²⁵I, ¹³¹I.

A multispecific antibody can also be derivatized with a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These groups may be useful to improve the biological characteristics of the antibody, such as to increase serum half-life or to increase tissue binding. IV. Polynucleotides and Expression

Nucleic acids encoding the amino acid sequences of the disclosed multispecific antibodies (for example, bispecific and trispecific antibodies), or a component thereof, can readily be produced by one of skill in the art. For example, these nucleic acids can be produced using the amino acid sequences provided herein (such as the CDR sequences, heavy chain and light chain sequences). Thus, nucleic acids encoding the disclosed antibodies and their components, conjugates and fusion proteins are provided herein. It will be appreciated that, in the case of a multispecific antibody including multiple polypeptide chains, one or more separate nucleic acid molecules can be used to encode the polypeptide chains of the multispecific antibody.

One of skill in the art can readily use the genetic code to construct a variety of functionally equivalent nucleic acids, such as nucleic acids which differ in sequence but which encode the same antibody sequence, or encode a conjugate or fusion protein including the V_L and/or V_H nucleic acid sequence of one or more of the antigen binding domains included in the disclosed antibodies.

The nucleic acid molecules can encode a heavy chain variable domain and/or a light chain variable domain of the first and second (and optionally the third) antigen binding domain of the disclosed antibodies. An exemplary nucleic acid sequence is set forth as SEQ ID NO: 1474. Recombinant nucleotide acid molecules encoding the disclosed antibodies, or a component thereof, can readily be produced by one of skill in the art, using the amino acid sequences provided herein, and the genetic code. In addition, one of skill can readily construct a variety of clones containing functionally equivalent nucleic acids, such as nucleic acids which differ in sequence but which encode the same protein sequence.

Nucleic acid sequences encoding the disclosed antibodies, or a component thereof, can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et al, Meth. Enzymol. 68: 109-151, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett. 22: 1859-1862, 1981 ; the solid phase phosphoramidite triester method described by Beaucage & Caruthers, Tetra. Letts. 22(20): 1859-1862, 1981, for example, using an automated synthesizer as described in, for example, Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168, 1984; and, the solid support method of U.S. Patent No. 4,458,066. Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill would recognize that while chemical synthesis of DNA is generally limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.

Exemplary nucleic acids encoding the disclosed multispecific antibodies, or a component thereof can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Sambrook et al., supra, Berger and Kimmel (eds.), supra, and Ausubel, supra. Product information from

manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA Chemical Company (Saint Louis, MO), R&D Systems (Minneapolis, MN), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (San Diego, CA), and Applied Biosystems (Foster City, CA), as well as many other commercial sources known to one of skill.

Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to persons of skill.

Any of the nucleic acids encoding any of the multispecific antibodies, V_H and/or V_L, disclosed herein (or fragment thereof) can be expressed in a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells. These antibodies can be expressed as individual V_H and/or V_L chain, or can be expressed as a fusion protein. An immunoadhesin can also be expressed. Thus, in some examples, nucleic acids encoding a V_H and V_L, and immunoadhesin are provided. The nucleic acid sequences can optionally encode a leader sequence.

In one example, a disclosed multispecific antibody, or a component thereof, is prepared by inserting the cDNA which encodes one or more variable regions from an antibody into a vector which includes the cDNA encoding an effector molecule (EM), such as an enzyme or label. The insertion is made so that the variable region and the EM are read in frame so that one continuous polypeptide is produced. Thus, the encoded polypeptide contains a functional Fv region and a functional EM region. In one embodiment, cDNA encoding an enzyme is ligated to a scFv so that the enzyme is located at the carboxyl terminus of the scFv. In several embodiments, cDNA encoding a horseradish peroxidase or alkaline phosphatase, or a polypeptide marker of interest is ligated to a scFv so that the enzyme (or polypeptide marker) is located at the amino terminus of the scFv. In another example, the label is located at the amino terminus of the scFv. In a further example, cDNA encoding the protein or polypeptide marker is ligated to a heavy chain variable region of an antibody, so that the enzyme or polypeptide marker is located at the carboxyl terminus of the heavy chain variable region. The heavy chain-variable region can subsequently be ligated to a light chain variable region of the antibody using disulfide bonds. In yet another example, cDNA encoding an enzyme or a polypeptide marker is ligated to a light chain variable region of an antibody, so that the enzyme or polypeptide marker is located at the carboxyl terminus of the light chain variable region. The light chain-variable region can subsequently be ligated to a heavy chain variable region of the antibody using disulfide bonds.

Once the nucleic acids encoding a multispecific antibody, or a component thereof, that specifically binds an HIV-1 envelope protein (such as gpl20 or gp41) and CD3 is isolated and cloned, the protein can be expressed in a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells using a suitable expression vector. One or more DNA sequences encoding the antibody, or a component thereof, can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art. Hybridomas expressing the antibodies of interest are also encompassed by this disclosure.

Polynucleotide sequences encoding the multispecific antibody, or a component thereof, that specifically bind an HIV-1 envelope protein (such as gpl20 or gp41) and CD3, can be operatively linked to expression control sequences. An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. The expression control sequences include, but are not limited to appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.

The polynucleotide sequences encoding the multispecific antibody, labeled multispecific antibody, or component thereof, can be inserted into an expression vector including, but not limited to a plasmid, virus or other vehicle that can be manipulated to allow insertion or incorporation of sequences and can be expressed in either prokaryotes or eukaryotes. Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art.

Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl₂ method using procedures well known in the art.

Alternatively, MgCl₂ or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can also be cotransformed with polynucleotide sequences encoding the antibody, labeled antibody, or functional fragment thereof, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).

One of skill in the art can readily use an expression systems such as plasmids and vectors of use in producing proteins in cells including higher eukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines.

Isolation and purification of recombinantly expressed polypeptide can be carried out by conventional means including preparative chromatography and immunological separations. Once expressed, the multispecific antibody, labeled multispecific antibody or component thereof can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, R. Scopes, Protein Purification, Springer- Verlag, N.Y., 1982). Substantially pure compositions of at least about 90 to 95% homogeneity are disclosed herein, and 98 to 99% or more homogeneity can be used for pharmaceutical purposes. Once purified, partially or to homogeneity as desired, if to be used therapeutically, the polypeptides should be substantially free of endotoxin.

Methods for expression of antibodies and/or refolding to an appropriate active form, including single chain antibodies, from bacteria such as E. coli have been described and are well-known and are applicable to the multispecific antibodies disclosed herein. See, Buchner et al., Anal. Biochem. 205:263- 270, 1992; Pluckthun, Biotechnology 9:545, 1991; Huse et al., Science 246: 1275, 1989 and Ward et al., Nature 341 :544, 1989, all incorporated by reference herein.

Often, functional heterologous proteins from E. coli or other bacteria are isolated from inclusion bodies and require solubilization using strong denaturants, and subsequent refolding. During the solubilization step, as is well known in the art, a reducing agent must be present to separate disulfide bonds. An exemplary buffer with a reducing agent is: 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol). Reoxidation of the disulfide bonds can occur in the presence of low molecular weight thiol reagents in reduced and oxidized form, as described in Saxena et al., Biochemistry 9: 5015- 5021, 1970, incorporated by reference herein, and especially as described by Buchner et al., supra.

Renaturation is typically accomplished by dilution (for example, 100-fold) of the denatured and reduced protein into refolding buffer. An exemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.

As a modification to the two chain antibody purification protocol, the heavy and light chain regions are separately solubilized and reduced and then combined in the refolding solution. An exemplary yield is obtained when these two proteins are mixed in a molar ratio such that a 5 fold molar excess of one protein over the other is not exceeded. Excess oxidized glutathione or other oxidizing low molecular weight compounds can be added to the refolding solution after the redox-shuf fling is completed.

In addition to recombinant methods, the multispecific antibodies, or components thereof, that are disclosed herein can also be constructed in whole or in part using standard peptide synthesis. Solid phase synthesis of the polypeptides of less than about 50 amino acids in length can be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by Barany & Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. pp. 3-284; Merrifield et al, J. Am. Chem. Soc. 85:2149-2156, 1963, and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. , Pierce Chem. Co., Rockford, 111., 1984. Proteins of greater length may be synthesized by condensation of the amino and carboxyl termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxyl terminal end (such as by the use of the coupling reagent N, N'-dicylohexylcarbodimide) are well known in the art.

V. Compositions and Therapeutic Methods

Methods are disclosed herein for the prevention or treatment of an HIV infection, such as an

HIV-1 infection. Prevention can include inhibition of infection with HIV-1. The methods include contacting a cell with an effective amount of the multispecific antibodies disclosed herein that specifically bind gpl20 or gp41, specifically bind CD3, and optionally bind CD28, CD40L, CD 137 or CD137L. Any of the multispecific antibodies disclosed herein can be used in these methods, including both bispecific and trispecific antibodies. The method can also include administering to a subject a therapeutically effective amount of the multispecific antibody to a subject.

In several embodiments, treating an HIV infection includes reducing and/or eliminating the latent reservoir of HIV-1 virus in a subject, for example, by administering a therapeutically effective amount of one or more of the disclosed multispecific antibodies to the subject. In some examples, treating an HIV infection includes reducing and/or eliminating the latent reservoir of HIV-1 virus in CD3 expressing T cells in the subject.

HIV infection does not need to be completely eliminated for the composition to be effective. For example, treatment with one or more of the provided multispecific antibodies can decrease HIV infection by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination of detectable HIV infected cells), as compared to HIV infection in the absence of the composition. In some embodiments, the cell is also contacted with an effective amount of an additional agent, such as anti-viral agent. The cell can be in vivo or in vitro. The methods can include administration of one on more additional agents known in the art. In additional examples, HIV replication can be reduced or inhibited by similar methods. HIV replication does not need to be completely eliminated for the composition to be effective. For example, a composition can decrease HIV replication by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination of detectable HIV), as compared to HIV replication in the absence of the composition. In one example, the cell is also contacted with an effective amount of an additional agent, such as anti-viral agent. The cell can be in vivo or in vitro.

To successfully reproduce itself, HIV must convert its RNA genome to DNA, which is then imported into the host cell's nucleus and inserted into the host genome through the action of HIV integrase. Because HIV's primary cellular target, CD4+ T cells, can function as the memory cells of the immune system, integrated HIV can remain dormant for the duration of these cells' lifetime. Memory T cells may survive for many years and possibly for decades. This latent HIV reservoir can be measured, for example, by co-culturing CD4+ T cells from infected patients with CD4+ T cells from uninfected donors and measuring HIV protein or RNA (See, e.g. , Archin et al., AIDS, 22: 1131-1135, 2008).

Additional methods for measuring the latent DNA reservoir are provided in Example 2, below. In some embodiments, the provided methods of treating HIV include reduction or elimination of the latent reservoir of HIV infected cells in a subject. For example, a reduction of at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination of detectable HIV) of the latent reservoir of HIV infected cells in a subject, as compared to the latent reservoir of HIV infected cells in a subject in the absence of the treatment with one or more of the provided multispecific antibodies.

Compositions are provided that include one or more of the multispecific antibodies, that are disclosed herein in a carrier. The compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration are at the discretion of the treating physician to achieve the desired purposes. The multispecific antibody can be formulated for systemic or local administration. In one example, the multispecific antibody that specifically binds and HIV-1 envelope protein (such as gpl20 or gp41) and CD3, and optionally binds CD28, CD40L, CD 137 or CD137L, is formulated for parenteral administration, such as intravenous administration. In other examples, the pharmaceutical composition is formulated for intramuscular administration.

The compositions for administration can include a solution of the multispecific antibody that specifically binds and HIV-1 envelope protein (such as gpl20 or gp41) and CD3 (and optionally binds CD28, CD40L, CD 137 or CD137L ) dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of multispecific antibody in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject's needs.

A typical pharmaceutical composition for intravenous administration includes about 0.1 to 10 mg of multispecific antibody per subject per day. Dosages from 0.1 up to about 100 mg per subject per day may be used, particularly if the agent is administered to a secluded site and not into the circulatory or lymph system, such as into a body cavity or into a lumen of an organ. Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA (1995).

The multispecific antibodies may be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration. The antibody solution is then added to an infusion bag containing 0.9% sodium chloride, USP, and typically administered at a dosage of from 0.5 to 15 mg/kg of body weight. Considerable experience is available in the art in the administration of antibody drugs, which have been marketed in the U.S. since the approval of RlTUXAN® in 1997. Antibodies can be administered by slow infusion, rather than in an intravenous push or bolus. In one example, a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level. For example, an initial loading dose of 4 mg/kg may be infused over a period of some 90 minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kg infused over a 30 minute period if the previous dose was well tolerated.

A therapeutically effective amount of a nucleic acid encoding a multispecific antibody can also be administered to a subject. One approach to administration of nucleic acids is direct immunization with plasmid DNA, such as with a mammalian expression plasmid. The nucleotide sequence encoding the disclosed multispecific antibodies can be placed under the control of a promoter to increase expression of the molecule. Immunization by nucleic acid constructs is well known in the art and taught, for example, in U.S. Patent No. 5,643,578, and U.S. Patent No. 5,593,972 and U.S. Patent No. 5,817,637. U.S. Patent No. 5,880,103 describes several methods of delivery of nucleic acids encoding immunogenic peptides or other antigens to an organism. The methods include liposomal delivery of the nucleic acids.

In another approach to using nucleic acids for immunization, a disclosed multispecific antibody can also be expressed by attenuated viral hosts or vectors or bacterial vectors. Recombinant vaccinia virus, adeno-associated virus (AAV), herpes virus, retrovirus, cytomegalovirus, poxvirus or other viral vectors can be used to express the antibody. For example, vaccinia vectors and methods useful in immunization protocols are described in U.S. Patent No. 4,722,848. BCG (Bacillus Calmette Guerin) provides another vector for expression of the disclosed antibodies (see Stover, Nature 351 :456-460, 1991).

In one embodiment, a nucleic acid encoding a disclosed multispecific antibody is introduced directly into cells. For example, the nucleic acid can be loaded onto gold microspheres by standard methods and introduced into the skin by a device such as Bio-Rad' s Heliosa Gene Gun. The nucleic acids can be "naked," consisting of plasmids under control of a strong promoter.

Typically, the DNA is injected into muscle, although it can also be injected directly into other sites. Dosages for injection are usually around 0.5 mg/kg to about 50 mg/kg, and typically are about 0.005 mg/kg to about 5 mg/kg (see, e.g. , U.S. Patent No. 5,589,466).

In another embodiment, the invention provides a method for inhibiting HIV infection by administering a therapeutically effective amount of a multispecific antibody that specifically bindsgpl20 and CD3 and optionally binds CD28, CD40L, CD137 or CD137L ), or a nucleic acid encoding the multispecific antibody, to a subject in need thereof. Thus, the multispecific antibodies disclosed herein can be used therapeutically. In one example, the subject is human. The antibody may be administered to a non-human mammal infected with HIV with which the antibody cross-reacts (such as a primate, or a cynomolgus or rhesus monkey). It should be noted that animal models, such as primate models, can be useful for evaluating the therapeutic efficacy of antibodies of this invention.

A therapeutically effective amount of a multispecific antibody that specifically binds and HIV-1 envelope protein (such as gpl20 or gp41) and CD3, and optionally binds CD28, CD40L, CD 137 or CD137L,will depend upon the severity of the disease and/or infection and the general state of the patient's health. A therapeutically effective amount of the multispecific antibody is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer. These compositions can be administered in conjunction with another therapeutic agent, either simultaneously or sequentially.

In one embodiment, administration of the multispecific antibody results in a reduction in the establishment of HIV infection, reducing subsequent HIV disease progression in a subject and/or reduction in the reservoir of latently HIV infected cells in a subject. A reduction in the establishment of HIV infection and/or a reduction in subsequent HIV disease progression encompass any statistically significant reduction in HIV activity. In some embodiments, methods are disclosed for treating a subject with an HIV-1 infection. These methods include administering to the subject a therapeutically effective amount of an antibody, or a nucleic acid encoding the antibody, thereby preventing or treating the HIV-1 infection.

Studies have shown that the rate of HIV transmission from mother to infant is reduced significantly when zidovudine is administered to HIV-infected women during pregnancy and delivery and to the offspring after birth (Connor et al., 1994 Pediatr Infect Dis J 14: 536-541). Several studies of mother-to-infant transmission of HIV have demonstrated a correlation between the maternal virus load at delivery and risk of HIV transmission to the child. The present disclosure provides isolated human monoclonal antibodies that are of use in decreasing HIV -transmission from mother to infant. Thus, In some embodiments a therapeutically effective amount of a multispecific antibody that specifically binds and HIV-1 envelope protein (such as gpl20 or gp41) and CD3, and optionally binds CD28, CD40L, CD 137 or CD137L, is administered in order to prevent transmission of HIV, or decrease the risk of transmission of HIV, from a mother to an infant. In some embodiments, a therapeutically effective amount of the multispecific antibody is administered to mother and/or to the child at childbirth. In other examples, a therapeutically effective amount of the multispecific antibody is administered to the mother and/or infant prior to breast feeding in order to prevent viral transmission to the infant or decrease the risk of viral transmission to the infant. In some embodiments, both a therapeutically effective amount of the multispecific antibody and a therapeutically effective amount of another agent, such as zidovudine, is administered to the mother and/or infant.

For any application, the multispecific antibody can be combined with anti-retroviral therapy (such as HAART). Antiretro viral drugs are broadly classified by the phase of the retrovirus life-cycle that the drug inhibits. The disclosed multispecific antibodies can be administered before, during, concurrent to and/or after retroviral therapy. In some embodiments, the multispecific antibodies are administered following a course of retroviral therapy. The multispecific disclosed antibodies can be administered in conjunction with Nucleoside and nucleotide reverse transcriptase inhibitors (nRTI), Non- nucleoside reverse transcriptase inhibitors (NNRTI), Protease inhibitors, Entry inhibitors (or fusion inhibitors), Maturation inhibitors, or a Broad spectrum inhibitors, such as natural antivirals. Exemplary agents include lopinavir, ritonavir, zidovudine, lamivudine, tenofovir, emtricitabine and efavirenz. The anti-retroviral therapy (e.g., HAART) can be initiated before, during or after treatment with the disclosed multispecific antibodies.

Single or multiple administrations of the compositions including the antibodies disclosed herein are administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of at least one of the antibodies disclosed herein to effectively treat the patient. The dosage can be administered once but may be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy. The dosage of the antibody can be infused for 15, 30, 45, 60, 90, or 120 minutes. In one example, the dosage is infused over one hour at four day intervals. The subject can be treated at regular intervals, such as daily, every two days, every three days, every four days, every five days, every six days, weekly, bi-weekly, monthly, bi-monthly, or quarterly, until a desired therapeutic result is achieved. In another embodiment, the dosage is administered by IV infusion over a one hour at four day intervals. Generally, the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the patient.

Controlled release parenteral formulations can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems see, Banga, A.J., Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing

Company, Inc., Lancaster, PA, (1995) incorporated herein by reference. Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the therapeutic protein, such as a cytotoxin or a drug, as a central core. In microspheres the therapeutic is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μιη are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 μιη so that only nanoparticles are administered intravenously. Microparticles are typically around 100 μιη in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315- 339, (1992) both of which are incorporated herein by reference.

Polymers can be used for ion-controlled release of the compositions disclosed herein. Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer, polaxamer 407, exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has been shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm.112:215-224, 1994). In yet another aspect, liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug (Betageri et al.,

Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous additional systems for controlled delivery of therapeutic proteins are known (see U.S. Patent No.

5,055,303; U.S. Patent No. 5,188,837; U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; U.S. Patent No. 4,957,735; U.S. Patent No. 5,019,369; U.S. Patent No. 5,055,303; U.S. Patent No. 5,514,670; U.S. Patent No. 5,413,797; U.S. Patent No. 5,268,164; U.S. Patent No. 5,004,697; U.S. Patent No. 4,902,505; U.S. Patent No. 5,506,206; U.S. Patent No. 5,271,961; U.S. Patent No.

5,254,342 and U.S. Patent No. 5,534,496).

VI. Kits

Kits are also provided. For example, kits for treating or preventing an HIV infection in a subject. The kits will typically include one or more of the disclosed multispecific antibodies, or a nucleic acid or a viral vector encoding, expressing or including a disclosed multispecific antibody.

The kit can include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container typically holds a composition including one or more of the disclosed bi- or tri-specific antibodies, or a nucleic acid or a viral vector encoding, expressing or including the antibody, which is effective for treating or preventing HIV infection. In several embodiments the container may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the composition is used for treating the particular condition.

The label or package insert typically will further include instructions for use of a disclosed multispecific antibody, or a nucleic acid or a viral vector encoding, expressing or including the antibody, for example, in a method of treating or preventing a HIV infection. The package insert typically includes instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. The instructional materials may be written, in an electronic form (such as a computer diskette or compact disk) or may be visual (such as video files). The kits may also include additional components to facilitate the particular application for which the kit is designed. The kits may additionally include buffers and other reagents routinely used for the practice of a particular method. Such kits and appropriate contents are well known to those of skill in the art.

VII. Summary of Sequences

SEQ ID NOs: 1, 3, and 27 are the amino acid sequences of the V_H regions of the VRCOl, VRC02, and VRC03 antibodies, respectively.

SEQ ID NOs: 2, 4, and 28 are the amino acid sequences of the V_L regions of the VRCOl, VRC02, and VRC03 antibodies, respectively.

SEQ ID NOs: 29, 31, and 33 are exemplary nucleic acid sequences encoding the V_H regions of the VRCOl, VRC02, and VRC03 antibodies, respectively.

SEQ ID NOs: 30, 32, and 34 are exemplary nucleic acid sequences encoding the V_L regions of the VRCOl, VRC02, and VRC03 antibodies, respectively.

SEQ ID NOs: 61-759 are nucleic acid sequences encoding V_Hregions of VRCOl-like antibodies.

SEQ ID NOs: 760-1459 are amino acid sequences including the heavy chains variable regions of VRCOl-like antibodies.

SEQ ID NO: 1460 is the amino acid sequence of the consensus V_H of the VRC07, VRC07b and

VRC07c gpl20 specific monoclonal antibodies with certain amino acid substitutions at positions 130, G54 and S58 (Kabat numbering).

SEQ ID NO: 1461 is the amino acid sequence of the VRC07 V_H region.

SEQ ID NO: 1462 is the amino acid sequence of the VRC07b V_H region.

SEQ ID NO: 1463 is the amino acid sequence of the VRC07c V_H region.

SEQ ID NO: 1464 is a consensus V_L region amino acid sequence for the VRC07, VRC07b and VRC07c antibodies.

SEQ ID NO: 1465 is the amino acid sequence of VRC07b V_L region.

SEQ ID NO: 1466 is the amino acid sequence of the VRC07c V_L region.

SEQ ID NO: 1467 is an exemplary nucleic acid sequence encoding the VRC07 V_H region.

SEQ ID NO: 1468 is an exemplary nucleic acid sequence encoding the VRC07b V_H region. SEQ ID NO: 1469 is an exemplary nucleic acid sequence encoding the VRC07c V_H region. SEQ ID NO: 1470 is an exemplary nucleic acid sequence encoding the VRC07b V_L region. SEQ ID NO: 1471 is an exemplary nucleic acid sequence encoding the VRC07c V_L region.

SEQ ID NO: 1472 is an exemplary nucleic acid sequence of an expression vector encoding the amino acid sequence of the VRC2678 bispecific antibody (VRCOl scFv + anti-rhCD3 scFv (C207)).

SEQ ID NO: 1473 is the amino acid sequence of the VRC2678 bispecific antibody (VRCOl scFv + anti-rhCD3 scFv(C207)).

SEQ ID NO: 1474 is an exemplary nucleic acid sequence of an expression vector encoding the VRC2455 bispecific antibody (VRCOl scFv + anti-hCD3 TR66 scFv).

SEQ ID NO: 1475 is the amino acid sequence of the VRC2455 bispecific antibody (VRCOl scFv + anti-hCD3 TR66 scFv).

SEQ ID NO: 1476 is an exemplary nucleic acid sequence of an expression vector encoding the

VRC2442 bispecific antibody (5B8 scFv + anti-hCD3 TR66 scFv).

SEQ ID NO: 1477 is the amino acid sequence of the VRC2442 bispecific antibody (5B8 scFv + anti-hCD3 TR66 scFv).

SEQ ID NO: 1478 is the amino acid sequence of a consensus V_H region of the VRC07, VRC07b and VRC07c gpl20 specific monoclonal antibodies with and without certain amino acid substitutions at positions 130, G54 and S58 (Kabat numbering).

SEQ ID NO: 1479 is the amino acid sequence of a consensus V_H region for VRC07 gpl20 specific monoclonal antibodies with certain amino acid substitutions at positions 130, G54 and S58 (Kabat numbering).

SEQ ID NO: 1480 is the amino acid sequence of the VRC07 (G54W, I30Q) V_H region

SEQ ID NO: 1481 is the amino acid sequence of the VRC07 (G54W, I30R) V_H region.

SEQ ID NO: 1482 is the amino acid sequence of the VRC07 (G54W, S58N) V_H region.

SEQ ID NO: 1483 is the amino acid sequence of the VRC07 (G54W) V_H region.

SEQ ID NO: 1484 is the amino acid sequence of the VRCOl (N72A) V_L region.

SEQ ID NO: 1485 is the amino acid sequence of 10E8 V_H region.

SEQ ID NO: 1486 is the amino acid sequence of the 10E8 V_L region.

SEQ ID NO: 1487 is an exemplary nucleic acid sequence of an expression vector encoding a variant 10E8 light chain joined to humanized anti-hCD3 (TR66) scFv with a (GGGGS)₃ linker

(VRC3111 ; 10E8gL03-hCD3 v2).

SEQ ID NO: 1488 is the amino acid sequence of a variant 10E8 light chain joined to humanized anti-hCD3 (TR66) scFv with a (GGGGS)₃ linker (VRC3111 ; 10E8gL03-hCD3 v2).

SEQ ID NO: 1489 an exemplary nucleic acid sequence of an expression vector encoding a variant 10E8 light chain joined to humanized anti-hCD3 (TR66) scFv vl with a (GGGGS)₂ linker

(VRC3112; 10E8gL03-hCD3 vl).

SEQ ID NO: 1490 is the amino acid sequence of a variant 10E8 light chain joined to humanized anti-hCD3 (TR66) scFv with a (GGGGS)₂ linker (VRC3112; 10E8gL03-hCD3 vl). SEQ ID NO: 1491 an exemplary nucleic acid sequence of an expression vector encoding a variant 10E8 light chain joined to anti-RhCD3(C207) scFv with a (GGGGS)₃ linker (VRC3113;

10E8gL03-RhCD3 v2).

SEQ ID NO: 1492 is the amino acid sequence of a variant 10E8 light chain joined to anti- RhCD3(C207) scFv with a (GGGGS)₃ linker (VRC3113; 10E8gL03-RhCD3 v2).

SEQ ID NO: 1493 an exemplary nucleic acid sequence of an expression vector encoding a variant 10E8 light chain joined to anti-RhCD3(C207) scFv with a (GGGGS)₂ linker (VRC3114;

10E8gL03-RhCD3 vl).

SEQ ID NO: 1494 is the amino acid sequence of a variant gp41-specific 10E8 light chain joined to anti-RhCD3(C207) scFv with a (GGGGS)₂ linker (VRC3114; 10E8gL03-RhCD3 vl).

SEQ ID NO: 1495 an exemplary nucleic acid sequence of an expression vector encoding a variant 10E8 heavy chain variable and CHI regions with C-terminal HRV3c proteinase cleavage site and HisTag (VRC3115; 10E8gH03_4 Fab).

SEQ ID NO: 1496 is the amino acid sequence of a variant gp41-specific 10E8 heavy chain variable and CHI regions with C-terminal HRV3c proteinase cleavage site and HisTag (VRC3115; 10E8gH03_4 Fab).

SEQ ID NO: 1497 an exemplary nucleic acid sequence of an expression vector encoding the humanized anti-VRCOl antibody 5B8 light chain linked to humanized anti-hCD3 (TR66) scFv with a (GGGGS)₃ linker (VRC3116; hu5B8L-anti-hCD3 v2).

SEQ ID NO: 1498 is the amino acid sequence of humanized anti-VRCOl antibody 5B8 light chain linked to humanized anti-hCD3 (TR66) scFv with a (GGGGS)₃ linker (VRC3116; hu5B8L-anti- hCD3 v2).

SEQ ID NO: 1499 an exemplary nucleic acid sequence of an expression vector encoding the amino acid sequence of humanized anti-VRCOl antibody 5B8 light chain linked to humanized anti-hCD3 (TR66) scFv with a (GGGGS)₂ linker (VRC3117; hu5B8L-anti-hCD3 vl).

SEQ ID NO: 1500 is the amino acid sequence of humanized anti-VRCOl antibody 5B8 light chain linked to humanized anti-hCD3 (TR66) scFv with a (GGGGS)₂ linker (VRC3117; hu5B8L-anti- hCD3 vl).

SEQ ID NO: 1501 an exemplary nucleic acid sequence of an expression vector encoding the humanized anti-VRCOl antibody 5B8 light chain linked to anti-RhCD3(C207) scFv with a (GGGGS)₃ linker (VRC3118; hu5B8L-anti-rhCD3 v2).

SEQ ID NO: 1502 is the amino acid sequence of humanized anti-VRCOl antibody 5B8 light chain joined to anti-RhCD3(C207) scFv with a (GGGGS)₃ linker (VRC3118; hu5B8L-anti-rhCD3 v2).

SEQ ID NO: 1503 an exemplary nucleic acid sequence of an expression vector encoding the humanized anti-VRCOl antibody 5B8 light chain joined to anti-RhCD3(C207) scFv with a (GGGGS)₂ linker (VRC3119; hu5B8L-anti-rhCD3 vl).

SEQ ID NO: 1504 is the amino acid sequence of humanized anti-VRCOl antibody 5B8 light chain joined to anti-RhCD3(C207) scFv with a (GGGGS)₂ linker (VRC3119; hu5B8L-anti-rhCD3 vl). SEQ ID NO: 1505 an exemplary nucleic acid sequence of an expression vector encoding the humanized anti-VRCOl antibody 5B8 heavy chain variable and CHI region with C-terminal HRV3c proteinase cleavage site and HisTag (VRC3120; hu5B8H Fab).

SEQ ID NO: 1506 is the amino acid sequence of humanized anti-VRCOl antibody 5B8 heavy chain variable and CHI region with C-terminal HRV3c proteinase cleavage site and HisTag (VRC3120; hu5B8H Fab).

SEQ ID NO: 1507 an exemplary nucleic acid sequence of an expression vector encoding the VRCOl light chain joined to humanized anti-hCD3 (TR66) scFv with a (GGGGS)₃ linker (VRC3121; VRCOl L-anti-hCD3 v2).

SEQ ID NO: 1508 is the amino acid sequence of VRCOl light chain joined to humanized anti- hCD3 (TR66) scFv with a (GGGGS)₃ linker (VRC3121 ; VRCOl L-anti-hCD3 v2).

SEQ ID NO: 1509 an exemplary nucleic acid sequence of an expression vector encoding the VRCOl light chain joined to humanized anti-hCD3 (TR66) scFv with a (GGGGS)₂ linker (VRC3122; VRCOl L-anti-hCD3 vl).

SEQ ID NO: 1510 is the amino acid sequence of VRCOl light chain joined to humanized anti- hCD3 (TR66) scFv with a (GGGGS)₂ linker (VRC3122; VRCOl L-anti-hCD3 vl).

SEQ ID NO: 1511 an exemplary nucleic acid sequence of an expression vector encoding the VRCOl light chain joined to humanized anti-RhCD3(C207) scFv with a (GGGGS)₃ linker (VRC3123; VRCOl L-anti-rhCD3 v2).

SEQ ID NO: 1512 is the amino acid sequence of VRCOl light chain joined to humanized anti-

RhCD3(C207) scFv with a (GGGGS)₃ linker (VRC3123; VRCOl L-anti-rhCD3 v2).

SEQ ID NO: 1513 an exemplary nucleic acid sequence of an expression vector encoding the VRCOl light chain joined to humanized anti-RhCD3(C207) scFv with a (GGGGS)₂ linker (VRC3124; VRCOl L-anti-rhCD3 vl).

SEQ ID NO: 1514 is the amino acid sequence of VRCOl light chain linked to humanized anti-

RhCD3(C207) scFv with a (GGGGS)₂ linker (VRC3124; VRCOl L-anti-rhCD3 vl).

SEQ ID NO: 1515 an exemplary nucleic acid sequence of an expression vector encoding the VRC07 G54W V_H and CHI regions with C-terminal HRV3c proteinase cleavage site and His-Tag (VRC3125; VRC07H G54W).

SEQ ID NO: 1516 is the amino acid sequence of the VRC07 G54W V_H and CHI regions with

C-terminal HRV3c proteinase cleavage site and His-Tag (VRC3125; VRC07H G54W).

SEQ ID NOs: 1517 and 1519 are the amino acid sequences of the heavy chains of the gp41- specific 7H6 and 7N16 antibodies, respectively.

SEQ ID NOs: 1518 and 1520 are the amino acid sequences of the light chains of the gp41- specific 7H6 and 7N16 antibodies, respectively.

SEQ ID NO: 1525 is the amino acid sequence of the heavy chain of a gp41-specific antibody. SEQ ID NO: 1526 is the amino acid sequence of the light chain of a gp41-specific antibody. SEQ ID NOs: 1549-1629 are the nucleic acid sequences of variant 10E8 heavy chains. SEQ ID NOs: 1630-1659 are the nucleic acid sequences of variant 10E8 light chains. SEQ ID NO: 1660 is the consensus amino acid sequence of variant 10E8 V_H regions. SEQ ID NOs: 1661-1663 are the amino acid sequences of the 10E8gH01, 10E8gH02, andE8gH03 variant 10E8 V_H regions, respectively.

SEQ ID NOs: 1664-1666 are the amino acid sequences of the 10E8gL01, 10E8gL02, andE8gL03 variant 10E8 V_L regions, respectively (also known as 10E8rLl, 10E8rL2, and 10E8rL3).

SEQ ID NOs: 1667-1677 are the amino acid sequences of variant 10E8 V_H regions.

SEQ ID NO: 1667 is the amino acid sequence of the HC10 variant 10E8 V_H region.

SEQ ID NO: 1668 is the amino acid sequence of the HC6 variant 10E8 V_H region.

SEQ ID NO: 1669 is the amino acid sequence of the HC9 variant 10E8 V_H region.

SEQ ID NO: 1670 is the amino acid sequence of the HC5 variant 10E8 V_H region.

SEQ ID NO: 1671 is the amino acid sequence of the HC7 variant 10E8 V_H region.

SEQ ID NO: 1672 is the amino acid sequence of the HC4 variant 10E8 V_H region.

SEQ ID NO: 1673 is the amino acid sequence of the HC3 variant 10E8 V_H region.

SEQ ID NO: 1674 is the amino acid sequence of the HC2 variant 10E8 V_H region.

SEQ ID NO: 1675 is the amino acid sequence of the HC11 variant 10E8 V_H region.

SEQ ID NO: 1676 is the amino acid sequence of the HC12 variant 10E8 V_H region.

SEQ ID NO: 1677 is the amino acid sequence of the HC1 variant 10E8 V_H region.

SEQ ID NO: 1678 is the amino acid sequences of the LC22 variant 10E8 V_L region.

SEQ ID NO: 1679 is the amino acid sequences of the LCI 6 variant 10E8 V_L region.

SEQ ID NO: 1680 is the amino acid sequences of the LC20 variant 10E8 V_L region.

SEQ ID NO: 1681 is the amino acid sequences of the LC23 variant 10E8 V_L region.

SEQ ID NO: 1682 is the amino acid sequences of the LC21 variant 10E8 V_L region.

SEQ ID NO: 1683 is the amino acid sequences of the LC4 variant 10E8 V_L region.

SEQ ID NO: 1684 is the amino acid sequences of the LC8 variant 10E8 V_L region.

SEQ ID NO: 1685 is the amino acid sequences of the LCI 5 variant 10E8 V_L region.

SEQ ID NO: 1686 is the amino acid sequences of the LCI 3 variant 10E8 V_L region.

SEQ ID NO: 1687 is the amino acid sequences of the LC6 variant 10E8 V_L region.

SEQ ID NO: 1688 is the amino acid sequences of the LCI 4 variant 10E8 V_L region.

SEQ ID NO: 1689 is the amino acid sequences of the LC3 variant 10E8 V_L region.

SEQ ID NO: 1690 is the amino acid sequences of the LCI 2 variant 10E8 V_L region.

SEQ ID NO: 1691 is the amino acid sequences of the LCI variant 10E8 V_L region.

SEQ ID NO: 1692 is the amino acid sequences of the LC27 variant 10E8 V_L region.

SEQ ID NO: 1693 is the amino acid sequences of the LC11 variant 10E8 V_L region.

SEQ ID NO: 1694 is the amino acid sequences of the LC26 variant 10E8 V_L region.

SEQ ID NO: 1695 is the amino acid sequences of the LC24 variant 10E8 V_L region.

SEQ ID NO: 1696 is the amino acid sequences of the LCI 9 variant 10E8 V_L region.

SEQ ID NO: 1697 is the amino acid sequences of the LC10 variant 10E8 V_L region. SEQ ID NO: 1698 is the amino acid sequences of the LC25 variant 10E8 V_L region.

SEQ ID NO: 1699 is the amino acid sequences of the LC18 variant 10E8 V_L region.

SEQ ID NO: 1700 is the amino acid sequences of the LC17 variant 10E8 V_L region.

SEQ ID NO: 1701 is a consensus amino acid sequence for variant 10E8 V_H regions.

SEQ ID NO: 1702 is a consensus amino acid sequence for variant 10E8 V_H regions.

SEQ ID NOs: 1703-1706 are the amino acid sequences of the HC6 S74A, HC6 S74R, HC6 S74V, and HC6 S74Y variant 10E8 V_H regions, respectively.

SEQ ID NOs: 1711-1713 are the amino acid sequences of the 10E8, 10E8gL03,

10E8gL03_hp_L01, 10E8gL03_hp_L02, and 10E8gL03_hp_L03 variant 10E8 V_L regions, respectively.

SEQ ID NOs: 1714-1718 are the amino acid sequences of the HC6_S74Y_hp_H01,

HC6_S74Y_hp_H02, HC6_S74Y_hp_H03, HC6_S74Y_hp_H04, HC6rH03S74Y variant 10E8 V_H regions, respectively.

SEQ ID NOs: 1719-1723 are the amino acid sequences of the 10E8, 10E8gL03,

10E8gL03_hp_L01 , 10E8gL03_hp_L02, and 10E8gL03_hp_L03 variant 10E8 V_L regions, respectively, further including a R23Q amino acid substitution.

SEQ ID NO: 1724 is the amino acid sequence of the VRC07 G54H V_H region.

SEQ ID NO: 1725 is the amino acid sequence of the VRC07 G54H, S58N V_H region.

SEQ ID NO: 1726 is the amino acid sequence of the VRC07 I37V, G54H, T93A V_H region.

SEQ ID NO: 1727 is the amino acid sequence of the VRC07 I37V, G54H, S58N, T93A V_H

SEQ ID NO: 1728 is the amino acid sequence of the VRCOl El/I2del V3E V_L region.

SEQ ID NO: 1729 is the amino acid sequence of the VRCOl El/I2del V3K V_L region.

SEQ ID NO: 1730 is the amino acid sequence of the VRCOl El/I2del V3S V_L region.

SEQ ID NO: 1731 is the amino acid sequence of the VRCOl El/I2del F97D V_L region.

SEQ ID NO: 1732 is the amino acid sequence of the VRCOl El/I2del F97K V_L region.

SEQ ID NO: 1733 is the amino acid sequence of the VRCOl El/I2del F97S V_L region.

SEQ ID NO: 1734 is the amino acid sequence of the VRCOl El/I2del F97H V_L region.

SEQ ID NO: 1735 is the amino acid sequence of the VRCOl El/I2del V3E, F97S V_L region.

SEQ ID NO: 1736 is the amino acid sequence of the VRCOl El/I2del V3E, F97H V_Lre£ ion.

SEQ ID NO: 1737 is the amino acid sequence of the VRC01hpL03 V_L region.

SEQ ID NO: 1738 is the amino acid sequence of the VRC01hpL04 V_L region.

SEQ ID NO: 1739 is the amino acid sequence of the VRCOl hpL05 V_L region.

SEQ ID NO: 1740 is the amino acid sequence of the VRC01hpL06 V_L region.

SEQ ID NO: 1741 is the amino acid sequence of the VRC01hpL02 El/I2del V3S V_L region.

SEQ ID NO: 1742 is the amino acid sequence of the VRC01LhpL03 El/I2del V3S V_Lrej jion.

SEQ ID NO: 1743 is the amino acid sequence of the VRC01LhpL04 El/I2del V3S V_Lrej jion.

SEQ ID NO: 1744 is the amino acid sequence of the VRC01LhpL05 El/I2del V3S V_Lrej jion.

SEQ ID NO: 1745 is the amino acid sequence of the VRC01LhpL06 El/I2del V3S V_Lre| jion. SEQ ID NO: 1746 is the amino acid sequence of the VRC01LhpL04 El/I2del V3E V_L region. SEQ ID NO: 1747 is the amino acid sequence of the VRCOl El/I2del V_L region.

SEQ ID NO: 1748 is the amino acid sequence of the VRC01hpL02 V_L region.

SEQ ID NO: 1749 is an amino acid sequence including VRC07 G54H Fab (V_H and CHI regions) with C-terminal HRV3c proteinase cleavage site and His-Tag (VRC 3487; VRC07HG54H Fab- HRV3c-His).

SEQ ID NO: 1750 is an exemplary nucleic acid sequence encoding an amino acid sequence including VRC07 G54H Fab (V_H and CHI regions) with C-terminal HRV3c proteinase cleavage site and His-Tag (VRC 3487; VRC07HG54H Fab-HRV3c-His).

SEQ ID NO: 1751 is an amino acid sequence including the VRC01L El/I2del V3S light chain joined to humanized anti-hCD3 (TR66) scFv with a (GGGGS)₂ linker (VRC 3485 (CMVR

VRC01L_dEI_V3S-(anti-hCD3ScFv2-3).

SEQ ID NO: 1752 an exemplary nucleic acid sequence encoding an amino acid sequence including the VRC01L El/I2del V3S light chain joined to humanized anti-hCD3 (TR66) scFv with a (GGGGS)₂ linker (VRC 3485 (CMVR VRC01L_dEI_V3S-(anti-hCD3ScFv2-3).

SEQ ID NO: 1753 is an amino acid sequence including the simVRC07rH04 G54H Fab (V_H and CHI regions) with C-terminal HRV3c proteinase cleavage site and His-Tag (VRC 3489; simVRC07rH04 G54H Fab-HRV3c-His).

SEQ ID NO: 1754 is an exemplary nucleic acid sequence encoding an amino acid sequence including the VRC07rH04 G54H Fab (V_H and CHI regions) with C-terminal HRV3c proteinase cleavage site and His-Tag (VRC 3489; simVRC07rH04 G54H Fab-HRV3c-His).

SEQ ID NO: 1755 is an amino acid sequence including the simVRC01rL3(RSR)_

El/I2del V3S linked to (anti-RhCD3ScFvsimC207_d3.2 with a (GGGGS)₃ linker (VRC 3484;

simVRC01rL3(RSR)_ El/I2d_V3S-(anti-RhCD3ScFvsimC207_d3.2)).

SEQ ID NO: 1756 is a nucleic acid sequence encoding an amino acid sequence including the simVRC01rL3 (RSR)_ El/I2del V3S linked to (anti-RhCD3ScFvsimC207_d3.2 with a (GGGGS)₃ linker (VRC 3484; simVRC01rL3 (RSR)_dEI_V3S-(anti-RhCD3ScFvsimC207_d3.2)).

SEQ ID NO: 1757 is an amino acid sequence including the 10E8 HC6 Fab (V_H and CHI regions) with C-terminal HRV3c proteinase cleavage site and His-Tag (VRC 3490; 10E8HC6 Fab- HRV3c-His).

SEQ ID NO: 1758 is an exemplary nucleic acid sequence encoding an amino acid sequence including 10E8HC6 Fab (V_H and CHI regions) with C-terminal HRV3c proteinase cleavage site and His- Tag (VRC 3490; 10E8HC6 Fab-HRV3c-His).

SEQ ID NO: 1759 is an amino acid sequence including the 10E8gH3simCHl Fab (V_H and CHI regions) joined to humanized anti-hCD28 scFv with a linker and including a with C-terminal HRV3c proteinase cleavage site and His-Tag (VRC 3492; 10E8gH3simCHl Fab-(anti-hCD28scFvHL)-HRV3c- His). SEQ ID NO: 1760 is an exemplary nucleotide sequence encoding an amino acid sequence including the 10E8gH3simCHl Fab (V_H and CHI regions) joined to humanized anti-hCD28 scFv with a linker and including a C-terminal HRV3c proteinase cleavage site and His-Tag (VRC 3492;

10E8gH3simCHl Fab-(anti-hCD28scFvHL)-HRV3c-His).

SEQ ID NO: 1761 is an amino acid sequence including the 10E8gL3simCL light chain linked to an anti-RhCD3scFvsimC207_d3.2 (VRC 3226; 10E8gL3simCL-(anti-RhCD3scFvsimC207_d3.2)).

SEQ ID NO: 1762 is an exemplary nucleic acid sequence encoding an amino acid sequence including the 10E8gL3simCL light chain linked to an anti-RhCD3scFvsimC207_d3.2 (VRC 3226;

10E8gL3simCL-(anti-RhCD3scFvsimC207_d3.2)).

SEQ ID NO: 1763-1765 are the amino acid sequences peptide linkers.

SEQ ID NO: 1766 is the amino acid sequence of a scFv that specifically binds to CD28.

SEQ ID NO: 1767 is the amino acid sequence of a scFv that specifically binds to CD 137.

Reference to "one of SEQ ID NOs: 1, 3, 27, and 760-1459" (VRCOl HC and variants thereof), refers to one of SEQ ID NOs: 1, 3, 27, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375, 1376, 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384, 1385, 1386, 1387, 1388, 1389, 1390, 1391, 1392, 1393, 1394, 1395, 1396, 1397, 1398, 1399, 1400, 1401, 1402, 1403, 1404, 1405, 1406, 1407, 1408, 1409, 1410, 1411, 1412, 1413, 1414, 1415, 1416, 1417, 1418, 1419, 1420, 1421, 1422, 1423, 1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432, 1433, 1434, 1435, 1436, 1437, 1438, 1439, 1440, 1441, 1442, 1443, 1444, 1445, 1446, 1447, 1448, 1449, 1450, 1451, 1452, 1453, 1454, 1455, 1456, 1457, 1458, or 1459.

Reference to "one of SEQ ID NOs: 1460-1463, 1478-1483, and 1724-1727" (VRC07 HC and variants thereof), refers to one of SEQ ID NOs: 1460, 1461, 1462, 1463, 1478, 1479, 1480, 1481, 1482,

1483, 1724, 1725, 1726, or 1727.

Reference to "one of SEQ ID NOs: 2, 4, 28, 1464, 1645, 1466, 1484, and 1728-1748" (VRCOl LC and VRC07 LC and variants thereof), refers to one of SEQ ID NOs: 2, 4, 28, 1464, 1645, 1466,

1484, 1728, 1729, 1730, 1731, 1732, 1733, 1734, 1735, 1736, 1737, 1738, 1739, 1740, 1741, 1742, 1743, 1744, 1745, 1746, 1747, or 1748

Reference to "one of SEQ ID NOs: 2, 4, 28, 1464, 1645, 1466, 1484, 1737-1740, and 1748" (VRCOl LC and VRC07 LC and variants thereof), refers to one of SEQ ID NOs: 2, 4, 28, 1464, 1645, 1466, 1484, 1737, 1738, 1739, 1740, or 1748

Reference to "one of SEQ ID NOs 1728-1736, and 1741-1747" (VRCOl LC and VRC07 LC and variants thereof), refers to one of SEQ ID NOs 1728, 1729, 1730, 1731, 1732, 1733, 1734, 1735, 1736, 1741, 1742, 1743, 1744, 1745, 1746, or 1747.

Reference to "one of SEQ ID NOs: 1485, 1517, 1519, 1525, 1660-1663, 1667-1677, 1701-1706, and 1714-1718" (10E8 HC and variants thereof), refers to one of SEQ ID NOs: 1485, 1517, 1519, 1525, 1660, 1661, 1662, 1663, 1667, 1668, 1669, 1670, 1671, 1672, 1673, 1674, 1675, 1676, 1677, 1701, 1702, 1703, 1704, 1705, 1706, 1714, 1715, 1716, 1717, or 1718

Reference to "one of SEQ ID NOs: 1486, 1518, 1520, 1526, 1664-1666, 1678-1700, 1711-1713, and 1719-1723" (10E8 LC and variants thereof) refers to one of SEQ ID NOs: 1486, 1518, 1520, 1526, 1664, 1665, 1666, 1678, 1679, 1680, 1681, 1682, 1683, 1684, 1685, 1686, 1687, 1688, 1689, 1690, 1691, 1692, 1693, 1694, 1695, 1696, 1697, 1698, 1699, 1700, 1711, 1712, 1713, 1719, 1720, 1721, 1722, or 1723. VIII. Description of Additional Embodiments

Clause 1. An isolated bispecific monoclonal antibody, comprising:

a first antigen binding domain comprising a heavy chain variable region and a light chain variable region comprising a heavy chain complementarity determining region (H-CDR)l, an H-CDR2, an H-CDR3, a light chain complementarity determining region (L-CDR)l, an L-CDR2, and an L-CDR3, and wherein the first antigen binding domain specifically binds to gpl20 or gp41 and is neutralizing; a second antigen binding domain a heavy chain variable region and a light chain variable region comprising a heavy chain H-CDRl, an H-CDR2, an H-CDR3, a L-CDRl, an L-CDR2, and an L-CDR3, wherein the second antigen binding domain specifically binds CD3, activates T cells, and activates expression of proviral HIV-1 DNA in T cells.

Clause 2. The isolated bispecific monoclonal antibody of clause 1 , wherein the first antigen binding domain comprises a Fab or a scFv, and wherein the second antigen binding domain comprising a Fab or a scFv.

Clause 3. The isolated bispecific antibody of clause 1 or 2, wherein the first antigen binding domain specifically binds to gpl20.

Clause 4. The isolated bispecific antibody of any one of clauses 1-3, wherein the first antigen binding domain specifically binds to the CD4 binding site of gpl20.

Clause 5. The isolated bispecific antibody of any one of clauses 1-4, wherein the H-CDRl, the H-CDR2, and the H-CDR3 are from the heavy chain of a VRCOl-like monoclonal antibody and the L- CDR1, the L-CDR2, and the L-CDR3 are from the light chain of a VRCOl-like monoclonal antibody.

Clause 6. The isolated bispecific monoclonal antibody of any one of clauses 1-5, wherein the heavy chain variable region of the first antigen binding domain comprises one of:

(a) amino acids 26-33 (CDR1), 51-58 (CDR2), and 97-110 (CDR3) of any one of SEQ ID NOs: 1, 3, 27, or 760-1459 (VRCOl and VRCOl variant IMGT CDRs).

(b) amino acids 31-35 (CDR1), 50-66 (CDR2), and 99-110 (CDR3) of any one of SEQ ID NOs: 1, 3, 27, or 760-1459 (VRCOl and VRCOl variant IMGT CDRs Kabat CDRs).

(c) amino acids 26-33 (CDR1), 51-58 (CDR2), and 97-114 (CDR3) of one of SEQ ID NOs: 1460-1463, 1478-1483, or 1724-1727 (VRC07 and VRC07 variant IMGT CDRs).

(d) amino acids 31-35 (CDR1), 50-66 (CDR2), and 99-114 (CDR3) of one of SEQ ID NOs:

1460-1463, 1478-1483, or 1724-1727 (VRC07 and VRC07 variant Kabat CDRs).

Clause 7. The isolated bispecific monoclonal antibody of any one of clauses 1-5, wherein the heavy chain variable region of the first antigen binding domain comprises one of:

(a) one of SEQ ID NOs: 1, 3, 27, or 760-1459 (VRCOl and VRCOl variant V_H); or

(b) one of SEQ ID NOs: 1460-1463, 1478-1483, or 1724-1727 (VRC07 and VRC07 variant V_H)

Clause 8. The isolated bispecific monoclonal antibody of any one of clauses 1-7, wherein the light chain variable region of the first antigen binding domain comprises one of (a) amino acids 27-30 (CDRl), 48-50 (CDR2), and 87-91 (CDR3) of one of SEQ ID NOs: 2, 4, 28, 1464, 1645, 1466, 1484, 1737-1740, or 1748 (VRCOl LC and VRC07 LC and variants IMGT CDRs).

(b) amino acids 24-32 (CDRl), 48-54 (CDR2), and 87-91 (CDR3) of one of SEQ ID NOs: 2, 4, 28, 1464, 1645, 1466, 1737-1740, or 1748 (VRCOl LC and VRC07 LC and variants Kabat CDRs).

(c) amino acids 25-28 (CDRl), 46-48 (CDR2), and 85-89 (CDR3) of one of SEQ ID NOs 1728- 1736, or 1741-1747 (VRCOl LC and VRC07 LC and variants w/El/I2 del IMGT CDRs).

(d) amino acids 22-30 (CDRl), 46-52 (CDR2), and 85-89 (CDR3) of one of SEQ ID NOs 1728- 1736, or 1741-1747 (VRCOl LC and VRC07 LC and variants w/El/I2 del Kabat CDRs).

Clause 9. The isolated bispecific monoclonal antibody of any one of clauses 1-7, wherein the light chain variable region of the first antigen binding domain comprises the amino acid sequence set forth as one of SEQ ID NOs: 2, 4, and 28, 1464, 1645, 1466, 1484, or 1728-1748

Clause 10. The isolated bispecific monoclonal antibody of any one of clauses 1-5, wherein the heavy chain variable region and the light chain variable region of the first antigen binding domain comprises one of:

(a) amino acids 26-33 (CDRl), 51-58 (CDR2), and 97-110 (CDR3) of one of SEQ ID NOs: 1, 3, 27, or 760-1459 (VRCOl and VRCOl variant IMGT CDRs), and amino acids 27-30 (CDRl), 48-50 (CDR2), and 87-91 (CDR3) of one of SEQ ID NOs: 2, 4, 28, 1464, 1645, 1466, 1484, 1737-1740, or 17481748 (VRCOl LC and VRC07 LC and variants IMGT CDRs), respectively;

(b) amino acids 26-33 (CDRl), 51-58 (CDR2), and 97-114 (CDR3) of one of SEQ ID NOs:

1460-1463, 1478-1483, or 1724-1727 (VRC07 and VRC07 variant IMGT), and amino acids 27-30 (CDRl), 48-50 (CDR2), and 87-91 (CDR3) of one of SEQ ID NOs: 2, 4, 28, 1464, 1645, 1466, 1484, 1737-1740, or 17481748 (VRCOl LC and VRC07 LC and variants IMGT CDRs), respectively;

(c) amino acids 31-35 (CDRl), 50-66 (CDR2), and 99-110 (CDR3) of one of SEQ ID NOs: 1, 3, 27, or 760-1459 (VRCOl and VRCOl variant CDRs KABAT), and amino acids 24-32 (CDRl), 48-54

(CDR2), and 87-91 (CDR3) of one of SEQ ID NOs: 2, 4, 28, 1464, 1645, 1466, 1737-1740, or

1748(VRC01 LC and VRC07 LC and variants Kabat CDRs), respectively;

(d) amino acids 31-35 (CDRl), 50-66 (CDR2), and 99-114 (CDR3) of one of SEQ ID NOs: 1460-1463, 1478-1483, or 1724-1727 (VRC07 and VRC07 variant CDRs KABAT), and amino acids 24-32 (CDRl), 48-54 (CDR2), and 87-91 (CDR3) of one of SEQ ID NOs: 2, 4, 28, 1464, 1645, 1466, 1737-1740, or 1748(VRC01 LC and VRC07 LC and variants Kabat CDRs, respectively;

(e) amino acids 26-33 (CDRl), 51-58 (CDR2), and 97-110 (CDR3) of one of SEQ ID NOs: 1, 3, 27, or 760-1459 (VRCOl and VRCOl variant CDRs IMGT), and amino acids 25-28 (CDRl), 46-48 (CDR2), and 85-89 (CDR3) of one of SEQ ID NOs 1728-1736, or 1741-1747 VRCOl LC and VRC07 LC and variants w/El/I2 del IMGT CDRs, respectively;

(f) amino acids 26-33 (CDRl), 51-58 (CDR2), and 97-114 (CDR3) of one of SEQ ID NOs: 1460-1463, 1478-1483, or 1724-1727 (VRC07 and VRC07 variant CDRs IMGT), and amino acids 25- 28 (CDR1), 46-48 (CDR2), and 85-89 (CDR3) of one of SEQ ID NOs 1728-1736, or 1741-1747 VRCOl LC and VRC07 LC and variants w/El/I2 del IMGT CDRs, respectively;

(g) amino acids 31-35 (CDR1), 50-66 (CDR2), and 99-110 (CDR3) of one of SEQ ID NOs: 1,

3, 27, or 760-1459 (VRCOl and VRCOl variant CDRs KABAT), and amino acids 22-30 (CDR1), 46-52 (CDR2), and 85-89 (CDR3) of one of SEQ ID NOs 1728-1736, or 1741-1747 VRCOl LC and VRC07

LC and variants w/El/I2 del Kabat CDRs, respectively;

(h) amino acids 31-35 (CDR1), 50-66 (CDR2), and 99-114 (CDR3) of one of SEQ ID NOs: 1460-1463, 1478-1483, or 1724-1727 (VRC07 and VRC07 variant CDRs KABAT), and amino acids 22-30 (CDR1), 46-52 (CDR2), and 85-89 (CDR3) of one of SEQ ID NOs 1728-1736, or 1741-1747 VRCOl LC and VRC07 LC and variants w/El/I2 del Kabat CDRs, respectively.

Clause 11. The isolated bispecific monoclonal antibody of any one of clauses 1-5, wherein the heavy chain variable region of the first antigen binding domain comprises one of:

(a) the amino acid sequence set forth as one of SEQ ID NOs: 1, 3, 27, or 760-1459 (VRCOl and VRCOl variant V_H), and the amino acid sequence set forth as one of SEQ ID NOs: 2, 4, and 28, 1464, 1645, 1466, 1484, or 1728-1748, respectively; or

(b) the amino acid sequence set forth as one of SEQ ID NOs: 1460-1463, 1478-1483, or 1724- 1727 (VRC07 and VRC07 variant V_H), and the amino acid sequence set forth as one of SEQ ID NOs: 2,

4, and 28, 1464, 1645, 1466, 1484, or 1728-1748, respectively.

Clause 12. The isolated bispecific monoclonal antibody of any one of clauses 1-5, wherein the heavy chain variable region and the light chain variable region of the first antigen binding domain respectively comprise one of SEQ ID NOs: 1 and 2; 1 and 1464; 1 and 1465; 1 and 1466; 1 and 1484; 1 and 1728; 1 and 1729; 1 and 1730; 1 and 1731; 1 and 1732; 1 and 1733; 1 and 1734; 1 and 1735; 1 and 1736; 1 and 1737; 1 and 1738; 1 and 1739; 1 and 1740; 1 and 1741; 1 and 1742; 1 and 1743; 1 and 1744; 1 and 1745; 1 and 1746; 1 and 1747; 1 and 1748; 1460 and 2; 1460 and 1464; 1460 and 1465; 1460 and 1466; 1460 and 1484; 1460 and 1728; 1460 and 1729; 1460 and 1730; 1460 and 1731; 1460 and 1732; 1460 and 1733; 1460 and 1734; 1460 and 1735; 1460 and 1736; 1460 and 1737; 1460 and 1738; 1460 and 1739; 1460 and 1740; 1460 and 1741; 1460 and 1742; 1460 and 1743; 1460 and 1744; 1460 and 1745; 1460 and 1746; 1460 and 1747; 1460 and 1748; 1461 and 2; 1461 and 1464; 1461 and 1465; 1461 and 1466; 1461 and 1484; 1461 and 1728; 1461 and 1729; 1461 and 1730; 1461 and 1731; 1461 and 1732; 1461 and 1733; 1461 and 1734; 1461 and 1735; 1461 and 1736; 1461 and 1737; 1461 and 1738; 1461 and 1739; 1461 and 1740; 1461 and 1741; 1461 and 1742; 1461 and 1743; 1461 and 1744; 1461 and 1745; 1461 and 1746; 1461 and 1747; 1461 and 1748; 1462 and 2; 1462 and 1464; 1462 and 1465; 1462 and 1466; 1462 and 1484; 1462 and 1728; 1462 and 1729; 1462 and 1730; 1462 and 1731; 1462 and 1732; 1462 and 1733; 1462 and 1734; 1462 and 1735; 1462 and 1736; 1462 and 1737; 1462 and 1738; 1462 and 1739; 1462 and 1740; 1462 and 1741; 1462 and 1742; 1462 and 1743; 1462 and 1744; 1462 and 1745; 1462 and 1746; 1462 and 1747; 1462 and 1748; 1463 and 2; 1463 and 1464; 1463 and 1465; 1463 and 1466; 1463 and 1484; 1463 and 1728; 1463 and 1729; 1463 and

1730; 1463 and 1731; 1463 and 1732; 1463 and 1733; 1463 and 1734; 1463 and 1735; 1463 and

1736; 1463 and 1737; 1463 and 1738; 1463 and 1739; 1463 and 1740; 1463 and 1741; 1463 and

1742; 1463 and 1743; 1463 and 1744; 1463 and 1745; 1463 and 1746; 1463 and 1747; 1463 and

1748; 1478 and 2; 1478 and 1464; 1478 and 1465; 1478 and 1466; 1478 and 1484; 1478 and

1728; 1478 and 1729; 1478 and 1730; 1478 and 1731; 1478 and 1732; 1478 and 1733; 1478 and

1734; 1478 and 1735; 1478 and 1736; 1478 and 1737; 1478 and 1738; 1478 and 1739; 1478 and

1740; 1478 and 1741; 1478 and 1742; 1478 and 1743; 1478 and 1744; 1478 and 1745; 1478 and

1746; 1478 and 1747; 1478 and 1748; 1479 and 2; 1479 and 1464; 1479 and 1465; 1479 and

1466; 1479 and 1484; 1479 and 1728; 1479 and 1729; 1479 and 1730; 1479 and 1731; 1479 and

1732; 1479 and 1733; 1479 and 1734; 1479 and 1735; 1479 and 1736; 1479 and 1737; 1479 and

1738; 1479 and 1739; 1479 and 1740; 1479 and 1741; 1479 and 1742; 1479 and 1743; 1479 and

1744; 1479 and 1745; 1479 and 1746; 1479 and 1747; 1479 and 1748; 1480 and 2; 1480 and

1464; 1480 and 1465; 1480 and 1466; 1480 and 1484; 1480 and 1728; 1480 and 1729; 1480 and

1730; 1480 and 1731; 1480 and 1732; 1480 and 1733; 1480 and 1734; 1480 and 1735; 1480 and

1736; 1480 and 1737; 1480 and 1738; 1480 and 1739; 1480 and 1740; 1480 and 1741; 1480 and

1742; 1480 and 1743; 1480 and 1744; 1480 and 1745; 1480 and 1746; 1480 and 1747; 1480 and

1748; 1481 and 2; 1481 and 1464; 1481 and 1465; 1481 and 1466; 1481 and 1484; 1481 and

1728; 1481 and 1729; 1481 and 1730; 1481 and 1731; 1481 and 1732; 1481 and 1733; 1481 and

1734; 1481 and 1735; 1481 and 1736; 1481 and 1737; 1481 and 1738; 1481 and 1739; 1481 and

1740; 1481 and 1741; 1481 and 1742; 1481 and 1743; 1481 and 1744; 1481 and 1745; 1481 and

1746; 1481 and 1747; 1481 and 1748; 1482 and 2; 1482 and 1464; 1482 and 1465; 1482 and

1466; 1482 and 1484; 1482 and 1728; 1482 and 1729; 1482 and 1730; 1482 and 1731; 1482 and

1732; 1482 and 1733; 1482 and 1734; 1482 and 1735; 1482 and 1736; 1482 and 1737; 1482 and

1738; 1482 and 1739; 1482 and 1740; 1482 and 1741; 1482 and 1742; 1482 and 1743; 1482 and

1744; 1482 and 1745; 1482 and 1746; 1482 and 1747; 1482 and 1748; 1483 and 2; 1483 and

1464; 1483 and 1465; 1483 and 1466; 1483 and 1484; 1483 and 1728; 1483 and 1729; 1483 and

1730; 1483 and 1731; 1483 and 1732; 1483 and 1733; 1483 and 1734; 1483 and 1735; 1483 and

1736; 1483 and 1737; 1483 and 1738; 1483 and 1739; 1483 and 1740; 1483 and 1741; 1483 and

1742; 1483 and 1743; 1483 and 1744; 1483 and 1745; 1483 and 1746; 1483 and 1747; 1483 and

1748; 1724 and 2; 1724 and 1464; 1724 and 1465; 1724 and 1466; 1724 and 1484; 1724 and

1728; 1724 and 1729; 1724 and 1730; 1724 and 1731; 1724 and 1732; 1724 and 1733; 1724 and

1734; 1724 and 1735; 1724 and 1736; 1724 and 1737; 1724 and 1738; 1724 and 1739; 1724 and

1740; 1724 and 1741; 1724 and 1742; 1724 and 1743; 1724 and 1744; 1724 and 1745; 1724 and

1746; 1724 and 1747; 1724 and 1748; 1725 and 2; 1725 and 1464; 1725 and 1465; 1725 and 1466; 1725 and 1484; 1725 and 1728; 1725 and 1729; 1725 and 1730; 1725 and 1731; 1725 and 1732; 1725 and 1733; 1725 and 1734; 1725 and 1735; 1725 and 1736; 1725 and 1737; 1725 and 1738; 1725 and 1739; 1725 and 1740; 1725 and 1741; 1725 and 1742; 1725 and 1743; 1725 and 1744; 1725 and 1745; 1725 and 1746; 1725 and 1747; 1725 and 1748; 1726 and 2; 1726 and 1464; 1726 and 1465; 1726 and 1466; 1726 and 1484; 1726 and 1728; 1726 and 1729; 1726 and 1730; 1726 and 1731; 1726 and 1732; 1726 and 1733; 1726 and 1734; 1726 and 1735; 1726 and 1736; 1726 and 1737; 1726 and 1738; 1726 and 1739; 1726 and 1740; 1726 and 1741; 1726 and 1742; 1726 and 1743; 1726 and 1744; 1726 and 1745; 1726 and 1746; 1726 and 1747; 1726 and 1748; 1727 and 2; 1727 and 1464; 1727 and 1465; 1727 and 1466; 1727 and 1484; 1727 and 1728; 1727 and 1729; 1727 and 1730; 1727 and 1731; 1727 and 1732; 1727 and 1733; 1727 and 1734; 1727 and 1735; 1727 and 1736; 1727 and 1737; 1727 and 1738; 1727 and 1739; 1727 and 1740; 1727 and 1741; 1727 and 1742; 1727 and 1743; 1727 and 1744; 1727 and 1745; 1727 and 1746; 1727 and 1747; or 1727 and 1748.

Clause 13. The isolated bispecific antibody of clause 1 or 2, wherein the first antigen binding domain specifically binds to gp41.

Clause 14. The isolated bispecific antibody of clause 13, wherein the first antigen binding domain specifically binds to the MPER of gp41.

Clause 15. The isolated bispecific monoclonal antibody of clause 14, wherein the heavy chain variable region of the first antigen binding domain comprises one of:

(a) amino acids 26-33 (CDR1), 51-60 (CDR2), and 99-120 (CDR3) of one of SEQ ID NOs:

1485, 1517, 1519, 1525, 1660-1663, 1667-1677, 1701-1706, or 1714-1718 (IMGT CDRs); or

(b) amino acids 26-33 (CDR1), 51-60 (CDR2), and 101-120 of one of SEQ ID NOs: 1485, 1517,

1519, 1525, 1660-1663, 1667-1677, 1701-1706, or 1714-1718 (Kabat CDRs).

Clause 16. The isolated bispecific monoclonal antibody of clause 14 or clause 15, wherein the heavy chain variable region of the first antigen binding domain comprises the amino acid sequence set forth as one of SEQ ID NOs: 1485, 1517, 1519, 1525, 1660-1663, 1667-1677, 1701-1706, or 1714-1718

Clause 17. The isolated bispecific monoclonal antibody of any one of clauses 14-16, wherein the light chain variable region of the first antigen binding domain comprises one of

(a) amino acids 26-33 (CDR1), 51-60 (CDR2), and 99-120 (CDR3) of one of SEQ ID NOs: 1486, 1518, 1520, 1526, 1664-1666, 1678-1700, 1711-1713, or 1719-1723 (IMGT CDRs); or

(b) amino acids 26-33 (CDR1), 51-60 (CDR2), and 101-120 of one of SEQ ID NOs: 1486, 1518,

1520, 1526, 1664-1666, 1678-1700, 1711-1713, or 1719-1723 (Kabat CDRs).

Clause 18. The isolated bispecific monoclonal antibody of any one of clauses 14-16, wherein the light chain variable region of the first antigen binding domain comprises the amino acid sequence set forth as one of SEQ ID NOs: 1486, 1518, 1520, 1526, 1664-1666, 1678-1700, 1711-1713, or 1719-1723 (10E8 LC and variants thereof). Clause 19. The isolated bispecific monoclonal antibody of clause 14, wherein the heavy and light chain variable regions of the first antigen binding domain comprises one of:

(a) amino acids 26-33 (CDR1), 51-60 (CDR2), and 99-120 (CDR3) of one of SEQ ID NOs: 1485, 1517, 1519, 1525, 1660-1663, 1667-1677, 1701-1706, or 1714-1718, and amino acids 26-33 (CDR1), 51-60 (CDR2), and 99-120 (CDR3) of one of SEQ ID NOs: 1486, 1518, 1520, 1526, 1664- 1666, 1678-1700, 1711-1713, or 1719-1723 (IMGT CDRs), respectively; or

(b) amino acids 26-33 (CDR1), 51-60 (CDR2), and 101-120 of one of SEQ ID NOs: 1485, 1517, 1519, 1525, 1660-1663, 1667-1677, 1701-1706, or 1714-1718, and amino acids 26-33 (CDR1), 51-60 (CDR2), and 101-120 of one of SEQ ID NOs: 1486, 1518, 1520, 1526, 1664-1666, 1678-1700, 1711- 1713 , or 1719- 1723 (Kabat CDRs) , respectively.

Clause 20. The isolated bispecific monoclonal antibody of clause 14, wherein the heavy chain variable region of the first antigen binding domain comprises the amino acid sequence set forth as one of SEQ ID NOs: 1485, 1517, 1519, 1525, 1660-1663, 1667-1677, 1701-1706, or 1714-1718, and the light chain variable region of the first antigen binding domain comprises the amino acid sequence set forth as one of SEQ ID NOs: 1486, 1518, 1520, 1526, 1664-1666, 1678-1700, 1711-1713, or 1719-1723.

Clause 21. The isolated bispecific monoclonal antibody of clause 20, wherein the heavy chain variable region of the second antigen binding domain comprises amino acids 265- 274 (H-CDR1), 289 - 305 (H-CDR2), and 338-347 (H-CDR3) of SEQ ID NO: 1510 (TR66), and the light chain variable region of the second antigen binding domain comprises amino acids 400 - 409 (L-CDR1), 425-431 (L-CDR2), and 464-472 (L-CDR3) of SEQ ID NO: 1510 (TR66).

Clause 22. The isolated bispecific monoclonal antibody of clause 20 or clause 21, wherein the heavy chain variable region of the second antigen binding domain comprises the amino acid sequence set forth as amino acids 240-360 of SEQ ID NO: 1510 (humanized TR66), and the light chain variable region of the second antigen binding domain comprises the amino acid sequence set forth as amino acids 375-482 of SEQ ID NO: 1510 (humanized TR66).

Clause 23. The isolated bispecific antibody of any one of clauses 1-22, wherein the first antigen binding domain is linked to the second antigen binding domain by a peptide linker.

Clause 24. The isolated bispecific antibody of any one of clauses 1-22, wherein

the first antigen binding domain is a Fab comprising the heavy chain variable region of the first antigen binding domain, a heavy chain constant 1(CH1) region, the light chain variable region of the first antigen binding domain, and a light chain constant (CL) region; and

the second antigen binding domain is a scFv.

Clause 25. The isolated bispecific antibody of clause 24, wherein the antibody comprises a first polypeptide and a second polypeptide, wherein

(A) the first polypeptide comprises, N-terminal to C-terminal, the heavy chain variable region of the Fab, the CHI region, and the scFv, and the second polypeptide comprises, N-terminal to C-terminal, the light chain variable region of the Fab and the CL region; or (B) the first polypeptide comprises, N-terminal to C-terminal, the heavy chain variable region of the Fab and the CHI region, and the second polypeptide comprises, N-terminal to C-terminal, the light chain variable region of the Fab, the CL region, and the scFv.

Clause 26. The isolated bispecific antibody of clause 25, wherein the first antigen binding domain specifically binds gpl20, and wherein the first and second polypeptides comprise the amino acid sequences set forth as one of:

(a) SEQ ID NO: 1516 (VRC3125; VRC07H G54W Fab-HRV3c-His) and SEQ ID NO: 1508 (VRC3121 ; VRC01L-(GGGGS)₃-hCD3), respectively;

(b) SEQ ID NO: 1749 (VRC 3487; VRC07H G54H Fab-HRV3c-His) and SEQ ID NO: 1508 (VRC3121 ; VRC01L-(GGGGS)₃-hCD3), respectively;

(c) SEQ ID NO: 1516 (VRC3125; VRC07H G54W Fab-HRV3c-His) and SEQ ID NO: 1510 (VRC3122; VRC01L-(GGGGS)₂-hCD3), respectively;

(d) SEQ ID NO: 1749 (VRC 3487; VRC07H G54H Fab-HRV3c-His) and SEQ ID NO: 1510 (VRC3122; VRC01L-(GGGGS)₂-hCD3), respectively;

(e) SEQ ID NO: 1516 (VRC3125; VRC07H G54W Fab-HRV3c-His) and SEQ ID NO: 1751

(VRC 3485; VRC01L El/I2del V3S-(GGGGS)₂-hCD3), respectively; or

(f) SEQ ID NO: 1749 (VRC 3487; VRC07H G54H Fab-HRV3c-His) and SEQ ID NO: 1751 (VRC 3485; VRC01L El/I2del V3S-(GGGGS)₂-hCD3), respectively.

Clause 27. The isolated bispecific antibody of clause 26, wherein the first antigen binding domain specifically binds gp41, and wherein the first and second polypeptides comprise the amino acid sequences set forth as one of:

(a) SEQ ID NO: 1757 (VRC 3490; 10E8HC6 Fab-HRV3c-His) and SEQ ID NO: 1488

(VRC3111 ; 10E8gL03-(GGGGS)₃-hCD3), respectively;

(b) SEQ ID NO: 1496 (VRC3115; 10E8gH03_4 Fab-HRV3c-His) and SEQ ID NO: 1488 (VRC3111 ; 10E8gL03-(GGGGS)₃-hCD3), respectively;

(c) SEQ ID NO: 1757 (VRC 3490; 10E8HC6 Fab-HRV3c-His) and SEQ ID NO: 1490

(VRC3112; 10E8gL03-(GGGGS)₂-hCD3), respectively; or

(d) SEQ ID NO: 1496 (VRC3115; 10E8gH03_4 Fab-HRV3c-His) and SEQ ID NO: 1490 (VRC3112; 10E8gL03-(GGGGS)₂-hCD3), respectively.

Clause 28. The isolated bispecific monoclonal antibody of clause 1 or 2, wherein the first and second antigen binding domains are scFvs and the bispecific antibody comprises the amino acid sequence set forth as SEQ ID NO: 1475 (VRC2455; VRCOl scFv + anti-hCD3 TR66 scFv).

Clause 29. An isolated trispecific antibody, comprising the bispecific antibody of any one of clauses 1-28, linked to a third antigen binding domain that specifically binds to CD28, CD40L, CD 137 or CD137L.

Clause 30. The isolated trispecific antibody of clause 29, wherein the third antigen binding domain is linked to the bispecific antibody by a peptide linker.

Clause 31. The isolated trispecific antibody of any one of clauses 29-30, wherein the first antigen binding domain is a Fab comprising the heavy chain variable region of the first antigen binding domain, a heavy chain constant 1(CH1) region, the light chain variable region of the first antigen binding domain, and a light chain constant region;

the second antigen binding domain is a first scFv; and

the third antigen binding domain is a second scFv.

Clause 32. The isolated trispecific antibody of any one of clauses 29-31 , wherein the trispecific antibody is a tribody.

Clause 33. The isolated trispecific antibody of clause 31 , wherein the antibody comprises a first polypeptide and a second polypeptide, wherein

(A) the first polypeptide comprises, N-terminal to C-terminal, the heavy chain variable region of the Fab, the CHI region, and the first scFv, and the second polypeptide comprises, N-terminal to C- terminal, the light chain variable region of the Fab, the CL region and the second scFv; or

(B) the first polypeptide comprises, N-terminal to C-terminal, the heavy chain variable region of the Fab, the CHI region and the second scFv, and the second polypeptide comprises, N-terminal to C- terminal, the light chain variable region of the Fab, the CL region, and the first scFv.

Clause 34. The trispecific antibody of clause 33, wherein the scFv of the third antigen binding domain:

(a) specifically binds to CD28 and comprises the amino acid sequence set forth as SEQ ID NO:

1766;

(b) specifically binds to CD40L;

(c) specifically binds to CD137 and comprises the amino acid sequence set forth as SEQ ID NO: 1767; or

(d) specifically binds to CD137L.

Clause 35. The isolated antibody of any of clauses 1-34, wherein the antibody is labeled.

Clause 36. An isolated nucleic acid molecule encoding the bispecific antibody of any one of clauses 1-26 or the trispecific antibody of any one of clauses 29-34.

Clause 37. The isolated nucleic acid molecule of clause 36, operably linked to a promoter.

Clause 38. A vector comprising the one or more isolated nucleic acid molecules of any one of clauses 35-36.

Clause 39. A composition comprising an effective amount of the isolated bispecific antibody of any one of clauses 1-28, the trispecific antibody of any one of clauses 29-34, the nucleic acid molecule of clause 36 or clause 37, or the vector of clause 38; and a pharmaceutically acceptable carrier.

Clause 40. A method for treating a subject with a human immunodeficiency virus (HIV)-l infection, comprising administering to a subject infected with HIV-1 a therapeutically effective amount of the isolated bispecific antibody of any one of clauses 1-28, the trispecific antibody of any one of clauses 29-34, the nucleic acid molecule of clause 36 or clause 37, or the vector of clause 38, or the composition of clause 39, thereby treating the HIV-1 infection in the subject. Clause 41. The method of clause 40, wherein the subject has acquired immune deficiency syndrome (AIDS).

Clause 42. The method of clause 40 or clause 41, further comprising

administering to the subject an therapeutically effective amount of an anti-retroviral agent.

Clause 43. The method of any one of clauses 40-42, further comprising administering a therapeutically effective amount of highly active anti-retroviral therapy (HAART) to the subject.

Clause 44. The method of clause 43, wherein HAART is administered to the subject prior to administration the therapeutically effective amount of the bispecific antibody.

Clause 45. The method of any one of clauses 40-41, wherein treating the HIV-1 infection comprises reducing the latent reservoir of HIV-1 infected cells in the subject.

Clause 46. The method of any one of clauses 40-45, further comprising measuring HIV-1 viral titer in the subject.

Clause 47. The method of any one of clauses 40-45, further comprising measuring pro viral HIV- 1 DNA in the subject.

Clause 48. A kit comprising:

(a) the isolated bispecific antibody of any one of clauses 1-28, the trispecific antibody of any one of clauses 29-34, the nucleic acid molecule of clause 36 or clause 37, or the vector of clause 38, the composition of clause 39, or a combination of two or more thereof; and

(b) instructions for using the kit.

Clause 49. Use of the isolated bispecific antibody of any one of clauses 1-28, the trispecific antibody of any one of clauses 29-34, the one or more nucleic acid molecules of clause 36 or clause 37, or the one or more vectors of clause 38, or the composition of clause 39, or a combination of two or more thereof, to treat Human Immunodeficiency Virus type 1 infection in a subject.

III. EXAMPLES

The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.

Example 1

Bispecific antibodies that specifically bind to HIV-1 envelope proteins and CD3 clear

latent HIV infection

This example illustrates construction and use of a bispecific single chain antibody that combines an antigen binding domain specific for gpl20 or gp41 and a second antigen binding domain specific for CD3. This bispecific construct is designed to activate and eliminate the latent reservoir of infected T cells by tethering cytotoxic T cells to the activated latent HIV envelop expressing cells.

Bispecific scFv construction: A DNA vector for expressing the VRC2455 bispecific single chain antibody is set forth as SEQ ID NO: 1474, which includes an scFv including heavy and light chain variable domains from the VRCOl monoclonal antibody joined to an scFv including heavy and light chain variable domains from a human CD3 specific monoclonal antibody TR66.

A DNA vector for expressing the VRC2678 bispecific single chain antibody set forth as SEQ ID NO: 1473, which includes an scFv including heavy and light chain variable domains from the VRCOl monoclonal antibody joined to an scFv including heavy and light chain variable domains from the rhesus macaque CD3 specific monoclonal antibody C207. VRCOl is described above. C207 is an affinity matured anti-rhCD3 mutant antibody selected from a library of FN18 scFvs randomly mutagenized with nucleotide analogs, displayed on yeast and selected by sorting flow cytometry using dye-labeled monkey CD3ey ectodomain heterodimer (Wang et al, J. BioConjug. Chem., 18:947-955, 2007).

A DNA vector for expressing the VRC2442 bispecific single chain antibody set forth as SEQ ID

NO: 1476, which includes an scFv including heavy and light chain variable domains from the humanized (hu) 5B8 monoclonal antibody joined to an scFv including heavy and light chain variable domains from a human CD3 specific monoclonal antibody TR66. hu5B8 is a humanized version of the monoclonal antiidiotype antibody 5B8 raised against VRCOl. Thus, each of the VRC2455, VRC2678 and VRC2442 bispecific single chain antibodies includes two scFvs joined together by a linker to form a single polypeptide chain of approximately 55 kDa.

Additional bispecific antibodies were also constructed. VRC07 (G54W) Fab-anti-CD3 scFv bispecific antibody was made by co-transfection of 293F cells with cDNA encoding VRC07 (G54W) heavy chain variable and CHI regions with C-terminal HisTag (VRC3125) and VRCOl light chain fused to anti-CD3 scFv (VRC3121, VRC3122, VRC3123 or VRC3124). Version 2 (v2) has a (GGGGS)₃ linker (VRC3121 and VRC3123), while Version 1 (vl) has a (GGGGS)₂ linker (VRC3122 and 3124 ), between the light chain and the anti-CD3 scFv. 10E8Fab-CD3 bispecific antibody was made by co- transfection of 293F cells with cDNA encoding partial germline reverted 10E8 heavy chain variable and CHI regions with C-terminal HisTag (VRC 3115), and partial germline reverted 10E8 light chain fused to anti-CD3 scFv (VRC3111, VRC3112, VRC3113 or VRC3114). VRC3111 and 3113 have a

(GGGGS)₃ linker , while VRC3112 and 3114 have a (GGGGS)₂ linker, between the light chain and the anti-CD3 scFv. Similarly, Hu5B8Fab-CD3 bispecific antibody was made by co-transfection into 293F cells of cDNA encoding humanized 5B8 heavy chain variable and CHI regions with C-terminal HisTag (VRC 3120) and humanized 5B8 light chain fused to anti-CD3 scFv (VRC3116, VRC3117, VRC3118 or VRC3119). VRC3116 and 3118 have a (GGGGS)₃ linker, while VRC3117 and 3119 have a (GGGGS)₂ linker between the light chain and the anti-CD3 scFv.

Purification and characterization of bispecific scFvs: The bispecific proteins were expressed by transient transfection of 293F cells and the bispecific Ab was purified from the culture supernatant using Ni-affinity column (the bispecific constructs encode a HIS -tag), followed by size exclusion column chromatography (see FIG. 2A). The purified monomeric based bispecific Ab fraction was ran on SDS- PAGE gel to characterize the purity and size of the protein. The VRCOl -hCD3 bispecific construct is shown in FIG. 2B. Detection of bound bispecific scFvs to T cells: Fluorophore-conjugated antibodies were obtained from eBioscience BD Pharmingen and Qiagen. To determine the absorbance of bispecific scFVs by monkey PBMCs in vivo, PBMCs were isolated from the treated animals and stained with anti-CD3- APC-Cy7, RSC3-FITC and anti-His-PE for 25 min. Free dyes were washed out and cells were acquired on a LSR-II cytometer. The data was analyzed with Flowjo software. The binding of bispecific scFvs to normal human donor PBMCs was assessed similarly, in which 20 g/ml of the bispecific scFv was incubated with PBMCs for 20 minutes and bound bispecific scFvs were stained with anti-CD3-APC-Cy7 and either RSC3-FITC or anti-His-PE as above.

Measurement of T cell activation: To measure activation of T cells by the bispecific scFvs, intracellular cytokine staining was performed. In this assay, normal human donor PBMCs were co- cultured with CEM-NKr-CCR5 cells that are chronically infected with HIV-1 or uninfected CEM-NKr- CCR5 cells at an E:T ratio of 10: 1, in the presence of Brefeldin A and 1 g/ml of three different bispecific single chain antibodies - VRC2455, VRC2678 and VRC2442. After 20-24 hours of co-culture, the PBMCs were then stained using fluorescent antibody conjugates for surface CD3, CD4 and CD8 and intracellular cytokines after fixation/permeabilization of the cells using the BD Fix/perm solution. The percentage of CD4+ and CD8+ T cells expressing IFN-gamma was quantified using LSR-II cytometer and the data was analyzed with Flowjo software.

Quantification of cytotoxicity activity: HIV-1 infected cell lines as target cells and human PBMCs as effector cells. The indicated HIV-1 infected cell lines were labeled with a fluorescent membrane dye (PKH26) and incubated with human PBMCs from normal donors at an E:T ratio of 10:1 in the presence of 10-fold serial dilutions of VRC2455 bispecific scFv (VRC01-hCD3) or VRCOl IgGl for 20-24 hours. The percentage of dead cells was determined by flow cytometry using a live/dead cell stain to measure amount of cell lysis for each protein.

Antiretroviral treatment and bispecific scFv administration: Two rhesus macaques

(A8V091 andA8V102) were infected with SHIV-BaL and were first treated with a highly active antiretroviral therapy (HAART) for six weeks until the plasma viremia was below the detection limit (FIG. 5). The HAART used was either 20 mg/Kg/day tenofovir (PMPA, given subcutaneously; from Gilead Sciences, CA, USA or 100 mg/Kg/BID raltegravir (Isentress, mixed with food; from Merck, NJ, USA). The animals were then infused intravenously with either VRC2678 bispecific single chain antibody (n=l) or control bispecific single chain antibody VRC2442 (n=l) at 5μg/kg, using daily dosing for seven consecutive days. Blood samples were collected every day for the first seven days and once a week thereafter. Plasma viremia and proviral DNA level in the PBMCs were examined at each time point.

Plasma viremia quantitation: Plasma samples were diluted and centrifuged at 23000g for 1 hr to pellet virions. RNA was extracted for the pellets and reversely transcribed to cDNA. cDNA was quantified with gag specific primers and probe. Copy number per ml of plasma was determined by interpolation into a serial dilution curve of known copy numbers using a gag gene cDNA construct. A threshold of 500 copies/ml was set according to the detection limit of the assay, Proviral DNA quantitation: Genomic DNA was extracted from about 20 million PBMCs with Qiagen DNeasy tissue and blood kit. A total of 500-1000ng of genomic DNA was used as template to amplify the gag sequence using qPCR. Copy numbers of gag in each sample was determined by a standard set of gag plasmid DNA of known copy numbers.

To assess the bispecific activity of VRC2455 bispecific scFv, we first tested the binding of it to human PBMCs (FIG. 2C; FIG. 2E). Normal human donor PBMCs were incubated in the presence or absence of the indicated bispecific antibody and then stained with either a FITC conjugated RSC3 (VRCOl cognate antigen) or a PE conjugated anti-His tag probe to detect the bispecific antibody (such as VRC2455). As shown in FIG. 2C, VRC2455 was able to bind to human T cells present in normal donor PBMCs.

After confirmation of the bispecific binding activity of VRC2455, we tested its ability to activate T cells using an intra-cellular cytokine staining assay (FIG. 3). Normal human donor PBMCs were co cultured with CEM-NKr-CCR5 cells chronically infected with HIV-1 (HIV+) or uninfected CEM-NKr- CCR5 cells (HIV-) at an E:T ratio of 10: 1, in the presence of Brefeldin A and 1 g/ml of three different bispecific scFvs. It was observed that VRC2455 was able to stimulate the production of IFN-gamma in both CD4+ and CD8+ T cells in the presence of HIV-1 infected target cells, whereas VRC2678 and VRC2442 did not stimulate any cytokine production. To test the functional activity of VRC2455, we measured the cytotoxic activity of this construct by using HIV-1 infected cell lines as target cells and human PBMCs as effector cells (FIG. 4). We used four different cell lines that infected with HIV-1 (CEM-IIIb, ACH2, Jl.l or OM10). We observed a dose dependent cytotoxic activity for VRC2455 against all three target cell lines, whereas in comparison VRCOl IgG did not demonstrate any significant cytotoxic activity at the same concentrations. Similar results were obtained using the VRC07 -based bispecific mAb (FIG. 11).

We used a nonhuman primate lentiviral immunodeficiency virus infection model to assess the in vivo efficacy of the bispecific single chain antibodies. In this experiment, two rhesus macaques (A8V091and A8V102) were infected with SHIV-BaL and were first treated with a highly active antiretroviral therapy (HAART) drug for six weeks until the plasma viremia was below the detection limit (FIG. 5). The HAART drug used was either 20 mg/Kg/day tenofovir (PMPA, given

subcutaneously; from Gilead Sciences, CA,USA) or 100 mg/Kg/BID raltegravir (Isentress, mixed with food; from Merck, NJ, USA). The animals were then immunized with either VRC2678 bispecific single chain antibody (n=l) or control VRC2442 bispecific single chain antibody (n=l) at 5μg/kg, using daily dosing for seven consecutive days. Blood samples were collected every day for the first seven days and once a week thereafter. Plasma viremia and proviral load in the PBMCs were examined at each time point.

As shown in FIG. 6, after two doses of VRC2678 bispecific single chain antibody (day 44 post-

ART), the PBMC proviral DNA of A8V102 had declined to baseline levels and remained undetectable by dayl4 post-treatment (day 56 post-ART). No significant change in proviral load was seen in the control bispecific single chain antibody-treated animal A8V091. Notably, the animal treated with VRC2678 showed a proviral spike at 6h after the first dose (FIG. 6, day 42 on the scale), suggesting an activation of the latent virus in the T cell reservoir that may result in the surface expression of HIV envelope for VRC2678 bispecific single chain antibody targeting.

To demonstrate that the VRC2678 bispecific single chain antibody binds to PBMCs in vivo after infusion, PBMCs were isolated at different time points and double-stained with the VRCOl cognate antigen RSC3 and anti-His tag antibody (the VRC2678 bispecific single chain antibody includes a histidine-tag). As shown in FIG. 7, a dramatic increase the RSC3+His tag-i- cell population was detected at only six hours following the first dose of VRC2678 bispecific single chain antibody, which then dropped to the baseline level afterwards.

Thus, the bispecific single chain antibody, combining a heavy and light chain variable region from VRCOl, a broad and potent anti- HIV neutralization antibody that binds to the CD4 binding site of gpl20, and a heavy and light chain variable region from C207, an anti-CD3 antibody, acted on the latent reservoir of SHIV infected cells in an animal model. Elimination of HIV-1 infected cells in vivo involves activated T cells through specific and non-specific mechanisms. Further, the anti-CD3 antibody can activate cytotoxic T cells and also activate the latent reservoir of HIV-1 infected CD4 T cells. Thus, the bispecific single chain antibodies provided herein activate and eliminate the latent reservoir of infected cells in chronic HIV-1 infection by tethering cytotoxic T cells to activated latent HIV envelope expressing cells.

A non-limiting explanation for these results is that when the bispecific single chain antibody is administered to chronic Simian HIV (SHIV) infected rhesus monkeys, the latent SHIV infected cell reservoir is activated by the anti-CD3 portion of the bispecific antibody, resulting in the expression of HIV-1 envelope (env) protein, encoded by the SHIV genome, on those cells. The HIV env-i- cells can then be recognized by the VRCOl portion of the bispecific antibody. The binding of the bispecific antibody to these env-i- infected cells can then transiently tether resting T cells through its anti-CD3 specificity, leading to concomitant T cell activation and subsequent lysis of these infected cells, and ultimately the latent reservoir.

The binding of the VRC07 (G54W)- and 10E8-based bispecific antibodies to their corresponding antigen was also tested (FIG. 8 and FIG. 9). ELISA plates were coated with 200 ng/well RSC3(gpl20 antigen) for VRC07 (G54W)-based antibodies or 100 ng/well MPER peptide

(RRRNEQELLELDKWASLWNWFDITNWLWYIRRRR; SEQ ID NO: 1539) for 10E8-based antibodies. The plates were then incubated with serial diluted VRC07 (G54W) or 10E8-based bispecific antibodies, and detected using an HRP-labeled anti-Fab conjugate. As shown in Fig 10 and 11, all Fab- scFv bispecific antibodies bound to their corresponding antigen.

Binding of the VRC07 -based bispecific antibody to human PBMCs was also tested (FIG. 10). Normal human donor PBMCs were incubated in the presence or absence of the indicated bispecific antibody and then stained with either a FITC conjugated RSC3 (VRCOl cognate antigen) or a PE conjugated anti-His tag probe to detect the bispecific antibody. As shown in FIG. 10, the VRC07 -based bispecific antibody was also able to bind to human T cells present in normal donor PBMCs. Example 2

Reduction of peripheral latent HIV infection with a bispecific antibody

Highly active antiretro viral therapy has reduced viral burden and ameliorated HIV-1 infection in infected individuals but does not eliminate lentiviral infection because of its inability to target virus in latent reservoirs. The ability to eliminate latently infected cells could potentially reduce long term antiviral drug dependence and facilitate immune reconstitution. A variety of broadly neutralizing antibodies to HIV-1 have recently been identified that facilitate immune recognition of naturally circulating strains. It is shown that a novel immunotherapeutic protein can activate latent viral gene expression and direct T lymphocytes to lyse these cells in vitro and in vivo. To achieve this effect, bispecific antibodies were generated by linking the Fab fragment of a neutralizing Ab to the HIV-1 CD4 binding site, VRC07, to anti-hCD3. This bispecific antibody, VRC07(G54W)(Fab)-anti-hCD3, bound to CD3 on T cells and Env on HIV-1 infected cells. Through the CD3 interaction, this protein activated CD4 and CD8 T cells and, importantly, stimulated proviral gene expression in latently infected T cell lines and primary T cells. It further stimulated CD8 T cell effector function and mediated T cell- dependent lysis of these previously latently infected cells. Treatment of SHIV-Bal infected Indian rhesus macaques suppressed with anti-retroviral therapy significantly reduced the levels of proviral DNA in PBMCs compared to controls. This modified antibody could help to eliminate the latently infected cells and thus reduce viral load, drug dependence, and deplete the viral reservoir in HIV-1 infected individuals.

Persistence of latently infected cells even after prolonged periods of highly active antiretroviral therapy (HAART) in HIV-1 infected patients remains a major hurdle towards finding a cure for HIV. Although HIV-1 preferentially infects actively replicating cells, it can also infect quiescent cells like resting CD4⁺ T cells at lower frequencies. Latent HIV-1 infection of resting memory CD4⁺ T cells is established when activated CD4⁺ T cells return to a quiescent state, or through infection of quiescent T cells. Latency leads to the integration of the viral genome into the host chromosome generally without any active transcription of viral genes. Since the HAART drugs target viral proteins involved in the viral replication cycle, they are unable to eliminate these quiescent cells that harbor proviral DNA. During therapy, active viral replication is potently limited by these drugs, but whenever an interruption in therapy occurs, there is a spontaneous reactivation of the latent virus leading to active viral replication in most cases. Due to this fact, most infected individuals have to be under therapy for the rest of their lives to limit HIV replication and have an improved health prognosis. Despite the overall anti-viral benefits of HAART, the patients under prolonged therapy have increased risk for the development of drug-induced diseases including cardiovascular disease, metabolic disorders and bone disorders. Also, there remains a high prevalence of HIV-associated neurocognitive disorders (HAND) in the HAART era. Therefore, eliminating the latently infected cells in HIV-1 infected individuals would limit the dependence on HAART drugs for treating HIV-1 infection. Activation and elimination of the latently infected cells in HIV-1 infection has become a major focus of HIV research. The initial use of anti-CD3 and IL-2 treatment to purge the latent HIV-1 reservoir in patients on therapy led to deleterious effects on the immune system, and also failed to eliminate the latently infected cells (Chun et al, Nature 401, 874-875, 1999; Abadie et al, Astron Astrophys 539, 2012; Prins et al, AIDS 13, 2405-2410, 1999; Fraser et al, AIDS 14, 659-669, 2000; van Praag et al, Journal of clinical immunology 21, 218-226, 2001 ; Kulkosky et al, The Journal of infectious diseases 186, 1403-1411, 2002).

More recently, the use of his tone deacetylase 1 (HDAC1) inhibitors to target latent HIV-1 infection have demonstrated the reactivation of latently infected cells in HIV-1 infected patients, but the effect in clearing the latent reservoir was modest (Archin et al, Nature 487, 482-485, 2012). Apart from the HDAC1 inhibitors, other molecules like Bryostatin, a protein kinase C activator, and disulfiram have also been shown to activate latent HIV-1 expression (Perez et al, Current HIV research 8, 418-429, 2010; Xing et al, Journal of virology 85, 6060-6064, 2011). All of these strategies are targeted towards activating latent cells but not elimination of the reactivated HIV-1 infected cells, which may require stimulation of HIV-1 -specific cytolytic T lymphocytes (Shan et al, Immunity 36, 491-501, 2012).

The use of bispecific antibodies for the treatment of tumors has been shown to be successful in model systems (Abadie et al, Astron Astrophys 539, 2012; Baeuerle et al, Current opinion in molecular therapeutics 11, 22-30, 2009) and in the case of one has also shown efficacy in clinical trials (Bargou et al, Science 321, 974-977,2008; Handgretinger et al, Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 25, 181-184, 2011; Topp et al, Journal of clinical oncology : official journal of the American Society of Clinical Oncology 29, 2493-2498, 2011 ; Klinger et al, Blood 119, 6226-6233, 2012). These bispecific antibodies consist of a tumor-targeting antibody fragment linked to an anti-T cell activating antibody fragment that enable T cells to come in close proximity to tumor cells, undergo activation and mediate lysis of the tumor cells. However, the bispecific antibodies used for tumor treatment target a tumor associated antigen that is constitutively expressed on the surface of tumor cells. Here, the generation of a bispecific antibody that can activate latent HIV-1 infected cells as well as mediate their lysis in vivo and in vitro is reported.

Characterization of bispecific antibodies

The recent identification of VRCOl and its clonal lineage members that bind the conserved CD4 binding site (CD4bs) on the HIV envelope glycoprotein enables targeting of a majority of the circulating strains of HIV-1. VRC07 is clonally related to VRCOl with higher potency than VRCOl and was identified using deep sequencing of antibody sequences from the same donor. Here, bispecific antibodies were generated by linking the Fab fragment of its mutant VRC07(G54W) to anti-hCD3. In this construct, a humanized form of the scFv fragment of an anti-hCD3 monoclonal antibody (Traunecker et al, The EMBO journal 10, 3655-3659, 1991) was linked by a 16 amino acid GS linker to the light chain of VRC07 and the VH/CH1 domains of VRC07(G54W) were kept intact to form VRC07(G54W)(Fab)- anti-hCD3. For targeting rhesus T cells, a similar bispecific construct was generated, simVRC07(G54W)(Fab)-anti-rhCD3, by replacing the anti-hCD3 scFv with an anti-rhCD3 scFv (Nooij et al, European journal of immunology 16, 975-979, 1986) and using the Fab from a simianized version of the human VRC07(G54W). The bispecific antibodies were monomeric as determined by performing size exclusion chromatography of the purified antibodies and consisted of heavy and light chains of appropriate sizes (FIG. 12A). In addition, two control bispecific antibodies were generated by replacing the VRC07 Fab with a non-HIV Fab that can bind either human or rhesus CD3. These bispecific antibodies were able to bind to a soluble form of the resurfaced HIV envelope core protein (RSC3) that has an intact CD4bs (FIG. 12B). Their ability to bind to CD3 and HIV Env expressed on the cell surface was also assayed, it was observed that the human and rhesus bispecific antibodies were able to bind to CD3 on the surface of human and rhesus T cells respectively (FIG. 12C). Also, it was found that bispecific antibodies with VRC07 Fab were only able to bind to HIV Env expressed on the surface of chronically HIV-1 infected cells (FIG. 12C) and retain their neutralizing activity against HIV-1 (Table 2) These data demonstrate the successful generation of bispecific antibodies with specificity for CD3 and HIV Env.

Table 2: Neutralization of HIV-1 by VRC07-based antibodies. Neutralizing IC50 titers ^g/ml) for indicated VRC07 -based mono- and bispecific antibodies against HIV-1 strains, one each from Clades A, B and C, are reported.

VRC07(Fab)- simVRC07(Fab)-

HlV Clade VRC071gG VRC07Fab anti-hCD3 anti-rhCD3

A 0.036 0.056 0.025 0.133

B 0.114 0.576 0.346 1.050

C 0.217 0.524 0.240 0.737 Activation of T cells by VRC07(Fab)-anti-hCD3

To assess the ability of the bispecific antibodies to activate T cells in an antigen-specific manner, in vitro T cell activation assays were performed. In this assay, T cells were co-cultured with either uninfected or HIV-1 infected CEM cells in the presence of the bispecific antibodies. After 14-16 hours of co-culture, the expression of cytokines and activation markers in the T cells by flow cytometry were measured. It was observed that in the presence of HIV-infected cells, VRC07(G54W)(Fab)-anti-hCD3 can activate T cells, whereas in the absence of HIV, minimal activation was observed (FIG. 13). It was also found that none of the control bispecific antibodies, including the one with specificity for human CD3 but not HIV Env, could activate T cells indicating the requirement for binding of both specificities for T cell activation. The increased expression of IFN-γ, TNF-OC and CD69 was observed in both CD4⁺ (FIG. 13 A) and CD8⁺ (FIG. 13B) T cells, indicating that VRC07(G54W)(Fab)-anti-hCD3 can induce activation of both T cell subsets. Higher expression of IFN-γ and TNF-OC in CD8⁺ T cells was observed compared to CD4⁺ T cells, but this may be due to previously observed delayed kinetics in expression of effector molecules by CD4⁺ T cells on activation by bispecific antibodies (Brischwein et al, Molecular immunology 43, 1129-1143, 2006). Also, the increased expression of CD69, which is an early pan-T cell activation marker, was similar for both T cell subsets. These results demonstrate that

VRC07(G54W)(Fab)-anti-hCD3 is able to mediate activation of T cells only in the presence of infection.

Lysis of chronic and latent HIV⁺ cells

VRC07(Fab)-anti-hCD3 was tested for cytotoxic function using HIV⁺ cells as targets. The HIV⁺ cells were used as targets are either chronically infected CEM cells (CEM-IIIb) or latently infected cell lines (ACH2, Jl.l, OM10). The CEM-IIIb cells constitutively express HIV Env on their cell surface, whereas the latent cell lines have to be activated to express HIV Env indicative of HIV latency (FIG. 14A). Despite the difference in constitutive and inducible expression of HIV Env on these chronic and latent cell lines, VRC07(G54W)(Fab)-anti-hCD3 is equally able to redirect T cells to mediate lysis of these cell lines in a dose-dependent manner (FIG. 14B). These results indicate that

VRC07(G54W)(Fab)-anti-hCD3 is able to activate latent cells to express HIV Env which enables the lysis of HIV Env⁺ cells by activated primary T cells.

To further evaluate the activity of VRC07(Fab)-anti-hCD3 on HIV latency, a primary T cell model for HIV latency was used (Saleh et al, Blood 110, 4161-4164, 2007; Saleh et al, Retrovirology 8, 80, 2011). In this assay, CCL19-treated resting CD4⁺ T cells that were infected with HIV-1 to establish latency were co-cultured with syngeneic CD8⁺ T cells in the presence of VRC07(G54W)(Fab)-anti-hCD3 or a control bispecific antibody. After overnight co-culture, there was a significant reduction in the expression of HIV Env on the surface of the CD4⁺ T cells in the presence of VRC07(G54W)(Fab)-anti- hCD3 compared to the control (FIGs. 4c, d). This indicates that VRC07(G54W)(Fab)-anti-hCD3 is also able to mediate lysis of latently infected primary CD4⁺ T cells. Overall these in vitro results indicate that the bispecific antibody that is capable of activating HIV latency and targeting these reactivated latent cells for T cell-mediated lysis.

In vivo effects of VRC07(G54W)(Fab)-anti-rhCD3

To evaluate the function and in vivo effect of the bispecific antibody, a latency model was established after infection of rhesus macaques with SHIV-BaL (Methods, FIG. 17). After pilot studies optimizing the antibody dosages and treatment schema, it was evaluated whether VRC07(G54W)(Fab)- anti-rhCD3 is able to target T cells and activate latent provirus in vivo. Anti-retroviral therapy (ART) treated SHIV-BaL infected rhesus macaques were administered a 25 g/kg dose of either

VRC07(G54W)(Fab)-anti-rhCD3 (Treatment, n=5) or a control bispecific antibody (Control, n=4) at 3-4 day intervals for a total of 6 doses. All animals were treated with ART throughout the study to repress viremia and plasma SHIV gag RNA was undetectable for three consecutive weeks prior to antibody infusion (FIG. 17B).

To determine the in vivo biological effects of VRC07(G54W)(Fab)-anti-rhCD3, PBMCs at 0, 1 and 24 hr after each dosing and assessed the frequency of CD3⁺ cells in PBMCs were collected. A significant decline in CD3⁺ population (from 30% to 5% on average) was observed at 1 hr post-infusion in VRC07(G54W)(Fab)-anti-rhCD3-treated animals, followed by a quick rebound to the original level by 24 hr (FIG. 15 A, right), suggesting a redistribution of T cells targeted by VRC07(G54W)(Fab)-anti- rhCD3. In contrast, regardless of a slight fluctuation, the frequency of CD3⁺ T cells in control animals was unchanged for up to 20 days (FIG. 15A, left). The T cell-activating effect of VRC07(G54W)(Fab)- anti-rhCD3 was assessed by measuring cytokine/chemokine release in plasma after each dosing. While the control bispecific antibody failed to drive any detectable cytokine release, VRC07(Fab)-anti-rhCD3- treated animals showed substantial production of TNF-a (300-3000 pg/mL), ΜΙΡ-1β (200-2000 pg/mL), IL-10 (10-60 pg/mL) and IFN-γ (10-70 pg/mL) at 1 hr post-infusion; levels then dropped to steady state by 24 hr (FIG. 15B, C; FIG. 18B, C). This result demonstrates successful in vivo activation of the monkey T cells, presumably including the cells harboring latent virus, by the anti-CD3 arm of the bispecific antibody, which may result in the surface expression of HIV Env for bispecific antibody targeting by its VRC07 arm. Notably, both T cell depletion and cytokine peaks were less remarkable after dose 4; this blunted effect may be explained by the emergence of immune responses against VRC07(G54W)(Fab)-anti-rhCD3 after 14 days (data not shown). To assess the bioavailability of

VRC07(G54W)(Fab)-anti-rhCD3 over time, plasma concentrations of VRC07(G54W)(Fab)-anti-rhCD3 were measured serially up to 20 days post-administration. Maximum antibody concentration of 80 ng/mL occurred at day 4 (post-dose 2), then gradually declined to baseline level by day 20 (FIG. 18 A). Reduction in proviral load

Several attempts have been made to activate HIV latency and purge the virus out of latently infected cells for immune targeting (Prins et al, AIDS 13, 2405-2410, 1999; Archin et al, Nature 487, 482-485, 2012; Lehrman et al, Lancet 366, 549-555, 2005; Margolis et al, Current opinion in HIV and AIDS 6, 25-29, 2011). However, none of these approaches has proven capable of eliminating the infection completely or preventing viral rebound after termination of therapy. It is demonstrated in latently infected T cell lines and primary T cells that VRC07(G54W)(Fab)-anti- rhesus CD3 is able to activate T cells by engaging CD3, and bind to Env on HIV-1 infected cells to stimulate proviral gene expression. Infection can then be eliminated through T cell-dependent lysis of latently infected cells (FIGs. 3, 4). To validate this virus purging / latency elimination effect in animals, PBMCs were collected at 0, 1 and 24 hr after each dosing and assessed for proviral DNA levels by real-time PCR. As shown in FIG. 16 A, while no significant change in proviral load was detected in the control group (FIG. 16A, left), a 4-fold proviral reduction was seen in animals treated with VRC07(Fab)-anti-rhCD3 by the time the second dose was administered and remained low for up to 20 days (FIG. 16 A, right). A significantly greater reduction was observed at day 20 in the VRC07(G54W)(Fab)-anti-rhCD3 group compared to the controls (p = 0.002, FIG. 16B). The animals were followed beyond 20 days and provirus in the treatment group remained low throughout this time; however, because the control group started clearing provirus after 20 days post-infusion (which was 50 days post-infection) due to the self- limiting nature of the SHIV-BaL infection model, the difference between the two groups was no longer prominent. To test whether VRC07(G54W)(Fab)-anti-rhCD3 can target HIV reservoirs in tissues and lymph nodes, axillary lymph node and rectum biopsies before and after infusion were collected. 4 out of 5 treatment animals showed 3-4 fold proviral reduction in rectum and 2 out of 5 showed reduction in lymph nodes. In contrast, only 1 out of 4 control animals showed reduction of around 2 fold in these tissue compartments (data not shown). In summary, these data validate the in vitro study by showing that prolonged (1 hour) infusion of VRC07(G54W)(Fab)-anti-rhCD3 is able to repress the pro virus in various tissue reservoirs for at least 20 days in ART-pre-treated, aviremic animals.

Discussion

In this study, a novel anti-HIV bispecific antibody that can target the latent reservoir of HIV infected cells shown. This bispecific antibody can bind to HIV Env, activate T cells via CD3 binding and mediate the T cell dependent lysis of chronically or latently infected cell lines. Treatment of chronically SHIV -infected Indian rhesus macaques with a rhesus homolog of this bispecific antibody led to a significant reduction in the number of latently infected cells in these animals. Therefore the ability of this anti-HIV bispecific antibody to activate and mediate elimination of latent HIV infection creates a new class of novel immunotherapeutic proteins that can be used in conjunction with HAART to treat HIV infection.

Bispecific antibodies have been designed to redirect T cells for targeting multiple tumors or infections including HIV-1 infection (Traunecker et al, The EMBO journal 10, 3655-3659, 1991 ; Lum et al., Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation 18, 1012-1022, 2012; Fernandez-Sesma et al, J Immunol 160, 1841-1849, 1998; Chamow et al, J Immunol 153, 4268-4280, 1994; Berg et al, Proceedings of the National Academy of Sciences of the United States of America 88, 4723-4727, 1991 ; Okada et al., Immunology letters 38, 195-199, 1993; Yin et al, Microbiology and immunology 45, 101-108, 2001 ; Newell et al, Annals of the New York Academy of Sciences 636, 279-287, 1991 ; Fernandez-Sesma et al., Journal of virology 70, 4800-4804, 1996). Multiple formats have been used to generate bispecific antibodies including quadromas, F(ab')₂, heterodimer scFv, heterodimeric Fab, diabody and tandem scFv (Kufer et al, Trends in biotechnology 22, 238-244, 2004). A bispecific protein in the tandem scFv format that targets B cells has become a viable therapy for the treatment of B-cell leukemias (Bargou et al, Science 321, 974-977, 2008; Klinger et al, Blood 119, 6226-6233, 2012) with many other bispecific antibodies that target different tumors been currently tested in phase I clinical trials (May et al, Biochemical pharmacology 84, 1105-1112, 2012). The use of bispecific antibodies for eliminating HIV-1 infected cells has been limited to either using soluble CD4 or use of anti-HIV- 1 antibodies that were limited in their coverage of HIV-1 strains (Traunecker et al, The EMBO journal 10, 3655-3659, 1991 ; Chamow et al, J Immunol 153, 4268-4280, 1994; Okada et al, Immunology letters 38, 195-199, 1993; Yin et al, Microbiology and immunology 45, 101-108, 2001). Here, an anti-HIV bispecific antibody that links the Fab of VRC07, a highly potent broadly neutralizing antibody to the scFv of an activating anti-hCD3 antibody was produced. The use of VRC07 makes use of its much higher affinity for HIV Env than soluble CD4 or other anti-HIV antibodies, and enables the targeting of most of the circulating strains of HIV-1 by this bispecific antibody. This bispecific antibody retains the potent neutralization property of VRC07 and also binds to human CD3 on the surface of T cells. The use of the slightly larger Fab-scFv format for this bispecific antibody may lead to longer half lives compared to the extremely short half lives observed for the smaller double scFv constructs. More variants of these bispecific antibodies can also be generated that target other conserved sites on the HIV Env, such as the MPER, that can be used in combination with the current VRC07 -based bispecific antibody to facilitate the recognition of almost all circulating strains of HIV-1 and prevent any viral escape.

The activation of T cells by anti-CD3 antibodies has been well documented (Newell et al, Annals of the New York Academy of Sciences 636, 279-287, 1991 ; Tsoukas et al., J Immunol 135, 1719- 1723, 1985; Van Wauwe et al, J Immunol 124, 2708-2713, 1980; Chang et al, Proceedings of the National Academy of Sciences of the United States of America 78, 1805-1808, 1981 ; von Wussow et al, J Immunol 127, 1197-1200, 1981 ; Tsoukas et al., Advances in experimental medicine and biology 184, 365-385, 1985; Ellenhorn et al, Transplantation 50, 608-612, 1990). This activation is a result of the TCR signaling machinery involving the initial induction of transcription factors like NF-kappa B

(Jamieson et al, J Immunol 147, 416-420, 1991) as well as AP-1 and NFAT (Williams et al, Cytokine 39, 63-74, 2007) followed by production of effector molecules like cytokines and chemokines by these activated T cells. In the case of HIV-1 infection, anti-CD3 antibodies have been shown to induce HIV-1 replication in latently infected CD4+ T cells (Williams et al, Cytokine 39, 63-74, 2007; Chun et al, The Journal of experimental medicine 188, 83-91, 1998) and have been tested in clinical trials for the treatment of latent HIV-1 infection (Prins et al, AIDS 13, 2405-2410, 1999; Fraser et al, AIDS 14, 659- 669, 2000). Although some reduction in HIV-1 infected T cell reservoir was observed, significant cytotoxicities were associated with this treatment due to global T cell activation induced by this antibody. In the case of the bispecific antibody, which includes monovalent binding to CD3, nonspecific activation of T cells is avoided and only in the presence of HIV Env, activation of T cells was observed. In addition, the bispecific antibody also could reactivate latently infected T cells by binding to CD3 and HIV Env expressed on the cell surface. This multifunctional ability to both reactivate latently infected cells and target them for T cell mediated lysis makes this class of bispecific antibodies attractive therapeutic reagents against HIV compared to the current strategies that reactivate latent cells but rely on indirect mechanisms for their eradication (Lafeuillade et al, Current HIV/AIDS reports 9, 121-131, 2012).

The marginal effect on the tissue reservoirs of latent infection may be a result of the short plasma half -life of these bispecific antibodies and the inability to fully activate the latent reservoirs in the tissue compartments. Alternative ways of administering these antibodies with constant infusion via osmotic pumps, as was employed to administer the bispecific antibodies in clinical trials (Klinger et al., Blood 119, 6226-6233, 2012), might aid in better targeting of these tissue reservoirs. Also, drug-based strategies that have been used to activate latent HIV infection with moderate success (Chun et al., AIDS 26, 1261-1268, 2012; Archin et al., Nature 487, 482-485, 2012; Margolis et al., Current opinion in HIV and AIDS 6, 25-29, 2011) may have some benefit. For example, a combination of drugs such as vorinostat with these bispecific antibodies may lead to much better activation and elimination of HIV latency in both the peripheral and tissue reservoirs. Thus, these bispecific antibodies that target HIV Env and CD3 will aid in the development of novel strategies to eliminate latent HIV reservoirs and clear HIV infection.

METHODS

Summary. The bispecific antibodies were constructed by linking the Fab fragment sequence of VRC07 to anti- CD3 scFv sequence, using overlapping PCR. The bispecific antibodies were then produced in bulk quantities by recombinant expression in mammalian cells. The binding of bispecific antibodies to soluble and cell surface antigens was measured using either ELISA or flow cytometric assays as described in Methods. To measure T cell activation, naive human T cells were co-cultured with either uninfected or HIV-infected cells in the presence of bispecific antibodies and the expression of intracellular cytokines and activation markers was quantitated by flow cytometric analysis. For cytotoxicity assays, a chronically infected cell line (CEM-IIIb) and latent cell lines (ACH2, Jl. l and OM10) were used as targets and enriched human T cells as effectors. For a primary cell model of HIV latency, HIV-BaL infection of CCL19-treated resting human CD4⁺ T cells was performed as described previously (Saleh et al, Blood 110, 4161-4164, 2007; Saleh et al, Retrovirology 8, 80, 2011). After 3 days of infection, syngeneic CD8⁺ T cells were co-cultured with the infected CD4⁺ T cells in the presence of the bispecific antibodies for 14-16 hours and the expression of HIV Env and Gag in the CD4⁺ T cells was measured by flow cytometric analysis. All animal experiments were reviewed and approved by the Animal Care and Use Committee of the Vaccine Research Center, NIAID, NIH. Naive rhesus macaques were intrarectally challenged with SHIV-BaL and after 7 weeks of infection, were treated with a combination of three anti-retroviral drugs for another 11 weeks before administration of the bispecific antibodies (25 μg/kg) every 3-4 days. The proportion of T cells in peripheral blood of the treated animals was quantitated using flow cytometry. Multiple cytokines and chemokines in plasma were also measured using a bead-based luminex assay (Millipore). Real-time PCR was performed for absolute quantitation of proviral DNA load in PBMCs, lymph nodes and rectal biospsies as described previously (Bolton et al, J Immunol 184, 303-314, 2010).

Construction and purification of bispecific antibodies. The cDNAs for human and simianized versions of VRC07(G54W)(Fab) were PCR amplified from the IgG vector and assembled to anti-hCD3 or anti-rhCD3 scFv, respectively, using overlapping PCR. Anti-hCD3 and anti-rhCD3 scFv sequences were synthesized using human preferred codons (GenScrip). The control bispecific antibodies based on an anti-VRCOl idiotypic antibody 5B8 were similarly assembled by overlapping PCR. All bispecific antibodies were configured as Fab-scFv. Assembled cDNAs were cloned into mammalian expression vector VRC8400 that contains a 3C protease cleavage site and a 6xhis tag at the C-terminus.

To produce large quantity of the bispecific antibodies, 293F cells were transfected with different bispecific expression vectors using 293Fectin according to the manufacturer' s protocol (Life Technologies). 5 days post-transfection, cell culture supernatant was harvested, filtered, and buffer exchanged to Ni-chromatographic binding buffer (50 mM Tris pH 8.0, 150 mM NaCl). The proteins were initially purified using a Ni-Affinity Chromatography column (GE Healthcare Biosciences), followed by gel-filtration using a HiLoad 16/600 Superdex 200 pg column (GE Healthcare Biosciences). Only the monomer fractions were collected for further characterization.

The purified bispecific antibody was treated with 3C protease (Novagen) at 37°C to remove the 6xhis tag, followed by passing the products through a Ni-Affinity Chromatography column for removing the cleaved 6xhis tag. The flow-through was collected, buffer-exchanged to PBS, and concentrated. The endotoxin level of all purified antibodies was measured and samples with high levels of endotoxin were passed through an endotoxin-removal column to remove excess endotoxin (Hyglos). The endotoxin level in all samples used in the in vitro and in vivo studies was <1 EU/mg.

Binding of bispecific antibodies to soluble and cell surface antigens. For binding to soluble antigen, microtiter plates were coated with a resurfaced HIV envelope core protein (RSC3) overnight at 4°C. The next day, plates were blocked with 5% BSA and after washing, increasing amounts of bispecific antibodies were allowed to bind to the coated RSC3. Bound bispecific antibodies were detected with a peroxidase-conjugated anti-human Fab (Jackson Immunoresearch) and tetramethyl benzidine detection (KPL). The binding of the bispecific antibodies to CD3 and HIV Env on the cell surface was performed using either human (HPB-ALL) or monkey (HSC-F) T cell lines and chronically HIV-infected CEM cells (CEM-IIIb), respectively. The cells were incubated with the bispecific antibodies (20 μg/ml) for 20 min and bound antibodies were detected by flow cytometry using a FITC- conjugated anti-human Fab (Jackson Immunoresearch).

Neutralization assay. Neutralization of HIV-1 envelope pseudotyped viruses by the mono- and bispecific antibodies was measured using Tzm-bl target cells by modification of previously described methods (Wei et al, Antimicrobial agents and chemotherapy 46, 1896-1905, 2002; Arkhipov et al, Biophysical journal 97, 2061-2069, 2009).

Activation of T cells by bispecific antibodies. Human T cells were enriched from buffy coats obtained from naive donors (NIH Blood Bank) using magnetic beads (Miltenyi Biotec). These cells were co-cultured for 14-16 hours with either uninfected or HIV-infected CEM cells in the presence of the bispecific antibodies (1 μg/ml) and brefeldin A. The cells were then stained for surface expression of T cell markers (CD3, CD4, and CD8) and activation markers (CD25 and CD69) followed by intracellular staining for cytokines (IFN-γ, TNF-OC and IL-2) using fluorescently conjugated antibodies (BD

Biosciences, eBioscience, Biolegend). The number of CD4 and CD8 T cells expressing each cytokine or activation marker was determined by running the samples on an LSRII flow cytometer and analysis using Flowjo software (Treestar).

Activation and targeted lysis of HIV-infected cell lines. Latent cell lines (ACH2, J 1.1 and

OM10) were obtained from the NIH AIDS Reagent Program. The activation of these cells was performed by culturing in the presence or absence of TNF-OC (10 ng/ml) for 14-16 hours. Activation was measured by determining the expression of cell surface HIV envelope protein by flow cytometry using an allophycocyanin-conjugated anti-HIV Env antibody (2G12). The CEM-IIIb, ACH2, Jl.l and OM10 cells were labeled with the membrane dye PKH-26 (Sigma) and used as target cells in a cytotoxicity assay. These labeled target cells were co-cultured for 14-16 hours at a E:T ratio of 10: 1 with enriched human T cells as effector cells in the presence of increasing amounts of the bispecific antibodies. The extent of cell lysis in the target cells was determined by staining with a live/dead cell marker (Invitrogen) and measuring the number of dead cells in the labeled target cell population by running the samples on an LSRII flow cytometer followed by analysis using Flowjo software (Treestar).

Latent infection of primary human T cells. Human PBMCs were obtained from naive donors (NIH Blood Bank) and resting CD4⁺ and CD8⁺ T cells were magnetically enriched from them. The CD8⁺ T cells were kept in culture in IL-2 (10 IU/ml) containing media. The resting CD4⁺ T cells were first cultured in the presence of CCL19 (100 nM) for 3 days. These cells were then infected with HIV BaL (MOI = 0.1) by spinoculation with centrifugation of the cells with the virus at 1200 g for 2 hours at room temperature. These cells were then washed twice with media and cultured for 3 days in the presence of IL-2 (10 IU/ml). The infected resting CD4⁺ T cells were then co-cultured with allogeneic CD8⁺ T cells in the presence of the bispecific antibodies (5 μg/ml) for 14-16 hours. The co-cultures were then stained with fluorescently conjugated antibodies against T cell markers - CD3, CD4 and CD8, and HIV envelope (2G12) and gag (KC57, Coulter) proteins, followed by flow cytometric analysis.

Treatment of nonhuman primates with bispecific antibodies. Naive rhesus macaques were screened for the high binding allele for the CD3 gene by PCR on the genomic DNA using primers specific for the CD3 gene (Liu et al, Immunology and cell biology 85, 357-362, 2007) and the selected animals were intrarectally challenged with SHIV-BaL. The infection was confirmed by measuring plasma viremia weekly by a quantitative real time RT PCR and allowed to progress for 7 weeks. Then a combination of three anti-retroviral drugs - tenofovir (30-40 mg/kg/day, Gilead Sciences), emtricitabine (20 mg/kg/day, Gilead Sciences) and raltegravir (100 mg BID, Merck), was administered to these animals to lower viremia to undetectable levels. 11 weeks after the start of ART administration, the bispecific antibodies (25 μg/kg) were administered over an hour period to these infected animals every 3- 4 days. Whole blood samples, lymph node and rectal biopsies were obtained at different time points from all animals to assay for various immunological and virological parameters.

Quantitation of bispecific antibodies during treatment. Plasma samples were obtained from rhesus macaques at various times during treatment to quantitate the amount of functional bispecific antibodies. Recombinant soluble rhesus CD38 protein (Sino Biological Inc.) was coated onto the wells of ELISA plates (Nunc) overnight followed by blocking of the wells with 5% BSA solution. The plasma samples were then allowed to bind to the CD38 protein and bound bispecific antibodies were detected using a peroxidase-conjugated RSC3 probe (Jackson Immunoresearch) and tetramethyl benzidine detection (KPL). The concentration of bispecific antibodies was quantitated from a standard curve plotted using known concentrations of bispecific antibodies.

Measurement of peripheral T cell levels during treatment. Whole blood samples were obtained from rhesus macaques at various times during treatment with the bispecific antibodies. The whole blood was directly stained with fluorescently conjugated antibodies against CD3, CD4, CD8, CD69 and human Kappa chain to assess the distribution of T cells and detect any bound bispecific antibodies on the T cells. After incubation with the antibodies, the blood was then lysed with RBC lysing buffer (BD Biosciences) and run on an LSRII flow cytometer to assess the proportions of peripheral T cells at different times during treatment.

Quantitation of cytokines and chemokines in plasma. Cytokines (TNF-a, IFN-γ, IL-Ιβ, IL-6, IL-10) and eta (β) -chemokines (ΜΙΡ-Ιβ, MCP-1 and eotaxin) were measured simultaneously with either Milliplex Non-human Primate Cytokine/Chemokine kit (Millipore) or Fluorokine^® Multianalyte Profiling kit (R&D) using the Luminex xMAP™ multiplexed bead system (Millipore), according to the manufacturer's instructions. Results obtained from the Luminex xMAP™ system were analyzed automatically by the Luminex^® xPONENT^® software program (Millipore) using a standard curve derived from recombinant cytokine and chemokine standards.

Proviral DNA quantitation. Blood samples were collected at 0, 1 and 24 hr post-infusion at each dosing. PBMCs and plasma were separated via Ficoll density centrifugation. Genomic DNA was extracted from 5xl0⁶ PBMCs with DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer's instructions. Real-time PCR amplification was performed using the Bio-Rad CFX96 cycler with 1 μg of genomic DNA as a template. For the absolute quantitation of provirus DNA load, the standard plasmids pTSIVgag and pT Albumin were constructed by PCR cloning. Serial dilutions of plasmid DNA were prepared and stored at -20 °C until use. Previously published primers and probes were used for real-time

CTTC(AG)TCAGT(CT)TGTTTCACTTTCTCTTCTGCG-3' TAMRA (SEQ ID NO: 1770); Monkey Albumin: forward primer: 5'- TGCATGAGAAAACGCC AGTAA-3 ' (SEQ ID NO: 1771); reverse primer: 5 ' -ATGGTCGCCTGTTC ACC AA-3 ' (SEQ ID NO: 1772); Probe: 5'FAM- DNA probe-3'BHQ- 1 (SEQ ID NO: 1773). The cycling conditions were as follows: 95°C for 2 minutes, followed by 43 cycles of: 95°C for 15 seconds, 60°C for 60 seconds.

Example 3

Bispecific antibodies to an HIV-1 envelope protein and CD3

for the treatment of HIV-1

This example describes a particular method that can be used to treat HIV in a human subject by administration of one or more bispecific antibodies that specifically bind an HIV-1 envelope protein (such as gpl20 or gp41) and CD3. Although particular methods, dosages, and modes of administrations are provided, one skilled in the art will appreciate that variations can be made without substantially affecting the treatment.

Based upon the teaching disclosed herein HIV-1 can be treated by administering a

therapeutically effective amount of one or more of the bispecific antibodies that specifically bind an HIV-1 envelope protein (such as gpl20 or gp41) and CD3 described herein, thereby reducing or eliminating HIV infection.

Screening subjects

In particular examples, the subject is first screened to determine if they have HIV. Examples of methods that can be used to screen for HIV include a combination of measuring a subject's CD4+ T cell count and the level of HIV in serum blood levels. Additional methods using the gpl20-specific mAbs disclosed herein can also be used to screen for HIV.

In some embodiments, HIV testing consists of initial screening with an enzyme-linked immunosorbent assay (ELISA) to detect antibodies to HIV, such as to HIV-1. Specimens with a nonreactive result from the initial ELISA are considered HIV-negative unless new exposure to an infected partner or partner of unknown HIV status has occurred. Specimens with a reactive ELISA result are retested in duplicate. If the result of either duplicate test is reactive, the specimen is reported as repeatedly reactive and undergoes confirmatory testing with a more specific supplemental test (e.g., Western blot or an immunofluorescence assay (IF A)). Specimens that are repeatedly reactive by ELISA and positive by IFA or reactive by Western blot are considered HIV-positive and indicative of HIV infection. Specimens that are repeatedly ELISA-reactive occasionally provide an indeterminate Western blot result, which may be either an incomplete antibody response to HIV in an infected person, or nonspecific reactions in an uninfected person. IFA can be used to confirm infection in these ambiguous cases. In some instances, a second specimen will be collected more than a month later and retested for subjects with indeterminate Western blot results. In additional examples, nucleic acid testing (e.g., viral RNA or proviral DNA amplification method) can also help diagnosis in certain situations.

The detection of HIV in a subject's blood is indicative that the subject has HIV and is a candidate for receiving the therapeutic compositions disclosed herein. Moreover, detection of a CD4+ T cell count below 350 per microliter, such as 200 cells per microliter, is also indicative that the subject is likely to have HIV.

Pre-screening is not required prior to administration of the therapeutic compositions disclosed herein

Pre-treatment of subjects

In particular examples, the subject is treated prior to administration of a therapeutic agent that includes one or more antiretroviral therapies known to those of skill in the art. However, such pre- treatment is not always required, and can be determined by a skilled clinician.

Administration of therapeutic compositions

Following subject selection, a therapeutically effective dose of one or more of the bispecific antibodies that specifically bind an HIV-1 envelope protein (such as gpl20 or gp41) and CD3 described herein is administered to the subject (such as an adult human or a newborn infant either at risk for contracting HIV or known to be infected with HIV). Additional agents, such as anti-viral agents, can also be administered to the subject simultaneously or prior to or following administration of the disclosed agents. Administration can be achieved by any method known in the art, such as oral administration, inhalation, intravenous, intramuscular, intraperitoneal, or subcutaneous.

The amount of the composition administered to prevent, reduce, inhibit, and/or treat HIV or a condition associated with it depends on the subject being treated, the severity of the disorder, and the manner of administration of the therapeutic composition. Ideally, a therapeutically effective amount of an agent is the amount sufficient to prevent, reduce, and/or inhibit, and/or treat the condition (e.g. , HIV) in a subject without causing a substantial cytotoxic effect in the subject. An effective amount can be readily determined by one skilled in the art, for example using routine trials establishing dose response curves. As such, these compositions may be formulated with an inert diluent or with an pharmaceutically acceptable carrier.

In one specific example, antibodies are administered at 5 mg per kg every two weeks or 10 mg per kg every two weeks depending upon the particular stage of HIV. In an example, the antibodies are administered continuously. In another example, antibodies or antibody fragments are administered at 50 μg per kg given twice a week for 2 to 3 weeks.

Administration of the therapeutic compositions can be taken long term (for example over a period of months or years).

Assessment

Following the administration of one or more therapies, subjects having HIV can be monitored for reductions in HIV levels, increases in a subjects CD4+ T cell count, or reductions in one or more clinical symptoms associated with HIV. In particular examples, subjects are analyzed one or more times, starting 7 days following treatment. Subjects can be monitored using any method known in the art. For example, biological samples from the subject, including blood, can be obtained and alterations in HIV or CD4+ T cell levels evaluated.

Additional treatments

In particular examples, if subjects are stable or have a minor, mixed or partial response to treatment, they can be re-treated after re-evaluation with the same schedule and preparation of agents that they previously received for the desired amount of time, including the duration of a subject's lifetime. A partial response is a reduction, such as at least a 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 70% in HIV infection, HIV replication or combination thereof. A partial response may also be an increase in CD4+ T cell count such as at least 350 T cells per microliter.

In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that illustrated embodiments are only examples and should not be considered a limitation on the scope of the claims. We therefore claim all that comes within the scope and spirit of these claims.

Claims

We claim:

1. An isolated bispecific monoclonal antibody, comprising:

a first antigen binding domain comprising a Fab or a scFv, wherein the Fab and the scFv comprise a heavy chain variable region and a light chain variable region comprising a heavy chain complementarity determining region (H-CDR)l, an H-CDR2, an H-CDR3, a light chain complementarity determining region (L-CDR)l, an L-CDR2, and an L-CDR3, and wherein the first antigen binding domain specifically binds to gpl20 or gp41 and is neutralizing;

a second antigen binding domain comprising a Fab or a scFv, wherein the Fab and the scFv comprise a heavy chain variable region and a light chain variable region comprising a heavy chain H- CDR1, an H-CDR2, an H-CDR3, a L-CDRl, an L-CDR2, and an L-CDR3, wherein the second antigen binding domain specifically binds CD3, activates T cells, and activates expression of proviral HIV-1 DNA in T cells.

2. The isolated bispecific antibody of claim 1 , wherein the first antigen binding domain specifically binds to gpl20.

3. The isolated bispecific antibody of claim 2, wherein the first antigen binding domain specifically binds to a CD4 binding site of gpl20.

4. The isolated bispecific antibody of any one of claims 1-3, wherein the H-CDR1, the H- CDR2, and the H-CDR3 are from the heavy chain of a VRCOl-like monoclonal antibody and the L- CDR1, the L-CDR2, and the L-CDR3 are from the light chain of a VRCOl-like monoclonal antibody.

5. The isolated bispecific monoclonal antibody of any one of claims 1-4, wherein the heavy chain variable region of the first antigen binding domain comprises one of:

(a) amino acids 26-33 (H-CDR1), 51-58 (H-CDR2), and 97-110 (H-CDR3) of SEQ ID NO: 1 (VRCOl HC);

(b) amino acids 26-33 (H-CDR1), 51-58 (H-CDR2), and 97-114 (H-CDR3) of SEQ ID NO: 1461 (VRC07 HC);

(c) amino acids 26-33 (H-CDR1), 51-58 (H-CDR2), and 97-114 (H-CDR3) of SEQ ID NO: 1483 (VRC07 HC G54W); or

(c) amino acids 26-33 (H-CDR1), 51-58 (H-CDR2), and 97-114 (H-CDR3) of SEQ ID NO: 1724 (VRC07 HC G54H).

6 The isolated bispecific monoclonal antibody of any one of claims 1-4, wherein the heavy chain variable region of the first antigen binding domain comprises one of:

(a) the amino acid sequence set forth as SEQ ID NO: 1 (VRCOl HC);

(b) the amino acid sequence set forth as SEQ ID NO: 1461 (VRC07 HC); (c) the amino acid sequence set forth as SEQ ID NO: 1483 (VRC07 HC G54W); or

(d) the amino acid sequence set forth as SEQ ID NO: 1724 (VRC07 HC G54H).

7. The isolated bispecific monoclonal antibody of any one of claims 1-6, wherein the light chain variable region of the first antigen binding domain comprises one of

(a) amino acids 27-30 (L-CDRl), 48-50 (L-CDR2), and 87-91 (L-CDR3) of SEQ ID NO: 2 (VRCOl LC); or

(b) amino acids 27-30 (L-CDRl), 48-50 (L-CDR2), and 87-91 (L-CDR3) of SEQ ID NO: 1730 (VRCOl El/12 del V3S).

8. The isolated bispecific monoclonal antibody of any one of claims 1-6, wherein the light chain variable region of the first antigen binding domain comprises one of

(a) the amino acid sequence set forth as SEQ ID NO: 2 (VRCOl LC); or

(b) the amino acid sequence set forth as SEQ ID NO: 1730 (VRCOl El/12 del V3S).

9. The isolated bispecific monoclonal antibody of any one of claims 1-4, wherein the heavy chain variable region and the light chain variable region of the first antigen binding domain comprise one of:

(a) amino acids 26-33 (H-CDR1), 51-58 (H-CDR2), and 97-110 (H-CDR3) of SEQ ID NO: 1 (VRCOl HC), and amino acids 27-30 (L-CDRl), 48-50 (L-CDR2), and 87-91 (L-CDR3) of SEQ ID NO:

2 (VRCOl LC), respectively;

(b) amino acids 26-33 (H-CDR1), 51-58 (H-CDR2), and 97-114 (H-CDR3) of SEQ ID NO: 1461 (VRC07 HC), and amino acids 27-30 (L-CDRl), 48-50 (L-CDR2), and 87-91 (L-CDR3) of SEQ ID NO: 2 (VRCOl LC), respectively;

(c) amino acids 26-33 (H-CDR1), 51-58 (H-CDR2), and 97-114 (H-CDR3) of SEQ ID NO: 1483

(VRC07 HC G54W), and amino acids 27-30 (L-CDRl), 48-50 (L-CDR2,) and 87-91 (L-CDR3) of SEQ ID NO: 2 (VRCOl LC), respectively;

(d) amino acids 26-33 (H-CDR1), 51-58 (H-CDR2), and 97-114 (H-CDR3) of SEQ ID NO: 1724 (VRC07 HC G54H), and amino acids 27-30 (L-CDRl), 48-50 (L-CDR2), and 87-91 (L-CDR3) of SEQ ID NO: 2 (VRCOl LC), respectively;

(e) amino acids 26-33 (H-CDR1), 51-58 (H-CDR2), and 97-110 (H-CDR3) of SEQ ID NO: 1 (VRCOl HC), and amino acids 27-30 (L-CDRl), 48-50 (L-CDR2), and 87-91 (L-CDR3) of SEQ ID NO: 1730 (VRCOl El/12 del V3S), respectively;

(f) amino acids 26-33 (H-CDR1), 51-58 (H-CDR2), and 97-114 (H-CDR3) of SEQ ID NO: 1461 (VRC07 HC), and amino acids 27-30 (L-CDRl), 48-50 (L-CDR2), and 87-91 (L-CDR3) of SEQ ID NO:

1730 (VRCOl El/12 del V3S), respectively; (g) amino acids 26-33 (H-CDRl), 51-58 (H-CDR2), and 97-114 (H-CDR3) of SEQ ID NO: 1483 (VRC07 HC G54W), and amino acids 27-30 (L-CDRl), 48-50 (L-CDR2), and 87-91 (L-CDR3) of SEQ ID NO: 1730 (VRCOl El/12 del V3S), respectively; or

(h) amino acids 26-33 (H-CDRl), 51-58 (H-CDR2), and 97-114 (H-CDR3) of SEQ ID NO: 1724 (VRC07 HC G54H), and amino acids 27-30 (L-CDRl), 48-50 (L-CDR2), and 87-91 (L-CDR3) of SEQ

ID NO: 1730 (VRCOl El/12 del V3S), respectively.

10. The isolated bispecific monoclonal antibody of any one of claims 1-4, wherein the heavy chain variable region of the first antigen binding domain comprises one of:

(a) the amino acid sequence set forth as SEQ ID NO: 1 (VRCOl HC), and the amino acid sequence set forth as SEQ ID NO: 2 (VRCOl LC), respectively;

(b) the amino acid sequence set forth as SEQ ID NO: 1461 (VRC07 HC), and the amino acid sequence set forth as SEQ ID NO: 2 (VRCOl LC), respectively;

(c) the amino acid sequence set forth as SEQ ID NO: 1483 (VRC07 HC G54W), and the amino acid sequence set forth as SEQ ID NO: 2 (VRCOl LC), respectively;

(d) the amino acid sequence set forth as SEQ ID NO: 1724 (VRC07 HC G54H), and the amino acid sequence set forth as SEQ ID NO: 2 (VRCOl LC), respectively;

(e) the amino acid sequence set forth as SEQ ID NO: 1 (VRCOl HC), and the amino acid sequence set forth as SEQ ID NO: 1730 (VRCOl El/12 del V3S), respectively;

(f) the amino acid sequence set forth as SEQ ID NO: 1461 (VRC07 HC), and the amino acid sequence set forth as SEQ ID NO: 1730 (VRCOl El/12 del V3S), respectively;

(g) the amino acid sequence set forth as SEQ ID NO: 1483 (VRC07 HC G54W), and the amino acid sequence set forth as SEQ ID NO: 1730 (VRCOl El/12 del V3S), respectively; or

(h) the amino acid sequence set forth as SEQ ID NO: 1724 (VRC07 HC G54H), and the amino acid sequence set forth as SEQ ID NO: 1730 (VRCOl El/12 del V3S), respectively.

11. The isolated bispecific antibody of claim 1 , wherein the first antigen binding domain specifically binds to gp41.

12. The isolated bispecific antibody of claim 11, wherein the first antigen binding domain specifically binds to the MPER of gp41.

13. The isolated bispecific monoclonal antibody of claim 11 or claim 12, wherein the heavy chain variable region of the first antigen binding domain comprises one of:

(a) amino acids 26-33 (H-CDRl), 51-60 (H-CDR2), and 99-120 (H-CDR3) of SEQ ID NO: 1485

(10E8 HC); or

(b) amino acids 26-33 (H-CDRl), 51-60 (H-CDR2), and 99-120 (H-CDR3) of SEQ ID NO: 1668 (10E8 HC6).

14 The isolated bispecific monoclonal antibody of claim 12 or claim 13, wherein the heavy chain variable region of the first antigen binding domain comprises one of:

(a) the amino acid sequence set forth as SEQ ID NO: 1485 (10E8 HC); or

(b) the amino acid sequence set forth as SEQ ID NO: 1668 (10E8 HC6).

15. The isolated bispecific monoclonal antibody of any one of claims 12-14, wherein the light chain variable region of the first antigen binding domain comprises one of

(a) amino acids 26-31 (L-CDRl), 49-51 (L-CDR2), and 87-98 (L-CDR3) of SEQ ID NO: 1486 (10E8 LC); or

(b) amino acids 26-31 (L-CDRl), 49-51 (L-CDR2), and 87-98 (L-CDR3) of SEQ ID NO: 1666 (10E8gL03 LC).

16. The isolated bispecific monoclonal antibody of any one of claims 12-14, wherein the light chain variable region of the first antigen binding domain comprises one of:

(a) the amino acid sequence set forth as SEQ ID NO: 1486 (10E8 LC); or

(b) the amino acid sequence set forth as SEQ ID NO: 1666 (10E8gL03 LC).

17. The isolated bispecific monoclonal antibody of claim 12, wherein the heavy and light chain variable regions of the first antigen binding domain comprises one of:

(a) amino acids 26-33 (H-CDR1), 51-60 (H-CDR2), and 99-120 (H-CDR3) of SEQ ID NO: 1485 (10E8 HC), and amino acids 26-31 (L-CDRl), 49-51 (L-CDR2), and 87-98 (L-CDR3) of SEQ ID NO: 1486 (10E8 LC), respectively;

(b) amino acids 26-33 (H-CDR1), 51-60 (H-CDR2), and 99-120 (H-CDR3) of SEQ ID NO: 1668 (10E8 HC6), and amino acids 26-31 (L-CDRl), 49-51 (L-CDR2), and 87-98 (L-CDR3) of SEQ ID NO:

1486 (10E8 LC), respectively;

(c) amino acids 26-33 (H-CDR1), 51-60 (H-CDR2), and 99-120 (H-CDR3) of SEQ ID NO: 1485 (10E8 HC), and amino acids 26-31 (L-CDRl), 49-51 (L-CDR2), and 87-98 (L-CDR3) of SEQ ID NO: 1666 (10E8gL03 LC), respectively; or

(d) amino acids 26-33 (H-CDR1), 51-60 (H-CDR2), and 99-120 (H-CDR3) of SEQ ID NO: 1668

(10E8 HC6), and amino acids 26-31 (L-CDRl), 49-51 (L-CDR2), and 87-98 (L-CDR3) of SEQ ID NO: 1666 (10E8gL03 LC), respectively.

18. The isolated bispecific monoclonal antibody of claim 12, wherein the heavy and light chain variable regions of the first antigen binding domain comprises one of:

(a) the amino acid sequence set forth as SEQ ID NO: 1485 (10E8 HC), and the amino acid sequence set forth as SEQ ID NO: 1486 (10E8 LC), respectively; (b) the amino acid sequence set forth as SEQ ID NO: 1668 (10E8 HC6), and the amino acid sequence set forth as SEQ ID NO: 1486 (10E8 LC), respectively;

(c) the amino acid sequence set forth as SEQ ID NO: 1485 (10E8 HC), and the amino acid sequence set forth as SEQ ID NO: 1666 (10E8gL03 LC), respectively; or

(d) the amino acid sequence set forth as SEQ ID NO: 1668 (10E8 HC6), and the amino acid sequence set forth as SEQ ID NO: 1666 (10E8gL03 LC), respectively.

19. The isolated bispecific monoclonal antibody of claim 18, wherein the heavy chain variable region of the second antigen binding domain comprises amino acids 265- 274 (H-CDR1), 289 - 305 (H-CDR2), and 338-347 (H-CDR3) of SEQ ID NO: 1510 (TR66), and the light chain variable region of the second antigen binding domain comprises amino acids 400 - 409 (L-CDR1), 425-431 (L-CDR2), and 464-472 (L-CDR3) of SEQ ID NO: 1510 (TR66).

20. The isolated bispecific monoclonal antibody of claim 18 or claim 19, wherein the heavy chain variable region of the second antigen binding domain comprises the amino acid sequence set forth as amino acids 240-360 of SEQ ID NO: 1510 (humanized TR66), and the light chain variable region of the second antigen binding domain comprises the amino acid sequence set forth as amino acids 375-482 of SEQ ID NO: 1510 (humanized TR66).

21. The isolated bispecific antibody of any one of claims 1-20, wherein the first antigen binding domain is linked to the second antigen binding domain by a peptide linker.

22. The isolated bispecific antibody of any one of claims 1-21, wherein

the second antigen binding domain is a scFv.

23. The isolated bispecific antibody of claim 22, wherein the antibody comprises a first polypeptide and a second polypeptide, wherein

(A) the first polypeptide comprises, N-terminal to C-terminal, the heavy chain variable region of the Fab, the CHI region, and the scFv, and the second polypeptide comprises, N-terminal to C-terminal, the light chain variable region of the Fab and the CL region; or

(B) the first polypeptide comprises, N-terminal to C-terminal, the heavy chain variable region of the Fab and the CHI region, and the second polypeptide comprises, N-terminal to C-terminal, the light chain variable region of the Fab, the CL region, and the scFv.

24. The isolated bispecific antibody of claim 23, wherein the first antigen binding domain specifically binds gpl20, and wherein the first and second polypeptides comprise the amino acid sequences set forth as one of:

(d) SEQ ID NO: 1749 (VRC 3487; VRC07H G54H Fab-HRV3c-His) and SEQ ID NO: 1510

(VRC3122; VRC01L-(GGGGS)₂-hCD3), respectively;

(e) SEQ ID NO: 1516 (VRC3125; VRC07H G54W Fab-HRV3c-His) and SEQ ID NO: 1751 (VRC 3485; VRC01L El/I2del V3S-(GGGGS)₂-hCD3), respectively; or

25. The isolated bispecific antibody of claim 24, wherein the first antigen binding domain specifically binds gp41, and wherein the first and second polypeptides comprise the amino acid sequences set forth as one of:

(a) SEQ ID NO: 1757 (VRC 3490; 10E8HC6 Fab-HRV3c-His) and SEQ ID NO: 1488

(VRC3111 ; 10E8gL03-(GGGGS)₃-hCD3), respectively;

(c) SEQ ID NO: 1757 (VRC 3490; 10E8HC6 Fab-HRV3c-His) and SEQ ID NO: 1490

(VRC3112; 10E8gL03-(GGGGS)₂-hCD3), respectively; or

26. The isolated bispecific monoclonal antibody of claim 1, wherein the first and second antigen binding domains are scFvs and the bispecific antibody comprises the amino acid sequence set forth as SEQ ID NO: 1475 (VRC2455; VRCOl scFv + anti-hCD3 TR66 scFv).

27. An isolated trispecific antibody, comprising the bispecific antibody of any one of claims 1-26 linked to a third antigen binding domain that specifically binds to CD28, CD40L, CD 137 or CD137L.

28. The isolated trispecific antibody of claim 27, wherein the third antigen binding domain is linked to the bispecific antibody by a peptide linker.

29. The isolated trispecific antibody of any one of claims 27-28, wherein

the first antigen binding domain is a Fab comprising the heavy chain variable region of the first antigen binding domain, a heavy chain constant 1(CH1) region, the light chain variable region of the first antigen binding domain, and a light chain constant region;

the second antigen binding domain is a first scFv; and

the third antigen binding domain is a second scFv.

30. The isolated trispecific antibody of any one of claims 27-29, wherein the trispecific antibody is a tribody.

31. The isolated trispecific antibody of claim 29, wherein the antibody comprises a first polypeptide and a second polypeptide, wherein

32. The trispecific antibody of claim 31 , wherein the scFv of the third antigen binding domain:

1766;

(b) specifically binds to CD40L;

(d) specifically binds to CD137L.

33. The isolated antibody of any of claims 1-32, wherein the antibody is labeled.

34. An isolated nucleic acid molecule encoding the bispecific antibody of any one of claims 1-26 or the trispecific antibody of any one of claims 27-32.

35. The isolated nucleic acid molecule of claim 34, operably linked to a promoter.

36. A vector comprising the one or more isolated nucleic acid molecules of any one of claims 34-35.

37. A composition comprising an effective amount of the isolated bispecific antibody of any one of claims 1-26, the trispecific antibody of any one of claims 27-32, the nucleic acid molecule of claim 34 or claim 35, or the vector of claim 36; and a pharmaceutically acceptable carrier.

38. A method for treating a subject with a human immunodeficiency virus (HIV)-l infection, comprising administering to a subject infected with HIV-1 a therapeutically effective amount of the isolated bispecific antibody of any one of claims 1-26, the trispecific antibody of any one of claims 27- 32, the nucleic acid molecule of claim 34 or claim 35, the vector of claim 36, or the composition of claim 37, thereby treating the HIV-1 infection in the subject.

39. The method of claim 38, wherein the subject has acquired immune deficiency syndrome (AIDS).

40. The method of claim 38 or claim 39, further comprising

41. The method of any one of claims 38-40, further comprising administering a

therapeutically effective amount of highly active anti-retroviral therapy (HAART) to the subject.

42. The method of claim 41, wherein administration of HAART to the subject is initiated prior to administration of the isolated bispecific antibody, the trispecific antibody, the nucleic acid molecule, the vector, or the composition.

43. The method of any one of claims 38-42, wherein the isolated bispecific antibody or the trispecific antibody, is administered to the subject by intravenous infusion for about one hour at four day intervals.

44. The method of any one of claims 38-43, wherein treating the HIV-1 infection comprises reducing the latent reservoir of HIV-1 infected cells in the subject.

45. The method of any one of claims 38-44, further comprising measuring HIV-1 viral titer in the subject.

46. The method of any one of claims 38-45, further comprising measuring pro viral HIV-1 DNA in the subject.

47. A kit comprising:

(a) the isolated bispecific antibody of any one of claims 1-26, the trispecific antibody of any one of claims 27-32, the nucleic acid molecule of claim 34 or claim 35, or the vector of claim 36, the composition of claim 37, or a combination of two or more thereof; and

(b) instructions for using the kit.

48. Use of the isolated bispecific antibody of any one of claims 1-26, the trispecific antibody of any one of claims 27-32, the one or more nucleic acid molecules of claim 34 or claim 35, or the one or more vectors of claim 36, or the composition of claim 37, or a combination of two or more thereof, to treat Human Immunodeficiency Virus type 1 infection in a subject.