BISPECIFIC ANTI-HIV BROADLY NEUTRALIZING

ANTIBODIES

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No. 62/262,091 filed on December 2, 2015. The content of the application is incorporated herein by reference in its entirety.

GOVERNMENT INTERESTS

The invention disclosed herein was made, at least in part, with Government support under Grant Nos. AI081677 and AI100148 from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health. Accordingly, the U.S. Government has certain rights in this invention.

FIELD OF THE INVENTION

This invention generally relates to bispecific antibodies and, in particular, relates to bispecific anti-HIV broadly neutralizing antibodies and uses thereof. BACKGROUND OF THE INVENTION

Human immunodeficiency virus (HIV) is a retrovirus that causes acquired immunodeficiency syndrome (AIDS). HIV-1 is the most common and pathogenic strain of the virus. For years, the development and use of potent neutralizing antibodies against HIV-1 has been a challenge for efforts to prevent or control HIV-1 infection. Indeed, HIV-1 presents several unique structural and functional determinants that conventional antibody strategies must overcome to block viral entry to target cells. These immune evasion mechanisms greatly compromise the host's capacity to mount broadly neutralizing potent antibody responses. However, early clinical studies have identified a small fraction of infected individuals that develop affinity matured antibodies with broad activity against diverse, cross-clade virus isolates (Simek, M.D. et al. J Virol 83, 7337-7348 (2009)). Over the past 5 years, several dozen monoclonal antibodies (mAbs) with potent and broad activity against the HIV-1 envelope glycoprotein (env) have been systematically isolated from these patients and extensively characterized (Klein, F. et al. Science 341, 1199-1204 (2013)). Passive administration of these broadly neutralizing mAbs (bNAbs) has been shown to confer sterilizing immunity against SHIV challenge in macaques and HIV-1 infection in humanized mouse models (Balazs, A.B. et al. Nature 481, 81-84 (2012); Hessell, A.J. et al. Nature 449, 101-104 (2007); and Mascola, J.R. et al. Nat Med 6, 207- 210 (2000)). More importantly, effective control of virus replication in HIV-1 -infected humanized mice and in SHIV-infected non-human primates by these bNAbs clearly suggested their potential clinical use to control HIV-1 infection in humans (Barouch, D.H. et al. Nature 503, 224-228 (2013); Horwitz, J.A. et al. Proc Natl Acad Sci U S A 110, 16538-16543 (2013); Klein, F. et al. Nature 492, 118-122 (2012); and Shingai, M. et al. Nature 503, 277-280 (2013)). Indeed, administration of the broadly neutralizing anti- CD4bs mAb, 3BNC117, in chronically infected HIV-1 patients successfully suppressed viremia for several days post-mAb infusion (Caskey, M. et al. Nature (2015)).

In vivo assessment of the activity of anti-HIV-1 bNAbs also revealed the limitations associated with this approach to effectively prevent or treat HIV-1 infection. For example, the therapeutic activity of bNAbs in murine and non-human primate models of HIV-1 infection was achieved only by the co-administration of at least two potent anti- HIV-1 bNAbs at relatively high and frequent dosing schedule. Indeed, administration of a single bNAb had limited capacity to suppress viremia and it was often associated with the emergence of virus escape mutants at the sites targeted by the administered anti-HIV-1 bNAb. These limitations directly reflect the immune evasion mechanisms that HTV-1 employs to escape antibody responses. Indeed, the relatively high virus mutation rate, its capacity to remain latent for several years even in chronically treated patients, as well as the chronicity of infection greatly favor the development of bNAb-resistant virus mutants resulting from selection pressure by the administered antibody. Additionally, the envelope glycoprotein of HIV-1 (env) is present at remarkably low density on the virus surface, thereby precluding high avidity concurrent interactions of both IgG Fab arms (Klein, J.S. & Bjorkman, P.J. PLoS Pathog 6, el000908 (2010)). This characteristic is unique to HTV- 1 and is a key immune evasion mechanism that greatly reduces the antiviral activity of passively administered bNAbs. Thus, there is a need for new antibody therapies for treating or preventing or controlling HIV infection.

SUMMARY OF INVENTION

This invention addresses the aforementioned unmet need by providing novel bispecific antibodies. In one aspect, the invention provides a bispecific antibody comprising (i) a first antigen-binding site that binds to a first epitope, said first antigen-binding site comprising a first heavy chain and a first light chain; (ii) a second antigen-binding site that binds to a second epitope, said second antigen-binding site comprising a second heavy chain and a second light chain. Either or both of the first heavy chain and the second heavy chain can comprise a variant hinge region that (a) is derived from a wild type IgG3 hinge region and (b) has fewer CH-1 proximal cysteine residues than the wild type IgG3 hinge region as compared to a reference antibody containing the wild type IgG3 hinge region.

In some embodiments, the wild type IgG3 hinge region can comprise the sequence of SEQ ID No. 2. In the bispecific antibody of this invention, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) or the majority (i.e., more than 50%) of the CH-1 proximal cysteine residues (i.e., cysteine residues proximal or closers to the variable regions) of the wild type IgG3 hinge region are substituted to another residue (i.e., any non-cysteine residue including but not limited to serine). The variant hinge region can be at least 60% (e.g., any number between 60% and 100%, inclusive, e.g., 60%, 70 %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%) identical to SEQ ID NO: 3. The variant hinge region can have a conservative modification as compared to SEQ ID NO: 3. In some examples, the variant hinge region can have a point mutation, an insertion, a deletion, a truncation, a fusion partner, or a combination thereof as compared to SEQ ID NO: 3. In one example, the variant hinge region can have one, two, or three of SEQ ID NOs: 6-8, or has SEQ ID NO: 9 or 10. In others, the variant hinge region can have one copy of a 17-mer sequence (e.g., SEQ ID NO: 6) and three copies of a 15 mer sequence (e.g., SEQ ID NO: 7 or 8). In that case, at least one of the 17-mer or 15-mer sequence can be selected the group consisting of SEQ ID NOs: 6-8. In general, the hinge region is longer than that of IgGl hinge region (19 amino acid residues) and at least 20 (e.g., at least 21, 22, 25, 30, 35, 40, 45, 50, 55, 60, 65, 66, 67, 68, 69, 70, 80, 90, or 100) amino acid residues in length. For example, the variant hinge region can comprise the sequence of SEQ ID NO: 3, where 9 of the 11 cysteine are changed to serines (S).

In some embodiment, the first antigen-binding site is derived from a first parent antibody, the CHI and CL domains of which are swapped in the first antigen-binding site. That is, each of the heavy chains is a fusion polypeptide having the VH-CL domains; and each of the light chain is fusion polypeptide having the VL-CH1 domains. Similarly, the second antigen-binding site can also be derived from a second parent antibody, the CHI and CL domains of which are swapped in the second antigen-binding site. As disclosed in FIG. 1 A here, such swapping of the CHI domain with the CL domain for the one but not for the other parental antibody allows one to pair correct light chains with the corresponding heavy chain. The first antigen-binding site has substantially the same antigen binding activity as the first parent antibody, and similarly, the second antigen- binding site has substantially the same antigen binding activity as the second parent antibody

In preferred embodiments, the first epitope and the second epitope mentioned above are non-overlapping epitopes on the envelope protein of HIV- 1. For example, the first epitope or the second epitope can be located in the CD4-binding site, the V3 loop, the V1/V2 loop, the gpl20/gp41 interface, or the MPER region of the envelope protein. For the bispecific antibody, the first antigen-binding site or the second antigen-binding site can be derived from a parent bNAb selected from the group consisting of 3BNC117, 8ANC131, CH103, 10-1074, PGT121, PGT128, PGT135, PG16, PGT145, PGDM1400, PGT151, 8ANC195, and 10E8.

In some examples, the first antigen-binding site and the second antigen-binding site can be derived from a pair of different parent bNAbs and show synergy as compared to the parent pair. For example, as shown in, e.g., FIG 3B, bispecific antibodies derived from the following pairs exhibit synergy on the neutralization activity against a small virus panel (wherein a positive number indicates synergy):

(a) if the first antigen-binding site is from 3BNC117, the second antigen-binding site is from the group consisting of PGT121, PGT135, PG16, 8ANC195, and 10E8;

(b) if the first antigen-binding site is from 8ANC131, the second antigen-binding site is from the group consisting of PGT128, PGT135, PGT145, PGDM1400 PGT151,

8ANC195, and 10E8;

(c) if the first antigen -binding site is from CHI 03, the second antigen-binding site is from the group consisting of PGT135, PGT145, PGT151, and 10E8;

(d) if the first antigen-binding site is from 10-1074, the second antigen-binding site is from the group consisting of PG16, PGT151, and 8ANC195;

(e) if the first antigen-binding site is from PGT121, the second antigen-binding site is from the group consisting of 3BNC117, PG16, PGT151, and 10E8; (f) if the first antigen-binding site is from PGT128, the second antigen-binding site is from the group consisting of 8ANC131, PG16, PGT151, and 8ANC195;

(g) if the first antigen-binding site is from PGT135, the second antigen-binding site is from the group consisting of 3BNC117, 8ANC131, CH103, PG16, PGT145, PGDM1400, PGT151, and 10E8;

(h) if the first antigen-binding site is from PG16, the second antigen-binding site is from the group consisting of 3BNC117, 10-1074, PGT121, PGT128, PGT135, PGT151, 8ANC195, and 10E8;

(i) if the first antigen-binding site is from PGT145, the second antigen-binding site is from the group consisting of 8ANC131, CH103, PGT135, PGT151, 8ANC195, and

10E8;

(j) if the first antigen-binding site is from PGDM1400, the second antigen-binding site is from the group consisting of 8ANC131, PGT135, PGT151, and 10E8;

(k) if the first antigen-binding site is from PGT151, the second antigen-binding site is from the group consisting of 8ANC131, CH103, 10-1074, PGT121, PGT128, PGT135, PG16, PGT145, PGDM1400, 8ANC195, and 10E8;

(1) if the first antigen-binding site is from 8ANC195, the second antigen-binding site is from the group consisting of 3BNC117, 8ANC131, 10-1074, PGT128, PG16, PGT145, PGT151, and 10E8; or

(m) if the first antigen-binding site is from 10E8, the second antigen-binding site is from the group consisting of 3BNC117, 8ANC131, CH103, PGT121, PGT135, PG16, PGT145, PGDM1400, PGT151, and 8ANC195.

Bispecific antibodies derived from the other pairs shown in FIG 3B and disclosed herein are also within the scope of this invention as they can show synergy for other virus strains. In some embodiments, the first antigen-binding site is derived from 3BNC117 and the second antigen-binding site is derived from 10-1074 or PGT 135, respectively.

The bispecific antibody described above can be used in a method of treating or preventing or controlling an HIV infection. The method includes administering to a subject in need thereof a therapeutically effective amount of the bispecific antibody. Accordingly, the invention also provides a pharmaceutical composition comprising (i) the bispecific antibody described above, or a bispecific antigen-binding fragment thereof, and (ii) a pharmaceutically acceptable carrier. In another aspect, the invention provides a kit comprising a pharmaceutically acceptable dose unit of a pharmaceutically effective amount of at least one bispecific antibody described above or a bispecific antigen-binding fragment thereof.

In a further aspect, the invention provides a method of detecting an HIV infection in a subject. The method includes contacting a biological sample from the subject with the antibody described above or bispecific antigen binding fragment thereof under conditions sufficient to form an immune complex; and detecting the presence of the immune complex in the sample from the subject. The presence of the immune complex in the sample from the subject indicates that the subject has an HIV infection.

Also provided are an isolated polypeptide comprising one or more of the following: a first sequence that is at least 75% identical to SEQ ID NO: 6 and has a non- cysteine residue (e.g., a serine) at position 13 or 16 or both of SEQ ID NO: 6; a second sequence that is at least 75% identical to SEQ ID NO: 7 and has a non-cysteine residue (e.g., a serine) at one or more of the positions 5, 1 1, and 14 of SEQ ID NO: 7, a third sequence that is at least 75% identical to SEQ ID NO: 8 and has a non-cysteine residue (e.g., a serine) at position 5 of SEQ ID NO: 8, a fourth sequence that is at least 75% identical to SEQ ID NO: 9 and has a non-cysteine residue (e.g., a serine) at one or more of positions 13, 16, 22, 28, 31, and 37 of SEQ ID NO: 9, and a fifth sequence that is at least 75%) identical to SEQ ID NO: 10 and has a non-cysteine residue (e.g., a serine) at one or more of positions 13, 16, and 22 of SEQ ID NO: 10 The isolated polypeptide can have two or three of the first sequence, the second sequence, and the third sequence. The isolated polypeptide can have different combination of the three sequences. In one example, the polypeptide is at least 60%> (e.g., any number between 60%> and 100%>, inclusive, e.g., 60%, 70 %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%) identical to SEQ ID NO: 3, 8 or 9. The first sequence can be 17 or more mer in length. The second and third sequence can be 15 mer or more in length. This polypeptide can be used as a hinge region for the antibody described herein. The polypeptide generally has the same length as or is longer than a reference sequence (e.g., one of SEQ ID Nos: 1-10).

Another aspect of the invention features an isolated nucleic acid comprising a sequence encoding a chain of the isolated antibody or antigen-binding fragment thereof or a polypeptide describe above. The nucleic acid can be used to express a polypeptide, a chain of the antibody, or antigen-binding fragment of this invention, or the antibody or fragment. For this purpose, one can operatively link the nucleic acid to suitable regulatory sequences to generate an expression vector. Accordingly, within the scope of this invention are a cultured host cell comprising the vector and a method for producing a polypeptide, the method comprising culturing the host cell under conditions in which the nucleic acid molecule is expressed.

In addition to bispecific anti-HIV antibodies, other bispecific antibodies such as anti-tumor bispecific antibodies and related methods are also provided.

The details of one or more embodiments of the invention are set forth in the description below. Other features, objectives, and advantages of the invention will be apparent from the description, the drawings, and he claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGs. 1A, IB, 1C, ID, IE, IF and 1G are an overview of bispecific antibody generation and neutralization activity of IgGl bispecific neutralizing antibodies (biNAbs). (FIG. 1A) Overview of the strategy for generating biNAbs to ensure proper heavy-light chain pairing and heterodimerization. (FIG. IB) Comparison of the in vitro neutralization activity of wild-type and CrossMab variants of 10-1074 mAbs. (FIG. 1C - FIG. 1G) In vitro neutralization breadth and potency plots of different combinations of biNAbs against an extended multiclade virus panel. Neutralization activity of their respective parental mAbs was included for comparison. All biNAb combinations exhibited marked reduction in neutralization potency compared to the activity of a mix of their parental mAbs.

FIGs. 2A, 2B and 2C show generation and characterization of biNAb hinge domain variants. (FIG.2A) Schematic representation and primary amino acid sequence of the hinge domain of IgGl, IgG3 and the generated variant, IgG3C- (SEQ ID NOs: 1-3). Disulphide bonds and Cys residues are depicted in red. IgGl hinge domain variants of 10- 1074 mAb were generated by switching the IgGl hinge region with the IgG3 or IgG3C- hinge. (FIG. 2B) In vivo stability and half-life was compared between the various 10-1074 hinge-domain variants (mean+SEM, n=4 mice/group). (FIG. 2C) Comparison of the in vitro neutralization activity of 3BNC117/10-1074 biNAbs expressed as IgGl or IgG3C- hinge variants. As control, the predicted neutralization of the mix of the two parental mAbs (3BNC117 and 10-1074) was included. FIGs. 3A, 3B and 3C show neutralization activity of hinge domain engineered (IgG3C-) biNAbs. A panel of mAbs were selected targeting different epitopes on Env. (FIG. 3A) epitope mapping on the surface of the HIV-1 Env trimer and biNAb combinations (encompassing the IgG3C- hinge variant) with non-overlapping epitope specificities were generated. In vitro neutralization activity against a cross-clade virus panel was assessed and combinations with variable degree of synergy were identified (FIG. 3B) grid showing the fold change (log) in activity of biNAb over the respective parental mAbs; (FIG. 3C) IC50 titers of example biNAb combinations exhibiting synergistic, neutral or inhibitory activity.

FIGs. 4 A, 4B, 4C, 4D, 4E, 4F and 4G show in vitro neutralization potency and in vivo protective activity of IgG3C- hinge biNAbs. In vitro neutralization breadth and potency against an extended multiclade virus panel was assessed for IgG3C- hinge variants of PGT151/10-1074 (FIG. 4A) 8ANC195/PGT128 (FIG. 4B) 3BNC117/PGT135 (FIG. 4C) biNAbs. (FIG. 4D - FIG. 4G) In vivo protective activity of 3BNC117/PGT135 IgG3C- biNAb was compared to that of a mix of 3BNC117 + PGT135 IgG3C- mAbs in HIV-1 -infected humanized mice. Mice with established viremia were treated for 4 weeks (red shaded area) with the corresponding mAb mix or biNAb and plasma viremia was monitored to assess the in vivo protective activity of antibodies. (FIG. 4F) Comparison of viremia levels or (FIG. 4G) change from baseline at the end of the treatment period revealed significant differences between the mix- and bispecific-treated mice (**p<0.005; mean+SEM, n=7 or 9).

FIGs. 5A, 5B, 5C and 5D show characterization of 3BNC117/10-1074 biNAbs. (FIG. 5 A) Binding specificity for gpl40 of wild-type and CrossMab variant of 10-1074 was assessed by ELISA using recombinant gpl40. (FIG. 5B - FIG. 5D) ELISA assays to determine dual specificity of the 3BNC117/10-1074 biAb. (FIG. 5B) 3BNC117/10-1074 biNAb, 3BNC117 and 10-1074 mAbs were immobilized to gpl40-coated microtiter plates and detected using Fc domain- or light chain (kappa or lambda) subclass-specific secondary IgG. BiAb was detected with both the anti-kappa and anti-lambda secondary antibodies, whereas 3BNC117 and 10-1074 only with anti -kappa or anti -lambda, respectively. (FIG. 5C) Competition ELISA with increasing concentrations of each of the parental mAbs or a mixture of the two mAbs to determine dual specificity of the bispecific mAb. (FIG. 5D) Epitope-specific ELISA using a CD4bs antigen (2-CC Core) for capture and an anti-lambda detection antibody to confirm bispecific activity.

FIGs. 6A and 6B show size exclusion chromatography (SEC) and thermostability of hinge domain variants of 10-1074. Hinge domain variants of 10-1074 were analyzed (FIG. 6A) by SEC to assess for protein aggregation and (FIG. 6B) by thermal shift protein assay to determine protein Tm.

FIGs. 7A, 7B, 7C, 7D, 7E and 7F show comparison of the neutralization activity of IgGl and IgG3C- hinge variants of anti-HIV-1 Env mAbs. In vitro neutralization breadth and potency plots of different anti-Env mAbs (FIG. 7A: 3BNC117; FIG. 7B: 10-1074; FIG. 7C: PGT128; FIG. 7D: PGT135; FIG. 7E: PGT151; FIG. 7F: 8ANC195) expressed as IgGl or IgG3C- hinge variants. In vitro neutralization was assessed by TZMbl assay against an extended multiclade virus panel.

FIGs. 8 A and 8B show crystal structure of the PGT135 and 3BNC117 Fabs bound to the Env trimer. (FIG. 8 A) Side and (FIG. 8B) top view of 3BNC117 (cyan; 4JPV) and PGT135 (red; 4JM2) Fabs bound to the Env trimer (4NCO). The distance between the ends of the two Fabs was calculated to be 67A, suggesting that the IgG3C- hinge variant might facilitate bivalent, intra-trimeric interactions of the 3BNC117/PGT136 biNAb.

FIGs. 9A, 9B, and 9C show in vivo IgG half-life and mutation analysis of biNAb- treated HIV-1 infected humanized mice. (FIG. 9 A) In vivo pharmacokinetics of 3BNC 117/PGT 135 biNAb and 3BNC 117+PGT 135 Mix (all in the IgG3C- format). Mice were injected (s.c; 0.5 mg) with either 3BNC117/PGT135 biNAb or a 1 : 1 mix of 3BNC117 and PGT135 mAbs and serum IgG concentration was detected by ELISA. Results are presented as the mean+SEM; n=4 mice/group. (FIG. 9B - FIG. 9C) HIV-1- infected humanized mice were treated for 4 weeks with either a mix of 3BNC117 + PGT135 IgG3C- mAbs or 3BNC117/PGT135 IgG3C- biNAb. (FIG. 9B) Serum anti-Env IgG concentration was assessed by gpl40 ELISA to determine the levels of antibody between the two groups (day 45 post-initiation of antibody treatment). Results are presented as the mean+SEM, n=5 or 7 (FIG. 9C) HIV-1 gpl20 from mice exhibiting viremia of >10 ⁴ copies/ml were cloned and their sequences were analyzed. Recurrent mutations (>50% of analyzed clones) and their epitope position (numbered based on HXBc2 reference sequence) are presented (color-matched to individual mice). All other mice showed no evidence of recurrent mutations within gpl20 ORF. Mutations within the V3 or the CD4bs epitopes (targeted by the PGT135 and 3BNC117 mAbs, respectively) were identified in both groups. However, no change in the frequency of recurrent mutations was noted between the two experimental groups.

FIGs. 10A and 10B show in vitro neutralization activity (IC50 titers ^g/ml)) of 3BNC117 + PGT135 bNAb mix or 3BNC117/PGT135 biNAb expressed with the following hinge domain structures: IgGl, IgG3 and IgG3C-. 3BNC117/PGT135 IgG3C- biNAb exhibited significantly improved neutralization activity (**p<0.001) compared to IgGl or IgG3 biNAb hinge variants. IC50 titer ^g/ml) results are presented as geometric mean±95%CI.

FIGs. 11A and 11B show IC50 and IC80 titers (ng/ml) of 3BNC117/PGT135

IgG3C- biNAbs with variable hinge domain length. Shorter variants of IgG3C- ("Full length") were generated by deleting either one ("-15mer") or two ("-2xl5mer") of the three 15-mer repeats. FIG 11B also shows comparison of the neutralization activity between 3BNC117/PGT135 IgG3C- biNAb (full length) and shortened hinge domain variants revealed that enhanced neutralization activity is correlated with hinge domain length. IC50 titer ^g/ml) results are presented as geometric mean±95%CI. *p<0.05; **p<0.001.

FIG 12 shows determination of half-life of 3BNC117/PGT135 IgG3C- biNAb in Rhesus macaques.

FIG. 13 A and 13B show comparison of the neutralization activity of the

3BNC117/PGT135 IgG3C-biNAb with previously characterized bNAbs and bNAb combinations, (FIG. 13 A) Neutralization breadth and potency plot comparing the activity of the 3BNC117/PGT135 IgG3C- biNAb to various bNAbs targeting distinct epitopes on HIV-1 Env. (FIG. 13B) Comparison of the neutralization potency of the 3BNC117/PGT135 IgG3C- biNAb with single bNAbs and bNAb combinations (2-4 bNAbs). Neutralization data for these bNAbs and biNAb combinations have been previously reported in Kong et al., (2015). Comparative analysis was performed using a panel of 115 viruses, for which neutralization data were available both for the biNAb and the bNAb combinations. DETAILED DESCRIPTION OF THE INVENTION

This invention provides novel bispecific anti-HIV bNAbs. As disclosed herein, to overcome the above-discussed limitations associated with virus escape mechanisms following antibody treatment, a panel of bispecific anti-HIV bNAbs were generated and characterized.

Broadly neutralizing monoclonal antibodies (bNAbs) against the envelope glycoprotein of HIV-1 (Env) (Klein, F. et al, Science 341, 1199-1204, doi: 10.1126/science. l241144 (2013) and Burton, D. R. & Mascola, J. R., Nat Immunol 16, 571-576, doi: 10.1038/ni.3158 (2015)) can suppress viremia in animal models of HIV-1 and humans (Barouch, D. H. et al., Nature 503, 224-228, doi: 10.1038/nature 12744 (2013); Bournazos, S. et al, Cell 158, 1243-1253, doi: 10.1016/j .cell.2014.08.023 (2014); Halper- Stromberg, A. et al, Cell 158, 989-999, doi: 10.1016/j .cell.2014.07.043 (2014); Horwitz, J. A. et al, Proc Natl Acad Sci U S A 110, 16538-16543, doi: 10.1073/pnas. l315295110 (2013); Klein, F. et al, Nature 492, 118-122, doi: 10.1038/nature 11604 (2012); Shingai, M. et al, Nature 503, 277-280, doi: 10.1038/nature 12746 (2013); and Caskey, M. et al, Nature, doi: 10.1038/naturel4411 (2015)), representing a promising prophylactic and therapeutic modality for the control of HIV-1 infection. To achieve potent therapeutic activity while avoiding the emergence of viral escape mutants, co-administration of cocktails of different bNAbs can be used to target distinct, non-overlapping epitopes essential for viral fitness (Horwitz, J. A. et al, Proc Natl Acad Sci U S A 110, 16538- 16543, doi: 10.1073/pnas. l315295110 (2013); Klein, F. et al, Nature 492, 118-122, doi: 10.1038/naturel l604 (2012)). Here the invention discloses the development and evaluation of novel bispecific anti-Env neutralizing antibodies (biNAbs) that exhibit potent in vitro and in vivo activity. Synergistic activity of biNAbs was achieved by engineering the hinge domain of IgGl through the use of variants based on the IgG3 hinge (Roux, K. H., Strelets, L. & Michaelsen, T. E., J Immunol 159, 3372-3382 (1997)) to increase Fab domain flexibility for hetero-bivalent binding to the Env trimer. Compared to unmodified biNAbs, hinge domain variants exhibited substantially improved neutralization activity, with particular combinations showing evidence of synergistic neutralization potency in vitro and enhanced in vivo therapeutic activity in HIV-1 -infected humanized mice. This invention provides novel strategies for generating biNAbs with improved in vitro and in vivo activity through hinge domain engineering. Such biNAbs exhibit remarkable neutralization breadth and potency and appear to be ideal candidate molecules for the prevention and treatment of HIV-1 infection. A. Bispecific Antibodies

Bispecific neutralizing antibodies (biNAbs) represent an attractive therapeutic strategy for the prevention and treatment of HIV-1 infection, given their unique advantages over conventional, monospecific antibodies. Indeed, biNAbs can overcome virus escape issues associated with the use of conventional bNAbs (Halper-Stromberg, A. et al, Cell 158, 989-999, doi: 10.1016/j .cell.2014.07.043 (2014); Horwitz, J. A. et al, Proc NatlAcadSci USA 110, 16538-16543, doi: 10.1073/pnas. l315295110 (2013); Klein, F. et al, Nature 492, 118-122, doi: 10.1038/naturel l604 (2012)), providing a compelling platform for the development of single therapeutic molecules that would combine the breadth and potency of two bNAbs for effective control of HIV-1 infection.

A key immune evasion mechanism of HIV-1 against host antibody responses is the remarkably low density of Env molecules on the viral surface, as well as the unique Env trimeric architecture, which preclude high-avidity bivalent interactions of IgG (Klein, J. S. & Bjorkman, P. J. F, PLoS Pathog 6, el000908, doi: 10.1371/journal.ppat.1000908 (2010).). It is therefore likely that conventional biNAbs would exhibit predominantly monovalent binding to their respective epitopes, possibly accounting for the lack of additive, synergistic activity. Indeed, given the relative rigidity and the short length of the hinge domain of IgGl, concurrent binding of the two Fab arms within the same Env trimer is largely restricted. Overcoming this limitation and favoring intra-trimeric, bivalent interactions of biNAbs should greatly augment their neutralization activity through enhanced avidity (Klein, J. S. & Bjorkman, P. J. F, PLoS Pathog 6, el000908, doi: 10.1371/journal.ppat.1000908 (2010); Galimidi, R. P. et al, Cell 760, 433-446, doi: 10.1016/j .cell.2015.01.016 (2015); and Klein, J. S. et al, Proc Natl Acad Sci U S A 706, 7385-7390, doi: 10.1073/pnas.0811427106 (2009)).

Among the human IgG subclasses, IgG3 encompasses an exceptionally long and flexible hinge domain, with distinct structural and functional characteristics (Roux, K. H., Strelets, L. & Michaelsen, T. E., J Immunol 159, 3372-3382 (1997) and Roux, K. H., et al, J Immunol 761, 4083-4090 (1998)) (FIG. 2A). It is comprised of a 17-mer amino acid sequence followed by three 15 mer repeats that are highly homologous to the IgGl hinge structure and represent genomic duplication events of the ancestral hinge-encoding exon, conserved among all other IgG subclasses (Roux, K. H., et al, J Immunol 161, 4083-4090 (1998)). Previous biophysical studies on the IgG3 hinge indicated that the hinge domain spans an over HOA distance and the unique primary amino acid composition of the CHI proximal 17mer confers increased flexibility of the Fab arms, allowing for greater degree of rotation compared to IgGl (117° vs. 136°) (Roux, K. H., et al, J Immunol 161, 4083- 4090 (1998)).

As disclosed herein, to further increase the inherent IgG3 hinge flexibility, the inventors generated an "open" IgG3-based hinge variant (IgG3C-), in which all the cysteine residues (C) have been replaced with a non-cysteine residue, such as serines (S) with the exception of the last two, CH2 proximal residues that are used to maintain the structural integrity of the Fc domain (FIG. 2A).

For example, as shown in the examples below, hinge domain variants of 10-1074

IgGl were expressed as chimeric molecules, in which the wild-type hinge domain (IgGl) was replaced with that of either IgG3 or the IgG3C- variant. With the exception of the hinge domain, all other domains of the constant region (CHI, CH2, CH3) were of the IgGl subclass to preserve the effector function and half-life of wild-type IgGl . Indeed, hinge domain variants of 10-1074 demonstrated comparable binding affinity to the different classes of human and mouse FcyRs, suggesting a minimal role for the hinge region in Fc-FcyR interactions (Extended Data Table 1). Likewise, no differences among the hinge domain variants were noted in terms of protein stability and in vivo pharmacokinetics (FIG. 2B, and FIG. 6).

This invention accordingly encompasses a bispecific antibody comprising (i) a first antigen-binding site that binds to a first epitope and (ii) a second antigen-binding site that binds to a second epitope. The first antigen-binding site comprises a first heavy chain and a first light chain while the second antigen-binding site comprises a second heavy chain and a second light chain. The first heavy chain and the second heavy chain comprises a variant hinge region that (a) is derived from a wild type IgG3 hinge region and (b) has fewer CH-1 proximal cysteine residues than the wild type IgG3 hinge region as compared to a reference antibody containing the wild type IgG3 hinge region. Shown below are exemplary hinge sequences and variants thereof. Variants of these hinge sequences with conservative modifications can also be used. SEQ Name Sequences Note

ID NO. :

1 IgGl hinge region EPKSCDKTHTCPPCP APEL

2 IgG3 hinge region ELKTPLGDTTHTCPRCP

EPKSCDTPPPCPRCP EPKSCDTPPPCPRCP EPKSCDTPPPCPRCP APEL

3 IgG3 C-hinge ELKTPLGDTTHTSPRSP This is the "Full-length" in FIG. region EPKSSDTPPPSPRSP 11B.

EPKSSDTPPPSPRSP EPKSSDTPPPCPRCP APEL

4 IgG3 hinge region ELKTPLGDTTHTCPRCP This is the same as aa 1-17 of 17-mer wild type SEQ ID NO: 2. It is a part of

SEQ ID NO: 2.

5 IgG3 hinge region EPKSCDTPPPCPRCP This correspond to aa 18-32 of 15-mer wild type SEQ ID NO: 2. It is a part of

SEQ ID NO: 2.

6 IgG3 C-hinge ELKTPLGDTTHTSPRSP This is a mutant version of SEQ region 17-mer ID NO: 4. It is a part of SEQ ID mutant NO: 3.

7 IgG3 C-hinge EPKSSDTPPPSPRSP This is a first mutant version of region 15-mer SEQ ID NO: 5. It is a part of mutant A SEQ ID NO: 3.

8 IgG3 C-hinge EPKSSDTPPPCPRCP This is a second mutant version region 15-mer of SEQ ID NO: 5.

mutant B It is a part of SEQ ID NO: 3.

9 "-15mer" ELKTPLGDTTHTSPRSP This is the "-15mer" in FIG.

EPKSSDTPPPSPRSP 11B.

EPKSSDTPPPCPRCP APEL

10 "-2xl5mer" ELKTPLGDTTHTSPRSP This is the "-2x 15mer" in FIG.

EPKSSDTPPPCPRCP APEL 11B.

To ensure proper pairing of the heavy and light chains from two different parent antibodies (e.g., bNAbs) and generation of heavy chain heterodimers, a combination of strategies, including that described in Schaefer et al. Proc Natl Acad Sci U S A 108, 11187-11192 (2011), can be used. In one example, heterodimerization of the heavy chains of the two mAbs was achieved by introducing mutations in the CH3 domain that alter the physical and chemical properties of the two heavy chains favoring heterodimerization. These mutations have no impact on the capacity of the Fc domain to interact with FcyRs and FcRn, having thereby no effect on Fc effector function and IgG half-life. Pairing of the correct heavy chain with the respective light chain was achieved by swapping the CHI domain with CL for one of the parental bNAbs, while maintaining the wild-type architecture for the other bA b. These approaches typically yielded >90% heterodimers, without evidence for protein aggregation (<10% IgG multimers).

Since the distance of most of the bNAb-targeting epitopes on the HIV-1 env trimer does not allow for concurrent intra-trimeric interactions that would augment neutralization activity through enhanced Fab-mediated avidity, antibodies with extended hinge regions that exhibit increased distance and flexibility of the two Fab arms were generated. These hinge domain-engineered bNAbs are based on the naturally occurring hinge domain of human IgG3. Human IgG3 encompasses an exceptionally long and flexible hinge domain compared to other IgG subclasses, which has been previously suggested to contribute to the improved effector activity of this IgG subclass. Based on the IgG3 hinge sequence, an "open" IgG3 hinge variant ("IgG3 C-") was generated by mutating most of the CHI proximal cysteine residues of the IgG3 hinge to increase the flexibility of the two Fab arms. This variant is expected to exhibit improved Fab arm flexibility and the capacity to span an over 20θΑ distance, which is sufficient for intra-trimeric interactions.

Several recent studies using anti-Env antibodies with broad and potent neutralizing activity revealed their capacity to confer both effective pre-exposure prophylaxis and therapeutic control of viremia in murine and non-human primate HIV-1 disease models, as well as in HIV-1 infected humans. Studies on animal models for HIV-1 infection suggested that sustained therapeutic viremia suppression is accomplished by coadministration of a cocktail of bNAbs targeting key epitopes on the HIV-1 Env that are essential for viral fitness, overcoming thereby the emergence of virus escape mutants. Bispecific anti-HIV-1 Env antibodies represent an ideal therapeutic approach that would combine the breadth, antigenic specificity and neutralization potency of two bNAbs, into a single molecule, facilitating preclinical evaluation and development. In addition to the additive effect on the neutralization breadth, favoring hetero-bivalent interactions of the two Fab arms could confer synergistic activity, yielding biNAbs with substantially improved neutralization potency compared to conventional bNAbs. Indeed, the use of DNA- and protein-based structures to link two Fab arms at optimal distances to achieve hetero-bivalent, intra-trimeric interactions resulted in enhanced in vitro neutralization potency.

Based on these observations, biNAbs with non-overlapping epitope specificities were developed and characterized to identify particular biNAb combinations that would demonstrate potent and synergistic neutralization activity. Given the significance of the FcyR-mediated pathways in the in vivo protective activity of anti-HIV-1 bNAbs (Bournazos et al, 2014, Cell 158, 1243-1253; Halper-Stromberg et al, 2014 Cell 158, 989-999; Hessell et al, 2007, Nature 449, 101-104), the inventors generated biNAbs with the wild-type IgG structure, while avoiding irregular, non-physiological architectures used in the past (Spiess et al, 2015, Mol Immunol 67, 95-106). This approach ensured that the resulting biNAb would have the capacity to interact with FcyRs and exhibit long and stable in vivo pharmacokinetics, with minimal immunogenicity potential. Indeed, all the generated biNAb variants exhibited identical in vivo half-life and affinity for the different classes of FcyRs. Attempts to generate anti-HIV-1 Env biNAbs based on the IgGl structure were generally characterized by lower neutralization potency compared to their corresponding parental bNAbs, irrespective of their epitope specificity. Similar findings have also been reported in a recent study that generated and characterized a number of anti-Env IgGl biNAbs (Asokan et al, 2015, J Virol 89, 12501-12512.), suggesting that IgGl biNAbs offer no advantages over conventional, monospecific bNAbs. Since the observed impairment in the neutralization potency possibly reflects the lack of sufficient flexibility of the two Fab arms to achieve bivalent, intra-trimeric interactions in the IgGl format, hinge domain engineered biNAbs were generated with improved Fab domain flexibility, based on the naturally-occurring hinge domain of IgG3, which is characterized by a uniquely long and flexible structure (Roux et al, 1998, J Immunol 161, 4083-4090.; Roux et al., 1997, J Immunol 159, 3372-3382). This approach incorporates minimal changes to the overall IgG structure, while it achieves significant enhancement in the neutralization breadth and potency.

Among all the hinge-engineered biNAb combinations tested and described in the examples below, 3BNC117/PGT135 showed evidence for synergistic activity, surpassing the potency of both parental bNAbs (3BNC117 and PGT135). Indeed, assessment of its neutralization potency indicated that for the vast majority of the tested viruses, the 3BNC117/PGT135 biNAb exhibits lower IC50 and IC80 titers, and for over a third of the tested viruses, the improvement in the neutralization potency exceed 10-fold compared to 3BNC117 and PGT135 bNAbs. Its enhanced activity was largely attributed to the hinge length and flexibility, as hinge domain variants with shorter length or decreased flexibility also exhibited reduced neutralization breadth and potency. These findings indicate that the improved neutralization activity of the 3BNC 117/PGT 135 IgG3C- biNAb is likely the result of bivalent, intra-trimeric interactions, accomplished by the unique structure of the engineered hinge domain of the IgG3C- variant. Intra-trimeric, heterobivalent crosslinking of the two Fab arms increases the overall avidity of the Env trimer - biNAb interaction, leading thereby to augmented neutralization potency.

Although several mechanisms of bNAb binding interference have been described, even for bNAbs with non-overlapping epitope specificities like 3BNC1 17 and PGT135 (Derking et al., 2015, PLoS Pathog 11, el004767), findings disclosed in this invention suggest that the observed effect of the 3BNC117/PGT135 biNAb could not be attributed to conformational changes induced upon bNAb binding, as no change in the neutralization activity was evident for the mix of the two bNAbs (3BNC 117 + PGT 135) (FIGs. 1 OA and 10B). However, a recent study that analyzed the binding profile of a panel of bNAbs to the soluble native BG505.SOSIP.664 gpl40 trimer revealed that PGT135 inhibited unidirectionally the binding of anti-CD4bs, VRCOl-like antibodies to the gpl40 trimer, due to reorientation of a glycan structure (predominantly Asn386 and/or Asn392 glycans), partly occluding the CD4bs epitope (Derking et al, 2015, PLoS Pathog 11, el004767). Since all VRCOl-like bNAbs exhibit substantially higher affinity for Env compared to PGT135, it is unlikely that such cross-bNAb inhibition would occur for strains sensitive to both bNAbs. Based on these findings, in the context of the 3BNC117/PGT135 biNAb, it is expected that for virus strains sensitive to 3BNC117, increased neutralization potency would be expected through initial binding of the 3BNC117 arm (due to higher affinity) followed by the PGT135. In contrast, for viruses that are resistant to 3BNC117, but sensitive to PGT135, no improvement in the neutralization activity is expected, due to PGT135-mediated occlusion of the CD4bs epitope. Indeed, assessment of the extended panel neutralization data indicates that for the few virus strains that are resistant to 3BNC117, but sensitive to PGT135 (like CNE20, CNE21, X2088_c9), no enhanced neutralization activity is evident for the 3BNC117/PGT135 biNAb and the observed neutralization potency is lower than that of the PGT135 bNAb, further supporting the notion that the enhanced neutralization activity of the 3BNC117/PGT135 biNAb is the result of bivalent, intra-trimeric interactions.

Despite the augmented neutralization potency of the 3BNC117/PGT135 IgG3C- biNAb, it was essential to determine whether its in vitro activity also translates to enhanced in vivo therapeutic efficacy. Therefore, its in vivo activity was assessed in HIV- 1 -infected humanized mice; a robust model that recapitulates human HIV-1 infection and has been systematically used in the past to accurately investigate the in vivo activity of bNAbs. Consistent with the in vitro findings, 3BNC 117/PGT 135 IgG3C- biNAb showed improved in vivo protective activity, probably reflecting its enhanced neutralization activity. However, a role for Fc effector functions in the in vivo protective activity of this biNAb could not be excluded. In addition to enhancing neutralization, hetero-bivalent biNAb binding to the Env trimer could also lead to more stable biNAb-Env interactions, facilitating thereby clearance of viral particles and infected cells through FcyR-mediated mechanisms.

Collectively, these findings suggest that the hinge-engineered 3BNC117/PGT135 biNAb represents one of the most potent anti-HIV-1 bNAbs developed to date, exhibiting high neutralization breadth and potency, as well as improved in vivo activity. To comparatively benchmark the activity of the 3BNC117/PGT135 biNAb with previously characterized bNAbs, its neutralization breadth and potency was compared to several bNAbs targeting distinct epitopes on gpl40. As shown in FIG 13 A, the 3BNC117/PGT135 biNAb displays increased neutralization potency, compared to the most potent bNAbs so far characterized, including VRC07, PGT121, and PG9. Additionally, to gain further insights on how the activity of this biNAb compares to physical combinations of bNAbs, the activity observed in this study for 3BNC117/PGT135 biNAb was directly compared with that previously reported for several bNAb combinations (Kong et al, 2015, J Virol 89, 2659-2671). Compared to all two- bNAb combinations, the 3BNC117/PGT135 biNAb demonstrated higher neutralization activity, neutralizing >80% of viral strains with IC50 <0.1 μg/ml (FIG. 13B). Its potency was only marginally lower compared to that observed for the three-bNAb combinations. This analysis provides a direct measure of the neutralization activity of the 3BNC117/PGT135 biNAb against other bNAbs and bNAb combinations, highlighting the improved potency of this biNAb over conventional, monospecific bNAbs. Development of bispecific anti-HIV-1 antibodies offers significant advantages over conventional monoclonal antibodies, as they combine the breadth and potency of two broadly neutralizing antibodies and overcome viral escape mechanisms associated with the use of single antibody preparations. To augment the activity of anti-HIV-1 bispecific antibodies, hinge domain variants were generated based on the IgG3 scaffold to optimize Fab domain flexibility, favoring intra-molecular Env interactions. Hinge-modified bispecific antibodies exhibited improved potency, with evidence for enhanced in vitro and in vivo activity.

As disclosed herein, a panel of IgG3 C- variants of bispecific bNAbs was generated and their in vitro neutralization activity was assessed against a small panel of HIV isolates, using a standardized TZM-bl assay. As control the activity of IgG3 C- monospecific bNAbs were assessed and no difference in their activity was evident when compared to their IgGl counterparts. However, comparison of the activity of IgG3 C- bispecific bNAbs revealed that particular bispecific combinations exhibit significant synergistic activity compared to the corresponding IgG3 C- parental bNAbs.

In summary, the present invention describes an approach for the generation of anti- HIV-1 Env biNAbs with physiological IgG architecture, FcyR binding profile and pharmacokinetic properties. Compared to conventional, monospecific bNAbs, biNAbs with hinge domain engineered structures exhibit potent neutralization activity with improved breadth and potency and enhanced in vivo activity. These unique advantages of hinge domain-optimized biNAbs represent a platform technology that can also be extended to other viral and cellular targets.

B. Parental Antibodies

The bispecific antibodies of the present invention can be made using or derived from the chains of any isolated antibodies {i.e., parental antibodies), in particular monoclonal antibodies such as human monoclonal antibodies that bind to different antigens or epitopes. Preferably the antibodies are human antibodies, although the antibodies can also be, for example, murine antibodies, chimeric antibodies, humanized antibodies, or a combination thereof.

Monoclonal antibody techniques allow for the production of specifically binding agents in the form of specifically binding monoclonal antibodies or fragments thereof. For creating monoclonal antibodies, or fragments thereof, one can use conventional hybridoma techniques. Alternatively, monoclonal antibodies, or fragments thereof, can be obtained by the use of phage libraries of scFv (single chain variable region), specifically human scFv (see e.g. U.S. Pat. No. 5,885,793, WO 92/01047, and WO 99/06587).

Anti-HIV Antibodies

In some embodiments, the invention disclosed herein involves bispecific anti-HIV bNAbs. In general, anti-HIV bNAbs antibodies refer to a class of neutralizing antibodies that neutralize multiple HIV-1 viral strains. Various bNAbs are known in the art and can be used in this invention. Examples include but are not limited to those described in U.S. Patent NO. 8673307, WO2014063059, WO2012158948, WO2015/117008, and PCT/US2015/41272, including antibodies 3BNC117, 3BNC60, 12A12, 12A21, NIH45- 46, bANC131, 8ANC134, IB2530, INC9, 8ANC195. 8ANC196, 10-259, 10-303, 10-410, 10- 847, 10-996, 10-1074, 10-1121, 10-1130, 10-1146, 10-1341, 10-1369, and 10- 1074GM. Additional examples include those described in Klein et al, Nature, 2012. 492(7427): p. 118-22, Horwitz et al, Proc Natl Acad Sci U S A, 2013. 110(41): p. 16538- -43, Scheid, et al. 2011. Science, 333 : 1633-1637, Scheid, et al. 2009. Nature, 458:636- 640, Eroshkin et al, Nucleic Acids Res. 2014 Jan;42 (Database issue):Dl 133-9, Mascola et al. Immunol Rev. 2013 Jul;254(l):225-44, such as those listed below.

Viral Epitope Antibody binding characteristics Antibody clonal family

MPER of gp41 Contiguous sequence 2F5

Contiguous sequence 4E10

Contiguous sequence M66.6

Contiguous sequence CAP206-CH12

Contiguous sequence 10E8 1

VlV2-glycan Peptidoglycan PG9, PG16

Peptidoglycan CHOl-04

Peptidoglycan PGT 141-145

Glycan only 2G12

V3-glycan Peptidoglycan PGT121-123

Peptidoglycan PGT125-131

Peptidoglycan PGT135-137

CD4 binding site CDRH3 loop bl2

No liganded structure HJ16

CDRH3 loop CH103-106 Mimics CD4 via CDRH2 VRCOl-03

Mimics CD4 via CDRH2 VRC-PG04, 04b

Mimics CD4 via CDRH2 VRC-CH30-34

No liganded structure 3BNC117, 3BNC60

Mimics CD4 via CDRH2 NIH45-46

No liganded structure 12A12, 12A21

No liganded structure 8ANC131, 134

No liganded structure 1NC9, 1B2530

In some embodiment, the anti-HIV bNAbs of this invention encompass the antigenic specificity of two potent bNAbs targeting distinct, non-overlapping epitopes on the HIV-1 Env necessary for viral fitness. Examples of these epitopes include: (i) the CD4 binding site, which is targeted by the 3BNC117, 8ANC131 and CHI 03 mAbs; (ii) the V3 region, which is recognized by the 10-1074, PGT121, PGT128, and PGT135 mAbs in a glycan dependent or independent fashion; (iii) the Vl/2 region, recognized by the PG16, PGT145 and PGDM1400 mAbs, (iv) the membrane proximal region (MPER), targeted by the 10E8 mAb, and (v) the gpl20/gp41 interface, which is recognized non-competitively by the PGT151 and 8ANC195 mAbs. Listed below are the heavy chain variable regions (VH) sequences and light chain variable regions (VL) of some of the antibodies mentioned above, where the CDRs are underlined.

CH103 VH QVQLQESGPGWKSSETLSLTCTVSGGSMGGTYWSWLRL 15 SPGKGLEWIGYI FHTGETNYSPSLKGRVS ISVDTSEDQF SLRLRSVTAADTAVYFCASLPRGQLVNAYFRNWGRGSLV SVSSAS

CH103 VL GVHSYELTQPPSVSVSPGQTATITCSGASTNVCWYQVKP 16

GQSPEWI FENYKRPSGIPDRFSGSKSGSTATLTIRGTQ AIDEADYYCQVWDSFSTFVFGSGTQVTVLRQPKAAP

10-1074 VH GVHSQVQLQESGPGLVKPSETLSVTCSVSGDSMNNYYWT 17

WIRQSPGKGLEWIGYISDRESATYNPSLNSRWISRDTS KNQLSLKLNSVTPADTAVYYCATARRGQRIYGWSFGEF FYYYSMDVWGKGTTVTVSSAS

10-1074 VL GSVTSYVRPLSVALGETARISCGRQALGSRAVQWYQHRP 18

GQAPILLIYNNQDRPSGIPERFSGTPDINFGTRATLTIS GVEAGDEADYYCHMWDSRSGFSWSFGGATRLTVLGQPKA AP

PGT121 VH QMQLQESGPGLVKPSETLSLTCSVSGASISDSYWSWIRR 19

SPGKGLEWIGYVHKSGDTNYSPSLKSRVNLSLDTSKNQV SLSLVAATAADSGKYYCARTLHGRRIYGIVAFNEWFTYF YMDVWGNGTQVTVSSAST

PGT121 VL GSVTHCTASVTSDISVAPGETARISCGEKSLGSRAVQWY 20

QHRAGQAPSLI IYNNQDRPSGIPERFSGSPDSPFGTTAT LTITSVEAGDEADYYCHIWDSRVPTKWVFGGGTTLTVLG QPKAAP

PGT128 VH QPQLQESGPTLVEASETLSLTCAVSGDSTAACNSFWGWV 21

RQPPGKGLEWVGSLSHCASYWNRGWTYHNPSLKSRLTLA LDTPKNLVFLKLNSVTAADTATYYCARFGGEVLRYTDWP KPAWVDLWGRGTLVTVSSA

PGT128 VL GSWAQSALTQPPSASGSPGQSITISCTGTSNNFVSWYQQ 22

HAGKAPKLVIYDVNKRPSGVPDRFSGSKSGNTASLTVSG LQTDDEAVYYCGSLVGNWDVI FGGGTKLTVLGQPKAAP

PGT135 VH GVHSQLQMQESGPGLVKPSETLSLSCTVSGDSIRGGEWG 23

DKDYHWGWVRHSAGKGLEWIGS IHWRGTTHYKESLRRRV SMSIDTSRNWFSLRLASVTAADTAVYFCARHRHHDVFML VPIAGWFDVWGPGVQVTVSSA

PGT135 VL GVHSEIVMTQSPDTLSVSPGETVTLSCRASQNINKNLAW 24

YQYKPGQSPRLVI FETYSKIAAFPARFVASGSGTEFTLT INNMQSEDVAVYYCQQYEEWPRTFGQGTKVDIKRTVAAP PG16 VH QEQLVESGGGWQPGGSLRLSCLASGFTFHKYGMHWVRQ 25 APGKGLEWVALISDDGMRKYHSDSMWGRVTISRDNSKNT LYLQFSSLKVEDTAMFFCAREAGGPIWHDDVKYYDFNDG YYNYHYMDVWGKGTTVTVSSAS

PG16 VL QSALTQPASVSGSPGQTITISCNGTSSDVGGFDSVSWYQ 26

QSPGKAPKVMVFDVSHRPSGISNRFSGSKSGNTASLTIS GLHIEDEGDYFCSSLTDRSHRI FGGGTKVTVLGQPKAAP

PGT145 VH QVQLVQSGAEVKKPGSSVKVSCKASGNSFSNHDVHWVRQ 27

ATGQGLEWMGWMSHEGDKTGLAQKFQGRVTITRDSGAST VYMELRGLTADDTAIYYCLTGSKHRLRDYFLYNEYGPNY EEWGDYLATLDVWGHGTAVTVSSAS

PGT145 VL EWITQSPLFLPVTPGEAASLSCKCSHSLQHSTGANYLA 28

WYLQRPGQTPRLLIHLATHRASGVPDRFSGSGSGTDFTL KISRVESDDVGTYYCMQGLHSPWTFGQGTKVEIKRTVAA P

PGDM1400 VH QAQLVQSGPEVRKPGTSVKVSCKAPGNTLKTYDLHWVRS 29

VPGQGLQWMGWISHEGDKKVIVERFKAKVTIDWDRSTNT AYLQLSGLTSGDTAVYYCAKGSKHRLRDYALYDDDGALN WAVDVDYLSNLEFWGQGTAVTVSSAS

PGDM1400 VL DFVLTQSPHSLSVTPGESAS ISCKSSHSLIHGDRNNYLA 30

WYVQKPGRSPQLLIYLASSRASGVPDRFSGSGSDKDFTL KISRVETEDVGTYYCMQGRESPWTFGQGTKVDIKRTVAA

P

10E8 VH EVQLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQ 31

PPGKGLEWVGRITGPGEGWSVDYAAPVEGRFTISRLNS I NFLYLEMNNLRMEDSGLYFCARTGKYYDFWSGYPPGEEY FQDWGRGTLVTVSSAS

10E8 VL SYELTQETGVSVALGRTVTITCRGDSLRSHYASWYQKKP 32

GQAPILLFYGKNNRPSGVPDRFSGSASGNRASLTISGAQ AEDDAEYYCSSRDKSGSRLSVFGGGTKLTVLGQPKAAP

PGT151 VH RVQLVESGGGWQPGKSVRLSCWSDFPFSKYPMYWVRQ 33

APGKGLEWVAAISGDAWHWYSNSVQGRFLVSRDNVKNT LYLEMNSLKIEDTAVYRCARMFQESGPPRLDRWSGRNYY YYSGMDVWGQGTTVTVSSAS

PGT151 VL DIVMTQTPLSLSVTPGQPAS ISCKSSESLRQSNGKTSLY 34

WYRQKPGQSPQLLVFEVSNRFSGVSDRFVGSGSGTDFTL RISRVEAEDVGFYYCMQSKDFPLTFGGGTKVDLKRTVAA

P 8ANC195 VH QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAVNWVR 35 QAPGQSLEYIGQIWRWKSSASHHFRGRVLISAVDLTGSS PPISSLEIKNLTSDDTAVYFCTTTSTYDRWSGLHHDGVM AFSSWGQGTLISVSAAS

8ANC195 VL DIQMTQSPSTLSASTGDTVRISCRASQSITGNWVAWYQQ 36

RPGKAPRLLIYRGAALLGGVPSRFRGSAAGTDFTLTIGN LQAEDFGTFYCQQYDTYPGTFGQGTKVEVKRTVAA

Other Parent Antibodies

The approach disclosed herein can be used for generating not only bispecific anti-

HIV bNAbs, but also other bispecific therapeutic antibodies. A number of therapeutic antibodies directed against cell surface molecules and/or their ligands are known which can be used for the selection and construction of tailor-made specific recognition binding moiety in the bispecific antibodies of this invention. Examples include

Blinatumomab/BLINCYTO, Rituxan/MabThera/Rituximab, H7/Ocrelizumab,

Zevalin/Ibrizumomab, Arzerra/Ofatumumab (CD20), HLL2/Epratuzumab, Inotuzomab (CD22), Zenapax/Daclizumab, Simulect/Basiliximab (CD25), Herceptin/Trastuzumab, Pertuzumab (Her2/ERBB2), Mylotarg/Gemtuzumab (CD33), Raptiva/Efalizumab (Cdl la), Erbitux/Cetuximab (EGFR, epidermal growth factor receptor), IMC-1121B (VEGF receptor 2), Tysabri/Natalizumab (a4-subunit of a4pi and α4β7 integrins), ReoPro/Abciximab (gpllb-gplla and avP3-integrin), Orthoclone OKT3/Muromonab-CD3 (CD3), Benlysta/Belimumab (BAFF), Tolerx/Oteliximab (CD3), Soliris/Eculizumab (C5 complement protein), Actemra/Tocilizumab (IL-6R), Panorex/Edrecolomab (EpCAM, epithelial cell adhesion molecule), CEA-CAM5/Labetuzumab (CD66/CEA, carcinoembryonic antigen), CT-11 (PD-1, programmed death-1 T-cell inhibitory receptor, CD-d279), H224G11 (c-Met receptor), SAR3419 (CD19), FMC-A12/Cixutumumab (IGF- 1R, insulin-like growth factor 1 receptor), MEDI-575 (PDGF-R, platelet-derived growth factor receptor), CP-675, 206/Tremelimumab (cytotoxic T lymphocyte antigen 4), R05323441 (placenta growth factor or PGF), HGS1012/Mapatumumab (TRAIL-R1), SGN-70 (CD70), Vedotin (SGN-35)/Brentuximab (CD30), and ARH460-16-2 (CD44).

The bispecific binding molecules/bispecific antibodies disclosed herein can be used in the preparation of medicaments for the treatment of e.g. an oncologic disease, a cardiovascular disease, an infectious disease, an inflammatory disease, an autoimmune disease, a metabolic (e.g., endocrine) disease, or a neurological (e.g. neurodegenerative) disease. Exemplary non-limiting examples of these diseases are Alzheimer's disease, non- Hodgkin's lymphomas, B-cell acute and chronic lymphoid leukemias, Burkitt lymphoma, Hodgkin's lymphoma, hairy cell leukemia, acute and chronic myeloid leukemias, T-cell lymphomas and leukemias, multiple myeloma, glioma, Waldenstrom's macroglobulinemia, carcinomas (such as carcinomas of the oral cavity, gastrointestinal tract, colon, stomach, pulmonary tract, lung, breast, ovary, prostate, uterus, endometrium, cervix, urinary bladder, pancreas, bone, liver, gall bladder, kidney, skin, and testes), melanomas, sarcomas, gliomas, and skin cancers, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonlein purpura, post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangitis obliterans, Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis, or fibrosing alveolitis.

A number of cell surface markers and their ligands are known. For example cancer cells have been reported to express at least one of the following cell surface markers and or ligands, including but not limited to, carbonic anhydrase IX, alpha-fetoprotein, alpha- ctinin-4, A3 (antigen specific for A33 antibody), ART -4, B7, Ba-733, BAGE, BrE3- antigen, CA125, CAMEL, CAP-1, CASP-8/m, CCCL19, CCCL21, CD 1, CDla, CD2, CD3, CD4, CDS, CD8, CD1-1A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD 126, CD 133, CD 138, CD 147, CD 154, CDC27, CDK-4/m, CDKN2A, CXCR4, CXCR7, CXCL12, HIF-1-α, colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, c-met, DAM, EGFR, EGFRvIII, EGP-1, EGP-2, ELF2-M, Ep-CAM, Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, GROB, HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and its subunits, HER2/neu, FDVlGB-1, hypoxia inducible factor (HIF-1), HSP70-2M, HST-2 or la, IGF-1R, IFN-γ, IFN-a, IFN-β, IL-2, IL-4R, IL-6R, IL- 13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, insulin-like growth factor- 1 (IGF-1), KC4-antigen, KS-1 -antigen, KS 1-4, Le-Y, LDR/FUT, macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART -2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-IA, MIP-IB, MIF, MUCl, MUC2, MUC3, MUC4, MUC5, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, pancreatic cancer mucin, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF, ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, 5100, survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptors, TNF-a, Tn-antigen, Thomson- Friedenreich antigens, tumor necrosis antigens, VEGFR, ED-B fibronectin, WT-1, 17-1A- antigen, complement factors C3, C3a, C3b, C5a, C5, an angiogenesis marker, bcl-2, bcl-6, Kras, cMET, an oncogene marker and an oncogene product (see, e.g., Sensi et al, Clin. Cancer Res. 12 (2006) 5023-5032; Parmiani et al, J. Immunol. 178 (2007) 1975-1979; Novellino et al, Cancer Immunol. Immunother. 54 (2005) 187-207). Thus, antibodies recognizing such specific cell surface receptors or their ligands can be used for specific and selective recognition binding moieties in the bispecific antibodies of this invention, targeting and binding to a number/multitude of cell surface markers or ligands that are associated with a disease.

The most widely used application of bispecific antibodies is in cancer immunotherapy, where bispecific antibodies are engineered that simultaneously bind to a cytotoxic cell {e.g., using a receptor like CD3) and a target like a tumor cell to be destroyed. See, e.g., Mueller et al, 2010). Biodrugs 24 (2): 89-98 and Chames et al, (2009). MAbs 1 (6): 539-547. Accordingly, the invention disclosed herein can also be used in cancer immunotherapy.

In some embodiments, for the treatment of cancer/tumors, bispecific antibodies are used to target tumor-associated antigens (TAAs), such as those reported in Herberman, "Immunodiagnosis of Cancer", in Fleisher ed., "The Clinical Biochemistry of Cancer", page 347 (American Association of Clinical Chemists, 1979) and in U.S. Pat. No. 4,150, 149; U.S. Pat. No. 4,361,544; and U.S. Pat. No. 4,444,744. Reports on tumor associated antigens include Mizukami et al., Nature Med. 11 (2005) 992-997; Hatfield et al, Curr. Cancer Drug Targets 5 (2005) 229-248; Vallbohmer et al., J. Clin. Oncol. 23 (2005) 3536-3544; and Ren et al., Ann. Surg. 242 (2005) 55-63), each incorporated herein by reference with respect to the TAAs identified. Where the disease involves a lymphoma, leukemia or autoimmune disorder, targeted antigens may be selected from the group consisting of CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD54, CD67, CD74, CD79a, CD80, CD126, CD138, CD154, CXCR4, B7, MUCl or la, HM1.24, HLA-DR, tenascin, VEGF, PIGF, ED-B fibronectin, an oncogene, an oncogene product (e.g., c-met or PLAGL2), CD66a-d, necrosis antigens, IL-2, T101, TAG, IL-6, MIF, TRAIL-R1 (DR4) and TRAIL-R2 (DR5).

Antibodies against the above-mentioned antigens can be used as the binding sites or moieties to make bispecific antibodies of this invention. A number of bispecific antibodies can be made against two different targets.

Examples of the antigen pairs include CD19/CD3, BCMA/CD3, different antigens of the HER family in combination (EGFR, HER2, HER3), IL17RA/IL7R, IL-6/IL-23, IL- l-p/IL-8, IL-6 or IL-6R/IL-21 or IL-21R, ANG2/VEGF, VEGF/PDGFR-beta, Vascular Endothelial Growth Factor (VEGF) acceptor 2/CD3, PSMA/CD3, EPCAM/CD3, combinations of antigens selected from a group consisting of VEGFR-l, VEGFR-2, VEGFR-3, FLT3, c-FMS/CSFIR, RET, c-Met, EGFR, Her2/neu, HER3, HER4, IGFR, PDGFR, c-KIT, BCR, integrin and MMPs with a water-soluble ligand is selected from the group consisting of VEGF, EGF, PIGF, PDGF, HGF, and angiopoietin, ERBB-3/C-MET, ERBB-2/C-MET, EGF receptor 1/CD3, EGFR/HER3, PSCA/CD3, C-MET/CD3, ENDOSIALIN/CD3, EPCAM/CD3, IGF-1R/CD3, FAPALPHA/CD3, EGFR/IGF-IR, IL 17A/F, EGF receptor 1/CD3, and CD19/CD16.

Additional examples of bispecific antibodies can have (i) a first specificity directed to a glycoepitope of an antigen selected from the group consisting of Lewis x-, Lewis b- and Lewis y-structures, Globo H-structures, KH1, Tn-antigen, TF-antigen and carbohydrate structures of Mucins, CD44, glycolipids and glycosphingolipids, such as Gg3, Gb3, GD3, GD2, Gb5, Gml, Gm2, and sialyltetraosylceramide and (ii) a second specificity directed to an ErbB receptor tyrosine kinase selected from the group consisting of EGFR, HER2, HER3 and HER4, GD2 in combination with a second antigen binding site is associated with an immunological cell chosen from the group consisting of T- lymphocytes K cell, B-lymphocytes, dendritic cells, monocytes, macrophages, neutrophils, mesenchymal stem cells, neural stem cells.

A monospecific antibody can be joined together with another using the method disclosed herein to make bispecific antibodies. By using already available specific therapeutic binding entities, such as those therapeutic antibodies described above, one can generate a desired combination. With this tailor-made generation of bispecific therapeutics by combining two single therapeutic molecules for simultaneous targeting and binding to two different epitopes, an additive/synergistic effect can be expected in comparison to the single therapeutic molecules.

An "antigen" refers to a substance that elicits an immunological reaction or binds to the products of that reaction. The term "epitope" refers to the region of an antigen to which an antibody or T cell binds.

The term "antibody" (Ab) as used herein includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies and polyreactive antibodies), and antibody fragments. Thus, the term "antibody" as used in any context within this specification is meant to include, but not be limited to, any specific binding member, immunoglobulin class and/or isotype (e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE and IgM); and biologically relevant fragment or specific binding member thereof, including but not limited to Fab, F(ab')2, Fv, and scFv (single chain or related entity). It is understood in the art that an antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. A heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CHI, CH2 and CH3). A light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The variable regions of both the heavy and light chains comprise framework regions (FWR) and complementarity determining regions (CDR). The four FWR regions are relatively conserved while CDR regions (CDR1, CDR2 and CDR3) represent hypervariable regions and are arranged from NH2 terminus to the COOH terminus as follows: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, and FWR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen while, depending of the isotype, the constant region(s) may mediate the binding of the immunoglobulin to host tissues or factors. Also included in the definition of "antibody" as used herein are chimeric antibodies, humanized antibodies, and recombinant antibodies, human antibodies generated from a transgenic non-human animal, as well as antibodies selected from libraries using enrichment technologies available to the artisan.

The term "variable" refers to the fact that certain segments of the variable (V) domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable regions. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" that are each 9-12 amino acids long. The variable regions of native heavy and light chains each comprise four FRs, largely adopting a beta sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the beta sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen- binding site of antibodies (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

The term "hypervariable region" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" ("CDR").

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The term "polyclonal antibody" refers to preparations that include different antibodies directed against different determinants ("epitopes").

The monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with, or homologous to, corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with, or homologous to, corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, for example, U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)). Chimeric antibodies include antibodies having one or more human antigen binding sequences (for example, CDRs) and containing one or more sequences derived from a non-human antibody, for example, an FR or C region sequence. In addition, chimeric antibodies included herein are those comprising a human variable region antigen binding sequence of one antibody class or subclass and another sequence, for example, FR or C region sequence, derived from another antibody class or subclass.

A "humanized antibody" generally is considered to be a human antibody that has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues often are referred to as "import" residues, which typically are taken from an "import" variable region. Humanization may be performed following the method of Winter and co-workers (see, for example, Jones et al, Nature 321 :522-525 (1986); Reichmann et al, Nature 332:323-327 (1988); Verhoeyen et al, Science 239: 1534-1536 (1988)), by substituting import hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (see, for example, U.S. Pat. No. 4,816,567), where substantially less than an intact human variable region has been substituted by the corresponding sequence from a non-human species.

An "antibody fragment" comprises a portion of an intact antibody, such as the antigen binding or variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see, for example, U.S. Pat. No. 5,641,870; Zapata et al, Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

"Fv" is the minimum antibody fragment that contains a complete antigen- recognition and antigen-binding site. This fragment contains a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable region (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

"Single-chain Fv" ("sFv" or "scFv") are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. The sFv polypeptide can further comprise a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. For a review of sFv, see, for example, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra.

The term "diabodies" refers to small antibody fragments prepared by constructing sFv fragments with short linkers (about 5-10 residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al, Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

Domain antibodies (dAbs), which can be produced in fully human form, are the smallest known antigen-binding fragments of antibodies, ranging from about 11 kDa to about 15 kDa. DAbs are the robust variable regions of the heavy and light chains of immunoglobulins (VH and VL, respectively). They are highly expressed in microbial cell culture, show favorable biophysical properties including, for example, but not limited to, solubility and temperature stability, and are well suited to selection and affinity maturation by in vitro selection systems such as, for example, phage display. DAbs are bioactive as monomers and, owing to their small size and inherent stability, can be formatted into larger molecules to create drugs with prolonged serum half-lives or other pharmacological activities. Examples of this technology have been described in, for example, W09425591 for antibodies derived from Camelidae heavy chain Ig, as well in US20030130496 describing the isolation of single domain fully human antibodies from phage libraries.

Fv and sFv are the only species with intact combining sites that are devoid of constant regions. Thus, they are suitable for reduced nonspecific binding during in vivo use. sFv fusion proteins can be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of a sFv. See, for example, Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment also can be a "linear antibody", for example, as described in U.S. Pat. No. 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

In certain embodiments, antibodies of the described invention are bispecific and can bind to two different epitopes of a single antigen. Other such antibodies can combine a first antigen binding site with a binding site for a second antigen. Alternatively, an anti- HIV arm can be combined with an arm that binds to a triggering molecule on a leukocyte, such as a T-cell receptor molecule (for example, CD3), or Fc receptors for IgG (Fc gamma R), such as Fc gamma RI (CD64), Fc gamma RII (CD32) and Fc gamma RIII (CD 16), so as to focus and localize cellular defense mechanisms to the infected cell. Bispecific antibodies also can be used to localize cytotoxic agents to infected cells. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (for example, F(ab')2 bispecific antibodies). See for example, WO 96/16673, U.S. Pat. No. 5,837,234, WO98/02463, U.S. Pat. No. 5,821,337, and Mouquet et al, Nature. 467, 591-5 (2010).

Methods for making bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (see, for example, Millstein et al., Nature, 305:537-539 (1983)). Similar procedures are disclosed in, for example, WO 93/08829, Traunecker et al., EMBO J., 10:3655-3659 (1991) and see also; Mouquet et al, Nature. 467, 591-5 (2010). Techniques for generating bispecific antibodies from antibody fragments also have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. See Brennan et al, Science, 229: 81 (1985).

Typically, the parent antibodies described in the invention can be produced using conventional hybridoma technology or made recombinantly using vectors and methods available in the art. Human antibodies also can be generated by in vitro activated B cells (see, for example, U.S. Pat. Nos. 5,567,610 and 5,229,275). General methods in molecular genetics and genetic engineering useful in the present invention are described in the current editions of Molecular Cloning: A Laboratory Manual (Sambrook, et al., Molecular Cloning: A Laboratory Manual (Fourth Edition) Cold Spring Harbor Lab. press, 2012), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), "Guide to Protein Purification" in Methods in Enzymology (M.P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R.I. Freshney. 1987. Liss, Inc. New York, NY), and Gene Transfer and Expression Protocols, pp. 109-128, ed. EJ. Murray, The Humana Press Inc., Clifton, N.J.). Reagents, cloning vectors, and kits for genetic manipulation are available from commercial vendors such as BioRad, Stratagene, Invitrogen, ClonTech and Sigma-Aldrich Co.

Human antibodies also can be produced in transgenic animals (for example, mice) that are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ- line mutant mice results in the production of human antibodies upon antigen challenge. See, for example, Jakobovits et al, Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immune, 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all of GenPharm); U.S. Pat. No. 5,545,807; and WO 97/17852. Such animals can be genetically engineered to produce human antibodies comprising a polypeptide of the described invention.

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al, Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of these fragments. Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (see, for example, Carter et al, Bio/Technology 10: 163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragment with increased in vivo half-life comprising a salvage receptor binding epitope residues are described in U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.

Other techniques that are known in the art for the selection of antibody fragments from libraries using enrichment technologies, including but not limited to phage display, ribosome display (Hanes and Pluckthun, 1997, Proc. Nat. Acad. Sci. 94: 4937-4942), bacterial display (Georgiou, et al., 1997, Nature Biotechnology 15: 29-34) and/or yeast display (Kieke, et al., 1997, Protein Engineering 10: 1303-1310) may be utilized as alternatives to previously discussed technologies to select single chain antibodies. Single- chain antibodies are selected from a library of single chain antibodies produced directly utilizing filamentous phage technology. Phage display technology is known in the art (e.g., see technology from Cambridge Antibody Technology (CAT)) as disclosed in U.S. Patent Nos. 5,565,332; 5,733,743; 5,871,907; 5,872,215; 5,885,793; 5,962,255; 6, 140,471; 6,225,447; 6,291650; 6,492,160; 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593, 081, as well as other U.S. family members, or applications which rely on priority filing GB 9206318, filed 24 May 1992; see also Vaughn, et al. 1996, Nature Biotechnology 14: 309- 314). Single chain antibodies may also be designed and constructed using available recombinant DNA technology, such as a DNA amplification method (e.g., PCR), or possibly by using a respective hybridoma cDNA as a template.

Variant antibodies also are included within the scope of the invention. Thus, variants of the sequences recited in the application also are included within the scope of the invention. Further variants of the antibody sequences having improved affinity can be obtained using methods known in the art and are included within the scope of the invention. For example, amino acid substitutions can be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of the nucleotide sequence can be used to improve the efficiency of translation in expression systems for the production of the antibody.

In certain embodiments, an antibody of the invention comprises a heavy chain variable region comprising CDRl, CDR2 and CDR3 sequences, and a light chain variable region comprising CDRl, CDR2 and CDR3 sequences, wherein one or more of these CDR sequences comprise specified amino acid sequences based on the preferred antibodies described herein, or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of neutralizing multiple HIV-1 viral strains. Similarly, an antibody of the invention can comprise a hinge region of the preferred antibodies described herein, e.g., SEQ ID NO: 3, a section thereof, or conservative modifications thereof.

A conservative modification or functional equivalent of a peptide, polypeptide, or protein disclosed in this invention (e.g., the hinge region or a heavy chain having the hinge region) refers to a polypeptide derivative of the peptide, polypeptide, or protein, e.g., a protein having one or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof. It retains substantially the activity to of the parent peptide, polypeptide, or protein (such as those disclosed in this invention). In general, a conservative modification or functional equivalent is at least 60% (e.g., any number between 60% and 100%, inclusive, e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%) identical to a parent (e.g., one of SEQ ID NOs: 1-36). Accordingly, within scope of this invention are hinge regions having one or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof, as well as heavy chains or antibodies having the variant hinge regions. For example, a variant hinge region can be a truncated form of IgG3 hinge region or IgG3 C-hinge region, such as one with fewer than three repeats (e.g., two or one copy) of the 15 mer or with repeats of a sequence shorter than the 15 mer or 17 mer mentioned above. In general, the hinge region is longer than that of IgGl hinge region (19 amino acid residues) and at least 20 (e.g., at least 21, 22, 25, 30, 35, 40, 45, 50, 55, 60, 65, 66, 67, 70, 80, 90, or 100) amino acid residues in length.

As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

Additionally or alternatively, the protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs {e.g., XBLAST and NBLAST) can be used. (See www.ncbi.nlm.nih.gov).

As used herein, the term "conservative modifications" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include

amino acids with basic side chains {e.g., lysine, arginine, histidine),

acidic side chains {e.g., aspartic acid, glutamic acid),

uncharged polar side chains {e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan),

nonpolar side chains {e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine),

beta-branched side chains {e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

Thus, one or more amino acid residues within the CDR or non-CDR regions of an antibody of the invention can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function using the functional assays described herein. In the same vein, the variant hinge region described herein can have one or more conservative amino acid substitutions. The hinge region of antibody molecules is a flexible domain that joins the Fab arms to the Fc piece. The flexibility of the hinge region in IgG and IgA molecules allows the Fab arms to adopt a wide range of angles, permitting binding to epitopes spaced variable distances apart. Since the variant hinge region is to provide flexibility, but not antigen-binding specificity or other function such as binding to a receptor, one skilled in the art would appreciate that various positions of the hinge regions disclosed herein can have conservative amino acid substitutions without comprising the flexibility.

Other modifications of the antibody are contemplated herein. For example, the antibody can be linked to one of a variety of nonproteinaceous polymers, for example, polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. The antibody also can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions. Such techniques are disclosed in, for example, Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).

As disclosed herein, bNAbs are significantly more effective than ART in blocking the establishment of the reservoir when given early in the infection. One of the key differences between antibodies and ART is that antibodies can engage a variety of host immune effector pathways by way of their Fc receptors (Nimmerjahn et al., Nat Rev Immunol, 2008. 8(1): p. 34-47). Consistent with this important difference, the mechanism by which antibodies interfere with the establishment of the reservoir is dependent on their ability to bind to Fc receptors.

The terms "Fc receptor" or "FcR" are used to describe a receptor that binds to the Fc region of an antibody. An Fc receptor is a protein found on the surface of certain cells - including, among others, B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils and mast cells - that contribute to the protective functions of the immune system. Its name is derived from its binding specificity for the Fc region (fragment crystallizable region) of an antibody.

Several antibody functions are mediated by Fc receptors. For example, Fc receptors bind to antibodies that are attached to infected cells or invading pathogens. Their activity stimulates phagocytic or cytotoxic cells to destroy microbes, or infected cells by antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity. It was also known in the art that the Fc region of an antibody ensures that each antibody generates an appropriate immune response for a given antigen, by binding to a specific class of Fc receptors, and other immune molecules, such as complement proteins. FcRs are defined by their specificity for immunoglobulin isotypes: Fc receptors for IgG antibodies are referred to as FcyR, for IgE as FcsFR, for IgA as FcaR and so on. Surface receptors for immunoglobulin G are present in two distinct classes-those that activate cells upon their crosslinking ("activation FcRs") and those that inhibit activation upon co-engagement ("inhibitory FcRs").

In all mammalian species studied to date, four different classes of IgG Fc-receptors have been defined: FcyRI (CD64), FcyRII (CD32), FcyRIII (CDI6) and FcylV. Whereas FcyRI displays high affinity for the antibody constant region and restricted isotype specificity, FcyRII and FcyRIII have low affinity for the Fc region of IgG but a broader isotype binding pattern (Ravetch and Kinet, 1991; Hulett and Hogarth, Adv Immunol 57, 1-127 (1994)). FcyRrV is a recently identified receptor, conserved in all mammalian species with intermediate affinity and restricted subclass specificity (Mechetina et al., Immunogenetics 54, 463-468 (2002); Davis et al, Immunol Rev 190, 123-136 (2002); Nimmerjahn et al, Immunity 23, 41-51 (2005)).

Functionally there are two different classes of Fc-receptors: the activation and the inhibitory receptors, which transmit their signals via immunoreceptor tyrosine based activation (ITAM) or inhibitory motifs (ITEVI), respectively (Ravetch, in Fundamental Immunology W. E. Paul, Ed. (Lippincott-Raven, Philadelphia, (2003); Ravetch and Lanier, Science 290, 84-89 (2000). The paired expression of activating and inhibitory molecules on the same cell is the key for the generation of a balanced immune response. Additionally, it has been appreciated that the IgG Fc-receptors show significant differences in their affinity for individual antibody isotypes rendering certain isotypes more strictly regulated than others (Nimmerjahn et al., 2005).

In one embodiment of the invention, FcR is a native sequence human FcR. In another embodiment, FcR, including human FcR, binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcyRII receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (IT AM) in its cytoplasmic domain. Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITFM) in its cytoplasmic domain (see review in Daron, Annu Rev Immunol, 15, 203-234 (1997); FcRs are reviewed in Ravetch and Kinet, Annu Rev Immunol, 9, 457-92 (1991); Capel et al, Immunomethods, 4, 25-34 (1994); and de Haas et al, J Lab Clin Med, 126, 330-41 (1995), Nimmerjahn and Ravetch 2006, Ravetch Fc Receptors in Fundamental Immunology, ed William Paul 5th Ed. each of which is incorporated herein by reference).

As used herein, the term "Fc fragment" or "Fc region" is used to define a C- terminal region of an immunoglobulin heavy chain. Such an Fc region is the tail region of an antibody that interacts with Fc receptors and some proteins of the complement system. The Fc region may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. A native sequence Fc region comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. A variant Fc region as appreciated by one of ordinary skill in the art comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one "amino acid modification."

In IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains; IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. The Fc regions of IgGs bear a highly conserved N-glycosylation site. Glycosylation of the Fc fragment is important for Fc receptor-mediated activity. The N-glycans attached to this site are predominantly core- fucosylated diantennary structures of the complex type. In addition, small amounts of these N-glycans also bear bisecting GlcNAc and a-2,6 linked sialic acid residues. See, e.g., US20080286819, US20100278808, US20100189714, US 2009004179, 20080206246, 20110150867, and WO2013095966, each of which is incorporated herein by reference.

As the Fc receptor function is involved in HIV-1 latent reservoir, the bNAb antibody of this invention can include antibody variable regions with the desired binding specificities (antibody-antigen combining sites) fused to immunoglobulin constant domain sequences. The fusion can be with an Ig heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. According to some embodiments, the first heavy-chain constant region (CHI) containing the site for light chain bonding, is present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired bispecific antibody. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios have no significant effect on the yield of the desired chain combination.

C. Other Agents

The above-described antibodies can be used in combination with one or more anti- retroviral agents for the treatment of HIV latency and/or infection. See, e.g., US 2010/0166806, US 2010/0324034, and US 2012/0203014, which are hereby incorporated in their entirety.

Compositions according to the present invention may also be administered in combination with other agents to enhance the biological activity of such agents. Such agents may include any one or more of the standard anti-HIV agents which are known in the art, including, but not limited to, azidothymidine (AZT), dideoxycytidine (ddC), and dideoxyinosine (ddl). Additional agents which have shown anti-HIV effects and may be combined with compositions in accordance to the invention include, for example, raltegravir, maraviroc, bestatin, human chorionic gonadotropin (hCG), levamisole, estrogen, efavirenz, etravirine, indomethacin, emtricitabine, tenofovir disoproxil fumarate, amprenavir, tipranavir, indinavir, ritonavir, darunavir, enfuvirtide, and gramicidin.

In some embodiments, such as those in connection with cancer therapy, a bispecific antibody of this invention can be further conjugated to one or more effector moieties, e.g. cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.

In some embodiments, the effector moiety can be a drug, including but not limited to a maytansinoid (see U.S. Pat. No. 5,208,020, U.S. Pat. No. 5,416,064, EP 0 425 235), an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF, see U.S. Pat. No. 5,635,483, U.S. Pat. No. 5,780,588, U.S. Pat. No. 7,498,298), a dolastatin, a calicheamicin or derivative thereof (see U.S. Pat. No. 5,712,374, U.S. Pat. No. 5,714,586, U.S. Pat. No. 5,739, 116, U.S. Pat. No. 5,767,285, U.S. Pat. No. 5,770,701, U.S. Pat. No. 5,770,710, U.S. Pat. No. 5,773,001, U.S. Pat. No. 5,877,296, Hinman, L. M, et al, Cancer Res. 53 (1993) 3336-3342, Lode, H. N., et al, Cancer Res. 58 (1998) 2925- 2928), an anthracycline such as daunomycin or doxorubicin (see Kratz, F., et al, Current Med. Chem. 13 (2006) 477-523, Jeffrey, S. C, et al, Bioorg. Med. Chem. Letters 16 (2006) 358-362, Torgov, M. Y., et al, Bioconjug. Chem. 16 (2005) 717-721, Nagy, A., et al, Proc. Natl. Acad. Sci. USA 97 (2000) 829-834, Dubowchik, G. M., et al, Bioorg. Med. Chem. Lett. 12 (2002) 1529-1532, King, H. D., et al, J. Med. Chem. 45 (2002) 4336-4343, and U.S. Pat. No. 6,630,579), methotrexate, vindesine, a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel, a trichothecene, and CC1065.

In other embodiments, the effector moiety can be an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. In yet some other embodiments, the effector moiety can be a radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugates. Examples include At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Rel ⁸⁸ , Sml ⁵³ , Bi ²¹² , P ³² , Pb ²¹² , and radioactive isotopes of Lu. When the radioconjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, for example Tc99 or 1123, or a spin label for nuclear magnetic resonance ( MR) imaging (also known as magnetic resonance imaging, MRI), such as I ¹²³ again, I ¹³¹ , In ¹¹¹ , F ¹⁹ , C ¹³ , N ¹⁵ , O ¹⁷ , gadolinium, manganese or iron.

D. Treatment Compositions and Methods HIV Infection Therapy

In one embodiment, the present invention provides a composition comprising at least one bNAb mentioned above alone or in combination with one of the other active agent mentioned above and a pharmaceutically acceptable carrier. The composition may include a plurality of the antibodies having the characteristics described herein in any combination and can further include antibodies neutralizing to HIV as are known in the art.

It is to be understood that compositions can be a single or a combination of antibodies disclosed herein, which can be the same or different, in order to prophylactically or therapeutically treat the progression of various subtypes of HIV infection. When an antibody or active agent is administered to an animal or a human, it can be combined with one or more pharmaceutically acceptable carriers, excipients or adjuvants as are known to one of ordinary skilled in the art.

Further, with respect to determining the effective level in a patient for treatment of HIV, in particular, suitable animal models are available and have been widely implemented for evaluating the in vivo efficacy against HIV of various therapy protocols. These models include mice, monkeys and cats. Even though these animals are not naturally susceptible to HIV disease, chimeric mice models (for example, SCID, bg/nu/xid, NOD/SCID, SCID-hu, immunocompetent SCID-hu, bone marrow-ablated BALB/c) reconstituted with human peripheral blood mononuclear cells (PBMCs), lymph nodes, fetal liver/thymus or other tissues can be infected with lentiviral vector or HIV, and employed as models for HIV pathogenesis. Similarly, the simian immune deficiency virus (SrV)/monkey model can be employed, as can the feline immune deficiency virus (FIV)/cat model. The pharmaceutical composition can contain other pharmaceuticals, in conjunction with a vector according to the invention, when used to therapeutically treat AIDS. These other pharmaceuticals can be used in their traditional fashion (i.e., as agents to treat HIV infection).

According to another embodiment, the present invention provides an antibody- based pharmaceutical composition comprising an effective amount of an isolated bNAb of the invention, or an affinity matured version, which provides a prophylactic or therapeutic treatment choice to reduce the latent reservoir and infection of the HIV virus. The pharmaceutical compositions of the present invention may be formulated by any number of strategies known in the art (e.g., see McGoff and Scher, 2000, Solution Formulation of Proteins/Peptides: In McNally, E.J., ed. Protein Formulation and Delivery. New York, NY: Marcel Dekker; pp. 139-158; Akers and Defilippis, 2000, Peptides and Proteins as Parenteral Solutions. In: Pharmaceutical Formulation Development of Peptides and Proteins. Philadelphia, PA: Talyor and Francis; pp. 145-177; Akers, et al., 2002, Pharm. Biotechnol. 14:47-127). A pharmaceutically acceptable composition suitable for patient administration will contain an effective amount of the bNAb antibody in a formulation which both retains biological activity while also promoting maximal stability during storage within an acceptable temperature range. The pharmaceutical compositions can also include, depending on the formulation desired, pharmaceutically acceptable diluents, pharmaceutically acceptable carriers and/or pharmaceutically acceptable excipients, or any such vehicle commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate- buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. The amount of an excipient that is useful in the pharmaceutical composition or formulation of this invention is an amount that serves to uniformly distribute the antibody throughout the composition so that it can be uniformly dispersed when it is to be delivered to a subject in need thereof. It may serve to dilute the antibody or other active agent to a concentration which provides the desired beneficial palliative or curative results while at the same time minimizing any adverse side effects that might occur from too high a concentration. It may also have a preservative effect. Thus, for an active ingredient having a high physiological activity, more of the excipient will be employed. On the other hand, for any active ingredient(s) that exhibit a lower physiological activity, a lesser quantity of the excipient will be employed.

The above described bNAb antibodies and antibody compositions, comprising at least one or a combination of the antibodies described herein, can be administered for the prophylactic and therapeutic treatment of HIV viral infection.

The composition can be a pharmaceutical composition that contains a pharmaceutically acceptable carrier. The term "pharmaceutical composition" refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo. A "carrier" as used herein includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include, but not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including, but not limited to, ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as, but not limited to, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as, but not limited to, polyvinylpyrrolidone; amino acids such as, but not limited to, glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including, but not limited to, glucose, mannose, or dextrins; chelating agents such as, but not limited to, EDTA; sugar alcohols such as, but not limited to, mannitol or sorbitol; salt-forming counterions such as, but not limited to, sodium; and/or nonionic surfactants such as, but not limited to, TWEEN.; polyethylene glycol (PEG), and PLURONICS.

The term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions, and various types of wetting agents. The compositions also can include stabilizers and preservatives. A pharmaceutically acceptable carrier, after administered to or upon a subject, does not cause undesirable physiological effects. The carrier in the pharmaceutical composition must be "acceptable" also in the sense that it is compatible with the active ingredient and, preferably, capable of stabilizing it. One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an active agent. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate.

A "subject" refers to a human and a non-human animal. Examples of a non-human animal include all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), dog, rodent (e.g., mouse or rat), guinea pig, cat, and non-mammals, such as birds, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or animal suitable as a disease model (such as non-human primates). A subject to be treated can be identified by standard diagnosing techniques for the disorder.

According to another embodiment, the present invention provides a method of reducing or preventing the establishment of a latent reservoir of HIV infected cells in a subject in need thereof (e.g., a subject infected with HIV or at risk of infection with HIV), thereby treating infection with a HIV infection, comprising administering to the subject a pharmaceutical composition comprising the HIV antibodies disclosed herein. The compositions of the invention can include more than one antibody having the characteristics disclosed (for example, a plurality or pool of antibodies). It also can include other HIV neutralizing antibodies and/or active agent known in the art.

Subjects at risk for HIV-related diseases or disorders include patients who have come into contact with an infected person or who have been exposed to HIV in some other way. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of HIV-related disease or disorder, such that a disease or disorder is prevented or, alternatively, delayed in its progression.

For in vivo treatment of human and non-human patients, the patient is administered or provided a pharmaceutical formulation including an HIV antibody of the invention. When used for in vivo therapy, the antibodies of the invention are administered to the patient in therapeutically effective amounts (i.e., amounts that eliminate or reduce the patient's latent viral reservoir). The antibodies are administered to a human patient, in accord with known methods, such as intravenous administration, for example, as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. The antibodies can be administered parenterally, when possible, at the target cell site, or intravenously. In some embodiments, antibody is administered by intravenous or subcutaneous administration. Therapeutic compositions of the invention may be administered to a patient or subject systemically, parenterally, or locally. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.

For parenteral administration, the antibodies may be formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable, parenteral vehicle. Examples of such vehicles include, but are not limited, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles include, but are not limited to, fixed oils and ethyl oleate. Liposomes can be used as carriers. The vehicle may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, such as, for example, buffers and preservatives. The antibodies can be formulated in such vehicles at concentrations of about 1 mg/ml to 10 mg/ml.

The dose and dosage regimen depends upon a variety of factors readily determined by a physician, such as the nature of the infection, for example, its therapeutic index, the patient, and the patient's history. Generally, a therapeutically effective amount of an antibody is administered to a patient. In some embodiments, the amount of antibody administered is in the range of about 0.1 mg/kg to about 50 mg/kg of patient body weight. Depending on the type and severity of the infection, about 0.1 mg/kg to about 50 mg/kg body weight (for example, about 0.1-15 mg/kg/dose) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. The progress of this therapy is readily monitored by conventional methods and assays and based on criteria known to the physician or other persons of skill in the art. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.

Other therapeutic regimens may be combined with the administration of the bNAb HIV antibody of the present invention. The combined administration includes coadministration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities. Such combined therapy can result in a synergistic therapeutic effect. The parameters for assessing successful treatment and improvement in the disease are also readily measurable by routine procedures familiar to a physician.

The terms "treating" or "treatment" or "alleviation" are used interchangeably and refer to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. In particular, it refers to administration of a compound or agent to a subject, who has a disorder (such as an HIV infection), with the purpose to cure, alleviate, relieve, remedy, delay the onset of, prevent, or ameliorate the disorder, the symptom of the disorder, the disease state secondary to the disorder, or the predisposition toward the disorder.

Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject or mammal is successfully "treated" for an infection if, after receiving a therapeutic amount of an antibody according to the methods of the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of infected cells or absence of the infected cells; reduction in the percent of total cells that are infected; and/or relief to some extent, one or more of the symptoms associated with the specific infection; reduced morbidity and mortality, and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.

Eliminating the HIV-1 reservoir in chronic infection is key to curing the disease, but direct measurement of the latent reservoir to evaluate therapeutic eradication strategies remains difficult (Siliciano et al, Curr Opin HIV AIDS, 2013. 8(4): p. 318-25). Quantitative viral outgrowth assays and PCR-based assays of integrated DNA yield variable results (Eriksson et al., PLoS Pathog, 2013. 9(2): p. el003174) in part because PCR cannot distinguish between inactive and permanently disabled proviruses, and outgrowth assays underestimate reservoir size (Ho et al., Cell, 2013. 155(3): p. 540-51). To that end, the most effective way to evaluate the reservoir in vivo is to measure viral rebound after terminating therapy as disclosed in the examples below. Cancer Therapy

In one aspect, the present invention relates to treatment of a subject in vivo using the above-described bispecific antibody such that growth and/or metastasis of cancerous tumors is inhibited. In one embodiment, the invention provides a method of inhibiting growth and/or restricting metastatic spread of tumor cells in a subject, comprising administering to the subject a therapeutically effective amount of a bispecific antibody.

Non-limiting examples of preferred cancers for treatment include chronic or acute leukemia including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphocytic lymphoma, breast cancer, ovarian cancer, melanoma {e.g., metastatic malignant melanoma), renal cancer {e.g. clear cell carcinoma), prostate cancer {e.g. hormone refractory prostate adenocarcinoma), colon cancer and lung cancer {e.g. non-small cell lung cancer). Additionally, the invention includes refractory or recurrent malignancies whose growth may be inhibited using the antibodies of the invention. Examples of other cancers that may be treated using the methods of the invention include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of said cancers.

The terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition. A "therapeutically effective amount" refers to the amount of an agent sufficient to effect beneficial or desired results. A therapeutically effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.

Administration "in combination with" one or more further therapeutic agents include simultaneous (concurrent) and consecutive administration in any order.

Pharmaceutically effective compositions of this invention may be administered to humans and other animals by a variety of methods that may include continuous or intermittent administration. Examples of methods of administration may include, but are not limited to, oral, rectal, parenteral, intracisternal, intrasternal, intravaginal, intraperitoneal, topical, transdermal, buccal, or as an oral or nasal spray. Accordingly, the pharmaceutically effective compositions may also include pharmaceutically acceptable additives, carriers or excipients. Such pharmaceutical compositions may also include the active ingredients formulated together with one or more non-toxic, pharmaceutically acceptable carriers specially formulated for oral administration in solid or liquid form, for parenteral injection or for rectal administration according to standard methods known in the art.

The term "parenteral" administration refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intracisternal, intrasternal, subcutaneous and intraarticular injection and infusion. Injectable mixtures are known in the art and comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), vegetable oils (such as olive oil), injectable organic esters (such as ethyl oleate) and suitable mixtures thereof.

The terms "peptide," "polypeptide," and "protein" are used herein interchangeably to describe the arrangement of amino acid residues in a polymer. A peptide, polypeptide, or protein can be composed of the standard 20 naturally occurring amino acid, in addition to rare amino acids and synthetic amino acid analogs. They can be any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation). A "recombinant" peptide, polypeptide, or protein refers to a peptide, polypeptide, or protein produced by recombinant DNA techniques; i.e., produced from cells transformed by an exogenous DNA construct encoding the desired peptide. A "synthetic" peptide, polypeptide, or protein refers to a peptide, polypeptide, or protein prepared by chemical synthesis. The term "recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Within the scope of this invention are fusion proteins containing one or more of the afore-mentioned sequences and a heterologous sequence. A heterologous polypeptide, nucleic acid, or gene is one that originates from a foreign species, or, if from the same species, is substantially modified from its original form. Two fused domains or sequences are heterologous to each other if they are not adjacent to each other in a naturally occurring protein or nucleic acid.

An "isolated" peptide, polypeptide, or protein refers to a peptide, polypeptide, or protein that has been separated from other proteins, lipids, and nucleic acids with which it is naturally associated. The polypeptide/protein can constitute at least 10% {i.e., any percentage between 10% and 100%, e.g., 20%, 30%, 40%, 50%, 60%, 70 %, 80%, 85%, 90%, 95%, and 99%) by dry weight of the purified preparation. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. An isolated polypeptide/protein described in the invention can be purified from a natural source, produced by recombinant DNA techniques, or by chemical methods.

The term "about" generally refers to plus or minus 10% of the indicated number. For example, "about 10%" may indicate a range of 9% to 11%, and "about 1" may mean from 0.9-1.1. Other meanings of "about" may be apparent from the context, such as rounding off, so, for example "about 1" may also mean from 0.5 to 1.4.

The term "biological sample" refers to a sample obtained from an organism (e.g., patient) or from components (e.g., cells) of an organism. The sample may be of any biological tissue, cell(s) or fluid. The sample may be a "clinical sample" which is a sample derived from a subject, such as a human patient. Such samples include, but are not limited to, saliva, sputum, blood, blood cells (e.g., white cells), amniotic fluid, plasma, semen, bone marrow, and tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells therefrom. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes. A biological sample may also be referred to as a "patient sample." A biological sample may also include a substantially purified or isolated protein, membrane preparation, or cell culture.

As used herein, the term "contacting" and its variants, when used in reference to any set of components, includes any process whereby the components to be contacted are mixed into same mixture (for example, are added into the same compartment or solution), and does not necessarily require actual physical contact between the recited components. The recited components can be contacted in any order or any combination (or subcombination), and can include situations where one or some of the recited components are subsequently removed from the mixture, optionally prior to addition of other recited components. For example, "contacting A with B and C" includes any and all of the following situations: (i) A is mixed with C, then B is added to the mixture; (ii) A and B are mixed into a mixture; B is removed from the mixture, and then C is added to the mixture; and (iii) A is added to a mixture of B and C. "Contacting a template with a reaction mixture" includes any or all of the following situations: (i) the template is contacted with a first component of the reaction mixture to create a mixture; then other components of the reaction mixture are added in any order or combination to the mixture; and (ii) the reaction mixture is fully formed prior to mixture with the template.

A "non-cysteine residue" refers to any natural or non-natural amino acid residue that is not cysteine and/or that does not form an SS-bond. Examples include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

EXAMPLES Example 1

This example describes general methods for carrying out the assays described in Examples 2-7 below. Recombinant Protein Expression and Purification

BiNAbs were generated using previously described approaches (Merchant, A. M. et al, Nat Biotechnol 16, 677-681, doi: 10.1038/nbt0798-677 (1998); Ridgway, J. B.,

Presta, L. G. & Carter, P., Protein Eng 9, 617-621 (1996); and Schaefer, W. et al, Proc Natl Acad Sci U S A 108, 11187-11192, doi: 10.1073/pnas.1019002108 (2011)). For correct heavy-light chain pairing, one of the parental mAbs was expressed in the

CrossMab format (CH1-CL swapping), while for the other mAb, the wild-type domain architecture was maintained (Schaefer, W. et al, Proc Natl Acad Sci U S A 108, 11187-

11192, doi: 10.1073/pnas.1019002108 (2011)). For heavy chain heterodimerization, point mutations were introduced in the CH3 domain: Y349C/T366S/L368A/Y407V for the 1 ^st mAb; S354C/T366W for the 2 ^nd mAb (Merchant, A. M. et al, Nat Biotechnol 16, 677- 681, doi: 10.1038/nbt0798-677 (1998); Ridgway, J. B., Presta, L. G. & Carter, P., Protein Eng 9, 617-621 (1996)). Purification tags were added to the C terminus of each heavy chain (His or Strep-Tagil) for determining heterodimer formation efficiency. Antibodies, gpl40, and 2CC-core were generated by transient transfection of HEK293T or 293E cells, as previously described (Bournazos, S. et al, Cell 158, 1243-1253, doi: 10.1016/j .cell.2014.08.023 (2014)). Antibodies were purified using PROTEF G SEPHAROSE 4 FAST FLOW (GE Healthcare); Strep-tagged and His-tagged proteins were purified using the STREP-TACTJN SUPERFLOW PLUS RESIN (Qiagen) and His- Tag isolation and pull-down dynabeads (ThermoFisher), respectively. Purified proteins were dialyzed in PBS and sterile filtered (0.22 μπι). Purity was assessed by SDS-PAGE and Coomassie staining and was estimated to be >90%. Size exclusion chromatography (SEC) was performed using a SUPEROSE 6 INCREASE 10/300GL column (GE Healthcare) on an Akta Pure 25 HPLC system. Protein Tm was determined using the PROTEFN THERMAL SHIFT DYE Kit (ThermoFisher) following manufacturer's instructions on a QUANT STUDIO 12K FLEX real-time thermal cycler.

In Vitro Neutralization Assay

In vitro neutralizing activity of antibodies was assessed against different HFV-1 envelope pseudoviruses, using previously described protocols (Montefiori, D. C, Curr Protoc Immunol 12, 12.11, doi: 10.1002/0471142735.iml211s64 (2005)). Neutralization assays were performed by the Collaboration for AIDS Vaccine Discovery (CAVD) core neutralization facility. IC50 and ICso values reflect the amount of antibody sufficient to reduce luciferase activity (as measured by relative luminescence units (RLU)) by 50% and 80%), respectively. For comparison of the activity of biNAbs with the corresponding parental mAbs, fold change values were calculated by dividing the IC50 or ICso titer of the most potent (lowest IC50/80) of the two parental mAbs by the IC50/80 titer of the biAb. Predicted neutralization activity of a mix of two mAbs was determined by selecting the lowest IC50/80 titer of the two mAbs for a given virus. For combinations involving 2 mAbs with non-overlapping epitope specificities, this predicted activity (theoretical IC50/80) has been shown to be comparable to the neutralization activity of a 1 : 1 mAb mix determined experimentally (experimental IC50/80) (Kong, R. et al, J Virol 89, 2659-2671, doi: 10.1128/JVI.03136-14 (2015)). Neutralization activity data of human IgGl mAbs that were used for comparison in this study were obtained for the CATNAP database (www. hi v .1 anl . gov/).

ELISA Assays and Quantification of Serum Anti-HIV-1 IgG Levels

For epitope-specific ELISA, gpl40- or 2CC-Core-coated (1 μg/ml) microtiter plates (NUNC) were used, as previously described (Bournazos, S. et al, Cell 158, 1243- 1253, doi: 10.1016/j .cell.2014.08.023 (2014)). Plate-bound IgG was detected by HRP- conjugated goat anti-human IgG (Fcy-specific, Jackson Immunoresearch), or goat anti- human kappa or lambda (Bethyl Laboratories). For competition ELISA, binding of biotinylated biAb (1 μg/ml) to gpl40-coated plates was competed with increasing amounts of mAbs, mAb mix (1 : 1) or biAb (0.001-100 and detected using HRP -conjugated neutravidin (ThermoFisher). For the quantification of serum concentration of anti-HIV-1 mAbs, goat anti-human IgG (mouse IgG absorbed, Jackson Immunoresearch)-coated or gpl40-coated plates were used (Bournazos, S. et al, Cell 158, 1243-1253, doi: 10.1016/j .cell.2014.08.023 (2014)). IgG binding was detected using HRP-conjugated goat anti-human IgG (mouse IgG absorbed; Jackson Immunoresearch), as previously described (Bournazos, S. et al, Cell 158, 1243-1253, doi: 10.1016/j .cell.2014.08.023 (2014)).

Surface Plasmon Resonance (SPR)

All experiments were performed with a BIACORE T200 SPR system (BIACORE, GE Healthcare) at 25°C in HBS-EP+ buffer (10 mM HEPES, pH 7.4; 150 mM NaCl; 3.4 mM EDTA; 0.005% (v/v) surfactant P20). For the measurement of the affinity of IgG hinge domain variants for mouse and human FcyRs, IgG antibodies (diluted at 25 μg/ml in 10 mM sodium acetate, pH 4.5) were immobilized on Series S CM5 chips by amine coupling at a density of 1000 RU. Recombinant human or mouse soluble FcyR ectodomains (Sinobiological) were injected through flow cells at a flow rate of 20 μΐ/min, with the concentration ranging from 15.625 - 2000 nM (1 :2 successive dilutions). Association time was 120 s followed by a 300-s dissociation step. At the end of each cycle, sensor surface was regenerated with 50 mM NaOH (50 μΐ/min, 30 s). Background binding to blank immobilized flow cells was subtracted and affinity constants were calculated using BIACORE T200 evaluation software (GE Healthcare) using the 1 : 1 Langmuir binding model .

Virus Production

HIV- 1 ^T251-1 was generated by cloning the T251-18 env gene (CRF02_AG; NIH AIDS Reagent program) to the NL4-3 HIV-1 vector backbone and produced by transfection in 293T cells, as previously described (Klein, F. et al, Nature 492, 118-122, doi: 10.1038/naturel l604 (2012)). HIV-1 virus preparations were quantified by p24 ELISA (Lenti-X p24 Rapid Titer Kit, Clontech), following manufacturer's recommendations.

In Vivo Experiments

All in vivo experiments were performed in compliance with federal laws and institutional guidelines and have been approved by the Rockefeller University Institutional Animal Care and Use Committee. Humanized NRG ( OD.Cg-Ragl ^tmlMom Il2rg ^mlWjl ISz5) mice were generated by intrahepatic human CD34 ⁺ HSCs injection of sublethally irradiated neonatal NRG mice, as previously described in Klein, F. et al, Nature 492, 118- 122, doi: 10.1038/naturel l604 (2012). Mice were screened at 6-8 wk of age for human leukocyte reconstitution by flow cytometry (as described in Bournazos, S. et al, Cell 158, 1243-1253, doi: 10.1016/j .cell.2014.08.023 (2014) and Klein, F. et al, Nature 492, 118- 122, doi: 10.1038/naturel l604 (2012)) and mice with a measurable human CD45 ⁺ graft (10-15 weeks, males and females) were infected following i.p. injection of HIV-1 ^T251-18 (180 ng p24). Viral load was quantified 3-weeks post-infection and mice with viral loads >10 ⁴ copies/ml were included in treatment experiments. Mice were randomly assigned to experimental groups and both groups had comparable baseline average viremia levels. Antibodies (either 1 mg of 3BNC 117/PGT 135 biAb or 1 mg 1 : 1 mix of 3BNC117 (0.5 mg) and PGT135 (0.5mg)) were administered biweekly s.c. for 4 weeks. Each experimental group consisted of 9 mice; a group size previously determined to sufficiently detect response to antibody therapy (Bournazos, S. et al, Cell 158, 1243-1253, doi: 10.1016/j .cell.2014.08.023 (2014) and Klein, F. et al, Nature 492, 118-122, doi: 10.1038/naturel l604 (2012). Plasma HIV-1 viral load was determined by quantitative reverse-transcriptase PCR as previously described in Bournazos, S. et al, Cell 158, 1243- 1253, doi: 10.1016/j .cell.2014.08.023 (2014) and Klein, F. et al, Nature 492, 118-122, doi: 10.1038/naturel l604 (2012. The lower limit of detection for this assay was previously determined to be 800 copies/ml (Klein, F. et al, Nature 492, 118-122, doi: 10.1038/naturel l604 (2012).

HIV gp 120 Sequence Analysis

Plasma-extracted viral RNA was reverse transcribed using the SUPERSCRIPT III first strand cDNA synthesis kit (Life Technologies) and the gpl20 sequence-specific primer 5'- T AGC A AT AGTTGTGTGGTC C-3 ' (SEQ ID NO: 37). Resulting cDNA was used as the template for PCR amplification using the following primer pairs: 5'-

TAGC AATAGTTGTGTGGTCC-3 ' and 5'- ATTGCTCTGCTGTTGC ACTATAC-3 '

(SEQ ID NOs: 38 and 39). Products from this PCR reaction were subjected to a second

PCR round using the following nested primer pairs: 5'- AG AA AG AGC AGAAGAC AGTGGC-3 ' and 5 ' -TACCGTC AGCGTC ATTG ACGC-3 '

(SEQ ID Nos: 40 and 41). All PCR reactions were performed using the PLATINUM TAQ

HIGH FIDELITY DNA POLYMERASE (ThermoFisher) and PCR amplicons were ligated into pCR4-TOPO vectors Inserts from individual colonies were sequenced

(Genewiz) using M13F (5 ' -TGT AAAACGACGGCC AGT-3 ' , SEQ ID NO: 42), M13R (5 ' -C AGGAAAC AGCT ATGAC-3 ' , SEQ ID NO: 43), and gpl20-specific primers (5'-

GTCTGGGCTAC AC ATGCCTGC-3 ' ; 5 ' -CC ATGC AAAAATGTC AGC AC A-3 ' ; 5 '-

GATGATATAACTCTCCAATGCAG-3'; SEQ ID NOs: 44-46). Sequence reads were aligned to gpl20 ^T251"18 and mutations were numbered using HXBc2 numbering.

Statistical Analysis

Results from multiple experiments are presented as mean ± standard error of the mean (SEM). IC50 titers are presented as geometric mean ± 95% CI. For comparison of the in vitro neutralization activity, Kruskal-Wallis test was used to test for differences in the IC50/80 titers, and where statistically significant effects were found, post hoc analysis using Dunn's multiple comparison test was performed. For comparison of viremia between the two experimental groups, Mann-Whitney non-parametric test (two-sided) was used. Data were analyzed with Graphpad Prism software (Graphpad) and P values of <0.05 were considered to be statistically significant.

Example 2

To study the activity of anti-Env biNAbs, inventors initially selected two mAbs with exceptionally broad and potent in vitro and in vivo activity: 3BNC117 and 10-1074, which target the CD4bs and the V3 sites of Env, respectively (Scheid, J. F. et al, Science 333, 1633-1637, doi: science.1207227 [pii] 10.1126/science.1207227 (2011) and Mouquet, H. et al, Proc Natl Acad Sci U S A 109, E3268-3277, doi: 10.1073/pnas.1217207109 (2012)). For the generation of 3BNC117/10-1074 biNAbs, inventors employed a combination of previously described strategies (Merchant, A. M. et al, Nat Biotechnol 16, 677-681, doi: 10.1038/nbt0798-677 (1998); Ridgway, J. B., Presta, L. G. & Carter, P., Protein Eng 9, 617-621 (1996); and Schaefer, W. et al, Proc Natl Acad Sci U S A 108, 11187-11192, doi: 10.1073/pnas.1019002108 (2011)) to ensure proper pairing of the heavy and light chains of the two parental mAbs and generation of heavy chain heterodimers (FIG. 1 A). Pairing of the correct heavy chain with the respective light chain was achieved by swapping the CHI domain with the constant domain of the light chain (CL) for one mAb (10-1074), while preserving the wild-type domain organization for the other mAb (3BNC117) (Schaefer, W. et al, Proc Natl Acad Sci U S A 108, 11187-11192, doi: 10.1073/pnas.1019002108 (2011)). This approach (CrossMab) had no measurable effect on the antigenic specificity and neutralization activity of the 10-1074 mAb (FIG. IB, FIG. 5 A, Table SI). Heterodimerization of the heavy chain was achieved by introducing mutations in the CH3 domain that alter the physical and chemical properties of the two mAb heavy chains, favoring heterodimer formation (Merchant, A. M. et al, Nat Biotechnol 16, 677-681, doi: 10.1038/nbt0798-677 (1998); Ridgway, J. B., Presta, L. G. & Carter, P., Protein Eng 9, 617-621 (1996)). Consistent with previous reports (Merchant, A. M. et al, Nat Biotechnol 16, 677-681, doi: 10.1038/nbt0798-677 (1998) and Asokan, M. et al. Bispecific Antibodies Targeting Different Epitopes on the HIV-1 Envelope Exhibit Broad and Potent Neutralization. J Virol 89, 12501-12512, doi: 10.1128/JVI.02097-15 (2015), this approach resulted in efficient expression of biNAbs, typically yielding >90% heterodimers. By taking advantage of the differences in the light chain isotype (κ for 3BNC117 and λ for 10-1074) and the Env epitope specificity (CD4bs and V3 for 3BNC117 and 10-1074, respectively), competition and epitope-specific ELISA assays confirmed the dual specificity of the resulting 3BCN117/10-1074 biNAb ( FIGs. 5B-D).

Example 3

Using a standardized TZMbl assay (Montefiori, D. C, Curr Protoc Immunol 12, 12.11, doi: 10.1002/0471142735.iml211s64 (2005)), evaluation of the in vitro neutralization activity of the 3BNC117/10-1074 biAb against an extended multiclade virus panel revealed a marginal increase in the neutralization breadth over 3BNC117 and 10- 1074 mAbs (FIG. 1C, Table S2). However, neutralization potency was markedly reduced when compared to the activity of the parental mAbs (either alone or as a mix, calculated based on the lowest IC50 titer of the two bNAbs for a given virus). For the majority of the viruses tested, the observed IC50 titers of the biNAb were the average from the two parental bNAb s (Tabl e S2) .

Table SI: IC50 titers fag/ml) of 10-1074 hlgGl and 10-1074 CrossMab variant determined by TZMbl neutralization assay

10-1074

Virus ID Clade Wild-type CrossMab

% Breadth 62.0 62.0

Geometric mean IC50 0.122 0.122

Table S2: Comparison of the in vitro neutralization activity of human IgGl 3BNC117 and 10-1074 mAbs and 3BNC117/10-1074 biAb.

Total Viruses 120 120 120

%Breadth 87.5 58.3 91.7

Geometric mean 0.103 0.242

In vitro neutralization activity was determined by standardized TZM-bl assay.

IC50 values are expressed as μ^ηιΐ.

Fold change values were calculated by dividing the IC50 titer ofthe most potent (lowest IC50) of the two parental mAbs (3BNC117 or 10-1074) by the IC50 titer ofthe biAb (3BNC117/10-1074).

ND: not determined Example 4

To determine whether the observed reduction in the neutralization potency was related to the epitope specificity of the selected mAbs, the inventors extended their study by focusing on different regions of Env, including the Vl/2 epitope (targeted by the PG16 mAb), the V3 epitope (targeted by the 10-1074, PGT121 and PGT128 mAb), as well as antibodies against the gpl20/41 interface (PGT151 and 35022). Similar to the 3BNC 117/10-1074 combination, V1/2-V3 biNAbs (PG16/10-1074, PG16/PGT121, PG16/PGT128) as well as the gpl20/41 interface biNAb, PGT151/35022 exhibited compromised neutralization potency and none of the combinations achieved the activity of their respective parental bNAbs (FIGs. 1D-G, Tables S3-S6). Comparable findings have also been recently reported when the neutralization activity of biNAbs targeting similar epitopes on Env has been tested (Asokan, M. et al, J Virol 89, 12501-12512, doi: 10.1128/JVI.02097-15 (2015).). These findings clearly suggest that biNAbs, irrespective of their epitope specificities, fail to recapitulate the neutralization activity of their respective parental bNAbs, offering no advantages over conventional, monospecific bNAbs.

Table S3: Comparison of the in vitro neutralization activity of human IgGl PG16 and PGT121 mAbs and PG16/PGT121 biAb.

Virus ID Clade PG16 PGT121 „~ _ Fold

Change

Total Viruses 116 116 116

%Breadth 82.8 63.8 91.4

Geometric mean IC50 0.073 0.083 0.207

In vitro neutralization activity was determined by standardized TZM-bl assay.

IC50 values are expressed as μ^ηιΐ.

Fold change values were calculated by dividing the IC50 titer of the most potent (lowest IC50) ofthe two parental mAbs (PG16 or PGT121) by the IC50 titer of the biAb (PG16/PGT121).

ND: not determined. Table S4: Comparison of the in vitro neutralization activity of human IgGl PG16 and PGT128 mAbs and PG16/PGT128 biAb.

Ce704809221 1 B3 C (T/F) mm mmm mmm -0.109 3817.v2.c59 CD 1β 1 mnmn . -0.637

6480.v4.c25 CD >50 0.003 7.987 -3.425

6952.V1.c20 CD 21.865 >50 36,891 -0.218

6811.v7.c18 CD >50 lt§lll|| >40 Ιϋϋϋΐΐ

89-F1 2 25 CD ■i 1 >50 >40 ND

3301.v1.c24 AC 0.020 0.067 0 030 -0.176

6041.v3.c23 AC 0.048 >50 0.1 uO -0.319

6540.v4.c1 AC 0.035 1 .798 U U29 0.082

6545.v4.c1 AC 0 044 >50 0135 -0.487

0815.v3.c3 ACD >50 0.030 24 937 !!!!lllll!

3103.v3.c10 ACD 25 391 0.014 5.523 -2536

Total Viruses 117 117 117

% Breadth 83.8 60.7 88.0

Geometric mean IC50 0.071 0.043 0.167

In vitro neutralization activity was determined by standardized TZM-bl assay.

IC50 values are expressed as μ^ηιΐ.

Fold change values were calculated by dividing the IC50 titer of the most potent (lowest IC50) of the two parental mAbs (PG16 or PGT128) by the IC50 titer of the biAb (PG16 PGT128).

ND: not determined

Table S5: Comparison of the in vitro neutralization activity of human IgGl PG16 and 10- 1074 mAbs and PG16/10-1074 biAb.

i _n PG16/ log

Virus ID Clade PG16 10- Fold

1074 Change

R3265.C06 CRF01_AE 0.016 >50 0.424 - 1 423

C2101.C01 CRF01_AE >50 0.164 -1.136

X1193 c1 G 0.091 IIHHHI -0.336

X2131_C1_B5 G 0024 lltiiili KB 0.283

3016.v5.c45 D >50 >50 >50 ND

6952.V1.c20 CD 21.865 O087 -0.349

6041.v3.c23 AC 21.720 2.038 l!il!jlgBII

6540.v4.c1 AC §iilliill >50 0.258 -0.868

Total Viruses 32 32 32

%Breadth 87.5 56.3 87.5

Geometric mean IC50 s 0.139 0.183 0.192

In vitro neutralization activity was determined by standardized TZM-bl assay.

IC50 values are expressed as μ^ηιΐ.

Fold change values were calculated by dividing the IC50 titer of the most potent (lowest IC50) of the two parental mAbs (PG16 or 10-1074) by the IC50 titer of the biAb (PG16/10-1074).

ND: not determined.

Table S6; Comparison of the in vitro neutralization activity of human IgGl PGT151 and 35022 mAbs and PGT151/35022 biAb.

log

Virus ID Clade PGT151 35Q22 PGT151/35Q22 Fold

Change

Total Viruses 120 120 120

% Breadth 64.2 27.5 72.5

Geometric mean IC50 « 0,077 2.840 0.220

In vitro neutralization activity was determined by standardized TZM-bl assay.

IC50 values are expressed as μ^ηιΐ.

Fold change values were calculated by dividing the IC50 titer of the most potent (lowest IC50) of the two parental mAbs (PGT151 or 35022) by the IC50 titer of the biAb (PGT151/35022).

ND: not determined.

Example 5

A key immune evasion mechanism of HIV-1 against host antibody responses is the remarkably low density of Env molecules on the viral surface, as well as the unique Env trimeric architecture, which preclude high-avidity bivalent interactions of IgG (Klein, J. S. & Bjorkman, P. J. F, PLoS Pathog 6, el000908, doi: 10.1371/journal.ppat.1000908 (2010)). It is therefore likely that the tested biNAbs would exhibit predominantly monovalent binding to their respective epitopes, possibly accounting for the lack of additive, synergistic activity. Indeed, given the relative rigidity and the short length of the hinge domain of IgGl, concurrent binding of the two Fab arms within the same Env trimer is largely restricted. Overcoming this limitation and favoring intra-trimeric, bivalent interactions of biNAbs should greatly augment their neutralization activity through enhanced avidity (Klein, J. S. & Bjorkman, P. J. F, PLoS Pathog 6, el000908, doi: 10.1371/journal.ppat.1000908 (2010); Galimidi, R. P. et al, Cell 760, 433-446, doi: 10.1016/j .cell.2015.01.016 (2015); and Klein, J. S. et al, Proc Natl Acad Sci U S A 706, 7385-7390, doi: 10.1073/pnas.0811427106 (2009)).

Among the human IgG subclasses, IgG3 encompasses an exceptionally long and flexible hinge domain, with distinct structural and functional characteristics (Roux, K. H., Strelets, L. & Michaelsen, T. E., J Immunol 159, 3372-3382 (1997) and Roux, K. H., et al, J Immunol 761, 4083-4090 (1998)) (FIG. 2A). It is comprised of a 17mer amino acid sequence followed by three 15-mer repeats that are highly homologous to the IgGl hinge structure and represent genomic duplication events of the ancestral hinge-encoding exon, conserved among all other IgG subclasses (Roux, K. H., et al, J Immunol 761, 4083-4090 (1998)). Previous biophysical studies on the IgG3 hinge indicated that the hinge domain spans an over HOA distance and the unique primary amino acid composition of the CHI proximal 17-mer confers increased flexibility of the Fab arms, allowing for greater degree of rotation compared to IgGl (117° vs. 136°) (Roux, K. H., et al, J Immunol 761, 4083- 4090 (1998)).

Based on these unique properties of the IgG3 hinge domain structure, the inventors aimed to develop biNAbs with increased Fab domain flexibility, using the IgG3 hinge as template. To further increase the inherent IgG3 hinge flexibility, the inventors generated an "open" IgG3-based hinge variant (IgG3C-), in which all the cysteine residues have been replaced with serines with the exception of the last two, CH2 proximal residues that are used to maintain the structural integrity of the Fc domain through inter-heavy chain disulfide bonding (FIG. 2A). Hinge domain variants of 10-1074 IgGl were expressed as chimeric molecules, in which the wild-type hinge domain (IgGl) was replaced with that of either IgG3 or the IgG3C- variant. With the exception of the hinge domain, all other domains of the constant region (CHI, CH2, CH3) were of the IgGl subclass to preserve the effector function and half-life of wild-type IgGl . Indeed, hinge domain variants of 10- 1074 demonstrated comparable binding affinity to the different classes of human and mouse FcyRs, suggesting a minimal role for the hinge region in Fc-FcyR interactions (Extended Data Table 1). Likewise, no differences among the hinge domain variants were noted in terms of protein stability and in vivo pharmacokinetics (FIG. 2B and FIG. 6).

5 Extended Data Table 1: Comparison of affinity of IgG hinge variants for human and mouse FcyRs

Values represent KD {M) determined by surface plasmon resonance using soluble human or mouse FcyR ectodomains.

Having established that modifications in the hinge domain had no impact on FcyR binding and IgG half-life and stability, the neutralization activity of 3BNC117/10-1074 biNAb hinge variants was assessed in TZM-bl assays against an extended multiclade virus

10 panel. Compared to the wild-type, unmodified IgGl biNAb, IgG3C- hinge variant of the 3BNC117/10-1074 biNAb presented substantially improved activity. For the vast majority of the tested virus strains (120 stains), a consistent decrease in both the IC50 and ICso titers was evident for the IgG3C- biNAb, indicating improved neutralization activity compared to IgGl biNAb (IC50 ^g/ml): 0.242 vs. 0.110; ICso ^g/ml): 0.717 vs. 0.388 for IgGl vs.

15 IgG3C-, respectively). In addition, the observed neutralization breadth and potency of the IgG3C-biNAb was comparable to that expected from the mix of the two parental antibodies (FIG. 2C, Tables S7-S8). Indeed, whereas IgGl biNAb exhibited the average neutralization potency of the two parental bNAbs (3BNC117 and 10-1074), IgG3C- biNAb demonstrated activity equal or in some cases better than that of the parental

20 bNAbs. In contrast, no difference in the neutralization activity was noted among hinge domain variants of the parental, monospecific antibodies (3BNC117 and 10-1074), demonstrating activity similar to that of their wild-type IgGl counterparts (FIGs. 7A-B, Table S9). These findings indicate that alterations in the Fab arm flexibility through modifications in the hinge domain structure led to improved neutralization activity of

25 biNAbs, suggesting that hinge structures engineered for increased length and flexibility could lead to the development of biNAbs with enhanced breadth and potency. Table S7: In vitro neutralization activity (ICso and ICso titers) of hinge variants 3BNC117/10-1074 biAb.

Fold lgG1 lgG3C- Change log

231966x02 D 4 740 1.263 6 726 0 574 0 430

I y I o i_tu_ I

D (T/F) 0.902 3.110 0.448 3.137 0 304 0.004

3817.v2.c59 CD 0.916 4.500 0.471 2.275 0 289 0.296

6480.v4.c25 CD eiiii 0.11 1 em 0.385 0.355

6952.V1.c20 CD 0.251 0.684 0.073 0.261 0.536 0.418

6811.v7.c18 CD W ll iiiiit 1 0,508 Q.602

89-F1_2_25 CD >20 >20 >20 >20 ND ND

3301.v1.c24 AC mumiiiiii 0.342 0.345

6041.v3.c23 AC 0.197 1.241 0.046 0,22-3 0JB32 0.745

6540.v4.c1 AC >20 >20 >20 >20 ND ND

6545.v4.c1 AC >20 >20 >20 >20 ND ND

0815.v3.c3 ACD

.c10 ACD 0.265 ■ 0.7l16l BHH ΙβΙΙ Ι 0 383 0.256

3103.v3 0.085 0 296 0 494 0 384

lgG1 lgG3C

Total Viruses 120 120 120

IC50 IC80 IC50 IC80

% Breadth 91.7 87.5 95.8 90.0

Geometric mean 0.242 0.717 0.110 0.388

In vitro neutralization activity was determined by standardized TZM-bl assay.

IC50 and ICso values are expressed as μ^ηιΐ.

Fold change values were calculated by dividing the IC50 or ICso titer of the IgG3C- with that of IgGl hinge variant biAb.

ND: not determined.

Table S8: Comparison of the in vitro neutralization activity of IgG3C5 hinge variants of 3BNC117and 10-1074 mAbs and 3BNC117/10-1074 biAb.

3BNC117/ Fold Change

Virus ID Clade 3BNC117 10-1074 10-1074 ^Lo 9

Total Viruses 119 119 119 119 119 119

% Breadth 83.2 80.7 58.8 54.6 95.8 89.9

Geometric mean 0.158 0.619 <K072 0.222 0.110 0.386

In vitro neutralization activity was determined by standardized TZM-bl assay. IC50 and ICso values are expressed as μ^ηιΐ.

Fold change values were calculated by dividing the IC50 or ICso titer of the most potent (lowest IC50/80) of the two parental mAbs (3BNC117 or 10-1074) by the ICso _/ so titer of the biAb (3BNC117/10-1074).

ND: not determined. Table S9: Comparison of the in vitro neutralization activity of hinge variants (IgGl, and IgG3C-) of 3BNC117, 10-1074, PGT128, PGT135, PGT151, and 8ANC 195 mAbs.



231965x01 D 8.038 0.122 >30 >30 >30 >30 >30 >30 mm 0.011 0.463 0 371

231966x02 D 0.290 0.643 >30 >30 3.197 5.107 NT >30 0. 159 0.077 0 540 0 646

191821 E6 1 D (T/F) 0.090 0.334 >30 >30 0 016 0.83S >30 0.276 0 203 0.360 0.099

3817.v2.c59 CD 0.191 0.617 1.151 w srnm liliiill >30 >30 >30 >30 0 883 0 379

6480.v4.c25 CD eiiiil 0.Θ42 0 005 0.0.1 0012 0.577 3.782 >30 >30 0.160 0 156

6952.V1 x20 CD 0.150 0.203 >30 >30 llliiii 0043 : >30 1.076 1.380 0 556

6811.v7.c18 CD 0.030 0.091 0.002 0,052 0 285 >30 >30 5 340 4 808

89-F1 2 25 CD >30 >30 >30 >30 >30 >30 w m >30 >30 >30 >30 >30

3301.v1.c24 AC Illiill 0.066 iiiiiiii llllilill 0.067 0.224 >30 >30 mm >30 >30

6041.v3.c23 AC ϋϋϋ 2) 720 >30 >30 >30 >30 >30 ΰ.ύΜ 0.011 >30 >30

6540.v4.c1 AC >30 >30 >30 >30 1 1 798 6.596 >30 >30 0.024 >30 >30

6545.v4.c1 AC >30 >30 >30 >30 >30 >30 >30 >30 >30 >30

13.65

3 Q23 0.088 0038 0038 >30 >30 >30

0815.v3.c3 ACD Ϊ1111 ( 2 1 582 0.746

3103.v3.c10 ACD 0.220 1.502 o4i tarn 0.014 0 014 2.001 7.981 0. 115 0 055 >30 >30

Total Viruses 116 117 117 117 117 117 113 117 117 117 116 116

%Breadth 87.9 82.9 59.8 59.8 59.0 65.8 29.2 26.5 63.2 68.4 62.9 65.5 Geometric mean IC50 0.111 0.160 0.082 0.071 0.035 0.067 0.358 0.552 0.050 0.029 0.999 0.620

In vitro neutralization activity was determined by standardized TZM-bl assay.

IC50 values are expressed as μg/ml.

Example 6

Given the intrinsic flexibility of the Env trimer (Ward, A. B. & Wilson, I. Trends Biochem Sci 40, 101-107, doi: 10.1016/j .tibs.2014.12.006 (2015)), as well as the differences in the angle and orientation by which the Fab domains of antibodies target their respective epitopes on Env, it is difficult to accurately predict the distance required for two Fab arms of a biNAb combination to preferentially exhibit bivalent, intra-trimeric binding and thus confer synergistic activity. To add to this complexity, there are multiple examples of bNAbs that recognize conformational epitopes on Env, or inhibit recognition by other bNABs upon binding, either by inducing structural changes to the Env trimer or by steic inhibition mechanism. In an attempt to identify particular biNAb combinations that would exhibit potent synergistic activity, a panel of broadly neutralizing bNAbs targeting various distinct epitopes on the Env trimer with different angle and orientation (Burton, D. R. & Mascola, J. R., Nat Immunol 16, 571-576, doi: 10.1038/ni.3158 (2015)) was selected (3BNC117, CH103, and 8ANC131 for CD4bs, PG16, PGT145, and PGDM1400 for Vl/2, 10-1074, PGT121, PGT128, and PGT135 for V3, PGT151, and 8ANC195 for the gpl20/gp41 interface and 10E8 for MPER)(FIG. 3A). These bNAbs were cloned as IgG3C- hinge domain variants and biNAbs were generated with non- overlapping epitope specificities. No difference in the neutralization activity was observed between IgGl and IgG3C- hinge variants for all the parental, monospecific antibodies (FIG. 7, Extended Data Table 2 below, and Table S9 above).

Analysis of the neutralization activity of the various biNAb combinations against a panel of 7 cross-clade tier 2/3 viruses identified particular combinations exhibiting higher (synergistic), equal (neutral) or lower (inhibitory) activity compared to their respective parental bNAb counterparts (also in the IgG3C- hinge variant format) (FIGs 3B-C, Table S 10 below).

Three particular biNAb combinations that showed evidence for synergistic activity were selected for further analysis, based on their epitope specificities, evidence for efficient expression, sufficient in vivo half-life, and long-term protein stability. These combinations included: PGT151/10-1074, 8ANC195/PGT128, and 3BNC117/PGT135 biNAbs. Evaluation of their neutralization activity against an extended multiclade virus panel revealed that the 8ANC195/PGT128 biNAb combination presented modest synergistic activity, while no differences were noted for the PGT151/10-1074 biNAb (FIGs. 4A-B, Tables S11-S12 below).

In contrast, the 3BNC1 17/PGT135 biNAb exhibited augmented neutralization breadth and potency, surpassing the activity of the parental bNAbs (3BNC117 and PGT135) (FIG. 4C, Extended Data Table 3). For the vast majority of the tested virus strains (119 strains), 3BNC 117/PGT 135 biNAb demonstrated lower IC50 and IC80 titers compared to the most potent of the parental bNAbs (3BNC117 or PGT135) for each tested virus strain. Overall, the 3BNC117/PGT135 IgG3C- biNAb was capable of neutralizing over 93% of the tested viruses, with an average (geometric mean) IC50 of 0.036 μg/ml, representing one of the most potent anti-HIV-1 Env antibodies characterized so far.

The synergistic activity observed for the 3BNC117/PGT135 IgG3C- biNAb might actually reflect the capacity of this biNAb combination for bivalent binding of the two Fab arms accomplished by the flexible IgG3C- hinge structure. Indeed, comparison of the gpl20-bound PGT135 Fab crystal structure to other anti-V3 mAbs (PGT122, or PGT128) revealed a unique orientation and angle for PGT135 (Kong, L. et al, Nat Struct Mol Biol 20, 796-803, doi: 10.1038/nsmb.2594 (2013)) that when combined with the 3BNC117 mAb, as in the case of 3BNC117/PGT135 IgG3C- biNAb, would facilitate intra-trimeric bivalent interactions (FIG. 8), as the length and flexibility of the IgG3C- hinge could sufficiently span the distance between the two Fab arms bound to the Env trimer. To test this assumption and provide additional mechanistic insights into the requirements for the enhanced synergistic neutralization activity of the 3BNC117/PGT135 IgG3C- biNAb, we assessed the in vitro neutralization activity of different hinge domain variants with variable lengths and properties.

Extended Data Table 2: In vitro neutralization activity of hinge variants of anti-HIV-1 Env mAbs against a multiclade virus panel.

In vitro neutralization activity was determined by standardized TZM-bl assay. IC50 values are expressed as μ^ηιΐ.

Table S10: In vitro neutralization activity oflgGSC- hinge variants of mAbs and biAbs against a multiclade virus panel.

Q461.e2 T251 -18 16845-2.22 CNE30 TRJ04551.58 ZM109F.PB4 T278-50

Clade A CRF02_AG C C C CRF02_AG Tier 2 2/3 2 2 2 2/3

In vitro neutralization activity was determined by standardized TZM-bl assay. IC50 and ICso values are expressed as μ^ηιΐ.

Table Sll: Comparison of the in vitro neutralization activity oflgGSC- hinge variants of 8ANC195 andPGT128 mAbs and 8ANC195/PGT128 biAb

8ANC195/ tog Fold

Virus ID Clade 8ANC195 PGT128 PGT128 Change

IC50 IC80 IC50 IC80 IC50 IC80 IC50 IC80

191821 E6 1 D (T/F) 0.09& 0.571 lltjlltll 0,068 lltllltll 0,07S 0.000 -0.043

3817.v2.c59 CD 0.379 1.471 Q.084 : 0.118 -0.229 -0.099

6480.v4.c25 CD 0.156 1.000 o,oe ■a 0.091 -0.222 -0.174

6952.V1.c20 CD 0.556 3.141 >30 >30 0.500 2.616 0.046 0.079

6811.v7.c18 CD 4 808 illillB::: ΙϋϋΙΙ 0,079 Ιϋϋϋ 0.052 0.051 0.182

89-F1_2_25 CD >30 >30 >30 >30 >30 >30 ND ND

3301.v1.c24 AC >30 >30 0.224 1.296 0 097 0.359 0.363 0 558

6041.v3.c23 AC >30 >30 >30 >30 13.831 >30 0.336 ND

6540.v4.c1 AC >30 >30 6 596 >30 0.257 1.661 inmn numi

6545.v4.c1 AC >30 >30 >30 >30 >30 >30 ND ND

0815.v3.c3 ACD 0.746 9.202 lllliill 0.170 0038 0.234 0.023 -0.139

3103.v3.c10 ACD >30 >30 0.0.4 0053 0 045 0.126 -0,507 -0.37§

Total Viruses 116 116 116 116 116 116

%Breadth 65.5 56.9 65.5 58.6 90.5 81.9

Geometric mean 0.645 2.373 S.O&fS ; 0.164 0.084 0.237

In vitro neutralization activity was determined by standardized TZM-bl assay.

IC50 and ICso values are expressed as μ^ηιΐ.

Fold change values were calculated by dividing the IC50 or ICso titer of the most potent (lowest IC50/80) of the two parental mAbs (8ANC195 or PGT128) by the ICso/so titer of the biAb (8ANC195/PGT128).

ND: not determined.

Table SI 2: Comparison of the in vitro neutralization activity of IgGSCl hinge variants of PGT151 and 10-1074 mAbs and PGT151/10-1074 biAb.

PGT151/ Fold Change

Virus ID Clade PGT151 10-1074 10-1074 ^Lo 9

IC50 IC80 IC50 IC80 IC50 IC80 IC50 IC80

Total Viruses 119 119 0 119 119 0 119 119

% Breadth 67.2 51.3 58.8 54.6 85.7 79.0

Geometric mean i 0.029 0.091 0.072 0.222 0.041 0.126

In vitro neutralization activity was determined by standardized TZM-bl assay.

IC50 and ICso values are expressed as μ^ηιΐ.

Fold change values were calculated by dividing the IC50 or ICso titer of the most potent (lowest ICso/so) of the two parental mAbs (PGT151 or 10-1074) by the IC ₅ o/so titer of the biAb (PGT151/10-1074).

ND: not determined. Extended Data Table 3: Comparison of the in vitro neutralization activity ofIgG3C- hinge variants of 3BNC117 and PGT135 mAbs and 3BNC117/PGT135 biAb

3BNC117/ Fold Change

Virus ID Clade 3BNC117 PGT135 PGT135 Log

3016.v5.c45 D 4 872 >30 >30 >30 0.146 1 .983 ND

A07412M1.WC1 2 D 8.05? 0.291 11.815 >30 0.0.7 : Q.08& 0.525 0.51

231965x01 D 0.122 0.556 >30 >30 0.218 0.581 mm

231966x02 D 0.643 6.279 >30 >30 0.243 1111111111 llllii

191821_E6_1 D (T/F) 0.334 2.256 >30 >30 0.152 LOGS 1111

3817.v2.c59 CD 0.617 2.055 >30 >30 0 066 0.299 0.83 ^'

6480.v4.c25 CD iliil 0.197 3.782 >30 B.0S4 0.544 0.48!

6952.V1 x20 CD 0.203 0.889 iBiii 0.288 0.213 -0.010 0.13

681 1.v7.c18 CD 0 091 0.297 0.285 4 842 0.06* 0 704 0 68i

89-F1_2_25 CD >30 >30 >30 >30 >30 >30 ND ND

3301.v1 .c24 AC 0 066 0.185 >30 >30 ^Q .054- 0.615 :885»

6041.v3.c23 AC 1111111111 0.204 >30 >30 111111111 0 646 0.84 ^'

6540.v4.c1 AC >30 >30 >30 >30 >30 ND ND

6545.v4.c1 AC >30 >30 >30 >30 >30 >30 ND ND

0815.v3.c3 ACD 0.087 >30 >30 0.459 m m

3103.v3.c10 ACD 1.502 3.823 7.981 llillll 0.06? 0.186 351 1.313

Total Viruses 119 119 0 119 119 0 119 119

% Breadth 83.2 80.7 25.2 16.0 93.3 89.1

Geometric mean 0.158 0.619 0.614 0.872 ¾ 0.036 0.159

In vitro neutralization activity was determined by standardized TZM~bl assay. IC50 and ICso values are expressed as μg/ml. Fold change values were calculated by dividing the IC50 or ICso titer of the most potent (lowest ICso _/ so) of the two parental mAbs (3BNC117 or PGT135) by the ICso _/ so titer of the biAb (3BNC117/PGT135). ND: not determine.

Example 7

In order to determine whether the observed increase in neutralization potency of the 3BNC117/PGT135 IgG3C- biNAb also translates to improved therapeutic efficacy in vivo, inventors evaluated its capacity to suppress viremia in humanized mice (human CD34 ⁺ -reconstituted NRG mice, Klein, F. et al, Nature 492, 118-122, doi: 10.1038/naturel l604 (2012)) infected with HIV-1. Based on the in vitro neutralization activity data, a tier 2 HIV-1 virus stain (T251-18) was selected that was highly sensitive (IC50: <0.023 to the 3BNC117/PGT135 biNAb, while exhibiting modest sensitivity or resistance for 3BNC117 (ICso: 0.219 and PGT135 (ICso: >50 μ^πιΐ) respectively. HIV-l ^T251"18 -infected humanized mice were treated either with a mix (1 : 1) of 3BNC117 and PGT135 bNAbs or with 3BNC 117/PGT 135 biNAb (all in IgG3C- hinge variant format). Among the two experimental groups, no differences in the in vivo half-life or the serum antibody levels were noted between the two experimental groups (FIGs. 9A- B). Quantitation of plasma viremia revealed that 3BNC117/PGT135 biNAb treatment decreased viremia by an average of 1.5 logw during the treatment period (FIGs. 4E-G) with the majority (7/9) of treated animals exhibiting substantially reduced plasma viremia levels (Δ logw viremia <-1.0). Mice that failed to demonstrate robust response to biNAb therapy were often associated with recurrent mutations in the biNAb-targeting epitopes (V3 or CD4bs; FIG. 9C). In contrast, administration of the 3BNC117 + PGT135 bNAb mix had minimal effect on plasma viremia (mean Δ logw viremia: 0.15; FIG. 4D; FIG. 9C), thereby confirming the in vitro neutralization data and establishing this unique biAb as a candidate for future therapeutic development. The findings strongly suggest that hinge domain variants optimized for enhanced Fab arm flexibility can augment the neutralization potency of biNAbs, leading to the development of biAb molecules with improved in vivo therapeutic efficacy and thus represent a platform technology that can be extended to other viral and cellular targets.

Example 8

The synergistic activity observed for the 3BNC 117/PGT 135 IgG3C- biNAb might actually reflect the capacity of this biNAb combination for bivalent binding of the two Fab arms accomplished by the flexible IgG3C- hinge structure. Indeed, comparison of the gpl20-bound PGT135 Fab crystal structure to other anti-V3 bNAbs (PGT122, or PGT128) revealed a unique orientation and angle for PGT135 (Kong, L. et al. 2013, Nat Struct Mol Biol 20, 796-803) that when combined with the 3BNC117 bNAb, as in the case of 3BNC117/PGT135 IgG3C- biNAb, would facilitate intra-trimeric bivalent interactions (FIGs. 8A and 8B), as the length and flexibility of the IgG3C- hinge could sufficiently span the distance between the two Fab arms bound to the Env trimer. To test this assumption and provide additional mechanistic insights into the requirements for the enhanced synergistic neutralization activity of the 3BNC117/PGT135 IgG3C- biNAb, assays were carried out to assess the in vitro neutralization activity of different hinge domain variants with variable lengths and properties.

3BNC117/PGT135 biNAbs and their respective parental bNAbs (3BNC117 and PGT135) were expressed as variants encompassing the hinge domain structure of wild- type IgGl, IgG3, or IgG3C- (open hinge structure based on wild-type IgG3 sequence) and their neutralization activity was assessed against a multiclade, 20-strain panel. When the activity of a 1 : 1 mix of the two parental bNAbs was compared among the different hinge domain variants, no significant differences were noted, with all the hinge variants exhibiting comparable neutralization activity (FIGs. 10A and 10B). In contrast, 3BNC117/PGT135 biNAb demonstrated significantly augmented activity only in the IgG3C-, but not in the IgGl or IgG3 hinge domain format, indicating that the synergistic activity of the 3BNC117/PGT135 biNAb is dependent upon the unique structure and flexibility of the IgG3C- hinge variant.

To provide further evidence on the role of the hinge length in the improved neutralization activity of the 3BNC117/PGT135 biNAb, two different variants of the IgG3C- hinge structure were generated, which lacked either one (-15mer) or two (- 2xl5mer) of the three 15 amino acid repeats (EPKSSDTPPPSPRSP or EPKSSDTPPPCPRCP, SEQ ID No: 7 or 8) that comprise part of the IgG3C-hinge sequence (FIG. 2A). 3BNC117/PGT135 biNAbs encompassing the shortened hinge domain variants were generated and their activity was compared to that of IgG3C-. Assessment of the in vitro neutralization activity revealed that hinge length is correlated with the neutralization potency of the 3BNC117/PGT135 biNAb, with shorter hinge variants ("-15mer" or "-2xl5mer") exhibiting impaired neutralization activity (FIGs. 11A and 11B). These findings suggest that the observed enhancement in the in vitro neutralization activity of the 3BNC117/PGT135 biNAb is dependent upon hinge length and flexibility, that likely favors intra-trimeric, bivalent Env interactions.

The half-life of the 3BNC117/PGT135 IgG3C- biNAb was then determined in in Rhesus macaques. Briefly, in vivo half-life of 3BNC117/PGT135 IgG3C- biNAb was determined from rhesus macaques (n=3) that had been injected with 5 mg/kg i.v. 3BNC117/PGT135 IgG3C- biNAb. The antibody ti/ ₂ for 3BNC117/PGT135 IgG3C- biNAb was calculated to range from 86.37 - 87.46 h (3.5-3.6 d), which is comparable to that of anti-HIV Env IgGl monospecific bNAbs (previously determined in Shingai et al, J. Exp. Med. 2014 Vol. 211 No. 10 2061-2074). See FIG. 12.

The foregoing examples and description of the preferred embodiments should be taken as illustrating, rather than as limiting the present invention as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. Such variations are not regarded as a departure from the scope of the invention, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated by reference in their entireties.