WO2017011413A1

WO2017011413A1 - Bispecific molecules comprising an hiv-1 envelope targeting arm

Info

Publication number: WO2017011413A1
Application number: PCT/US2016/041808
Authority: WO
Inventors: Chia-Ying Kao LAM; Jeffrey Lee NORDSTROM; Barton F. Haynes; Mattia Bonsignori; Ryan MEYERHOFF
Original assignee: Duke University
Priority date: 2015-07-10
Filing date: 2016-07-11
Publication date: 2017-01-19

Abstract

The invention is directed to multispecific molecules comprising an HIV-1 envelope targeting arm and an arm targeting an effector cell, compositions comprising these molecules and methods of use.

Description

Bispecific MoleculesComprising an HIV‐1 Envelope Targeting Arm [0001] This application claims the benefit of and priority to U.S. Application Serial No.

62/191,054 filed July 10, 2015, U.S. Application Serial No.62/222,175 filed September 22, 2015, U.S. Application Serial No.62/261,233 filed November 30, 2015, and International Application No. PCT/US16/23380 filed March 21, 2016 the entire contents of each of which are hereby incorporated by reference in their entireties.

[0002] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

[0003] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosure of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described herein. FIELDOF THE INVENTION

[0004] The invention is directed to multispecific molecules, such as but not limited to bispecific and trispecific molecules (e.g. bispecific antibodies, bispecific diabodies, and trivalent binding molecules) comprising an HIV-1 binding domain and an effector cell binding domain, and their uses. BACKGROUND

[0005] Highly Active Antiretroviral Therapy (HAART) has been effective in reducing the viral burden and ameliorating the effects of HIV-1 infection in infected individuals. However, despite this therapy the virus persists in the individual due to latent reservoir of HIV-1 infected cells which evade this treatment. Thus, there is a need for therapeutic agents for treatment of HIV-1 infected individuals, as well as agents that target virus infected cells and have the potential to reduce the latent reservoir of HIV-1 infected cells. SUMMARY OF THE INVENTION

[0006] In some aspects the invention is directed to multispecific molecules, such as but not limited to bispecific and trispecific molecules (e.g., bispecific antibodies, bispecific diabodies, trivalent binding molecules, etc.) which comprise epitope-binding fragments of antibodies (e.g., VL and VH Domains) that enable them to coordinately bind immunospecifically to at least one target on HIV-1 envelope (e.g. but not limited to a V3 glycan or a CD4 binding site epitope) and at least one epitope of a second molecule that is not HIV-1 Env, for example but not limited to an effector cell which expresses CD2, CD3, CD8, CD16, T-Cell Receptor (TCR), NKG2D, etc. epitope.

[0007] Selection of the VL and VH Domains of the polypeptide domains of the multispecific molecules of the invention is coordinated so that the polypeptides chains that make up such multispecific molecules assemble to form at least one functional epitope-binding site that is specific for at least one epitope of HIV-1 Env and at least one functional epitope-binding site that is specific for at least one epitope of a molecule that is not HIV-1 Env. In some embodiments, the multispecific molecules of the invention comprise an Fc Domain (Fc bearing multispecific molecules of the invention).

[0008] In non-limiting embodiments, the multispecific molecules comprise 1, 2 or all 3 of the CDR_Hs of a VH Domain with the specificity of the V3 glycan binding antibody DH542 (also referred to as DH270.6), a variant of DH542 called DH542_QSA, DH542_L4, and/or other antibodies from the DH542 lineage (DH542-like antibodies, e.g, DH270.UCA, DH270.IA4, DH270.IA3, DH270.IA2, DH270.IA1, DH270 (also referred to as DH270.1), DH473 (also referred to as DH270.2), DH391 (also referred to as DH270.3), DH429 (also referred to as DH270.4); DH471 (also referred to as DH270.5)), and/or 1, 2 or all 3 of the CDR_Ls of a VL Domain of the V3 glycan binding antibody DH542 (also referred to as DH270.6), a variant of DH542 called DH542_QSA, DH542_L4 (the VL of DH542_L4 comprises the VL from DH429 (also referred to as DH270.4)), and/or other antibodies from the DH542 lineage (DH542-like antibodies, e.g., DH270.UCA, DH270.IA4, DH270.IA3, DH270.IA2,

DH270.IA1, DH270 (also referred to as DH270.1), DH473 (also referred to as DH270.2), DH391 (also referred to as DH270.3), DH429 (also referred to as DH270.4); DH471 (also referred to as DH270.5)).

[0009] In non-limiting embodiments, the multispecific molecules comprise the VH Domain with the specificity of the V3 glycan binding antibody DH542, a variant of DH542 called DH542_QSA, DH542_L4, and/or other antibodies from the DH542 lineage (DH542-like antibodies , e.g., DH270.UCA, DH270.IA4, DH270.IA3, DH270.IA2, DH270.IA1, DH270 (also referred to as DH270.1), DH473 (also referred to as DH270.2), DH391 (also referred to as DH270.3); DH429 (also referred to as DH270.4), DH471 (also referred to as DH270.5)), and/or the VL Domain, of the V3 glycan binding antibody DH542, a variant of DH542 called DH542_QSA, DH542_L4 (the VL of DH542_L4 comprises the VL from DH429 (also referred to as DH270.4)), and/or other antibodies from the DH542 lineage (DH542-like antibodies, e.g., DH270.UCA, DH270.IA4, DH270.IA3, DH270.IA2, DH270.IA1, DH270 (also referred to as DH270.1), DH473 (also referred to as DH270.2), DH391 (also referred to as DH270.3), DH429 (also referred to as DH270.4); DH471 (also referred to as DH270.5)).

[0010] In non-limiting embodiments, the multispecific molecules comprise 1, 2 or all 3 of the CDR_Hs of a VH Domain with the specificity of the CD4 binding site antibody CH557 (also referred to as CH235.12) or any of the antibodies of the CH235 lineage (CH557-like antibodies, e.g., CH556 (also referred to as CH235.11), CH555 (also referred to as

CH235.10), CH493 (also referred to as CH235.9), CH492 (also referred to as CH235.8), CH491 (also referred to as CH235.7), or CH490 (also referred to as CH235.6)), and/or 1, 2 or all 3 of the CDR_Ls of a VL Domain with the specificity of the CD4 binding site antibody CH557 (also referred to as CH235.12) or any of the antibodies of the CH235 lineage (CH557- like antibodies, e.g., CH556 (also referred to as CH235.11), CH555 (also referred to as CH235.10), CH493 (also referred to as CH235.9), CH492 (also referred to as CH235.8), CH491 (also referred to as CH235.7), or CH490 (also referred to as CH235.6)).

[0011] In non-limiting embodiments, the multispecific molecules comprise the VH Domain with the specificity of the CD4 binding site antibody CH557 (also referred to as CH235.12) or any of the antibodies of the CH235 lineage (CH557-like antibodies, e.g., CH556 (also referred to as CH235.11), CH555 (also referred to as CH235.10), CH493 (also referred to as

CH235.9), CH492 (also referred to as CH235.8), CH491 (also referred to as CH235.7), or CH490 (also referred to as CH235.6)) and/or the VL Domain with the binding specificity of the CD4 binding site antibody CH557 (also referred to as CH235.12) or any of the antibodies of the CH235 lineage (CH557-like antibodies, e.g., CH556 (also referred to as CH235.11), CH555 (also referred to as CH235.10), CH493 (also referred to as CH235.9), CH492 (also referred to as CH235.8), CH491 (also referred to as CH235.7), or CH490 (also referred to as CH235.6)).

[0012] In certain embodiments an antibody (or a molecule comprising the CDRs, or the variable domains of such antibody) binds specifically to a particular target, peptide, or polysaccharide (such as an antigen present on the surface of a pathogen, for example gpl20, gp41, or CD3), even where the specific epitope may not be known, and do not bind in a significant amount to other proteins or polysaccharides present in the sample or subject.

Specific binding can be determined by methods known in the art. Various competitive binding assays are known in the art. With reference to an antibody antigen complex, in certain embodiments specific binding of the antigen and antibody has a KD of less than about 10⁶ Molar, such as less than about 10⁶ Molar, 10⁷ Molar, 10⁸ Molar, 10⁹, or even less than about 10¹⁰ Molar.

[0013] In some aspects, the present invention is directed to bispecific molecules, e.g.

covalently linked polypeptide chains to form bispecific antibodies, covalently linked diabodies and/or trivalent binding molecules and their use in the treatment of HIV-1. In certain aspects, the bispecific molecules of the present invention can bind to two different targets or epitopes on two different cells wherein the first epitope is expressed on a different cell type than the second epitope, such that the bispecific molecules can bring the two cells together. In certain aspects, the bispecific molecules of the present invention can bind to two different cells, wherein the bispecific molecules comprises an arm with the binding specificity for an HIV-1 envelope, for example as provided by the binding specificity of the V3 glycan binding antibody DH542, a variant of DH542 called DH542_QSA, DH542_L4 and/or other antibodies from the DH542 lineage, and/or provided by the binding specificity of the CD4 binding site antibody CH557 (also referred to as CH235.12) or any of the antibodies of the CH235 lineage, which arm binds to the HIV-1 envelope expressed on a first cell, e.g. HIV-1 infected cell, and a second arm with the binding specificity for an epitope expressed on a different cell type than the first cell, such that the bispecific molecules can bring the two cells together. In certain embodiment, the second cell is in effector cell which expresses CD2, CD3, CD8, CD16, T-Cell Receptor (TCR), NKG2D, etc. epitope.

[0014] In certain aspects, the invention provides a bispecific molecule comprising a first polypeptide chain and a second polypeptide chain, covalently bonded to one another, wherein: (I) the first polypeptide chain comprises in the N- to C-terminal direction:

(i) a domain (A) comprising a binding region of the light chain variable domain of a first immunoglobulin (VL1) having the binding specificity of a CD4 binding site HIV-1 antibody or a V3 glycan binding antibody (1);

(ii) a domain (B) comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for an epitope (2); and

(iii) a domain (C) comprising a heterodimer promoting domain;

(II) the second polypeptide chain comprises in the N- to C-terminal direction:

(i) a domain (D) comprising a binding region of a light chain variable domain of the second immunoglobulin (VL2) specific for the epitope (2); (ii) a domain (E) comprising a binding region of a heavy chain variable domain of the first immunoglobulin (VH1) having the binding specificity of a CD4 binding site HIV-1 antibody or a V3 glycan binding antibody (1); and

(iii) a domain (F) comprising a heterodimer promoting domain; and wherein:

the domains (A) and (B) are linked so that they do not associate with one another to form an epitope binding site;

the domains (D) and (E) are linked so that they do not associate with one another to form an epitope binding site; and

the domains (A) and (E) associate to form a binding site that binds the HIV-1 envelope like a CD4 binding site HIV-1 antibody or like a V3 glycan binding antibody (1); and

the domains (B) and (D) associate to form a binding site that binds the epitope (2).

[0015] In certain aspects the invention provides such bispecific molecules wherein:

(i) domains (A) and (B) are separated by a peptide linker 1;

(ii) domains (C) and (B) are separated by a peptide linker 2;

(iii) domains (D) and (E) are separated by a peptide linker 1; and

(iv) domains (F) and (E) are separated by a peptide linker 2.

[0016] In certain aspects the invention provides bispecific molecules comprising a first polypeptide chain and a second polypeptide chain, covalently bonded to one another, wherein: (I) the first polypeptide chain comprises in the N- to C-terminal direction:

(i) a domain (A) comprising a binding region of the light chain variable domain of a first immunoglobulin (VL1) having the binding specificity of the V3 glycan binding antibody DH542, a variant of DH542 called DH542_QSA, DH542_L4 and/or other antibodies from the DH542 lineage, and/or the binding specificity of the CD4 binding site antibody CH557 (also referred to as CH235.12) or any of the antibodies of the CH235 HIV-1 antibody lineage (1);

(ii) a domain (B) comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for an epitope (2), wherein domains (A) and (B) are separated from one another by a peptide linker 1; and

(iii) a domain (C) comprising a heterodimer promoting domain, including but not limited to a K coil or E coil; wherein domain (C) and domain (B) are separated by a peptide linker 2;

(II) the second polypeptide chain comprises in the N- to C-terminal direction: (i) a domain (D) comprising a binding region of a light chain variable domain of the second immunoglobulin (VL2) specific for the epitope (2);

(ii) a domain (E) comprising a binding region of a heavy chain variable domain of the first immunoglobulin (VH1) having the binding specificity of the V3 glycan binding antibody DH542, a variant of DH542 called DH542_QSA, DH542_L4 and/or other antibodies from the DH542 lineage, and/or the binding specificity of the CD4 binding site antibody CH557 (also referred to as CH235.12) or any of the antibodies of the CH235 HIV-1 antibody lineage (1), wherein domains (D) and (E) are separated from one another by a peptide linker 1; and

(iii) a domain (F) comprising a heterodimer promoting domain, including but not limited to a K coil or E coil; wherein domain (F) and domain (E) are separated by a peptide linker 2; and wherein:

the domains (A) and (B) do not associate with one another to form an epitope binding site; the domains (D) and (E) do not associate with one another to form an epitope binding site; and

the domains (A) and (E) associate to form a binding site that binds the HIV-1 envelope like the V3 glycan binding antibody DH542, a variant of DH542 called DH542_QSA, DH542_L4 and/or other antibodies from the DH542 lineage, and/or like the CD4 binding site antibody CH557 (also referred to as CH235.12) or any of the antibodies of the CH235 HIV-1 antibody lineage (1); and the domains (B) and (D) associate to form a binding site that binds the epitope (2).

[0017] In certain aspects the invention provides such bispecific molecules, wherein the first or second polypeptide chain further comprises an Fc Domain. The invention also provides such bispecific molecules wherein the first or second polypeptide chain further comprises an Fc Domain and the bispecific molecule further comprises a third polypeptide chain.

[0018] In certain aspects, the invention provides bispecific molecules comprising a first polypeptide chain and a second polypeptide chain, covalently bonded to one another, wherein: (I) the first polypeptide chain comprises in the N- to C-terminal direction:

(i) a domain (A) comprising SEQ ID NO: 553, 565, 567, 570, 574, 578, 582, 586, 590, or 594;

(ii) a domain (B) comprising SEQ ID NO: 500 or 504; and

(iii) a domain (C) comprising SEQ ID NO: 520, 521, 518, or 519;

(II) the second polypeptide chain comprises in the N- to C-terminal direction: (i) a domain (D) comprising SEQ ID NO: 502, or 506;

(ii) a domain (E) comprising SEQ ID NO: 551, 564, 566, 568, 572, 576, 580, 584, 588, 592; and

(iii) a domain (F) comprising SEQ ID NO: 518, 519, 520, or 521; and wherein:

the domains (B) and (D) associate to form a binding site that binds an epitope (2). [0019] In certain aspects the invention provides such bispecific molecules wherein:

(i) domains (A) and (B) are separated by SEQ ID NO: 508;

(ii) domains (C) and (B) are separated by SEQ ID NO: 509 or 510;

(iii) domains (D) and (E) are separated by SEQ ID NO: 508; and

(iv) domains (F) and (E) are separated by SEQ ID NO: 509 or 510. [0020] In certain aspects the invention provides bispecific molecules comprising a first polypeptide chain and a second polypeptide chain, covalently bonded to one another, wherein: (I) the first polypeptide chain comprises in the N- to C-terminal direction:

(ii) a domain (B) comprising SEQ ID NO:500 or 504, wherein domains (A) and (B) are separated from one another by SEQ ID NO: 508; and

(iii) a domain (C) comprising SEQ ID NO: 520, 521, 518, or 519; wherein domain (C) and domain (B) are separated by SEQ ID NO: 509 or 510;

(II) the second polypeptide chain comprises in the N- to C-terminal direction:

(i) a domain (D) comprising SEQ ID NO: 502, or 506;

(ii) a domain (E) comprising SEQ ID NO: 551, 564, 566, 568, 572, 576, 580, 584, 588, 592, wherein domains (D) and (E) are separated from one another by SEQ ID NO: 508; and (iii) a domain (F) comprising SEQ ID NO: 518, 519, 520, or 521; wherein domain (F) and domain (E) are separated by SEQ ID NO: 509 or 510; and wherein:

wherein if the domain (A) comprises SEQ ID NO: 553 and domain (E) comprises SEQ ID NO: 551 they associate to form a binding site that binds the HIV-1 envelope like antibody DH557;

wherein if the domain (A) comprises SEQ ID NO: 565 and domain (E) comprises SEQ ID NO: 564 they associate to form a binding site that binds the HIV-1 envelope like antibody DH556;

wherein if the domain (A) comprises SEQ ID NO: 567 and domain (E) comprises SEQ ID NO: 566 they associate to form a binding site that binds the HIV-1 envelope like antibody DH555;

wherein if the domain (A) comprises SEQ ID NO: 570 and domain (E) comprises SEQ ID NO: 568 they associate to form a binding site that binds the HIV-1 envelope like antibody DH493;

wherein if the domain (A) comprises SEQ ID NO: 574 and domain (E) comprises SEQ ID NO: 572 they associate to form a binding site that binds the HIV-1 envelope like antibody DH492;

wherein if the domain (A) comprises SEQ ID NO: 578 and domain (E) comprises SEQ ID NO: 576 they associate to form a binding site that binds the HIV-1 envelope like antibody DH491;

wherein if the domain (A) comprises SEQ ID NO: 582 and domain (E) comprises SEQ ID NO: 580 they associate to form a binding site that binds the HIV-1 envelope like antibody DH490;

wherein if the domain (A) comprises SEQ ID NO: 586 and domain (E) comprises SEQ ID NO: 584 they associate to form a binding site that binds the HIV-1 envelope like antibody DH542;

wherein if the domain (A) comprises SEQ ID NO: 590 and domain (E) comprises SEQ ID NO: 588 they associate to form a binding site that binds the HIV-1 envelope like antibody DH542_QSA; or wherein if the domain (A) comprises SEQ ID NO: 594 and domain (E) comprises SEQ ID NO: 592 they associate to form a binding site that binds the HIV-1 envelope like antibody DH542_L4; and

wherein if the domain (B) comprises SEQ ID NO: 500 and domain (D) comprises SEQ ID NO: 502 they associate to form a binding site that binds CD3; or

wherein if the domain (B) comprises SEQ ID NO: 504 and domain (D) comprises SEQ ID NO: 506 they associate to form a binding site that binds CD16.

[0021] In certain aspects the invention provides a bispecific molecule comprising a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein some of the polypeptides are covalently bonded, and wherein:

(I) the first polypeptide chain comprises in the N- to C-terminal direction:

(ii) a domain (B) comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for an epitope (2);

(iii) a domain (C) comprising a heterodimer promoting domain; and

(iv) a CH2-CH3 domain;

(II) the second polypeptide chain comprises in the N- to C-terminal direction:

(i) a domain (D) comprising a binding region of a light chain variable domain of the second immunoglobulin (VL2) specific for the epitope (2);

(ii) a domain (E) comprising a binding region of a heavy chain variable domain of the first immunoglobulin (VH1) having the binding specificity of a CD4 binding site HIV-1 antibody or a V3 glycan binding antibody (1); and

(iii) a domain (F) comprising a heterodimer promoting domain;

(III) the third polypeptide chain comprises in the N- to C-terminal direction:

(i) a CH2-CH3 domain, and wherein:

the domains (D) and (E) are linked so that they do not associate with one another to form an epitope binding site;

the domains (A) and (E) associate to form a binding site that binds the HIV-1 envelope like a CD4 binding site HIV-1 antibody or like a V3 glycan binding antibody (1); the domains (B) and (D) associate to form a binding site that binds the epitope (2); and the CH2-CH3 domains of the first and third polypeptide form an Fc Domain.

[0022] In certain aspects the invention provides such bispecific molecules wherein:

(i) the third polypeptide chain further comprises a peptide linker 3 N-terminal to the

CH2-CH3 domain;

(ii) domains (A) and (B) are separated by a peptide linker 1;

(iii) domains (C) and (B) are separated by a peptide linker 2;

(iv) the CH2-CH3 domain and domain (C) are separated by a peptide linker 3 or a spacer linker 3;

(v) domains (D) and (E) are separated by a peptide linker 1; and

(vi) domains (F) and (E) are separated by a peptide linker 2. [0023] In certain aspects the invention provides bispecific molecules comprising a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein some of the polypeptides are covalently bonded (See Figure 34), and wherein: (I) the first polypeptide chain comprises in the N- to C-terminal direction:

(i) a domain (A) comprising a binding region of the light chain variable domain of a first immunoglobulin (VL1) having the binding specificity of the V3 glycan binding antibody DH542, a variant of DH542 called DH542_QSA, DH542_L4 and/or other antibodies from the DH542 lineage, and/or the binding specificity of the CD4 binding site antibody CH557 (also referred to as CH235.12) or any of the antibodies of the CH235 lineage HIV- 1 antibody (1);

(ii) a domain (B) comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for an epitope (2), wherein domains (A) and (B) are separated from one another by a peptide linker 1;

(iv) a CH2-CH3 domain, wherein the CH2-CH3 domain and domain (C) are separated by a peptide linker 3 or a spacer-linker 3;

(II) the second polypeptide chain comprises in the N- to C-terminal direction:

(i) a domain (D) comprising a binding region of a light chain variable domain of the second immunoglobulin (VL2) specific for the epitope (2); (ii) a domain (E) comprising a binding region of a heavy chain variable domain of the first immunoglobulin (VH1) having the binding specificity of the V3 glycan binding antibody DH542, a variant of DH542 called DH542_QSA, DH542_L4 and/or other antibodies from the DH542 lineage, and/or the binding specificity of the CD4 binding site antibody CH557 (also referred to as CH235.12) or any of the antibodies of the CH235 lineage HIV- 1 antibody (1), wherein domains (D) and (E) are separated from one another by a peptide linker 1; and

(iii) a domain (F) comprising a heterodimer promoting domain, including but not limited to a K coil or E coil; wherein domain (F) and domain (E) are separated by a peptide linker 2;

(III) the third polypeptide chain comprises in the N- to C-terminal direction:

(i) a peptide linker 3,

(ii) a CH2-CH3 domain, and wherein:

the domains (A) and (B) do not associate with one another to form an epitope binding site; the domains (D) and (E) do not associate with one another to form an epitope binding site; the domains (A) and (E) associate to form a binding site that binds the HIV-1 envelope like the V3 glycan binding antibody DH542, a variant of DH542 called DH542_QSA, DH542_L4 and/or other antibodies from the DH542 lineage, and/or like the CD4 binding site antibody CH557 (also referred to as CH235.12) or any of the antibodies of the CH235 lineage HIV-1 antibody (1);

the domains (B) and (D) associate to form a binding site that binds the epitope (2); and the CH2-CH3 domains of the first and third polypeptide form an Fc Domain. [0024] In certain aspects said first and second polypeptide chains are covalently bonded to one another; and said second and third polypeptide chains are covalently bonded to one another.

[0025] In certain aspects the invention provides a bispecific molecule comprising a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first and second polypeptide chains are covalently bonded and the second and third polypeptide chains are covalently bonded, and wherein:

(I) the first polypeptide chain comprises in the N- to C-terminal direction:

(i) a domain (A) comprising SEQ ID NO: 553, 565, 567, 570, 574, 578, 582, 586, 590, or 594; (ii) a domain (B) comprising SEQ ID NO:500 or 504;

(iii) a domain (C) comprising SEQ ID NO: 520, 521, 518, or 519; and

(iv) a CH2-CH3 domain comprising SEQ ID NO: 531, 532, 533, or 534;

(II) the second polypeptide chain comprises in the N- to C-terminal direction:

(i) a domain (D) comprising SEQ ID NO: 502, or 506;

(iii) a domain (F) comprising SEQ ID NO: 518, 519, 520, or 521;

(III) the third polypeptide chain comprises in the N- to C-terminal direction:

(i) a CH2-CH3 domain comprising SEQ ID NO: 531, 532, 533, or 534; and wherein: the domains (A) and (B) are linked so that they do not associate with one another to form an epitope binding site;

the domains (A) and (E) associate to form a binding site that binds the HIV-1 envelope like a CD4 binding site HIV-1 antibody or like a V3 glycan binding antibody (1);

the domains (B) and (D) associate to form a binding site that binds an epitope (2); and the CH2-CH3 domains of the first and third polypeptide form an Fc Domain.

[0026] In certain aspects the invention provides such bispecific molecules wherein:

(i) the third polypeptide chain further comprises SEQ ID NO: 523 N-terminal to the

CH2-CH3 domain;

(ii) domains (A) and (B) are separated by SEQ ID NO: 508;

(iii) domains (C) and (B) are separated by SEQ ID NO: 509 or 510;

(iv) the CH2-CH3 domain and domain (C) are separated by SEQ ID NO: 523 or 522; (v) domains (D) and (E) are separated by SEQ ID NO: 508; and

(vi) domains (F) and (E) are separated by SEQ ID NO: 509 or 510. [0027] In certain aspects the invention provides bispecific molecules comprising a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first and second polypeptide chains are covalently bonded and the second and third polypeptide chains are covalently bonded, and wherein: (I) the first polypeptide chain comprises in the N- to C-terminal direction: (i) a domain (A) comprising SEQ ID NO: 553, 565, 567, 570, 574, 578, 582, 586, 590, or 594;

(ii) a domain (B) comprising SEQ ID NO:500 or 504, wherein domains (A) and (B) are separated SEQ ID NO: 508;

(iii) a domain (C) comprising SEQ ID NO: 513, 520, 511, or 518; wherein domain (C) and domain (B) are separated by SEQ ID NO: 509 or 510;

(iv) a CH2-CH3 domain comprising SEQ ID NO: 531, 532, 533, or 534, wherein the CH2-CH3 domain and domain (C) are separated by SEQ ID NO: 523 or 522;

(II) the second polypeptide chain comprises in the N- to C-terminal direction:

(i) a domain (D) comprising SEQ ID NO: 502, or 506;

(ii) a domain (E) comprising SEQ ID NO: 551, 564, 566, 568, 572, 576, 580, 584, 588, 592, wherein domains (D) and (E) are separated from one another by SEQ ID NO: 508; and

(iii) a domain (F) comprising SEQ ID NO: 513, 520, 511, or 518; wherein domain (F) and domain (E) are separated by SEQ ID NO: 509 or 510;

(III) the third polypeptide chain comprises in the N- to C-terminal direction:

(i) SEQ ID NO: 523; and

(ii) a CH2-CH3 domain comprising SEQ ID NO: 531, 532, 533, or 534; and wherein: the domains (A) and (B) do not associate with one another to form an epitope binding site; the domains (D) and (E) do not associate with one another to form an epitope binding site; and

wherein if the domain (A) comprises SEQ ID NO: 570 and domain (E) comprises SEQ ID NO: 568 they associate to form a binding site that binds the HIV-1 envelope like antibody DH493; wherein if the domain (A) comprises SEQ ID NO: 574 and domain (E) comprises SEQ ID NO: 572 they associate to form a binding site that binds the HIV-1 envelope like antibody DH492;

wherein if the domain (A) comprises SEQ ID NO: 590 and domain (E) comprises SEQ ID NO: 588 they associate to form a binding site that binds the HIV-1 envelope like antibody DH542_QSA; or

wherein if the domain (A) comprises SEQ ID NO: 594 and domain (E) comprises SEQ ID NO: 592 they associate to form a binding site that binds the HIV-1 envelope like antibody DH542_L4; and

wherein if the domain (B) comprises SEQ ID NO: 504 and domain (D) comprises SEQ ID NO: 506 they associate to form a binding site that binds CD16; and

the CH2-CH3 domains of the first and third polypeptide form an Fc Domain. [0028] In certain aspects the invention provides bispecific molecules comprising a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein some of the polypeptides are covalently bonded (See Figure 34), and wherein:

(I) the first polypeptide chain comprises in the N- to C-terminal direction:

(i) a CH2-CH3 domain;

(ii) a domain (A) comprising a binding region of the light chain variable domain of a first immunoglobulin (VL1) having the binding specificity of a CD4 binding site HIV- 1 antibody or a V3 glycan binding antibody (1); (iii) a domain (B) comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for an epitope (2); and

(iv) a domain (C) comprising a heterodimer promoting domain;

(II) the second polypeptide chain comprises in the N- to C-terminal direction:

(ii) a domain (E) comprising a binding region of a heavy chain variable domain of the first immunoglobulin (VH1) having the binding specificity of a CD4 binding site HIV- 1 antibody or a V3 glycan binding antibody (1); and

(iii) a domain (F) comprising a heterodimer promoting domain;

(III) the third polypeptide chain comprises in the N- to C-terminal direction:

(i) a CH2-CH3 domain, and wherein:

the domains (A) and (E) associate to form a binding site that binds the HIV-1 envelope like a CD4 binding site HIV-1 antibody or like a V3 glycan binding antibody (1); the domains (B) and (D) associate to form a binding site that binds the epitope (2); and

the CH2-CH3 domains of the first and third polypeptide form an Fc Domain. [0029] In certain aspects the invention provides such bispecific molecules wherein:

(i) the CH2-CH3 domain and domain (A) are separated by a peptide linker 4;

(ii) domains (A) and (B) are separated by a peptide linker 1;

(iii) domains (C) and (B) are separated by a peptide linker 2;

(iv) domains (D) and (E) are separated by a peptide linker 1;

(v) domains (F) and (E) are separated by a peptide linker 2;

(vi) the first polypeptide chain further comprises a peptide linker 3 N-terminal to the CH2- CH3 domain; and

(vii) the third polypeptide chain further comprises a peptide linker 3 N-terminal to the

CH2-CH3 domain. [0030] In other aspects the invention provides bispecific molecules comprising a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein some of the polypeptides are covalently bonded (See Figure 34), and wherein:

(I) the first polypeptide chain comprises in the N- to C-terminal direction:

(i) a peptide linker 3 followed by a CH2-CH3 domain;

(ii) a domain (A) comprising a binding region of the light chain variable domain of a first immunoglobulin (VL1) having the binding specificity of the V3 glycan binding antibody DH542, a variant of DH542 called DH542_QSA, DH542_L4 and/or other antibodies from the DH542 lineage, and/or the binding specificity of the CD4 binding site antibody CH557 (also referred to as CH235.12) or any of the antibodies of the CH235 lineage (1), wherein the CH2-CH3 domain and domain (A) are separated by a peptide linker 4;

(iii) a domain (B) comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for an epitope (2), wherein domains (A) and (B) are separated by a peptide linker 1;

(iv) a domain (C) comprising a heterodimer promoting domain, including but not limited to a K coil or E coil; wherein domain (C) and domain (B) are separated by a peptide linker 2;

(II) the second polypeptide chain comprises in the N- to C-terminal direction:

(ii) a domain (E) comprising a binding region of a heavy chain variable domain of the first immunoglobulin (VH1) having the binding specificity of the V3 glycan binding antibody DH542, a variant of DH542 called DH542_QSA, DH542-L4 and/or other antibodies from the DH542 lineage, and/or the binding specificity of the CD4 binding site antibody CH557 (also referred to as CH235.12) or any of the antibodies of the CH235 lineage (1), wherein domains (D) and (E) are separated by a peptide linker 1; and

(III) the third polypeptide chain comprises in the N- to C-terminal direction:

(i) a peptide linker 3, (ii) a CH2-CH3 domain, and wherein:

the domains (A) and (B) do not associate with one another to form an epitope binding site; the domains (D) and (E) do not associate with one another to form an epitope binding site; the domains (A) and (E) associate to form a binding site that binds the HIV-1 envelope like the V3 glycan binding antibody DH542, a variant of DH542 called DH542_QSA, DH542_L4 and/or other antibodies from the DH542 lineage, and/or like the CD4 binding site antibody CH557 (also referred to as CH235.12) or any of the antibodies of the CH235 lineage (1); the domains (B) and (D) associate to form a binding site that binds the epitope (2); and the CH2-CH3 domains of the first and third polypeptide form an Fc Domain. [0031] In certain aspects said first and second polypeptide chains are covalently bonded to one another; and said first and third polypeptide chains are covalently bonded to one another. [0032] In certain aspects the invention provides bispecific molecules comprising a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first and second polypeptide chains are covalently bonded and the first and third polypeptide chains are covalently bonded (See Figure 34), and wherein:

(I) the first polypeptide chain comprises in the N- to C-terminal direction:

(i) a CH2-CH3 domain comprising SEQ ID NO: 531, 532, 533, or 534;

(ii) a domain (A) comprising SEQ ID NO: 553, 565, 567, 570, 574, 578, 582, 586, 590, or 594;

(iii) a domain (B) comprising SEQ ID NO:500 or 504; and

(iv) a domain (C) comprising SEQ ID NO: 520, 521, 518, or 519;

(II) the second polypeptide chain comprises in the N- to C-terminal direction:

(i) a domain (D) comprising SEQ ID NO: 502, or 506;

(iii) a domain (F) comprising SEQ ID NO: 518, 519, 520, or 521;

(III) the third polypeptide chain comprises in the N- to C-terminal direction:

(i) a CH2-CH3 domain comprising SEQ ID NO: 531, 532, 533, or 534, and wherein: the domains (A) and (B) are linked so that they do not associate with one another to form an epitope binding site; the domains (D) and (E) are linked so that they do not associate with one another to form an epitope binding site;

the domains (A) and (E) associate to form a binding site that binds the HIV-1 envelope like a CD4 binding site HIV-1 antibody or like a V3 glycan binding antibody (1); the domains (B) and (D) associate to form a binding site that binds an epitope (2); and

the CH2-CH3 domains of the first and third polypeptide form an Fc Domain. [0033] In certain aspects the invention provides such bispecific molecules wherein:

(i) the CH2-CH3 domain and domain (A) are separated by SEQ ID NO: 524 or 525; (ii) domains (A) and (B) are separated by SEQ ID NO: 508;

(iii) domains (C) and (B) are separated by SEQ ID NO: 509 or 510;

(iv) domains (D) and (E) are separated by SEQ ID NO: 508;

(v) domains (F) and (E) are separated by SEQ ID NO: 509 or 510;

(vi) the first polypeptide chain further comprises SEQ ID NO: 523 N-terminal to the CH2- CH3 domain; and

(vii) the third polypeptide chain further comprises SEQ ID NO: 523 N-terminal to the CH2-CH3 domain. [0034] In other aspects the invention provides bispecific molecules comprising a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first and second polypeptide chains are covalently bonded and the first and third polypeptide chains are covalently bonded (See Figure 34), and wherein:

(I) the first polypeptide chain comprises in the N- to C-terminal direction:

(i) SEQ ID NO: 523 followed by a CH2-CH3 domain comprising SEQ ID NO: 531, 532, 533, or 534;

(ii) a domain (A) comprising SEQ ID NO: 553, 565, 567, 570, 574, 578, 582, 586, 590, or 594, wherein the CH2-CH3 domain and domain (A) are separated SEQ ID NO: 524 or 525;

(iii) a domain (B) comprising SEQ ID NO:500 or 504, wherein domains (A) and (B) are separated by SEQ ID NO: 508;

(iv) a domain (C) comprising SEQ ID NO: 520, 521, 518, or 519; wherein domain (C) and domain (B) are separated by SEQ ID NO: 509 or 510;

(II) the second polypeptide chain comprises in the N- to C-terminal direction:

(i) a domain (D) comprising SEQ ID NO: 502, or 506; (ii) a domain (E) comprising SEQ ID NO: 551, 564, 566, 568, 572, 576, 580, 584, 588, 592, wherein domains (D) and (E) are separated by SEQ ID NO: 508; and

(iii) a domain (F) comprising SEQ ID NO: 518, 519, 520, or 521; wherein domain (F) and domain (E) are separated by SEQ ID NO: 509 or 510;

(III) the third polypeptide chain comprises in the N- to C-terminal direction:

(i) SEQ ID NO: 523,

(ii) a CH2-CH3 domain comprising SEQ ID NO: 531, 532, 533, or 534, and wherein: the domains (A) and (B) do not associate with one another to form an epitope binding site; the domains (D) and (E) do not associate with one another to form an epitope binding site; wherein if the domain (A) comprises SEQ ID NO: 553 and domain (E) comprises SEQ ID NO: 551 they associate to form a binding site that binds the HIV-1 envelope like antibody DH557;

wherein if the domain (A) comprises SEQ ID NO: 586 and domain (E) comprises SEQ ID NO: 584 they associate to form a binding site that binds the HIV-1 envelope like antibody DH542; wherein if the domain (A) comprises SEQ ID NO: 590 and domain (E) comprises SEQ ID NO: 588 they associate to form a binding site that binds the HIV-1 envelope like antibody DH542_QSA; or

the CH2-CH3 domains of the first and third polypeptide form an Fc Domain. [0035] In certain aspects the invention provides a bispecific molecule comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein some of the polypeptides are covalently bonded, and wherein: (I) the first and the third polypeptide chains each comprise in the N- to C-terminal direction:

(iii) a domain (C) comprising a heterodimer promoting domain; and

(iv) a CH2-CH3 domain;

(II) the second and fourth polypeptide chains each comprise in the N- to C-terminal direction:

(ii) a domain (E) comprising a binding region of a heavy chain variable domain of the first immunoglobulin (VH1) having the binding specificity of a CD4 binding site HIV-1 antibody or a V3 glycan binding antibody (1);

(iii) a domain (F) comprising a heterodimer promoting domain; and wherein

the domains (A) and (B) are linked so that they do not associate with one another to form an epitope binding site; the domains (D) and (E) are linked so that they do not associate with one another to form an epitope binding site;

the domains (B) and (D) associate to form a binding site that binds the epitope (2); and the CH2-CH3 domains of the first and third polypeptide form an Fc Domain.

[0036] In certain aspects the invention provides such bispecific molecules wherein:

(i) domains (A) and (B) are separated by a peptide linker 1;

(ii) domains (C) and (B) are separated by a peptide linker 2;

(iii) the CH2-CH3 domain and domain (C) are separated by a peptide linker 3 or a spacer linker 3;

(iv) domains (D) and (E) are separated by a peptide linker 1; and

(v) domains (F) and (E) are separated by a peptide linker 2. [0037] In certain aspects the invention provides a bispecific molecule comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein some of the polypeptides are covalently bonded, and wherein: (I) the first and the third polypeptide chains each comprise in the N- to C-terminal direction:

(i) a domain (A) comprising a binding region of the light chain variable domain of a first immunoglobulin (VL1) having the binding specificity of the V3 glycan binding antibody DH542, a variant of DH542 called DH542_QSA, DH542_L4 and/or other antibodies from the DH542 lineage, and/or the binding specificity of the CD4 binding site antibody CH557 (also referred to as CH235.12) or any of the antibodies of the CH235 lineage (1); (ii) a domain (B) comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for an epitope (2), wherein domains (A) and (B) are separated by a peptide linker 1;

(iii) a domain (C) comprising a heterodimer promoting domain, wherein domains (C) and (B) are separated by a peptide linker 2; and

(iv) a CH2-CH3 domain, wherein the CH2-CH3 domain and domain (C) are separated by a peptide linker 3 or a spacer linker 3;

(II) the second and fourth polypeptide chains each comprise in the N- to C-terminal direction: (i) a domain (D) comprising a binding region of a light chain variable domain of the second immunoglobulin (VL2) specific for the epitope (2);

(ii) a domain (E) comprising a binding region of a heavy chain variable domain of the first immunoglobulin (VH1) having the binding specificity of the V3 glycan binding antibody DH542, a variant of DH542 called DH542_QSA, DH542_L4 and/or other antibodies from the DH542 lineage, and/or the binding specificity of the CD4 binding site antibody CH557 (also referred to as CH235.12) or any of the antibodies of the CH235 lineage (1), wherein domains (D) and (E) are separated by a peptide linker 1;

(iii) a domain (F) comprising a heterodimer promoting domain, wherein domains (F) and (E) are separated by a peptide linker 2; and wherein

the domains (A) and (B) do not associate with one another to form an epitope binding site; the domains (D) and (E) do not associate with one another to form an epitope binding site; the domains (A) and (E) associate to form a binding site that binds the HIV-1 envelope like the V3 glycan binding antibody DH542, a variant of DH542 called DH542_QSA, DH542_L4 and/or other antibodies from the DH542 lineage, and/or like the CD4 binding site antibody CH557 (also referred to as CH235.12) or any of the antibodies of the CH235 lineage (1); the domains (B) and (D) associate to form a binding site that binds the epitope (2); and the CH2-CH3 domains of the first and third polypeptide form an Fc Domain.

[0038] In certain aspects said first and second polypeptide chains are covalently bonded to one another; said third and fourth polypeptide chains are covalently bonded to one another; and said first and third chains are covalently bonded to one another. [0039] In certain aspects the invention provides a bispecific molecule comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first and second polypeptide chains are covalently bonded, the third and fourth polypeptide chains are covalently bonded, and the first and third chains are covalently bonded, and wherein: (I) the first and the third polypeptide chains each comprise in the N- to C-terminal direction:

(ii) a domain (B) comprising SEQ ID NO:500 or 504;

(iii) a domain (C) comprising SEQ ID NO: 520, 521, 518, or 519; and (iv) a CH2-CH3 domain comprising SEQ ID NO: 527, 528, or 529;

(i) a domain (D) comprising SEQ ID NO: 502, or 506;

(ii) a domain (E) comprising SEQ ID NO: 551, 564, 566, 568, 572, 576, 580, 584, 588, 592;

(iii) a domain (F) comprising SEQ ID NO: 518, 519, 520, or 521; and wherein

the domains (B) and (D) associate to form a binding site that binds an epitope (2); and the CH2-CH3 domains of the first and third polypeptide form an Fc Domain. [0040] In certain aspects the invention provides such bispecific molecules wherein:

(i) domains (A) and (B) are separated by SEQ ID NO: 508;

(ii) domains (C) and (B) are separated by SEQ ID NO: 509 or 510;

(iii) the CH2-CH3 domain and domain (C) are separated by SEQ ID NO: 523 or 522; (iv) domains (D) and (E) are separated by SEQ ID NO: 508; and

(v) domains (F) and (E) are separated by SEQ ID NO: 509 or 510. [0041] In certain aspects the invention provides a bispecific molecule comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first and second polypeptide chains are covalently bonded, the third and fourth polypeptide chains are covalently bonded, and the first and third chains are covalently bonded, and wherein: (I) the first and the third polypeptide chains each comprise in the N- to C-terminal direction:

(ii) a domain (B) comprising SEQ ID NO:500 or 504, wherein domains (A) and (B) are separated by SEQ ID NO: 508; (iii) a domain (C) comprising SEQ ID NO: 520, 521, 518, or 519, wherein domains (C) and (B) are separated by SEQ ID NO: 509 or 510; and

(iv) a CH2-CH3 domain comprising SEQ ID NO: 527, 528, or 529, wherein the CH2- CH3 domain and domain (C) are separated by SEQ ID NO: 523 or 522;

(i) a domain (D) comprising SEQ ID NO: 502, or 506;

(ii) a domain (E) comprising SEQ ID NO: 551, 564, 566, 568, 572, 576, 580, 584, 588, 592, wherein domains (D) and (E) are separated by SEQ ID NO: 508;

(iii) a domain (F) comprising SEQ ID NO: 518, 519, 520, or 521, wherein domains (F) and (E) are separated by SEQ ID NO: 509 or 510; and wherein

the domains (A) and (B) do not associate with one another to form an epitope binding site; the domains (D) and (E) do not associate with one another to form an epitope binding site; wherein if the domain (A) comprises SEQ ID NO: 553 and domain (E) comprises SEQ ID NO: 551 they associate to form a binding site that binds the HIV-1 envelope like antibody DH557;

wherein if the domain (A) comprises SEQ ID NO: 582 and domain (E) comprises SEQ ID NO: 580 they associate to form a binding site that binds the HIV-1 envelope like antibody DH490; wherein if the domain (A) comprises SEQ ID NO: 586 and domain (E) comprises SEQ ID NO: 584 they associate to form a binding site that binds the HIV-1 envelope like antibody DH542;

the CH2-CH3 domains of the first and third polypeptide form an Fc Domain. [0042] In certain aspects the invention provides trivalent binding molecules comprising a first, second, third and fourth polypeptide chain, wherein some of the polypeptides are covalently bonded and wherein:

(I) the first polypeptide chain comprises in the N-terminus to C-terminus direction:

(i) a domain (A) comprising a binding region of a light chain variable domain of a first immunoglobulin (VL1) specific for an epitope (1);

(iii) a domain (C) comprising a heterodimer promoting domain; and

(iv) a CH2-CH3 Domain;

(II) the second polypeptide chain comprises, in the N-terminus to C-terminus direction:

(i) a domain (D) comprising a binding region of a light chain variable domain of the first immunoglobulin (VL1) specific for the epitope (2);

(ii) a domain (E) comprising a binding region of a heavy chain variable domain of the second immunoglobulin (VH1) specific for the epitope (1); and

(iii) a domain (F) comprising a heterodimer promoting domain;

(III) the third polypeptide chain that comprises, in the N-terminus to C-terminus direction: (i) a domain (G) comprising a binding region of a heavy chain variable domain of a third immunoglobulin (VH3) specific for an epitope (3); and

(ii) a CH1-Hinge Domain, and a CH2-CH3 Domain; and

(IV) the fourth polypeptide chain that comprises, in the N-terminus to C-terminus direction:

(i) a domain (H) comprising a binding region of a light chain variable domain of the third immunoglobulin (VL3) specific for the epitope (3); and

(ii) CL Kappa Domain or a CL Lambda Domain; and wherein

the domains (A) and (E) associate to form a binding site that binds the epitope (1);

the domains (B) and (D) associate to form a binding site that binds the epitope (2);

the domains (G) and (H) associate to form a binding site that bind the epitope (3);

at least one of epitope (1), epitope (2), and epitope (3) is an epitope bound by a CD4 binding site HIV-1 antibody or a V3 glycan binding antibody, and at least one of epitope (1), epitope (2), and epitope (3) is an epitope of, for example, but not limited to, CD3, CD8, or CD16, or an epitope on any suitable effector cell;

the CH2-CH3 domains of the first and third polypeptide form an Fc Domain;

said first and second polypeptide chains are covalently bonded to one another;

said first and third polypeptide chains are covalently bonded to one another; and

said third and fourth polypeptide chains are covalently bonded to one another. [0043] In certain aspects the invention provides such trivalent molecules wherein:

(i) domains (A) and (B) are separated by a peptide linker 1;

(ii) domains (C) and (B) are separated by a peptide linker 2 or a peptide linker 2-C;

(iv) domains (D) and (E) are separated by a peptide linker 1; and

(v) domains (F) and (E) are separated by a peptide linker 2 or a peptide linker 2-C. [0044] In certain aspects the invention provides trivalent binding molecules comprising a first, second, third and fourth polypeptide chain, wherein some of the polypeptides are covalently bonded and wherein:

(ii) a domain (B) comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for an epitope (2), wherein domains (A) and (B) are separated by a Peptide Linker 1;

(iii) a domain (C) comprising:

(a) a heterodimer promoting domain; wherein domain (C) and domain (B) are separated by a Peptide Linker 2-C; or

(b) a heterodimer promoting domain; wherein domain (C) and domain (B) are separated by a Peptide Linker 2; and

(ii) a domain (E) comprising a binding region of a heavy chain variable domain of the second immunoglobulin (VH1) specific for the epitope (1), wherein domains (D) and (E) are separated by a Peptide Linker 1;

(iii) a domain (F) comprising

(a) a heterodimer promoting domain; wherein domain (F) and domain (E) are separated by a peptide Peptide Linker 2-C; or

(b) a heterodimer promoting domain; wherein domain (F) and domain (E) are separated by a peptide Peptide Linker 2;

(III) the third polypeptide chain that comprises, in the N-terminus to C-terminus direction:

(i) a domain (G) comprising a binding region of a heavy chain variable domain of a third immunoglobulin (VH3) specific for an epitope (3); and

(ii) a CH1-Hinge Domain, and a CH2-CH3 Domain; and

(IV) the fourth polypeptide chain that comprises, in the N-terminus to C-terminus direction: (i) a domain (H) comprising a binding region of a light chain variable domain of the third immunoglobulin (VL3) specific for the epitope (3); and

(ii) CL Kappa Domain or a CL Lambda Domain; and wherein

the domains (A) and (B) do not associate with one another to form an epitope binding site; the domains (D) and (E) do not associate with one another to form an epitope binding site; the domains (A) and (E) associate to form a binding site that binds the epitope (1);

at least one of epitope (1), epitope (2), and epitope (3) is an epitope bound by the V3 glycan binding antibody DH542, a variant of DH542 called DH542_QSA, DH542_L4 and/or other antibodies from the DH542 lineage, and/or the CD4 binding site antibody CH557 (also referred to as CH235.12) or any of the antibodies of the CH235 lineage, and at least one of epitope (1), epitope (2), and epitope (3) is an epitope of for example, but not limited to, CD3, CD8, or CD16, or an epitope on any suitable effector cell;

the CH2-CH3 domains of the first and third polypeptide form an Fc Domain;

said first and second polypeptide chains are covalently bonded to one another;

said third and fourth polypeptide chains are covalently bonded to one another. [0045] In certain aspects the invention provides trivalent binding molecules comprising a first, second, third and fourth polypeptide chain, wherein some of the polypeptides are covalently bonded and wherein:

(ii) a domain (B) comprising SEQ ID NO:500;

(iii) a domain (C) comprising SEQ ID NO: 520, 521, 518, or 519; and

(iv) a CH2-CH3 Domain comprising SEQ ID NO: 531, 532, 533, or 534;

(i) a domain (D) comprising SEQ ID NO: 502;

(iii) a domain (F) comprising SEQ ID NO: 518, 519, 520, or 521; (III) the third polypeptide chain that comprises, in the N-terminus to C-terminus direction: (i) a domain (G) comprising SEQ ID NO: 551, 564, 566, 568, 572, 576, 580, 584, 588, 592, 543, or 547; and

(ii) a CH1-Hinge Domain comprising SEQ ID NO: 515, and a CH2-CH3 Domain comprising SEQ ID NO: 531, 532, 533, or 534; and

(i) a domain (H) comprising SEQ ID NO: 553, 565, 567, 570, 574, 578, 582, 586, 590, 594, 545, or 549; and

(ii) CL Kappa Domain comprising SEQ ID NO: 516 or a CL Lambda Domain comprising SEQ ID NO: 517; and wherein

the domains (A) and (E) associate to form a binding site that binds an epitope (1);

the domains (B) and (D) associate to form a binding site that binds an epitope (2);

the domains (G) and (H) associate to form a binding site that bind an epitope (3);

wherein epitope (1) is an epitope bound by a CD4 binding site HIV-1 antibody or a V3 glycan binding antibody, epitope (2) is an epitope of CD3, and epitope (3) is an epitiope bound by a CD4 binding site HIV-1 antibody or a V3 glycan binding antibody or is an epitope of CD8; the CH2-CH3 domains of the first and third polypeptide form an Fc Domain;

said first and second polypeptide chains are covalently bonded to one another;

said third and fourth polypeptide chains are covalently bonded to one another. [0046] In certain aspects the invention provides such trivalent molecules wherein:

(i) domains (A) and (B) are separated by SEQ ID NO: 508;

(ii) domains (C) and (B) are separated by SEQ ID NO: 509 or 510;

(iii) the CH2-CH3 domain and domain (C) are separated by SEQ ID NO: 522, or 523; (iv) domains (D) and (E) are separated by SEQ ID NO: 508; and

(v) domains (F) and (E) are separated by SEQ ID NO: 509 or 510. [0047] In certain aspects the invention provides trivalent binding molecules comprising a first, second, third and fourth polypeptide chain, wherein some of the polypeptides are covalently bonded and wherein:

(ii) a domain (B) comprising SEQ ID NO:500, wherein domains (A) and (B) are separated by SEQ ID NO: 508;

(iii) a domain (C) comprising SEQ ID NO: 520, 521, 518, or 519, wherein domains (C) and (B) are separated by SEQ ID NO: 509 or 510;

(iv) a CH2-CH3 Domain comprising SEQ ID NO: 531, 532, 533, or 534, wherein the CH2-CH3 domain and domain (C) are separated by SEQ ID NO: 522, or 523;

(i) a domain (D) SEQ ID NO: 502;

(ii) a domain (E) comprising SEQ ID NO: 551, 564, 566, 568, 572, 576, 580, 584, 588, 592, wherein domains (D) and (E) are separated from one another by SEQ ID NO: 508;

(iii) a domain (F) comprising SEQ ID NO: 518, 519, 520, or 521, wherein domain (F) and domain (E) are separated by SEQ ID NO: 509 or 510;

(i) a domain (G) comprising SEQ ID NO: 551, 564, 566, 568, 572, 576, 580, 584, 588, 592, 543, or 547; and

(ii) a CH1-Hinge Domain SEQ ID NO: 515, and a CH2-CH3 Domain comprising SEQ ID NO: 531, 532, 533, or 534; and

the domains (A) and (B) do not associate with one another to form an epitope binding site; the domains (D) and (E) do not associate with one another to form an epitope binding site; the domains (A) and (E) associate to form a binding site that binds an epitope (1);

the domains (B) and (D) associate to form a binding site that binds an epitope (2); the domains (G) and (H) associate to form a binding site that bind an epitope (3);

wherein if the domain (B) comprises SEQ ID NO: 500 and domain (D) comprises SEQ ID NO: 502 they associate to form a binding site that binds CD3; or wherein if the domain (H) comprises SEQ ID NO: 553 and domain (G) comprises SEQ ID NO: 551 they associate to form a binding site that binds the HIV-1 envelope like antibody DH557;

wherein if the domain (H) comprises SEQ ID NO: 565 and domain (G) comprises SEQ ID NO: 564 they associate to form a binding site that binds the HIV-1 envelope like antibody DH556;

wherein if the domain (H) comprises SEQ ID NO: 567 and domain (G) comprises SEQ ID NO: 566 they associate to form a binding site that binds the HIV-1 envelope like antibody DH555;

wherein if the domain (H) comprises SEQ ID NO: 570 and domain (G) comprises SEQ ID NO: 568 they associate to form a binding site that binds the HIV-1 envelope like antibody DH493;

wherein if the domain (H) comprises SEQ ID NO: 574 and domain (G) comprises SEQ ID NO: 572 they associate to form a binding site that binds the HIV-1 envelope like antibody DH492;

wherein if the domain (H) comprises SEQ ID NO: 578 and domain (G) comprises SEQ ID NO: 576 they associate to form a binding site that binds the HIV-1 envelope like antibody DH491;

wherein if the domain (H) comprises SEQ ID NO: 582 and domain (G) comprises SEQ ID NO: 580 they associate to form a binding site that binds the HIV-1 envelope like antibody DH490;

wherein if the domain (H) comprises SEQ ID NO: 586 and domain (G) comprises SEQ ID NO: 584 they associate to form a binding site that binds the HIV-1 envelope like antibody DH542;

wherein if the domain (H) comprises SEQ ID NO: 590 and domain (G) comprises SEQ ID NO: 588 they associate to form a binding site that binds the HIV-1 envelope like antibody DH542_QSA; or

wherein if the domain (H) comprises SEQ ID NO: 594 and domain (G) comprises SEQ ID NO: 592 they associate to form a binding site that binds the HIV-1 envelope like antibody DH542_L4;

wherein if the domain (H) comprises SEQ ID NO: 545 and domain (G) comprises SEQ ID NO: 543 they associate to form a binding site that binds CD8; or wherein if the domain (H) comprises SEQ ID NO: 549 and domain (G) comprises SEQ ID NO: 547 they associate to form a binding site that binds CD8; and wherein

epitope (1) is an epitope bound by the antibody CH557, CH556, CH555, CH493, CH492, CH491, CH490, DH542, DH542_QSA, or DH542_L4, epitope (2) is an epitope of CD3, and epitope (3) is is an epitope bound by the antibody CH557, CH556, CH555, CH493, CH492, CH491, CH490, DH542, DH542_QSA, or DH542_L4 or is an epitope of CD8; and wherein

the CH2-CH3 domains of the first and third polypeptide form an Fc Domain;

said first and second polypeptide chains are covalently bonded to one another;

said third and fourth polypeptide chains are covalently bonded to one another.

[0048] In certain aspects the invention also provides trivalent binding molecules comprising a first, second, and third polypeptide chain, wherein some of the polypeptides are covalently bonded and wherein:

(iii) a domain (C) comprising a heterodimer promoting domain; and

(iv) a CH2-CH3 Domain;

(ii) a domain (E) comprising a binding region of a heavy chain variable domain of the second immunoglobulin (VH1) specific for the epitope (1);

(iii) a domain (F) comprising a heterodimer promoting domain; and

(i) a domain (H) comprising a binding region of a light chain variable domain of a third immunoglobulin (VL3) specific for an epitope (3)

(ii) a domain (G) comprising a binding region of a heavy chain variable domain of the third immunoglobulin (VH3) specific for the epitope (3);

(iii) a CH2-CH3 Domain; and wherein the domains (A) and (B) are linked so that they do not associate with one another to form an epitope binding site;

at least one of epitope (1), epitope (2), and epitope (3) is an epitope bound by a CD4 binding site HIV-1 antibody or a V3 glycan antibody, and at least one of epitope (1), epitope (2), and epitope (3) is and epitope of for example, but not limited to, CD3, CD8, or CD16, or any other suitable epitope on an effector cell ;

the CH2-CH3 domains of the first and third polypeptide form an Fc Domain;

said first and second polypeptide chains are covalently bonded to one another;

said third and fourth polypeptide chains are covalently bonded to one another. [0049] In certain aspects the invention provides such trivalent molecules wherein:

(i) domains (A) and (B) are separated by a peptide linker 1;

(iv) domains (D) and (E) are separated by a peptide linker 1;

(v) domains (F) and (E) are separated by a peptide linker 2 or a peptide linker 2-C;

(vi) domains (H) and (G) are separated by a peptide linker 5; and

(vii) the CH2-CH3 domain and domain (G) are separated by a peptide linker 3. [0050] In certain aspects the invention also provides trivalent binding molecules comprising a first, second, and third polypeptide chain, wherein some of the polypeptides are covalently bonded and wherein:

(i) a domain (A) comprising a binding region of a light chain variable domain of a first immunoglobulin (VL1) specific for an epitope (1); (ii) a domain (B) comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for an epitope (2), wherein domains (A) and (B) are separated by a Peptide Linker 1;

(iii) a domain (C) comprising:

(iii) a domain (F) comprising:

(ii) a domain (G) comprising a binding region of a heavy chain variable domain of the third immunoglobulin (VH3) specific for the epitope (3), wherein domains (H) and (G) are separated by a Peptide Linker 5;

(iii) a Peptide Linker 3; and

(iv) a CH2-CH3 Domain; and wherein

the domains (B) and (D) associate to form a binding site that binds the epitope (2); the domains (G) and (H) associate to form a binding site that bind the epitope (3); at least one of epitope (1), epitope (2), and epitope (3) is an epitope bound by the V3 glycan binding antibody DH542, a variant of DH542 called DH542_QSA, DH542_L4 and/or other antibodies from the DH542 lineage, and/or the CD4 binding site antibody CH557 (also referred to as CH235.12) or any of the antibodies of the CH235 lineage, and at least one of epitope (1), epitope (2), and epitope (3) is and epitope of for example, but not limited to, CD3, CD8 or CD16, or an epitope on any suitable effector cell;

the CH2-CH3 domains of the first and third polypeptide form an Fc Domain;

said first and second polypeptide chains are covalently bonded to one another; and

said first and third polypeptide chains are covalently bonded to one another.

[0051] In certain aspects the invention also provides trivalent binding molecules comprising a first, second, and third polypeptide chain, wherein some of the polypeptides are covalently bonded and wherein:

(ii) a domain (B) comprising SEQ ID NO:500;

(iii) a domain (C) comprising SEQ ID NO: 520, 521, 518, or 519; and

(iv) a CH2-CH3 Domain comprising SEQ ID NO: 531, 532, 533, or 534;

(i) a domain (D) comprising SEQ ID NO: 502;

(ii) a domain (E) comprising SEQ ID NO: 551, 564, 566, 568, 572, 576, 580, 584, 588, or 592;

(iii) a domain (F) comprising SEQ ID NO: 518, 519, 520, or 521; and

(i) a domain (H) comprising SEQ ID NO: 545 or 549;

(ii) a domain (G) comprising SEQ ID NO: 543 or 547;

(iii) a CH2-CH3 Domain comprising SEQ ID NO: 531, 532, 533, or 534; and wherein

the domains (A) and (E) associate to form a binding site that binds an epitope bound by a CD4 binding site HIV-1 antibody or a V3 glycan antibody;

the domains (B) and (D) associate to form a binding site that binds CD3;

the domains (G) and (H) associate to form a binding site that binds CD8; wherein

the CH2-CH3 domains of the first and third polypeptide form an Fc Domain;

said first and second polypeptide chains are covalently bonded to one another;

said third and fourth polypeptide chains are covalently bonded to one another. [0052] In certain aspects the invention provides such trivalent molecules wherein:

(i) domains (A) and (B) are separated by SEQ ID NO: 508;

(ii) domains (C) and (B) are separated by SEQ ID NO: 509 or 510;

(iii) the CH2-CH3 domain and domain (C) are separated by SEQ ID NO: 522, or 523; (iv) domains (D) and (E) are separated by SEQ ID NO: 508;

(v) domains (F) and (E) are separated by SEQ ID NO: 509 or 510;

(vi) domains (H) and (G) are separated by SEQ ID NO: 526; and

(vii) the CH2-CH3 domain and domain (G) are separated by SEQ ID NO: 523 or a CH1- Hinge Domain comprising SEQ ID NO: 515. [0053] In certain aspects the invention also provides trivalent binding molecules comprising a first, second, and third polypeptide chain, wherein some of the polypeptides are covalently bonded and wherein:

(iii) a domain (C) comprising SEQ ID NO: 520, 521, 518, and 519, wherein domains (C) and (B) are separated by SEQ ID NO: 509 or 510; and

(II) the second polypeptide chain comprises, in the N-terminus to C-terminus direction: (i) a domain (D) comprising SEQ ID NO: 502;

(ii) a domain (E) comprising SEQ ID NO: 551, 564, 566, 568, 572, 576, 580, 584, 588, or 592, wherein domains (D) and (E) are separated by SEQ ID NO: 508;

(iii) a domain (F) comprising comprising SEQ ID NO: 518, 519, 520, or 521, wherein domains (F) and (E) are separated by SEQ ID NO: 509 or 510;

(i) a domain (H) comprising SEQ ID NO: 545 or 549;

(ii) a domain (G) comprising SEQ ID NO: 543 or 547, wherein domains (H) and (G) are separated by SEQ ID NO: 526;

(iii) SEQ ID NO: 523, or a CH1-Hinge Domain comprising SEQ ID NO: 515; and (iv) a CH2-CH3 Domain comprising SEQ ID NO: 531, 532, 533, or 534; and wherein

the domains (B) and (D) associate to form a binding site that binds CD3;

the domains (G) and (H) associate to form a binding site that binds CD8; and

wherein if the domain (A) comprises SEQ ID NO: 574 and domain (E) comprises SEQ ID NO: 572 they associate to form a binding site that binds the HIV-1 envelope like antibody DH492; wherein if the domain (A) comprises SEQ ID NO: 578 and domain (E) comprises SEQ ID NO: 576 they associate to form a binding site that binds the HIV-1 envelope like antibody DH491;

wherein if the domain (H) comprises SEQ ID NO: 545 and domain (G) comprises SEQ ID NO: 543 they associate to form a binding site that binds CD8; or

wherein if the domain (H) comprises SEQ ID NO: 549 and domain (G) comprises SEQ ID NO: 547 they associate to form a binding site that binds CD8;

wherein the CH2-CH3 domains of the first and third polypeptide form an Fc Domain;

said first and second polypeptide chains are covalently bonded to one another; and said first and third polypeptide chains are covalently bonded to one another.

[0054] In certain aspects the invention provides such trivalent binding molecules wherein one of epitope (1), epitope (2), and epitope (3) is an epitope of HIV-1 Envelope, one of epitope (1), epitope (2), and epitope (3) is an epitope of CD3, and one of epitope (1), epitope (2), and epitope (3) is an epitope of CD8. In particular aspects of such trivalent binding molecules:

(a) epitope (1) is an epitope of HIV-1 Envelope V3 glycan or an epitope of HIV-1 Envelope CD4 binding site, epitope (2) is an epitope of CD3, and epitope (3) is an epitope of CD8;

(b) epitope (1) is an epitope of HIV-1 Envelope V3 glycan or an epitope of HIV-1 Envelope CD4 binding site, epitope (2) is an epitope of CD8, and epitope (3) is an epitope of CD3; (c) epitope (1) is an epitope of CD3, epitope (2) is an epitope of HIV-1 Envelope V3 glycan or an epitope of HIV-1 Envelope CD4 binding site, and epitope (3) is an epitope of CD8;

(d) epitope (1) is an epitope of CD3, epitope (2) is an epitope of CD8, and epitope (3) is an epitope of HIV-1 Envelope V3 glycan or an epitope of HIV-1 Envelope CD4 binding site;

(e) epitope (1) is an epitope of CD8, epitope (2) is an epitope of CD3, and epitope (3) is an epitope of HIV-1 Envelope V3 glycan or an epitope of HIV-1 Envelope CD4 binding site; or

(f) epitope (1) is an epitope of CD8, epitope (2) is an epitope of HIV-1 Envelope V3 glycan or an epitope of HIV-1 Envelope CD4 binding site, and epitope (3) is an epitope of CD3. [0055] In certain aspects the invention provides such trivalent binding molecules wherein two of epitope (1), epitope (2), and epitope (3) are an epitopes of HIV-1 Envelope, and one of epitope (1), epitope (2), and epitope (3) is an epitope of CD3, where said epitopes of HIV-1 Envelope may be the same epitope or different epitopes. In particular aspects of such trivalent binding molecules:

(a) epitope (1) is an epitope of HIV-1 Envelope V3 glycan or an epitope of HIV-1 Envelope CD4 binding site, epitope (2) is an epitope of HIV-1 Envelope V3 glycan or an epitope of HIV-1 Envelope CD4 binding site, and epitope (3) is an epitope of CD3; (b) epitope (1) is an epitope of HIV-1 Envelope V3 glycan or an epitope of HIV-1 Envelope CD4 binding site, epitope (2) is an epitope of CD3, and epitope (3) is an epitope of HIV-1 Envelope V3 glycan or an epitope of HIV-1 Envelope CD4 binding site; or

(c) epitope (1) is an epitope of CD3, epitope (2) is an epitope of HIV-1 Envelope V3 glycan or an epitope of HIV-1 Envelope CD4 binding site, and epitope (3) is an epitope of HIV-1 Envelope V3 glycan or an epitope of HIV-1 Envelope CD4 binding site. [0056] In certain aspects, domain (H) comprises a binding region of a light chain variable domain of an anti-CD8 antibody, an anti-CD3 antibody, an anti-CD16 antibody, an HIV-1 envelope CD4 binding site antibody, or a HIV-1 envelope V3 glycan antibody. In certain aspects, domain (G) comprises a binding region of a heavy chain variable domain of an anti- CB8 antibody, an anti-CD3 antibody, an anti-CD16 antibody, an HIV-1 envelope CD4 binding site antibody, or a HIV-1 envelope V3 glycan antibody.

[0057] In certain aspects, domain (B) comprises the heavy chain variable domain of an anti- CD3 antibody, an anti-CD8 antibody, or an anti-CD16 antibody. In certain embodiment, domain (D) comprises the light chain variable domain of an anti-CD3 antibody, an anti-CD8 antibody, or an anti-CD16 antibody.

[0058] In certain aspects, the CH2-CH3 domain of the first polypeptide chain of any of the multispecific molecules of the invention is the of the“knob” design and the CH2-CH3 domain of the third polypeptide chain of any of the multivalent molecules of the invention is of the“hole” design.

[0059] In certain aspects, the CH2-CH3 domain of the third polypeptide chain of any of the multispecific molecules of the invention is the of the“knob” design and the CH2-CH3 domain of the first polypeptide chain of any of the multivalent molecules of the invention is of the“hole” design.

[0060] In certain aspects, the CH2-CH3 domain of the first polypeptide chain is the of the “knob” design (SEQ ID NOs: 531 or 532) and the CH2-CH3 domain of the third polypeptide chain is of the“hole” design (SEQ ID NOs: 533 or 534). In certain aspects, the CH2-CH3 domain of the first polypeptide comprises SEQ ID NO: 531 and the CH2-CH3 domain of the third polypeptide chain comprises SEQ ID NO: 533. In certain aspects, the CH2-CH3 domain of the third polypeptide chain is the of the“knob” design (SEQ ID NOs: 531 or 532) and the CH2-CH3 domain of the first polypeptide chain is of the“hole” design (SEQ ID NOs: 533 or 534). In certain aspects, the CH2-CH3 domain of the third polypeptide comprises SEQ ID NO: 531 and the CH2-CH3 domain of the first polypeptide chain comprises SEQ ID NO: 533.

[0061] In certain aspects, the epitope (2) is a CD3 epitope, CD8 epitope or a CD16 epitope. In certain embodiments, the bispecific or trivalent molecule binds HIV-1 envelope with the specificity of the V3 glycan binding antibody DH542, a variant of DH542 called

DH542_QSA, DH542_L4 and/or other antibodies from the DH542 lineage, and/or the binding specificity of CD4 binding site antibody CH557 (also referred to as CH235.12) or any of the antibodies from the CH235 HIV-1 antibody lineage and also binds CD3. In certain embodiments, the bispecific or trivalent molecule binds HIV-1 envelope with the specificity of the V3 glycan binding antibody DH542, a variant of DH542 called DH542_QSA,

DH542 L4 and/or other antibodies from the DH542 lineage, and/or the binding specificity of CD4 binding site antibody CH557 (also referred to as CH235.12) or any of the antibodies of the CH235 HIV-1 antibody lineage and also binds CD3, CD8, or CD16. [0062] In certain aspects, domain (A) comprises the CDR1, CDR2, and CDR3 of the light chain variable domain of immunoglobulin CH557, CH556, CH555, CH493, CH492, CH491, or CH490. In certain aspects, the domain (E) comprises the CDR1, CDR2, and CDR3 of the heavy chain variable domain of immunoglobulin CH557, CH556, CH555, CH493, CH492, CH491, or CH490. In certain aspects, domain (A) comprises the light chain variable domain of immunoglobulin CH557, CH556, CH555, CH493, CH492, CH491, or CH490. In certain aspects, domain (E) comprises the heavy chain variable domain of immunoglobulin CH557, CH556, CH555, CH493, CH492, CH491, or CH490. [0063] In certain aspects, domain (A) comprises the CDR1, CDR2, and CDR3 of the light chain variable domain of immunoglobulin DH542, DH542_QSA, DH542_L4, DH429. In certain aspects, the domain (E) comprises the CDR1, CDR2, and CDR3 of the heavy chain variable domain of immunoglobulin DH542, DH542_QSA, DH542_L4, DH429. In certain aspects, domain (A) comprises the light chain variable domain of immunoglobulin DH542, DH542_QSA, DH542_L4, DH429. In certain aspects, domain (E) comprises the heavy chain variable domain of immunoglobulin DH542, DH542_QSA, DH542_L4, DH429. [0064] In certain aspects, the first polypeptide comprises SEQ ID NO: 555. In certain aspects, the second polypeptide comprises SEQ ID NO: 557. In certain aspects, the third polypeptide comprises SEQ ID NO: 559. [0065] In certain aspects, the bispecific molecule comprises the first polypeptide of SEQ ID NO: 555, the second polypeptide of SEQ ID NO: 557, and the third polypeptide of SEQ ID NO: 559. [0066] In certain aspects, the bispecific molecule consists essentially of the first polypeptide of SEQ ID NO: 555, the second polypeptide of SEQ ID NO: 557, and the third polypeptide of SEQ ID NO: 559. In certain aspects, the bispecific molecule consists of the first polypeptide of SEQ ID NO: 555, the second polypeptide of SEQ ID NO: 557, and the third polypeptide of SEQ ID NO: 559. [0067] In certain aspects, a four chain trivalent binding molecule is a trispecific molecule and comprises the first polypeptide of SEQ ID NO: 555, the second polypeptide of SEQ ID NO: 557, the third polypeptide of SEQ ID NO: 561, the fourth polypeptide of SEQ ID NO: 562 (See Figure 35A). [0068] In certain aspects, a three chain trivalent binding molecule is a trispecific molecule and comprises the first polypeptide of SEQ ID NO: 555, the second polypeptide of SEQ ID NO: 557, the third polypeptide of SEQ ID NO: 563 (See Figure 35D). [0069] In certain aspects, the first polypeptide comprises SEQ ID NO: 596. In certain embodiments, the second polypeptide comprises SEQ ID NO: 597. In certain embodiments, the third polypeptide comprises SEQ ID NO: 559. An exemplary DH542 bispecific Fc bearing diabody is shown in Figure 34A. [0070] In certain aspects, the invention provides a composition comprising any one of the multispecific molecules or any combination thereof. In certain aspects, the composition comprises a composition comprising a bispecific molecule comprising a first arm with the binding specificity of a HIV-1 envelope CD4 binding site antibody or HIV-1 V3 glycan binding site antibody and a second arm targeting CD3, CD8, or CD16. In certain aspects, the bispecific molecule comprises an Fc portion or any other modification which extends its serum half-life. In certain aspects, the composition further comprises a second bispecific molecule or trivalent binding molecule comprising a first arm with an HIV-1 envelope binding specificity different from the HIV-1 binding specificity of the first multispecific molecule, and a second arm targeting CD3, CD8, or CD16, wherein the first and second multipecific molecules are different in either the HIV-1 binding specificity and/or the specificity of the second arm. [0071] In certain aspects, the invention provides a method to treat or prevent HIV-1 infection in a subject in need thereof comprising administering to the subject a composition comprising any one of the multispecific molecules of the invention or a combination of any one of the multispecific molecules in a therapeutically effective amount. In certain embodiments, the methods further comprise administering a latency activating agent. In some embodiments, the latency activating agent is vorinostat, romidepsin , panobinostat, disulfiram, JQ1, bryostatin, PMA, inonomycin, or any combination thereof. [0072] In certain aspects, the invention provides nucleic acids comprising nucleotides encoding the multispecific molecules of the invention. In certain aspects, the invention provides a vector comprising nucleic acids comprising nucleotides encoding the multispecific molecules of the invention. Provided are also compositions comprising a vector comprising a nucleic acid encoding the multispecific molecules. In certain aspects the invention provide a cell line comprising vectors or nucleic acids encoding the multispecific molecules of the invention, wherein the vectors encode polypeptide chains for expression of the multispecific molecules of the invention, e.g. but not limited to, polypeptide chain 1 and polypeptide chain 2, or polypeptide chain 1, polypeptide chain 2 and polypeptide chain 3. In certain

embodiments, the vector is suitable for gene delivery and expression. In certain embodiment, the vector is an adenoviral vector, an adeno associated virus based vector, or a combination thereof. [0073] In certain embodiments, the multispecific molecule binds to the HIV-1 envelope like the HIV-1 antibody from which it is derived. In certain embodiments, the multispecific molecule binds to the CH557-HIV-1 envelope epitope, i.e. the multispecific molecule binds to the HIV-1 envelope like the CH557 antibody, and also binds CD3, CD8, or CD16. [0074] In certain embodiments, the multispecific molecule binds to the DH542-HIV-1 envelope epitope, i.e. the multispecific molecule binds to the HIV-1 envelope like the DH542 antibody, and also binds CD3, CD8, or CD16. [0075] In certain embodiments a multispecific molecule of the invention comprises, consists essentially of or consists of sequences as described herein, (e.g., Table 4). [0076] In certain aspects the invention provides compositions comprising any of the multispecific molecules described herein, or a combination thereof. In certain embodiments, these compositions are formulated as pharmaceutical composition for therapeutic use. [0077] In certain aspects the invention is directed to nucleic acids which encode the multispecific molecule of the invention. In certain embodiments, these nucleic acids are comprised in a vector, and are operably linked to a promoter. In certain aspects the invention provides cell lines, or isolated cells, which comprise nucleic acids for the expression of the multispecific molecule of the invention. [0078] In certain aspects, the invention provides compositions comprising the multispecific molecule of the invention or nucleic acids encoding the same for use in methods of treating or preventing HIV-1 infection. In some embodiments, these methods further comprise administering a Latency Activating Reagent. Non-limiting examples of these include HDAC inhibitors, e,g, vorinostat, romidepsin, panobinostat, disulfiram, JQ1, bryostatin, PMA, inonomycin, or any combination thereof. In some embodiments, this combination therapy targets the pool of latently infected HIV-1 cells. [0079] In certain aspects, the invention provides methods of treating or preventing an HIV-1 infection in a subject, the method comprising administering to the subject a composition comprising any one of the multispecific molecules the invention, or a combination thereof in a therapeutically sufficient amount. In certain embodiments, the methods further comprise administering a latency activating agent. BRIEF DESCRIPTIONOF THE DRAWINGS

[0080] To conform to the requirements for PCT patent applications, many of the figures presented herein are black and white representations of images originally created in color.

[0081] Figure 1A shows index sorting of B cell that produced DH542 antibody, and Figure 1B shows the V3 peptide used as a hook to sort B cells from individual CH848 (SEQ ID NOS 286 and 286, respectively, in order of appearance).

[0082] Figure 2A shows the gene information of DH542 and Figure 2B shows DH542 sequences (CDRs are bolded and underlined) (SEQ ID NOs: 1-4). Figure 2A discloses SEQ ID NO: 287.

[0083] Figures 3A and 3B show that in an ELISA assay DH542 binding to HIV-Env depends on V3 loop glycans.

[0084] Figures 4A-4D show DH542 neutralization data. Figure 4A shows that DH542 neutralizes 71% of HIV-1 pseudoviruses tested in the TZM-bl assay. Figure 4B shows neutralization of a panel of 24 viruses. Figures 4C and 4D show summary of neutralization data from TZM-bl assay.

[0085] Figure 5 shows that DH542 binds high-mannose glycans. The data represent antibody binding measured with a custom oligomanose glycan array—average from three separate glycan arrays. The glycan array is printed by Zbiotech (Aurora, CO) on polymer coated glass slides. Each glycan is printed in triplicate at three different concentrations. The antibody DH542 was diluted in PBS supplemented with 1% BSA to a final concentration of 50 ug/mL, and incubated on the glycan array for 1 h at room temperature. Unbound antibody is washed away with 5 washes with PBS-T. The binding of DH542 is detected with an anti-IgG Fc antibody conjugated to Cy3. The Cy3 intensity for each glycan is read with a GenePix 4000B array scanner and the means and standard error of the three replicates are shown in the graph. The data show that DH542 is a direct glycan binding HIV-1 antibody. It binds most strongly to Man9GlcNAc2 which is similar to known bnAbs such as PGT128 and 2G12. DH542 bound well to Man8GlcNAc2 and Man7GlcNAc2, but at a lower level than Man9GlcNAc2. Binding was also detected for two other lower forms of oligomannose, but at decreased magnitudes. DH542 did not exhibit detectable binding to the GlcNAc2 alone, meaning that it requires mannose for glycan recognition, and more specifically, binds the highest forms of oligomannose (Man9GlcNAc2) with the greatest magnitude. Overall, DH542 is a glycan- reactive antibody that binds directly to the predominant glycan, high mannose, present on HIV-1 Envelope. The figure also shows the binding by the non-glycan reactive HIV antibody 19B. It shows no binding to 100 uM glycan printed on the array. The figure also shows that the prototypic glycan-dependent HIV-1 antibody PGT128 binds well to Man7GlcNAc2, Man8, and Man9. PGT128 is reported to bind best to Man8GlcNAc2and Man9GlcNAc2, which was confirmed here as well. DH542 is shown binding to 100 uM of high mannose glycans. DH542 also binds to Man7GlcNAc2D3, which is not bound by PGT128.

[0086] Figure 6 is summarized data showing that DH542 is not autoreactive by Athena ANA panel. Results are expressed as relative luminescence units. Readings <100 are considered negative, results between 100 and 120 are considered“indeterminate” and results >120 are considered positive.

[0087] Figure 7 shows that DH542 binds HEp2 cells as demonstrated by intracellular fluorescence staining—left panel shows DH542/293i 50ug/mL 40x obj 8 sec (2+), right panel shows DH542/293i 25ug/mL 40x obj 8 sec (1+).

[0088] Figures 8-11 show the amino acids sequences of VH and VL chains of antibodies of the DH270 lineage, and nucleic acid sequences encoding these amino acids. CDRs are highlighted in each antibody. Figure 8 shows SEQ ID NOs: 5-16 (Heavy chain nucleotide sequences in order of appearance from UCA-DH270H). Figure 9 shows SEQ ID NOs: 17-28 (Heavy chain amino acid sequences in order of appearance from UCA-DH270H). Figure 10 shows SEQ ID NOs: 29-40 (Light chain nucleotide sequences in order of appearance from UCA-DH270H). Figure 11 shows SEQ ID NOs: 41-52 (Light chain amino acid sequences in order of appearance from UCA-DH270H).

[0089] Figure 12 shows a clonal tree that was estimated using both the heavy and light chains of the listed lineage members.

[0090] Figure 13 shows summary results of neutralization data of DH542_L4, DH542, PGT128, PGT 121, 10-1074, DH270 and DH471 against a panel of HIV-1 isolates in the Luc/TZM-bl neutralization assay. Values represent IC50 in µg/ml.

[0091] Figure 14 shows the mean IC50 and percent of isolates neutralized at different IC50 values. Median and Geometric Mean titers are calculated only for samples with IC50 <50ug/ml. Values less than the lowest concentration assayed were assigned a value 2-fold less for calculation purposes. Indicated in italics.

[0092] Figure 15A shows summary results of neutralization data of DH542_L4, DH542, PGT128, PGT 121, 10-1074, DH270 and DH471 against a panel of HIV-1 isolates in the Luc/TZM-bl neutralization assay. Values represent IC80 in µg/ml. Figure 15B shows the mean IC80 and percent of isolates neutralized at different IC80 <50ug/ml values. Median and Geometric Mean titers are calculated only for samples with IC80 <50ug/ml. Values less than the lowest concentration assayed were assigned a value 2-fold less for calculation purposes. Indicated in italics.

[0093] Figure 16 shows DH542_QSA sequences (SEQ ID NO:113-116). DH542_QSA is a variant of DH542. The heavy chain is identical to that of DH542. The light chain has some variation in the N-terminus.

[0094] Figure 17A shows an alignment of amino acid and Figure 17B shows an alignment of nucleic acid sequences of VH and VL chains for antibodies from the DH270 lineage. CDRs are highlighted and underlined in the UCA. The Figure 17A shows SEQ ID NOs: 117-126 (Heavy chain nucleotide sequences in order of appearance from UCA-DH270), SEQ ID NOs: 127-136 (Heavy chain amino acid sequences in order of appearance from UCA-DH270). Figure 17B shows SEQ ID NOs: 137-146 (Light chain nucleotide sequences in order of appearance from UCA-DH270), SEQ ID NOs: 147-156 (Light chain amino acid sequences in order of appearance from UCA-DH270H)

[0095] Figure 18 shows neutralization by antibodies DH272, DH272_UCA, DH391 and DH542 identified from subject CH848 and DH563 identified from subject CH0765 measured in TZM-bl cells. Pseudoviruses were produced by transfection in 293T cells. Values are the antibody concentration (µg/ml) at which relative luminescence units (RLUs) were reduced 50% compared to virus control wells (no test sample). Values in bold are considered positive for neutralizing antibody activity. DH542 IC50 neutralization summary: mean IC50 = 0.21 µg/ml; geometric mean = 0.08 µg/ml; median IC50 = 0.06 µg/ml.

[0096] Figures 19A-1 and 19A-2 shows a phylogenetic tree of VH sequences from CH0848 donor. The tree includes VH chains from natural VH:VL pairs (identified by single cell sorts) and VH chains identified by Illumina sequencing. Figure 19B shows a detailed view of the bottom portion of the tree in 19A-2. Figure 19C shows the IDs of the VH chains.

[0097] Figure 20A shows sequences of VH chains identified from CH0848 donor by Illumina deep sequencing (SEQ ID NOs: 157-166). No natural VL sequence pairing was identified for these VH sequences. Figure 20B shows an alignment of the sequences in Figure 20A (SEQ ID NOS 162-166, respectively, in order of appearance).

[0098] Figures 21-22 show sequences of CH557 (SEQ ID NOs: 167-170). CDRs are bolded and underlined. Figure 21 shows the amino acid sequences. Figure 22 shows the nucleic acid sequences.

[0099] Figure 23 shows amino acid alignment of CH235 lineage antibody heavy chain and light chain (SEQ ID NOs: 179-190, in order of appearance from UCA HC to CH557_HC). Antibodies are listed in ascending order of somatic mutations and compared to the inferred unmutated common ancestor previously published (Gao, Bonsignori, Liao et al. Cell 2014)

[0100] Figure 24 shows amino acid alignment of CH235 lineage antibody light chain (SEQ ID NOs: 191-200, in order of appearance from UCA LC to CH556_LC). Antibodies are listed in ascending order of somatic mutations and compared to the inferred unmutated common ancestor previously published (Gao, Bonsignori, Liao et al. Cell 2014)

[0101] Figure 25 shows the names and VH:VLcomposition of chimeric antibodies of Example 10. Sequences for these antibodies are shown in Figures 8-11, 16 and 17.

[0102] Figures 26A and 26B show a summary of neutralization data for the chimeric antibodies of Example 10. Viruses used in this TZMBl assay are described in the side line of the table in Figures 26A and 26B. Antibodies are shown in the top line of the table.

[0103] Figure 27 shows a summary of neutralization data for the chimeric antibodies of Example 10. Dotted line shows median IC50 of all viruses including those not neutralized; <0.023 graphed as 0.01; >50 graphed as 50.

[0104] Figure 28A shows Phylogram of DH270 lineage. Sequences shown were isolated by NGS in longitudinal analysis that overlapped in two separate runs. The phylogeny was computed by collapsing on a radius of 8 base pairs and the 90 sequences shown are representative of a total of 1500 sequences. VH sequences of isolated DH270 lineage antibodies were added and the lineage was inferred using Cloanalyst. Figure 28B shows Heatmap analysis of neutralization of 24 pseudoviruses (row) by 11 DH270 lineage mAbs. Figure 28C shows Neutralization dendrograms display of DH270.6 (right) neutralization of a genetically diverse panel of 200 HIV-1 isolates. Coloration is by IC50 (red: <1 ug/ml; green: 1-50 ug/ml; black: >50 ug/ml).

[0105] Figure 29 shows V_H and VL mutation frequencies of the isolated antibodies.

[0106] Figure 30 shows Neutralization of wild-type and N332 mutated HIV-1 strains AC13.8, PVO4, TRO.11, AC10.0.29 and RHPA confirmed DH270 lineage N332 sensitivity of neutralization.

[0107] Figure 31 shows the neutralization profile of DH270.6 most closely paralleled that of 10-1074.

[0108] Figure 32 provides a schematic of a representative covalently bonded diabody having two epitope-binding sites composed of two polypeptide chains, each having an E-coil or K- coil Heterodimer-Promoting Domain (alternative Heterodimer-Promoting Domains are provided below). A cysteine residue may be present in a linker and/or in the Heterodimer- Promoting Domain as shown in Figure 33B. VL and VH Domains that recognize the same epitope are shown using the same shading or fill pattern. [0109] Figures 33A-33C provide schematics showing representative covalently bonded tetravalent diabodies having four epitope-binding sites composed of two heterodimer pairs of polypeptide chains (i.e., four polypeptide chains in all). One polypeptide of each heterodimer pair possesses a CH2 and CH3 Domain, such that the associated chains form all or part of an Fc Domain. VL and VH Domains that recognize the same epitope are shown using the same shading or fill pattern. The two pairs of polypeptide chains may be the same. In such embodiments wherein the two pairs of polypeptide chains are the same and the VL and VH Domains recognize different epitopes (as shown in Figures 33A-33B), the resulting molecule possesses four epitope-binding sites and is bispecific and bivalent with respect to each bound epitope. In such embodiments wherein the VL and VH Domains recognize the same epitope (e.g., the same VL Domain CDRs and the same VH Domain CDRs are used on all chains) the resulting molecule possesses four epitope-binding sites and is monospecific and tetravalent with respect to a single epitope. Alternatively, the two pairs of polypeptides may be different. In such embodiments wherein the two pairs of polypeptide chains are different and the VL and VH Domains of each pair of polypeptides recognize different epitopes (as shown by the different shading and patterns in Figure 33C), the resulting molecule possesses four epitope- binding sites and is tetraspecific and monovalent with respect to each bound epitope. Figure 33A shows an Fc Domain-containing diabody which contains a peptide Heterodimer- Promoting Domain comprising a cysteine residue. Figure 33B shows an Fc Domain- containing diabody, which contains E-coil and K-coil Heterodimer-Promoting Domains comprising a cysteine residue and a linker (with an optional cysteine residue). Figure 33C, shows an Fc-Region-Containing diabody, which contains antibody CH1 and CL domains which could serve as Heterodimer Promoting Domains. [0110] Figures 34A and 34B provide schematics of a representative covalently bonded Fc bearing diabody molecule having two epitope-binding sites composed of three polypeptide chains. Two of the polypeptide chains comprise a CH2 and CH3 Domain, such that the associated chains form all or part of an Fc Domain. The polypeptide chains comprising the VL and VH Domain further comprise a Heterodimer-Promoting Domain. VL and VH Domains that recognize the same epitope are shown using the same shading or fill pattern. [0111] Figures 35A-35F provide schematics of representative Fc Domain-containing trivalent binding molecules having three epitope-binding sites. Figures 35A and 35B, respectively, illustrate schematically the domains of trivalent binding molecules comprising two diabody-type binding domains and a Fab-type binding domain having different domain orientations in which the diabody-type binding domains are N-terminal or C-terminal to an Fc Domain. The molecules in Figures 35A and 35B comprise four chains. Figures 35C and 35D, respectively, illustrate schematically the domains of trivalent binding molecules comprising two diabody-type binding domains N-terminal to an Fc Domain, and a Fab-type binding domain in which the light chain and heavy chain are linked via a polypeptide spacer, or an scFv-type binding domain. The trivalent binding molecules in Figures 35E and 35F, respectively, illustrate schematically the domains of trivalent binding molecules comprising two diabody-type binding domains C-terminal to an Fc Domain, and a Fab-type binding domain in which the light chain and heavy chain are linked via a polypeptide spacer, or an scFv-type binding domain. The trivalent binding molecules in Figures 35C-35F comprise three chains. VL and VH Domains that recognize the same epitope are shown using the same shading or fill pattern. [0112] Figures 36A-36E provide schematics of a representative HIVxCD3 bispecific monovalent diabody comprising three polypeptide chains. Figure 36A shows the domains of each of the three polypeptide chains, dashed lines represent disulfide bonds which form between the chains, and the arrows indicate the interactions of the Variable Domains. Figure 5B provides a schematic of the assembled chains. Such diabodies contain an anti-HIV-1 binding arm (e.g., DH491 or CH493, DH542, CH557, CH558, or any of the CH235 lineage antibodies) combined with an anti-CD3 binding arm (e.g., hXR32). They are composed of two polypeptide chains: one with the VL of an anti-CD3 antibody linked to the VH of an anti- HIV-1 antibody; the second with the VL of an anti-HIV-1 antibody linked to the VH of an anti-CD3 antibody. The first and the second polypeptide chains are linked by interchain disulfide bond and paired via oppositely charged E-coil/K-coil Heterodimer-Promoting Domains. The amino acid and nucleotide sequences of Chain 1, 2 and 3 which form the bispecific monovalent diabody designated CH557xCD3 Fc are provided in Table 4 (SEQ ID NOs: 555, 556, 557, 558, 559, 560). Control molecules have one of the arms replaced by a non-HIV-1 envelope binding arm derived, for example, from an anti-FITC antibody (4420) or from an anti-RSV antibody (palivizumab). DETAILED^DESCRIPTION^

[0113] Highly active anti-retroviral therapy (HAART) alone or in combination with latency reversing agents fails to reduce the pool of latently infected cells. This is due to limited ability of the CD8+ T cells to eliminate HIV-1 latently infected cells. Dual Affinity Re-Targeting proteins (DARTs) are multispecific, antibody-based diabody molecules that can bind at least two distinct antigens simultaneously. HIV-1 diabodies contain an HIV-1 binding arm combined with an effector cell binding arm (e.g., but not limited to CD3 effector cells) are designed to redirect effector cells (e.g. but not limited to cytotoxic CD3+ T cells) to engage and kill HIV-1-infected cells. Additionally, multispecific molecules such as trivalent binding molecules containing one or more HIV-1 binding arms combined with one or more effector cell binding arms, may also be designed to redirected effector cells to engage and kill HIV-1- infected cells.

[0114] The provision of multispecific/non-mono-specific molecules provides a significant advantage over typical mono-specific antibodies: the capacity to co-ligate and co-localize cells that express different epitopes. Bivalent diabodies have wide-ranging applications including therapy and immunodiagnosis. Bi-valency allows for great flexibility in the design and engineering of the diabody in various applications, providing enhanced avidity to multimeric antigens, the cross-linking of differing antigens, and directed targeting to specific cell types relying on the presence of both target antigens. Due to their increased valency, low dissociation rates and rapid clearance from the circulation (for diabodies of small size, at or below ~50 kDa), diabody molecules known in the art have also shown particular use in the field of tumor imaging (Fitzgerald et al. (1997)“Improved Tumour Targeting By Disulphide Stabilized Diabodies Expressed In Pichia pastoris,” Protein Eng.10:1221). Of particular importance is the co-ligating of differing cells, for example, the cross-linking of cytotoxic T cells to tumor cells (Staerz et al. (1985)“Hybrid Antibodies Can Target Sites For Attack By T Cells,” Nature 314:628-631, and Holliger et al. (1996)“Specific Killing Of Lymphoma Cells By Cytotoxic T-Cells Mediated By A Bispecific Diabody,” Protein Eng.9:299-305).

[0115] Diabody epitope binding domains may also be directed to a surface determinant of a B cell, such as CD19, CD20, CD22, CD30, CD37, CD40, and CD74 (Moore, P.A. et al. (2011) “Application Of Dual Affinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing Of B-Cell Lymphoma,” Blood 117(17):4542-4551; Cheson, B.D. et al. (2008) “Monoclonal Antibody Therapy For B-Cell Non-Hodgkin’s Lymphoma,” N. Engl. J. Med. 359(6):613-626; Castillo, J. et al. (2008)“Newer monoclonal antibodies for hematological malignancies,” Exp. Hematol.36(7):755-768. In many studies, diabody binding to effector cell determinants, e.g., Fcγ receptors (FcγR), was also found to activate the effector cell (Holliger et al. (1996)“Specific Killing Of Lymphoma Cells By Cytotoxic T-Cells Mediated By A Bispecific Diabody,” Protein Eng.9:299-305; Holliger et al. (1999)“Carcinoembryonic Antigen (CEA)-Specific T-Cell Activation In Colon Carcinoma Induced By Anti-CD3 x Anti- CEA Bispecific Diabodies And B7 x Anti-CEA Bispecific Fusion Proteins,” Cancer Res. 59:2909-2916; WO 2006/113665; WO 2008/157379; WO 2010/080538; WO 2012/018687; WO 2012/162068). Normally, effector cell activation is triggered by the binding of an antigen bound antibody to an effector cell via Fc-FcγR interaction; thus, in this regard, diabody molecules may exhibit Ig-like functionality independent of whether they comprise an Fc Domain (e.g., as assayed in any effector function assay known in the art or exemplified herein (e.g., ADCC assay)). By cross-linking tumor and effector cells, the diabody not only brings the effector cell within the proximity of the tumor cells but leads to effective tumor killing (see e.g., Cao et al. (2003)“Bispecific Antibody Conjugates In Therapeutics,” Adv. Drug. Deliv. Rev.55:171-197). [0116] The formation of such non-mono-specific diabodies requires the successful assembly of two or more distinct and different polypeptides (i.e., such formation requires that the diabodies be formed through the heterodimerization of different polypeptide chain species). This fact is in contrast to mono-specific diabodies, which are formed through the

homodimerization of identical polypeptide chains. Because at least two dissimilar polypeptides (i.e., two polypeptide species) must be provided in order to form a non-mono- specific diabody, and because homodimerization of such polypeptides leads to inactive molecules (Takemura, S. et al. (2000)“Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System,” Protein Eng.13(8):583-588), the production of such polypeptides must be accomplished in such a way as to prevent covalent bonding between polypeptides of the same species (i.e., so as to prevent homodimerization)

(Takemura, S. et al. (2000)“Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System,” Protein Eng.13(8):583-588). The art has therefore taught the non-covalent association of such polypeptides (see, e.g., Olafsen et al. (2004) “Covalent Disulfide-Linked Anti-CEA Diabody Allows Site-Specific Conjugation And Radiolabeling For Tumor Targeting Applications,” Prot. Engr. Des. Sel.17:21-27; Asano et al. (2004)“A Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion Of Human Fc Domain,” Abstract 3P-683, J. Biochem.76(8):992; Takemura, S. et al. (2000)“Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System,” Protein Eng.13(8):583-588; Lu, D. et al. (2005)“A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity,” J. Biol. Chem.280(20):19665-19672).

[0117] The art has recognized that bispecific diabodies composed of non-covalently associated polypeptides are unstable and readily dissociate into non-functional monomers (see, e.g., Lu, D. et al. (2005)“A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity,” J. Biol. Chem.280(20):19665-19672).

[0118] In the face of this challenge, the invention provides stable, covalently bonded heterodimeric multispecific diabodies, termed DARTs™ (see, e.g., United States Patent Publications No.2014-0099318; 2013-0295121; 2010-0174053 and 2009-0060910; European Patent Publication No. EP 2714079; EP 2601216; EP 2376109; EP 2158221 and PCT Publications No. WO 2015/026894; WO2015/026892; WO 2015/021089; WO 2014/159940; WO 2012/162068; WO 2012/018687; WO 2010/080538; Moore, P.A. et al. (2011) “Application Of Dual Affinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing Of B-Cell Lymphoma,” Blood 117(17):4542-4551; Veri, M.C. et al. (2010) “Therapeutic Control Of B Cell Activation Via Recruitment Of Fcgamma Receptor IIb (CD32B) Inhibitory Function With A Novel Bispecific Antibody Scaffold,” Arthritis Rheum. 62(7):1933-1943; Johnson, S. et al. (2010)“Effector Cell Recruitment With Novel Fv-Based Dual-Affinity Re-Targeting Protein Leads To Potent Tumor Cytolysis And in vivo B-Cell Depletion,” J. Mol. Biol.399(3):436-449), the contents of which publications are herein incorporated by reference in their entirety). Such multispecific molecules comprise two or more covalently complexed polypeptides and involve engineering one or more cysteine residues into each of the employed polypeptide species. For example, the addition of a cysteine residue to the C-terminus of such constructs has been shown to allow disulfide bonding between the polypeptide chains, stabilizing the resulting heterodimer without interfering with the binding characteristics of the bivalent molecule.

[0119] The invention provides multispecific, antibody-based molecules that can bind at least two distinct antigens simultaneously, wherein at least one of the antigens is comprised in an HIV-1 envelope. In certain aspects, the present invention is directed to HIV-1 multispecific molecules that are capable of simultaneous binding to an epitope of HIV-1 envelope and an epitope of an antigen on a number of effector cells, e.g. but not limited to an effector cell which expresses CD2, CD3, CD8, CD16, T-Cell Receptor (TCR), NKG2D, etc. epitope) and to the uses of such molecules in the treatment of HIV-1 infection.

[0120] In certain embodiments the invention provides molecules with dual targeting specificity (including but not limited to bispecific antibodies, bispecific diabodies, and trivalent binding molecules). In certain aspects the invention provides bispecific molecules that are capable of localizing an immune effector cell to an HIV-1 envelope expressing cell, so as facilitate the killing of the HIV-1 envelope expressing cell. In this regard, bispecific molecules bind with one "arm" to an epitope of a surface antigen on target cells, and with the second "arm" to an epitope of an activating, invariant component of the T cell receptor (TCR) complex. The simultaneous binding of such a bispecific molecule to both of its targets will force a temporary interaction between target cell and T cell, causing activation of any cytotoxic T cell and subsequent lysis of the target cell. Hence, the immune response is re- directed to the target cells and is independent of peptide antigen presentation by the target cell or the specificity of the T cell as would be relevant for normal MHC-restricted activation of CTLs. In this context it is crucial that CTLs are only activated when a target cell is presenting the bispecific molecule to them, i.e. the immunological synapse is mimicked. Particularly desirable are bispecific molecules that do not require lymphocyte preconditioning or co- stimulation in order to elicit efficient lysis of target cells. In certain embodiments, such molecule may further comprise a third binding“arm” and be trivalent. In some embodiments, the third arm binds to an epitope of a surface antigen on target cells, which may be the same epitope or a different epitope as bound by the first arm. In some embodiments, the third arm binds to an epitope of an activating, invariant component of the TCR complex, which may be the same epitope or a different epitope as bound by the second arm. In alternative

embodiments, the third arm binds to a different epitope to that bound by the first arm or second arm, such as an epitope of a surface antigen on target cells or an epitope expressed on the surface of an effector cell (e.g., but not limited to an epitope of CD2, CD3, CD8, CD16, T-Cell Receptor (TCR), NKG2D, etc.).

[0121] In certain embodiments, such bispecific molecules comprise one portion which targets HIV-1 envelope and a second portion which binds a second target. In certain embodiments, the first portion comprises VH and VL sequences, or CDRs from the antibodies described herein. In certain embodiments, the second target could be, for example but not limited to an effector cell. In certain embodiments the second portion is a T-cell engager. In certain embodiments, the second portion comprises a sequence/paratope which targets CD3. In certain embodiments, the second portion is an antigen-binding region derived from a CD3 antibody, optionally a known CD3 antibody. In certain embodiments, the anti-CD antibody induces T cell-mediated killing. In certain embodiments, the bispecific molecules comprise whole antibodies. In other embodiments, the dual targeting bispecific molecules consist essentially of Fab fragments. In other embodiments, the dual targeting bispecific molecules comprise a heavy chain constant region (CH1). In certain embodiments, the bispecific molecule does not comprise Fc Domain. In certain embodiments, the bispecific molecules have improved effector function. In certain embodiments, the bispecific molecules have improved cell killing activity. Various methods and platforms for design of bispecific molecules (including but not limited to bispecific antibodies, bispecific diabodies, etc.) are known in the art. See for example US Pub.20140206846, US Pub.20140170149,

20100174053, US Pub.20090060910, US Pub 20130295121, US Pub.20140099318, US Pub.20140088295 which contents are herein incorporated by reference in their entirety. [0122] A bispecific or bifunctional molecule is an artificial hybrid antibody that can comprise two different heavy/light chain pairs and two different binding sites (see, e.g., Romain Rouet & Daniel Christ“Bispecific antibodies with native chain structure” Nature Biotechnology 32, 136–137 (2014); Byrne et al.“A tale of two specificities: bispecific antibodies for therapeutic and diagnostic applications” Trends in Biotechnology, Volume 31, Issue 11, November 2013, Pages 621–632 Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990);

Kostelny et al., J. Immunol.148:1547-53 (1992) (and references therein)). In certain embodiments the bispecific molecule is a whole antibody of any isotype. In other

embodiments the bispecific molecule is a bispecific fragment, for example but not limited to F(ab)₂ fragment. In some embodiments, the bispecific molecules do not include Fc portion, which makes these bispecific molecules relatively small in size and easy to penetrate tissues.

[0123] In some embodiments, the invention encompasses polypeptide chains, each of which polypeptide chains comprise a VH and VL domain, comprising CDRs as described herein. In certain embodiments, the VL and VH domains comprising each polypeptide chain have the same specificity, and the multimer molecule is bivalent and monospecific. In other embodiments, the VL and VH domains comprising each polypeptide chain have differing specificity and the multimer is bivalent and bispecific. In still other embodiments, the VH and VL domains of each polypeptide chain have differing specificity and the multimer is trivalent and bispecific or trivalent and trispecific.

[0124] The multispecific molecules of the invention can simultaneously bind two separate and distinct epitopes. In certain embodiments the epitopes are from the same antigen. In other embodiments, the epitopes are from different antigens. In non-limiting embodiments at least one epitope binding site is specific for a determinant expressed on an immune effector cell (e.g. CD3, CD16, CD32, CD64, etc.) which are expressed on T lymphocytes, natural killer (NK) cells or other mononuclear cells. In one embodiment, the multispecific molecule binds to the effector cell determinant and also activates the effector cell. In this regard, multispecific molecules of the invention (e.g., bispecific antibodies, bispecific diabodies, trivalent binding molecules, etc.) may exhibit Ig-like functionality independent of whether they further comprise an Fc domain (e.g., as assayed in any effector function assay known in the art or exemplified herein).

[0125] In certain embodiments, the multispecific molecule comprises an HIV-1 envelope binding fragment, for example but not limited to an HIV-1 envelope binding fragment from any of the antibodies described herein. In other embodiments, the multispecific molecule further comprises a second antigen-interaction-site/fragment. In other embodiments, the multispecific molecule further comprises at least one effector cell targeting arm.

[0126] In certain embodiments the multispecific molecules engage cells for Antibody- Dependent Cell-mediated Cytotoxicity (ADCC). In certain embodiments the multispecific molecules engage natural killer cells, neutrophil polymorphonuclear leukocytes, monocytes and macrophages. In certain embodiments the multispecific molecules are T-cell engagers. In certain embodiments, the bispecific molecule comprises an HIV-1 envelope binding fragment and CD3 binding fragment. Various CD3 antibodies are provided herein (see, e.g., Table 4) and others are known in the art. See for example US Patent 8,784,821, and United States Patent Publications No.2014-0099318 providing various disclosure on various CD3 antibodies, which disclosure is incorporated by reference in its entirety. In certain

embodiments, the bispecific molecule comprises an HIV-1 envelope binding fragment and CD16 binding fragment. Various CD16 antibodies are provided herein (see e.g., Table 4) and others are known in the art. See for example WO 03/101485, which disclosure is

incorporated by reference in its entirety.

[0127] In certain embodiments, the invention provides molecules or fragments comprising a CDR(s) of the VH and/or VL chains, or VH and/or VL chains of any suitable HIV-1 antibody, as the HIV-1 binding arm(s) of multispecific molecules, e.g., but not limited to bispecific antibodies, bispecific diabodies, trivalent binding molecules, etc., or toxin labeled HIV-1 binding molecules. Exemplary HIV-1 antibodies are provided in Table 4.

[0128] The invention also includes variants of the antibodies (and fragments) disclosed herein, including variants that retain the ability to bind to recombinant Env protein, the ability to bind to the surface of virus-infected cells and/or ADCC-mediating properties of the antibodies specifically disclosed, and methods of using same to, for example, reduce HIV-1 infection risk. Combinations of the antibodies, or fragments thereof, disclosed herein can also be used in the generation of the multispecific molecules of the invention.

[0129] Homologs and variants of a VL or a VH of an antibody that specifically binds a polypeptide are typically characterized by possession of at least about 75%, for example at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full length alignment with the amino acid sequence of interest.

Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.

[0130] In certain embodiments, the invention provides multispecific molecules comprising the VL and VH domains of antibodies which are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% identical to the VH and VL amino acid sequences of the antibodies described herein and still maintain their epitope binding breadth and/or potency. In certain embodiments, the invention provides multispecific molecules comprising the CDR 1, 2, and/or 3 of the VH and CDR1, 2, and/or 3 of the VL which are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% identical to the CDR1, 2, and/or 3 of VH and CDR1, 2, and/or 3 VL amino acid sequences of the antibodies described herein and still maintain their epitope binding breadth and/or potency.

[0131] In certain embodiments, the invention provides multispecific molecules comprising polypeptide chains which are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% identical to SEQ ID NOs: 555, 557, 559, 561, 562, 563, 596, or 597.

[0132] In certain embodiments, the invention provides multispecific molecules comprising polypeptide chains which are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% identical to SEQ ID NOs: 500-597.

[0133] In some aspects the invention provides recombinant, multispecific molecules, polyclonal or monoclonal antibodies, variants, fusion proteins comprising an antibody portion with an antigen recognition site of the required specificity, humanized antibodies, and chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity. Throughout this application, the numbering of amino acid residues of the light and heavy chains of antibodies is according to the EU index as in Kabat et al. (1992) SEQUENCES OF PROTEINS OF

IMMUNOLOGICAL INTEREST, National Institutes of Health Publication No.91-3242. Amino acids from the Variable Domains of the mature heavy and light chains of immunoglobulins are designated by the position of an amino acid in the chain. Kabat described numerous amino acid sequences for antibodies, identified an amino acid consensus sequence for each subgroup, and assigned a residue number to each amino acid. The Kabat numbering scheme is extendible to antibodies not included in his compendium by aligning the antibody in question with one of the consensus sequences in Kabat by reference to conserved amino acids. This method for assigning residue numbers has become standard in the field and readily identifies amino acids at equivalent positions in different antibodies, including chimeric or humanized variants.

[0134] In some embodiments, antigen-binding fragment of an antibody is a portion of an antibody that possesses an at least one antigen recognition site. Fragments include for example but not limited to, Fab, Fab', F(ab')₂ Fv), and single chain (scFv).

[0135] In certain embodiments the invention provides recombinant molecules. In certain embodiments, recombinant molecules encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')₂ Fv), single chain (scFv), mutants thereof, fusion proteins and multispecific molecules comprising an antibody portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity and the ability to bind to an antigen. Recombinant molecules are not limited as regards to the source of the molecule or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.).

[0136] Methods of making recombinant molecules are known in the art. In certain

embodiments, the molecules are produced recombinantly by any means known in the art. In one embodiment, the polynucleotide sequence encoding such a recombinant molecule is cloned into a vector for expression or propagation. The sequence encoding an antibody of interest may be maintained in a vector in a host cell and the host cell can then be expanded and frozen for future use. The polynucleotide sequence of such antibodies may be used for genetic manipulation to generate the multispecific molecules of the invention (e.g., bispecific antibodies, bispecific diabodies, trivalent binding molecules, etc.) as well as a chimeric antibody, a humanized antibody, or a caninized antibody, to improve the affinity, or other characteristics of the antibody. The general principle in humanizing an antibody involves retaining the basic sequence of the antigen-binding portion of the antibody, while swapping the non-human remainder of the antibody with human antibody sequences. There are four general steps to humanize a monoclonal antibody. These are: (1) determining the nucleotide and predicted amino acid sequence of the starting antibody light and heavy variable Domains (2) designing the humanized antibody or caninized antibody, i.e., deciding which antibody framework region to use during the humanizing or canonizing process (3) the actual humanizing or caninizing methodologies/techniques and (4) the transfection and expression of the humanized antibody. See, for example, U.S. Patents Nos.4,816,567; 5,807,715;

5,866,692; and 6,331,415.

[0137] The antibodies described herein, or fragments thereof, or molecules comprising such fragments, may be recombinantly produced in prokaryotic or eukaryotic expression systems. These systems are well described in the art. In general, protein therapeutics are produced from mammalian cells. The most widely used host mammalian cells are Chinese hamster ovary (CHO) cells and mouse myeloma cells, including NS0 and Sp2/0 cells. Two derivatives of the CHO cell line, CHO-K1 and CHO pro-3, gave rise to the two most commonly used cell lines in large scale production, DUKX-X11 and DG44. (See, e.g., Kim, J., et al.,“CHO cells in biotechnology for production of recombinant proteins: current state and further potential,” Appl. Microbiol. Biotechnol., 2012, 93:917-30, which is hereby incorporated-by-reference.) Other mammalian cell lines for recombinant protein expression include, but are not limited to, COS, HeLa, HEK293T, U2OS, A549, HT1080, CAD, P19, NIH 3T3, L929, N2a, HEK 293, MCF-7, Y79, SO-Rb50, HepG2, J558L, and BHK. If the aim is large-scale production, the most currently used cells for this application are CHO cells. Guidelines to cell engineering for mAbs production were also reported. (Costa et al., “Guidelines to cell engineering for monoclonal antibody production,” Eur J Pharm

Biopharm, 2010, 74:127–38, which is hereby incorporated-by-reference.) Using heterologous promoters, enhancers and amplifiable genetic markers, the yields of antibody and antibody fragments can be increased. Similar methods are utilized for the expression of the

multispecific molecules of the invention. Thus, in certain embodiments, the invention provides an antibody, or antibody fragment, or molecule comprising such fragment, that is recombinantly produced from a mammalian cell-line, including a CHO cell-line. In certain embodiments, the invention provides a composition comprising an antibody, or antibody fragment, or molecule comprising such fragment, wherein the antibody, or antibody fragment, or molecule comprising such fragment was recombinantly produced in a mammalian cell-line, and wherein the antibody, or antibody fragment, or molecule comprising such fragment is present in the composition at a concentration of at least 1, 10, 100, 1000 micrograms/mL, or at a concentration of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or 100 milligrams/mL.

[0138] Furthermore, large-scale production of therapeutic-grade molecules are much different than those for laboratory scale. There are extreme purity requirements for therapeutic- grade. Large-scale production of therapeutic-grade molecules requires multiples steps, including product recovery for cell-culture harvest (removal of cells and cell debris), one or more chromatography steps for antibody purification, and formulation (often by tangential filtration). Because mammalian cell culture and purification steps can introduce variants that are unique to the recombinant production process (i.e., protein aggregates, N- and C- terminal variants, acidic variants, basic variants, different glycosylation profiles), there are recognized approaches in the art for analyzing and controlling these variants. (See, Fahrner, et al., Industrial purification of pharmaceutical antibodies: Development, operation, and validation of chromatography processes, Biotech. Gen. Eng. Rev., 2001, 18:301-327, which is hereby incorporated-by-reference.) In certain embodiments of the invention, the composition comprises less than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 50, or 100 nanograms of host cell protein (i.e., proteins from the cell-line used to recombinantly produce the molecule). In other embodiments, the composition comprises less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 ng of protein A per milligram of molecule (i.e., protein A is a standard approach for purifying antibodies and other Fc bearing molecules from recombinant cell culture, but steps should be done to limit the amount of protein A in the composition, as it may be immunogenic). (See, e.g., U.S. Patent No.7,458,704, Reduced protein A leaching during protein A affinity chromatography; which is hereby incorporated-by-reference.)

[0139] Multispecific molecules

[0140] Various non-limiting multispecific molecule designs are provides in Figures 32-36.

[0141] As provided herein, the invention contemplates designs of multispecific molecules, which include, but are not limited to bispecific antibodies, bispecific diabodies, Fc Domain bearing diabodies, trivalent binding molecules, Fc Domain bearing trivalent bind molecules etc. The multispecific molecules provided herein comprise various domains, including, but not limited to peptide linkers, Heterodimer Promoting Domains, VL and VH domains, and Fc Domains. Specific non-limiting embodiments of exemplary multispecific molecules are provided herein. Alternative combinations of the various domains described herein can be employed in the multispecific molecules of the invention. [0142] As provided herein, the invention contemplates designs of multispecific molecules with various peptide linkers (also referred to herein as“intervening peptide linkers”) separating the domains comprised in the polypeptide chains. Any of a variety of peptide linkers can be used to separate the domains in the polypeptide chains of the multispecific molecules of the invention. Typically, such peptide linkers will comprise 1-20, 1-19, 1-18, 1- 17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues. Such polypeptide linkers can include a series of glycine residues (Gly) and/or Serine (Ser) residues and may optionally comprise cysteine residue(s). Specific non-limiting embodiments of exemplary polypeptide linkers (e.g., Peptide Linker 1, Peptide Linker 2, Spacer Linker 3, etc.) are provided herein. Alternative peptide linkers are well-known in the art and can be employed in the multispecific molecules of the invention. Other linkers can be readily determined. Some additional examples of linkers are disclosed in US Pub 20100174053, incorporated by reference in its entirety.

[0143] Typically, the VH and VL domains of a polypeptide chain of the multispecific molecules are linked so that they do not associate with each other. In certain embodiments the length of the peptide linker, which separates such VL and VH domains of a polypeptide chain is selected to substantially or completely prevent such VL and VH domains from binding to one another. Thus the VL and VH domains of a polypeptide chain are

substantially or completely incapable of binding to one another. In certain embodiments this is due to the peptide linker which separates the VH and VL domains. As provided herein, the invention also contemplates designs of multispecific molecules wherein the domains comprising the polypeptide chains (e.g., Heterodimer Promoting Domains, VL and VH domains, and Fc Domains etc.) are directly linked (i.e. no peptide linker is used between the domain). In such multispecific molecules the domains can be linked by a peptide bond.

[0144] One embodiment of the present invention relates to multispecific molecules, which are bispecific, that are capable of binding to a“first epitope” and a“second epitope,” such epitopes not being identical to one another. Such bispecific molecules comprise“VL1” / “VH1” domains that are capable of binding to the first epitope, and“VL2” /“VH2” domains that are capable of binding to the second epitope. The notation“VL1” and“VH1” denote respectively, the Variable Light Chain Domain and Variable Heavy Chain Domain of such bispecific molecules that bind the“first” epitope. Similarly, the notation“VL2” and“VH2” denote respectively, the Light Chain Variable Domain and Heavy Chain Variable Domain of such bispecific molecules that bind the“second” epitope. It is irrelevant whether a particular epitope is designated as the first vs. the second epitope; such notation having relevance only with respect to the presence and orientation of domains of the polypeptide chains of such multispecific molecules of the present invention. In one embodiment, one of such epitopes is an epitope of HIV-1 Env (for example but not limited to a V3 glycan and/or a CD4 binding site epitope), and the other is an epitope of a molecule that is not HIV-1 Env. In particular embodiments, one of such epitopes is an epitope of HIV-1 Env and the other is an epitope of a molecule (e.g., CD2, CD3, CD8, CD16, T-Cell Receptor (TCR), NKG2D, etc.) present on the surface of an effector cell, for example but not limited to a T lymphocyte, a natural killer (NK) cell or other mononuclear cell (see, e.g., Figures 32 and 34A-34B). In certain embodiments, a bispecific molecule comprises more than two epitope-binding sites (see, e.g., Figures 33A-33C). Such bispecific molecules will bind at least one epitope of HIV-1 Env and at least one epitope of a molecule that is not HIV-1 Env. [0145] One embodiment of the present invention also relates to trivalent binding molecules that are capable of binding to a“first epitope,” a“second epitope,” and a“third epitope,” wherein at least one of such epitopes is not identical to another. Such trivalent binding molecules comprise VL1 / VH1 domains that are capable of binding to the first epitope, VL2 / VH2 domains that are capable of binding to the second epitope, and further comprise“VL3” / “VH3” domains that are capable of binding to the third epitope, wherein the notation“VL3” and“VH3” denote respectively, the Variable Light Chain Domain and Variable Heavy Chain Domain of such trivalent binding molecules that bind the“third” epitope. The capacity to bind a third epitope provides additional and/or enhanced functionality. In one embodiment, one (or two) of such epitopes is an epitope of HIV-1 Env (particularly aV3 glycan or a CD4 binding site epitope), and two (or one) of such epitopes is an epitope of a molecule that is not HIV-1 Env. In particular embodiments, one (or two) of such epitopes is an epitope of HIV-1 Env and two (or one) of such epitopes is an epitope of a molecule (e.g., CD2, CD3, CD8, CD16, T-Cell Receptor (TCR), NKG2D, etc.) present on the surface of an effector cell, such as a T lymphocyte, a natural killer (NK) cell or other mononuclear cell (see, e.g., Figures 35A-35F). Such trivalent binding molecules will bind at least one epitope of HIV-1 Env and at least one epitope of a molecule that is not HIV-1 Env, and may bind two epitopes of HIV-1 Env and one epitope of a molecule that is not HIV-1 Env or may bind one epitope of HIV-1 Env and two epitopes that are not epitopes of HIV-1 Env. [0146] In some embodiments, such molecules comprise two polypeptide chains, wherein each of the two polypeptide chains comprises three Domains (Figure 32). The first polypeptide chain comprises: (i) a Domain that comprises a binding region of a light chain variable Domain of a first immunoglobulin (VL1), (ii) a second Domain that comprises a binding region of a heavy chain variable Domain of a second immunoglobulin (VH2), and (iii) a third Domain that serves to promote heterodimerization with the second polypeptide chain and to covalently bond the first polypeptide to the second polypeptide chain of the molecule. The second polypeptide chain contains a complementary first Domain (a VL2 Domain), a complementary second Domain (a VH1 Domain) and a third Domain that complexes with the third Domain of the first polypeptide chain in order to promote heterodimerization and covalent bonding with the first polypeptide chain. Such molecules are stable, potent and have the ability to simultaneously bind two or more antigens. They are able to promote redirected T cell (CD3) or NK (CD16) cell mediated killing of cells expressing target antigens.

[0147] In certain embodiments, the HIV-1 multispecific molecules of the present invention are composed of two polypeptide chains which associate with one another to form one binding site specific for an epitope of HIV-1, and one binding site specific for an epitope of CD2, CD3, CD8, CD16, T-Cell Receptor (TCR), NKG2D, etc., so as to be capable of simultaneously binding to HIV-1 and, for example, to CD3. Thus, such diabodies bind to a “first epitope,” which may be either an epitope of CD3 or HIV-1, and a“second epitope,” which is an epitope of HIV-1 when the first epitope is an epitope of CD3, and is an epitope of CD3 when the first epitope is from HIV-1. Alternatively, such diabodies bind to a“first epitope,” which may be either an epitope of CD16 or HIV-1, and a“second epitope,” which is an epitope of HIV-1 when the first epitope is of CD16, and is an epitope of CD16 when the first epitope is an epitope of HIV-1.

[0148] In certain embodiments, the first of such two polypeptide chains will contain, in the N- terminal to C-terminal direction, an N-terminus, the Antigen-Binding Domain of a Light Chain Variable Domain (VL) of an antibody that binds to a“first” epitope of a“first” antigen (e.g., either CD3 or HIV-1 envelope), the Antigen-Binding Domain of a Heavy Chain

Variable Domain (VH) of an antibody that binds to a“second” epitope of a“second” antigen (HIV-1, if the first antigen was CD3; CD3, if the first antigen was HIV-1), a

Heterodimerization-Promoting Domain, and a C-terminus. An intervening peptide linker (Peptide Linker 1) separates the Antigen-Binding Domain of the Light Chain Variable Domain from the Antigen-Binding Domain of the Heavy Chain Variable Domain. In certain embodiments the Antigen-Binding Domain of the Heavy Chain Variable Domain is linked to the Heterodimerization-Promoting Domain by an intervening peptide linker (Peptide Linker 2). In certain embodiments the first of the two polypeptide chains will thus contain, in the N- terminal to C-terminal direction: VL_{First Antigen}– Peptide Linker 1– VH_{Second Antigen}– Peptide Linker 2– Heterodimerization-Promoting Domain.

[0149] The terms VL/VH for first and second antigens, VL_{first antigen}/VH_{first antigen} VL_second _antigen/VH_{second antigen}, and VL1/VH1 and VL2/VH2 are used interchangeably throughout the application.

[0150] In certain embodiments, the second of such two polypeptide chains will contain, in the N-terminal to C-terminal direction, an N-terminus, the Antigen-Binding Domain of a Light Chain Variable Domain (VL) of an antibody that binds to the second epitope of the second antigen, the Antigen-Binding Domain of a Heavy Chain Variable Domain (VH) of an antibody that binds to the first epitope of the first antigen, a Heterodimerization-Promoting Domain and a C-terminus. An intervening peptide linker (Peptide Linker 1) separates the Antigen-Binding Domain of the Light Chain Variable Domain from the Antigen-Binding Domain of the Heavy Chain Variable Domain. In certain embodiments, the Antigen-Binding Domain of the Heavy Chain Variable Domain is linked to the Heterodimerization-Promoting Domain by an intervening peptide linker (Peptide Linker 2). In certain embodiments the second of the two polypeptide chains will thus contain, in the N-terminal to C-terminal direction: VL_{Second Antigen}– Peptide Linker 1– VH_{First Antigen}– Peptide Linker 2–

Heterodimerization-Promoting Domain.

[0151] The Antigen-Binding Domain of the Light Chain Variable Domain of the first polypeptide chain interacts with the Antigen-Binding Domain of the Heavy Chain Variable Domain of the second polypeptide chain in order to form a functional antigen-binding site that is specific for the first antigen (e.g., either HIV-1 envelope or CD3). Likewise, the Antigen- Binding Domain of the Light Chain Variable Domain of the second polypeptide chain interacts with the Antigen-Binding Domain of the Heavy Chain Variable Domain of the first polypeptide chain in order to form a second functional antigen-binding site that is specific for the second antigen (e.g., either CD3 or HIV-1 envelope, depending upon the identity of the first antigen). Thus, the selection of the Antigen-Binding Domain of the Light Chain Variable Domain and the Antigen-Binding Domain of the Heavy Chain Variable Domain of the first and second polypeptide chains are coordinated, such that the two polypeptide chains collectively comprise Antigen-Binding Domains of light and Heavy Chain Variable Domains capable of binding to the intended targets, in certain embodiments e.g. HIV-1 envelope and CD3, or CD16.

[0152] In certain embodiments the length of Peptide Linker 1, which separates such VL and VH domains of a polypeptide chain is selected to substantially or completely prevent such VL and VH domains from binding to one another. Thus the VL and VH domains of the first polypeptide chain are substantially or completely incapable of binding to one another.

Likewise, the VL and VH domains of the second polypeptide chain are substantially or completely incapable of binding to one another. In certain embodiments this is due to the peptide linker which separates the VH and VL domains. In certain embodiments, the peptide linker is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, but no more than 20 amino acids. In some embodiments, the peptide linker is less than 12 amino acids in length. In certain embodiments an intervening spacer peptide (Peptide Linker 1) has the sequence (SEQ ID NO:508): GGGSGGGG.

[0153] Peptide Linker 2 separates the VH Domain of a polypeptide chain from the

Heterodimer-Promoting Domain of that polypeptide chain. Any of a variety of linkers can be used for the purpose of Peptide Linker 2. The length and composition of Peptide Linker 2 may be selected based on the choice of heterodimer-promoting domains. Typically, the second intervening peptide linker (Peptide Linker 2) will comprise 1-20 amino acid residues. In certain embodiments, where the heterodimer-promoting domains do not comprise a cysteine residue a cysteine-containing second intervening peptide linker (Peptide Linker 2) is utilized. Optionally, both a cysteine-containing Peptide Linker 2 (Peptide Linker 2-C) and a cysteine-containing Heterodimer-Promoting Domain are used. In certain embodiments a sequence for such Peptide Linker 2 has the amino acid sequence: GGCGGG (SEQ ID NO:509), which has a cysteine residue that may be used to covalently bond the first and second polypeptide chains to one another via a disulfide bond. In certain embodiments, a sequence for Peptide Linker 2-C has the amino acid sequence: ASTKG (SEQ ID NO: 510). Peptide Linker 2 and Peptide Linker 2-C could be used interchangeably.

[0154] The formation of heterodimers of the first and second polypeptide chains can be driven by the inclusion of specific sequences referred to as Heterodimer-Promoting Domains (HPDs). Such domains include without limitation GVEPKSC (SEQ ID NO:511) or VEPKSC (SEQ ID NO:512) on one polypeptide chain and GFNRGEC (SEQ ID NO:513) or FNRGEC (SEQ ID NO:514) on the other polypeptide chain (See US2007/0004909 herein incorporated by reference in its entirety). Various HPD sequences are contemplated by the invention and disclosed in the specification. Table 4 discloses non-limiting embodiments of various HPDs. In some embodiments the HPD include E/K-coils (SEQ ID NOs: 518, 520) or cysteine engineered E/K-coils (SEQ ID NOs: 519, 521). In some embodiments HPD includes combinations of SEQ ID NOs: 511, 512, 513, and 514 (e.g., SEQ ID NOs: 511 and 513; SEQ ID NOs: 512 and 513; SEQ ID NOs: 511 and 514; SEQ ID NOs: 512 and 514). In some embodiments HPDs include any suitable sequences with a Cysteine residue to permit disulfide bond. In some embodiments HPDs includes suitable CH1 and CL domains (See for example CH domain (SEQ ID NO: 515) and kappa and lambda light chain constant domains (SEQ ID NOs: 516 and 517).

[0155] In certain embodiments, the Heterodimer-Promoting Domains of the present invention are formed from one, two, three or four tandemly repeated coil domains of opposing charge that comprise a sequence of at least six, at least seven or at least eight charged amino acid residues (Apostolovic, B. et al. (2008)“pH-Sensitivity of the E3/K3 Heterodimeric Coiled Coil,” Biomacromolecules 9:3173–3180; Arndt, K.M. et al. (2001)“Helix-stabilized Fv (hsFv) Antibody Fragments: Substituting the Constant Domains of a Fab Fragment for a Heterodimeric Coiled-coil Domain,” J. Molec. Biol.312:221-228; Arndt, K.M. et al. (2002) “Comparison of In Vivo Selection and Rational Design of Heterodimeric Coiled Coils,” Structure 10:1235-1248; Boucher, C. et al. (2010)“Protein Detection By Western Blot Via Coiled–Coil Interactions,” Analytical Biochemistry 399:138-140; Cachia, P.J. et al. (2004) “Synthetic Peptide Vaccine Development: Measurement Of Polyclonal Antibody Affinity And Cross-Reactivity Using A New Peptide Capture And Release System For Surface Plasmon Resonance Spectroscopy,” J. Mol. Recognit.17:540-557; De Crescenzo, G.D. et al. (2003) “Real-Time Monitoring of the Interactions of Two-Stranded de novo Designed Coiled-Coils: Effect of Chain Length on the Kinetic and Thermodynamic Constants of Binding,”

Biochemistry 42:1754-1763; Fernandez-Rodriquez, J. et al. (2012)“Induced

Heterodimerization And Purification Of Two Target Proteins By A Synthetic Coiled-Coil Tag,” Protein Science 21:511-519; Ghosh, T.S. et al. (2009)“End-To-End And End-To- Middle Interhelical Interactions: New Classes Of Interacting Helix Pairs In Protein

Structures,” Acta Crystallographica D65:1032-1041; Grigoryan, G. et al. (2008)“Structural Specificity In Coiled-Coil Interactions,” Curr. Opin. Struc. Biol.18:477-483; Litowski, J.R. et al. (2002)“Designing Heterodimeric Two-Stranded α-Helical Coiled-Coils: The Effects Of Hydrophobicity And α-Helical Propensity On Protein Folding, Stability, And Specificity,” J. Biol. Chem.277:37272-37279; Steinkruger, J.D. et al. (2012)“The d′--d--d′ Vertical Triad is Less Discriminating Than the a′--a--a′ Vertical Triad in the Antiparallel Coiled-coil Dimer Motif,” J. Amer. Chem. Soc.134(5):2626–2633; Straussman, R. et al. (2007)“Kinking the Coiled Coil– Negatively Charged Residues at the Coiled-coil Interface,” J. Molec. Biol. 366:1232-1242; Tripet, B. et al. (2002)“Kinetic Analysis of the Interactions between

Troponin C and the C-terminal Troponin I Regulatory Region and Validation of a New Peptide Delivery/Capture System used for Surface Plasmon Resonance,” J. Molec. Biol. 323:345–362; Woolfson, D.N. (2005)“The Design Of Coiled-Coil Structures And

Assemblies,” Adv. Prot. Chem.70:79-112; Zeng, Y. et al. (2008)“A Ligand-Pseudoreceptor System Based On de novo Designed Peptides For The Generation Of Adenoviral Vectors With Altered Tropism,” J. Gene Med.10:355-367).

[0156] Such repeated coil domains may be exact repeats or may have substitutions. For example, the Heterodimer-Promoting Domain of the first polypeptide chain may comprise a sequence of eight negatively charged amino acid residues and the Heterodimerization- Promoting Domain of the second polypeptide chain may comprise a sequence of eight negatively charged amino acid residues. It is immaterial which coil is provided to the first or second polypeptide chains, provided that a coil of opposite charge is used for the other polypeptide chain.

[0157] In certain embodiments a multispecific molecule of the present invention has a first polypeptide chain having a negatively charged coil. The positively charged amino acid may be lysine, arginine, histidine, etc. and/or the negatively charged amino acid may be glutamic acid, aspartic acid, etc. In certain embodiments the positively charged amino acid is lysine and/or the negatively charged amino acid is glutamic acid. It is possible for only a single Heterodimer-Promoting Domain to be employed (since such domain will inhibit

homodimerization and thereby promote heterodimerization). In certain embodiments both the first and second polypeptide chains of the multispecific molecules of the present invention contain Heterodimer-Promoting Domains.

[0158] In certain embodiments, one of the Heterodimer-Promoting Domains will comprise four tandem“E-coil” helical domains (SEQ ID NO:518 (EVAALEK-EVAALEK- EVAALEK-EVAALEK)), whose glutamate residues will form a negative charge at pH 7, while the other of the Heterodimer-Promoting Domains will comprise four tandem“K-coil” domains (SEQ ID NO:520 (KVAALKE-KVAALKE-KVAALKE-KVAALKE)), whose lysine residues will form a positive charge at pH 7. The presence of such charged domains promotes association between the first and second polypeptides, and thus fosters heterodimerization. In other embodiments, one of the four tandem“E-coil” helical domains of SEQ ID NO: 518 has been modified to contain a cysteine residue: EVAACEK- EVAALEK-EVAALEK-EVAALEK (SEQ ID NO: 519). In some embodiments, a

Heterodimer-Promoting Domain in which one of the four tandem“K-coil” helical domains of SEQ ID NO: 520 has been modified to contain a cysteine residue: KVAACKE-KVAALKE- KVAALKE-KVAALKE (SEQ ID NO: 521). Such cysteine modified Heterodimer- Promoting Domains may be used to covalently bond the first and second polypeptide chains to one another via a disulfide bond.

[0159] In some embodiments, the number of K coil and E coil domains can vary and a skilled artisan can readily determine whether a different number of K-coil or E-coil domain lead to heterodimerization.

[0160] In certain embodiments, the multispecific molecules of the invention, for example but not limited to bispecific monovalent diabodies and trivalent binding molecules, are engineered so that their first and second polypeptide chains covalently bond to one another via one or more cysteine residues positioned along their length. Such cysteine residues may be introduced into the intervening peptide linker that separates the VL and VH domains of the polypeptides. Alternatively, as provided above Peptide Linker 2 and/or the HPDs may contain such cysteine residues.

[0161] The invention also includes variants of the multispecific molecules, or fragments thereof disclosed herein, including variants that retain the ability to bind to recombinant Env protein, the ability to bind to the surface of virus-infected cells and/or ADCC-mediating properties of the antibodies specifically disclosed, and methods of using same to, for example, reduce HIV-1 infection risk. Combinations of the multispecific molecules, antibodies, or fragments thereof, disclosed herein can also be used in the methods of the invention.

[0162] Formation of multispecific molecule as described herein requires the interaction of differing polypeptide chains. Such interactions are difficult to achieve with efficiency within a single cell recombinant production system, due to the many variants of potential chain mispairings. One solution to decrease the probability of mispairings, is to engineer "knobs- into-holes" type mutations into the desired polypeptide chain pairs. Such mutations favor heterodimerization over homodimerization. For example, with respect to Fc-Fc-interactions, an amino acid substitution (preferably a substitution with an amino acid comprising a bulky side group forming a `knob`, e.g., tryptophan) can be introduced into the CH2 or CH3 domain such that steric interference will prevent interaction with a similarly mutated domain and will obligate the mutated domain to pair with a domain into which a complementary, or accommodating mutation has been engineered, i.e., `the hole` (e.g., a substitution with glycine). Such sets of mutations can be engineered into any pair of polypeptides comprising the multispecific molecule, and further, engineered into any portion of the polypeptides chains that comprise a multispecific molecule. Methods of protein engineering to favor

heterodimerization over homodimerization are well known in the art, in particular with respect to the engineering of immunoglobulin-like molecules, and are encompassed herein (see e.g., Ridgway et al. (1996) "`Knobs-Into-Holes` Engineering Of Antibody CH3 Domains For Heavy Chain Heterodimerization," Protein Engr.9:617-621, Atwell et al. (1997) "Stable Heterodimers From Remodeling The Domain Interface Of A Homodimer Using A Phage Display Library," J. Mol. Biol.270: 26-35, and Xie et al. (2005) "A New Format Of

Bispecific Antibody: Highly Efficient Heterodimerization, Expression And Tumor Cell Lysis," J. Immunol. Methods 296:95-101; each of which is hereby incorporated herein by reference in its entirety).

[0163] In some embodiments the invention provides multispecific molecules comprising variant Fc domain (or portions thereof), which variant Fc domain comprises at least one amino acid modification (e.g. substitution, insertion deletion) relative to a comparable wild- type Fc domain or hinge-Fc domain (or portion thereof). Molecules comprising variant Fc domains or hinge-Fc domains (or portion thereof) (e.g., antibodies) normally have altered phenotypes relative to molecules comprising wild-type Fc domains or hinge-Fc domains or portions thereof. The variant phenotype may be expressed as altered serum half-life, altered stability, altered susceptibility to cellular enzymes or altered effector function as assayed in an NK dependent or macrophage dependent assay. Fc domain variants identified as altering effector function are known in the art. For example International Application WO04/063351, U.S. Patent Application Publications 2005/0037000 and 2005/0064514.

[0164] In some embodiments the invention provides multispecific molecules comprising a hinge domain. The hinge domain be derived from any immunoglobulin isotype or allotype including IgA, IgD, IgG, IgE and IgM. In preferred embodiments, the hinge domain is derived from IgG, wherein the IgG isotype is IgG1, IgG2, IgG3 or IgG4, or an allotype thereof. The hinge domain may be engineered into a polypeptide chain comprising the multispecific molecule together with an Fc domain such that the multispecific molecule comprises a hinge-Fc domain. In certain embodiments, the hinge and Fc domain are independently selected from any immunoglobulin isotype known in the art or exemplified herein. In other embodiments the hinge and Fc domain are separated by at least one other domain of the polypeptide chain, e.g., the VL domain.

[0165] In another aspect, the invention provides multispecific molecules which include Fc domain(s)-- Fc bearing multispecific molecules. While some of the disclosure regarding Fc domain(s) refers to specific designs, a skilled artisan appreciates that the Fc disclosure is pertinent to any Fc bearing design of multispecific molecules, including but not limited to the designs described in Figures 32-36.

[0166] Fc bearing multispecific molecules, for example but not limited to Fc bearing diabodies are heavier, and could bind neonatal Fc receptor, increasing their circulating half- life. See Garber“Bispecific antibodies rise again” Nature Reviews Drug Discovery 13, 799– 801 (2014), Figure 1a; See US Pub 20130295121, US Pub 20140099318 incorporated by reference in their entirety. In certain embodiments, the invention encompasses multispecific molecules comprising an Fc domain or portion thereof (e.g. a CH2 domain, or CH3 domain). The Fc domain or portion thereof may be derived from any immunoglobulin isotype or allotype including, but not limited to, IgA, IgD, IgG, IgE and IgM. In some embodiments, the Fc domain (or portion thereof) is derived from IgG. In some embodiments, the IgG isotype is IgG1, IgG2, IgG3 or IgG4 or an allotype thereof. In some embodiments, the multispecific molecule comprises an Fc domain, which Fc domain comprises a CH2 domain and CH3 domain independently selected from any immunoglobulin isotype (i.e. an Fc domain comprising the CH2 domain derived from IgG and the CH3 domain derived from IgE, or the CH2 domain derived from IgG1 and the CH3 domain derived from IgG2, etc.). In some embodiments, the Fc domain may be engineered into a polypeptide chain comprising the multispecific molecule of the invention in any position relative to other domains or portions of the polypeptide chain (e.g., the Fc domain, or portion thereof, may be C-terminal to both the VL and VH domains of the polypeptide of the chain; may be N-terminal to both the VL and VH domains; or may be N-terminal to one domain and C-terminal to another (i.e., between two domains of the polypeptide chain)).

[0167] Other modifications of the multispecific molecules are contemplated to increase the half-life of the molecules. In some embodiments, these modifications include addition of a polypeptide portion of a serum binding protein. See US20100174053 A1, incorporated by reference.

[0168] In some embodiments, the Fc variants of the multispecific molecules of the invention are expected to have increased serum half-life compared to the non-Fc variants. Skilled artisan can readily carry out various assays, including pharmacokinetic studies, to determine the half-life of these molecules.

[0169] In some embodiments, the polypeptide chains in multispecific molecules further comprise an Fc domain. Dimerization of the Fc domains leads to formation of a multispecific molecule that exhibits immunoglobulin-like functionality, i.e., Fc mediated function (e.g., Fc- Fc.gamma.R interaction, complement binding, etc.).

[0170] As provided in Figures 33-34, one or both of the polypeptide chains of bispecific diabodies may additionally comprise the sequence of a CH2-CH3 Domain, such that complexing between the two diabody polypeptides forms an Fc Domain that may be capable of binding to the Fc receptor of cells (such as B lymphocytes, dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils and mast cells). Similarly, the first and third polypeptide chains of trivalent binding molecules can comprise the sequence of a CH2- CH3 Domain, such that complexing between these two polypeptide chains forms an Fc Domain. As provided in more detail below, the CH2 and/or CH3 Domains of such polypeptide chains need not be identical in sequence, and advantageously are modified to foster complexing between the two polypeptide chains. Many variations of such molecules have been described (see, e.g., United States Patent Publications No.2014-0099318; 2013- 0295121; 2010-0174053 and 2009-0060910; European Patent Publication No. EP 2714079; EP 2601216; EP 2376109; EP 2158221 and PCT Publications No. WO 2015/026894; WO 2015/026892; WO 2015/021089; WO 2014/159940; WO 2012/162068; WO 2012/018687; WO 2010/080538, the content of each of these publications in herein incorporated by reference in its entirety).

[0171] In some embodiments, Fc -bearing bispecific diabodies may comprise two pairs of polypeptide chains (or four different chains, as provided below). The first and third polypeptide chains of such a bispecific molecule (e.g., diabodies) contain three domains: (i) a VL1-containing Domain, (ii) a VH2-containing Domain, (iii) Heterodimer-Promoting Domain and (iv) a Domain containing a CH2-CH3 sequence. The second and fourth polypeptide chains contain: (i) a VL2-containing Domain, (ii) a VH1-containing Domain and (iii) a Heterodimer-Promoting Domain, where the Heterodimer-Promoting Domains promote the dimerization of the first/third chains with the second/fourth chains. The VL and/or VH Domains of the third and fourth polypeptide chains, and VL and/or VH Domains of the first and second polypeptide chains may be the same or different so as to permit tetravalent binding that is either monospecific, bispecific or tetraspecific. Such molecules are tetravalent and have enhanced potency. The general structure of the polypeptide chains of a representative four-chain Fc Domain-containing multispecific molecules of invention is provided in Table 1:

[0172] HIV-1 bispecific bivalent Fc bearing diabodies can be composed of two pairs of polypeptide chains (i.e., two first polypeptide chain and two second polypeptide chains) which associate with one another to form two binding sites specific for an epitope of HIV-1 and two binding sites specific for an epitope, for example but not limited to CD3 (see,

Figures 33A-33C), so as to be capable of simultaneously binding to HIV-1 and to CD3. Thus such molecules binding to a“first” epitope, on a“first” antigen which may be either CD3 or HIV-1, and a“second“ epitope on a“second” antigen, which is HIV-1 when the first epitope is CD3, and is CD3 when the first epitope is HIV-1. [0173] As shown in Figures 33A-33C, the first polypeptide chain comprises (in the N- terminal to C-terminal direction): an N-terminus, the Antigen-Binding Domain of a Light Chain Variable Domain (VL1) of an antibody that binds to a“first” epitope of a“first” antigen (either an effector cell epitope such as but not limited to CD3, or HIV-1), the Antigen- Binding Domain of a Heavy Chain Variable Domain (VH2) of an antibody that binds to a “second” epitope of a“second” antigen (HIV-1, if the first antigen as CD3; CD3, if the first antigen was HIV-1), a Heterodimer-Promoting Domain which may comprise a cysteine residue, the CH2-CH3 domains of an Fc Domain (“Fc Domain”) and a C-terminus. The second polypeptide contains (in the N-terminal to C-terminal direction): an N-terminus, the Antigen-Binding Domain the Light Chain Variable Domain (VL2) of an antibody that binds to the second epitope of the second antigen (VL2), the Antigen-Binding Domain of the Heavy Chain Variable Domain (VH1) of an antibody that binds to the first epitope of the first antigen, a Heterodimer-Promoting Domain that promotes heterodimerization with the first polypeptide chain, and a C-terminus. Here, two first polypeptides complex with each other to form an Fc Domain. An intervening peptide linker (Peptide Linker 1) separates the Antigen- Binding Domain of the Light Chain Variable Domain from the Antigen-Binding Domain of the Heavy Chain Variable Domain. In non-limiting embodiments the Antigen-Binding Domain of the Heavy Chain Variable Domain is linked to the Heterodimer-Promoting Domain by an intervening peptide linker (Peptide Linker 2). In other non-limiting embodiments, the Heterodimer-Promoting Domain is linked to the Fc Domain by an intervening peptide linker (Peptide Linker 3) or by an intervening spacer-linker peptide (Spacer-Linker 3). In certain embodiments, the first and second polypeptide chains form a disulfide bond between cysteine residues, which may be present in Peptide Linker 2 (e.g., but not limited to Peptide Linker 2-C) and/or in the Heterodimer-Promoting Domains (e.g. but not limited to E-coil-C/K-coil-C). Figures 33A-33C provide schematics of three variations of such diabodies utilizing different Heterodimer-Promoting Domains. In non-limiting embodiments, the first polypeptide chains will contain, in the N-terminal to C-terminal direction: VL1 - Peptide Linker 1– VH2– Peptide Linker 2– Heterodimer-Promoting Domain– Spacer-Linker 3– Fc Domain, and the second polypeptide chains will contain, in the N-terminal to C-terminal direction: VL2 - Peptide Linker 1– VH1– Peptide Linker 2– Heterodimer-Promoting Domain.

[0174] In some embodiments, Fc bearing diabodies may comprise three polypeptide chains. The first polypeptide of such a molecule contains three Domains: (i) a VL1-containing Domain, (ii) a VH2-containing Domain, (iii) a Domain that promotes heterodimerization and covalent bonding with the diabody’s first polypeptide chain and (iv) a Domain containing a CH2-CH3 sequence. The second polypeptide of such diabodies contains: (i) a VL2- containing Domain, (ii) a VH1-containing Domain and (iii) a Domain that promotes heterodimerization and covalent bonding with the diabody’s first polypeptide chain. The third polypeptide of such diabodies comprises a CH2-CH3 sequence. Thus, the first and second polypeptide chains of such diabodies associate together to form a VL1/VH1 binding site that is capable of binding to the epitope, as well as a VL2/VH2 binding site that is capable of binding to the second epitope. The first and second polypeptides are bonded to one another through a disulfide bond involving cysteine residues in their respective third Domains.

Notably, the first and third polypeptide chains complex with one another to form an Fc Domain that is stabilized via a disulfide bond. Such diabodies have enhanced potency. Such Fc bearing diabodies may have either of two orientations (Table 2):

[0175] HIV-1 bispecific monovalent Fc bearing diabodies can be composed of three polypeptide chains which associate with one another to form one binding site specific for an epitope of HIV-1 and one binding site specific for another epitope, for example but not limited to an epitope of CD3 (see, Figure 34A-34B), so as to be capable of simultaneously binding to HIV-1 and to CD3. Thus, such molecules bind to a“first” antigen, which may be either CD3 or HIV-1, and a“second” antigen, which is HIV-1 when the first epitope is CD3, and is CD3 when the first epitope is HIV-1.

[0176] As shown in Figure 34A, the first of such three polypeptide chains will contain, in the N-terminal to C-terminal direction, an N-terminus, the Antigen-Binding Domain of a Light Chain Variable Domain (VL) of an antibody that binds to a“first” epitope of a“first” antigen (for example but not limited to either CD3 or HIV-1), the Antigen-Binding Domain of a Heavy Chain Variable Domain (VH) of an antibody that binds to a“second” epitope of a “second” antigen (for example but not limited to HIV-1, if the first antigen was CD3; CD3, if the first antigen was HIV-1), a Heterodimerization-Promoting Domain, and a C-terminus. An intervening peptide linker (Peptide Linker 1) separates the Antigen-Binding Domain of the Light Chain Variable Domain from the Antigen-Binding Domain of the Heavy Chain Variable Domain. In non-limiting embodiments, the Antigen-Binding Domain of the Heavy Chain Variable Domain is linked to the Heterodimerization-Promoting Domain by an intervening peptide linker (Peptide Linker 2). In a non-limiting example of an HIV-1 bispecific monovalent Fc bearing diabody, the C-terminus of the Heterodimerization- Promoting Domain is linked to the CH2-CH3 domains of an Fc Domain (“Fc Domain”) by an intervening peptide linker (Peptide Linker 3) or by an intervening spacer-linker peptide (Spacer-Linker 3). In non-limiting embodiments, the first of the three polypeptide chains will thus contain, in the N-terminal to C-terminal direction: VL_{First Antigen}– Peptide Linker 1– VH_{Second Antigen}– Peptide Linker 2– Heterodimerization-Promoting Domain– Spacer-Linker 3 – Fc Domain.

[0177] Alternatively, as shown in Figure 34B, the first of such three polypeptide chains will contain, in the N-terminal to C-terminal direction, an N-terminus, Peptide Linker 3, the CH2-CH3 domains of an Fc Domain (“Fc Domain”), an intervening spacer peptide (Peptide Linker 4), having, for example the amino acid sequence: APSSS (SEQ ID NO:524) or the amino acid sequence APSSSPME (SEQ ID NO:525), the Antigen-Binding Domain of a Light Chain Variable Domain (VL) of an antibody that binds to the first epitope of the first antigen (for example but not limited to CD3 or HIV-1), the Antigen-Binding Domain of a Heavy Chain Variable Domain (VH) of an antibody that binds to the second epitope of the second antigen (for example but not limited to HIV-1, if the first antigen was CD3; CD3, if the first antigen was HIV-1), a Heterodimerization-Promoting Domain, and a C-terminus. An intervening peptide linker (Peptide Linker 1) separates the Antigen-Binding Domain of the Light Chain Variable Domain from the Antigen-Binding Domain of the Heavy Chain

Variable Domain. In non-limiting embodiments, the Antigen-Binding Domain of the Heavy Chain Variable Domain is linked to the Heterodimerization-Promoting Domain by an intervening peptide linker peptide (Peptide Linker 2). In non-limiting embodiments, the first of the three polypeptide chains will thus contain, in the N-terminal to C-terminal direction: Peptide Linker 3– Fc Domain– Peptide Linker 4– VL_{First Antigen}– Peptide Linker 1– VH_{Second Antigen}– Peptide Linker 2– Heterodimerization-Promoting Domain.

[0178] In non-limiting embodiments, the second of such three polypeptide chains will contain, in the N-terminal to C-terminal direction, an N-terminus, the Antigen-Binding Domain of a Light Chain Variable Domain (VL) of an antibody that binds to the second epitope of the second antigen, the Antigen-Binding Domain of a Heavy Chain Variable Domain (VH) of an antibody that binds to the first epitope of the first antigen, a

Heterodimerization-Promoting Domain and a C-terminus. An intervening peptide linker (Peptide Linker 1) separates the Antigen-Binding Domain of the Light Chain Variable Domain from the Antigen-Binding Domain of the Heavy Chain Variable Domain. In non- limiting embodiments, the Antigen-Binding Domain of the Heavy Chain Variable Domain is linked to the Heterodimerization-Promoting Domain by an intervening peptide linker

(Peptide Linker 2). In non-limiting embodiments, the second of the three polypeptide chains will thus contain, in the N-terminal to C-terminal direction: VL_{Second Antigen}– Peptide Linker 1 – VH_{First Antigen}– Peptide Linker 2– Heterodimerization-Promoting Domain.

[0179] In non-limiting embodiments, the third of such three polypeptide chains will contain a peptide linker (Peptide Linker 3) and the CH2-CH3 domains of an Fc Domain (“Fc

Domain”).

[0180] For the various Fc bearing multispecific molecules described herein, the Antigen- Binding Domain of the Light Chain Variable Domain of the first polypeptide chain interacts with the Antigen-Binding Domain of the Heavy Chain Variable Domain of the second polypeptide chain in order to form a functional antigen-binding site that is specific for the first antigen (e.g., either HIV-1 or CD3). Likewise, the Antigen-Binding Domain of the Light Chain Variable Domain of the second polypeptide chain interacts with the Antigen-Binding Domain of the Heavy Chain Variable Domain of the first polypeptide chain in order to form a second functional antigen-binding site that is specific for the second antigen (e.g., either CD3 or HIV-1, depending upon the identity of the first antigen). Thus, the selection of the

Antigen-Binding Domain of the Light Chain Variable Domain and the Antigen-Binding Domain of the Heavy Chain Variable Domain of the first and second polypeptide chains are coordinated, such that the two polypeptide chains collectively comprise Antigen-Binding Domains of light and Heavy Chain Variable Domains capable of binding to the first and second antigens (e.g., HIV-1 and CD3).

[0181] The Fc Domain of the Fc bearing multispecific molecules, including but not limited to bispecific and trispecific molecules (e.g. bispecific antibodies, bispecific diabodies, and trivalent binding molecules), of the present invention may be either a complete Fc Domain (e.g., a complete IgG Fc Domain) or only a fragment of a complete Fc Domain. In some embodiments the Fc Domain of the molecules of the present invention may possess the ability to bind to one or more Fc receptors (e.g., FcγR(s)). In non-limiting embodiments the Fc Domain will cause reduced binding to FcγRIA (CD64), FcγRIIA (CD32A), FcγRIIB

(CD32B), FcγRIIIA (CD16a) or FcγRIIIB (CD16b) (relative to the binding exhibited by a wild-type Fc Domain) or will substantially eliminate the ability of such Fc Domain to bind to such receptor(s). The Fc bearing multispecific molecules of the present invention may include some or all of the CH2 Domain and/or some or all of the CH3 Domain of a complete Fc Domain, or may comprise a variant CH2 and/or a variant CH3 sequence (that may include, for example, one or more insertions and/or one or more deletions with respect to the CH2 or CH3 domains of a complete Fc Domain). The Fc Domain of the Fc bearing multispecific molecules of the present invention may comprise non-Fc polypeptide portions, or may comprise portions of non-naturally complete Fc Domains, or may comprise non-naturally occurring orientations of CH2 and/or CH3 domains (such as, for example, two CH2 domains or two CH3 domains, or in the N-terminal to C-terminal direction, a CH3 Domain linked to a CH2 Domain, etc.).

[0182] Polymorphisms have been observed at a number of different positions within antibody constant regions (e.g., Fc positions, including but not limited to positions 270, 272, 312, 315, 356, and 358 as numbered by the EU index as set forth in Kabat), and thus slight differences between the presented sequence and sequences in the prior art can exist. Polymorphic forms of human immunoglobulins have been well-characterized. At present, 18 Gm allotypes are known: G1m (1, 2, 3, 17) or G1m (a, x, f, z), G2m (23) or G2m (n), G3m (5, 6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m (b1, c3, b3, b0, b3, b4, s, t, g1, c5, u, v, g5) (Lefranc, et. al.,“The Human IgG Subclasses: Molecular Analysis Of Structure, Function And

Regulation.” Pergamon, Oxford, pp.43-78 (1990); Lefranc, G. et. al., 1979, Hum. Genet.: 50, 199-211). It is specifically contemplated that the molecules of the present invention may be incorporate any allotype, isoallotype, or haplotype of any immunoglobulin gene, and are not limited to the allotype, isoallotype or haplotype of the sequences provided herein.

Furthermore, in some expression systems the C-terminal amino acid residue (bolded above) of the CH3 Domain may be post-translationally removed. Accordingly, the C-terminal residue of the CH3 Domain is an optional amino acid residue. Specifically encompassed by the instant invention are molecules lacking the C-terminal residue of the CH3 Domain. Also specifically encompassed by the instant invention are such constructs comprising the C- terminal lysine residue of the CH3 Domain.

[0183] In non-limiting embodiments the first and third polypeptide chains of the Fc bearing multispecific molecules of the present invention each comprise CH2-CH3 domains that complex together to form an immunoglobulin (IgG) Fc Domain. The amino acid sequence of the CH2-CH3 domain of human IgG1 is (SEQ ID NO: 527):

APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK

[0184] Thus the CH2 and/or CH3 Domains of the first and third polypeptide chains may both be composed of SEQ ID NO: 527, or a variant thereof (e.g., SEQ ID NO: 528, 529, 530). [0185] In non-limiting embodiments the CH2-CH3 domains of the first and third polypeptide chains of the Fc bearing multispecific molecules of the present invention exhibit decreased (or substantially no) binding to FcγRIA (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a) or FcγRIIIB (CD16b) (relative to the binding exhibited by the wild-type Fc Domain). Fc variants and mutant forms capable of mediating such altered binding are well known in the art and include amino acid substitutions at positions 234 and 235, a substitution at position 265 or a substitution at position 297 (see, for example, US Patent No.5,624,821, herein incorporated by reference). In non-limiting embodiments the CH2-CH3 Domain of the first and/or third polypeptide chains of the Fc bearing multispecific molecules of the present invention include a substitution at position 234 with alanine and 235 with alanine.

[0186] The CH2 and/or CH3 Domains of the first and third polypeptide chains need not be identical in sequence, and in some embodiment are modified to foster complexing between the two polypeptide chains. For example, an amino acid substitution (for example a substitution with an amino acid comprising a bulky side group forming a‘knob’, e.g., tryptophan) can be introduced into the CH2 or CH3 Domain such that steric interference will prevent interaction with a similarly mutated domain and will obligate the mutated domain to pair with a domain into which a complementary, or accommodating mutation has been engineered, i.e.,‘the hole’ (e.g., a substitution with glycine). Such sets of mutations can be engineered into any pair of polypeptides comprising Fc bearing multispecific molecules of the invention, and further, engineered into any portion of the polypeptides chains of the pair. Methods of protein engineering to favor heterodimerization over homodimerization are well known in the art, in particular with respect to the engineering of immunoglobulin-like molecules, and are encompassed herein (see e.g., Ridgway et al. (1996)“‘Knobs-Into-Holes’ Engineering Of Antibody CH3 Domains For Heavy Chain Heterodimerization,” Protein Engr. 9:617-621, Atwell et al. (1997)“Stable Heterodimers From Remodeling The Domain

Interface Of A Homodimer Using A Phage Display Library,” J. Mol. Biol.270: 26-35, and Xie et al. (2005)“A New Format Of Bispecific Antibody: Highly Efficient

Heterodimerization, Expression And Tumor Cell Lysis,” J. Immunol. Methods 296:95-101; each of which is hereby incorporated herein by reference in its entirety). In non-limiting embodiments the‘knob’ is engineered into the CH2-CH3 Domains of the first polypeptide chain and the‘hole’ is engineered into the CH2-CH3 Domains of the third polypeptide chain. Thus, the‘knob’ will help in preventing the first polypeptide chain from homodimerizing via its CH2 and/or CH3 Domains. In non-limiting embodiments, as the third polypeptide chain contains the‘hole’ substitution it will heterodimerize with the first polypeptide chain as well as homodimerize with itself. In non-limiting embodiments a knob is created by modifying a native IgG Fc Domain to contain the modification T366W. In non-limiting embodiments a hole is created by modifying a native IgG Fc Domain to contain the modification T366S, L368A and Y407V. To aid in purifying the third polypeptide chain homodimer from the final bispecific monovalent Fc bearing diabody comprising the first, second and third polypeptide chains, the protein A binding site of the CH2 and CH3 Domains of the third polypeptide chain is mutated by amino acid substitution at position 435 (H435R). Thus, the third polypeptide chain homodimer will not bind to protein A, whereas the bispecific monovalent Fc bearing diabody will retain its ability to bind protein A via the protein A binding site on the first polypeptide chain.

[0187] In non-limiting embodiments a sequence for the CH2 and CH3 Domains of the first polypeptide chain of the Fc bearing multispecific molecules of the present invention will have the“knob-bearing” sequence (SEQ ID NO: 531):

APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLWCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK

[0188] In non-limiting embodiments a sequence for the CH2 and CH3 Domains of the third polypeptide chain of the Fc bearing multispecific molecules of the present invention will have the“hole-bearing” sequence (SEQ ID NO: 533):

APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQG NVFSCSVMHE ALHNRYTQKS LSLSPGK

[0189] As will be noted, the CH2-CH3 Domains of SEQ ID NO: 531 and SEQ ID NO: 533 include a substitution at position 234 with alanine and 235 with alanine, and thus form an Fc Domain exhibit decreased (or substantially no) binding to FcγRIA (CD64), FcγRIIA

(CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a) or FcγRIIIB (CD16b) (relative to the binding exhibited by the wild-type Fc Domain (SEQ ID NO: 527).

[0190] In non-limiting embodiments, the first polypeptide chain will have a“knob-bearing” CH2-CH3 sequence, such as that of SEQ ID NO: 531 or 532. However, as will be recognized, a“hole-bearing” CH2-CH3 Domain (e.g., SEQ ID NO: 533 or 534) could be employed in the first polypeptide chain, in which case, a“knob-bearing” CH2-CH3 Domain (e.g., SEQ ID NO: 531 or 532) would be employed in the third polypeptide chain. [0191] In non-limiting embodiments, the Fc domain can be modified by amino acid substitution to increase binding to the neonatal Fc receptor and therefore the half-life of the antibody when administered to a subject. The Fc domain can be an IgA, IgM, IgD, IgE or IgG Fc domain. The Fc domain can be an optimized Fc domain, as described in U.S.

Published Patent Application No.20100093979, incorporated herein by reference. In certain embodiments the Fc bearing multispecific molecules comprise amino acid alterations, or combinations thereof, for example in the Fc domain(s) outside of epitope binding, which alterations can improve their properties. Various Fc modifications are known in the art. Amino acid numbering is according to the EU Index in Kabat. In some embodiments, the invention contemplates antibodies comprising mutations that affect neonatal Fc receptor (FcRn) binding, antibody half-life, and localization and persistence of antibodies at mucosal sites. See e.g. Ko SY et al., Nature 514: 642-45, 2014, at Figure 1a and citations therein; Kuo, T. and and Averson, V., mAbs 3(5): 422-430, 2011, at Table 1, US Pub 20110081347 (an aspartic acid at Kabat residue 288 and/or a lysine at Kabat residue 435), US Pub

20150152183 for various Fc Domain mutation, incorporated by reference in their entirety.

[0192] In certain embodiments, the Fc bearing multispecific molecules comprise AAAA substitution in and around the Fc Domain of the Fc bearing multispecific molecule that has been reported to enhance ADCC via NK cells (AAA mutations) containing the Fc Domain aa of S298A as well as E333A and K334A (Shields RI et al. JBC, 276: 6591-6604, 2001) and the 4^th A (N434A) is to enhance FcR neonatal mediated transport of the IgG to mucosal sites (Shields RI et al. ibid).

[0193] Other mutations have been reported to improve antibody half-life or function or both and can be incorporated into the Fc Domain of the Fc bearing multispecific molecules. These include the DLE set of mutations (Romain G, et al. Blood 124: 3241, 2014), the LS mutations M428L/N434S, alone or in a combination with other Fc Domain mutations, (Ko SY et al. Nature 514: 642-45, 2014, at Figure 1a and citations therein; Zlevsky et al., Nature

Biotechnology, 28(2): 157-159, 2010; US Pub 20150152183); the YTE Fc mutations (Robbie G et al Antimicrobial Agents and Chemotherapy 12: 6147-53, 2013) as well as other engineered mutations to the Fc Domain such as QL mutations, IHH mutations (Ko SY et al. Nature 514: 642-45, 2014, at Figure 1a and relevant citations; See also Rudicell R et al. J. Virol 88: 12669-82, 201). In some embodiments, modifications, such as but not limited to fucosylation, which may affect interaction with Fc receptors (See e.g. Moldt, et al. JVI 86(11): 66189-6196, 2012). In some embodiments, the Fc bearing multispecific molecules can comprise modifications, for example but not limited to glycosylation, which reduce or eliminate polyreactivity of such a molecule. See e.g. Chuang, et al. Protein Science 24: 1019- 1030, 2015. In some embodiments the Fc bearing multispecific molecules can comprise modifications in the Fc domain such that the Fc domain exhibits, as compared to an unmodified Fc domain enhanced antibody dependent cell mediated cytotoxicity (ADCC); increased binding to FcγRIIA or to FcγRIIIA; decreased binding to FcγRIIB; or increased binding to FcγRIIB. See e.g. US Pub 20140328836.

[0194] In another aspect, the invention provides trivalent structures incorporating two diabody-type binding domains and one non-diabody-type domain and an Fc Domain (see, e.g., Figures 35A-35F and PCT Publication Nos. WO 2015/184207 and WO 2015/184203). Such trivalent binding molecules may be utilized to generate monospecific, bispecific or trispecific molecules. The ability to bind three different epitopes provides enhanced capabilities.

[0195] A further embodiment of the present invention relates to trivalent binding molecules comprising an Fc Domain. The Fc Domain bearing trivalent binding molecules can simultaneously bind a first epitope, a second epitope, and a third epitope, wherein at least one of such epitopes is not identical to another. Such trivalent binding molecules comprise three epitope-binding sites, two of which are Diabody-Type Binding Domains, which provide binding Site A and binding Site B, and one of which is a Fab-Type Binding Domain, or an scFv-Type Binding Domain, which provides binding Site C (see, e.g., Figures 35A-35F, and PCT Publication Nos. WO 2015/184207 and WO 2015/184203). Such trivalent binding molecules thus comprise“VL1” /“VH1” domains that are capable of binding to the first epitope and“VL2” /“VH2” domains that are capable of binding to the second epitope and “VL3” and“VH3” domains that are capable of binding to the“third” epitope of such trivalent binding molecule. A“Diabody-Type Binding Domain” is the type of epitope-binding site present in a diabody, and especially, a DART® diabody, as described above. Each of a“Fab- Type Binding Domain” and an“scFv-Type Binding Domain” are epitope-binding sites that are formed by the interaction of the VL Domain of an immunoglobulin light chain and a complementing VH Domain of an immunoglobulin heavy chain. Fab-Type Binding Domains differ from Diabody-Type Binding Domains in that the two polypeptide chains that form a Fab-Type Binding Domain comprise only a single epitope-binding site, whereas the two polypeptide chains that form a Diabody-Type Binding Domain comprise at least two epitope- binding sites. Similarly, scFv-Type Binding Domains also differ from Diabody-Type Binding Domains in that they comprise only a single epitope-binding site. Thus, as used herein Fab- Type, and scFv-Type Binding Domains are distinct from Diabody-Type Binding Domains. [0196] Typically, the trivalent binding molecules of the present invention will comprise four different polypeptide chains (see Figures 35A-35B), however, the molecules may comprise fewer or greater numbers of polypeptide chains, for example by fusing such polypeptide chains to one another (e.g., via a peptide bond) or by dividing such polypeptide chains to form additional polypeptide chains, or by associating fewer or additional polypeptide chains via disulfide bonds. Figures 35C-35F illustrate this aspect of the present invention by schematically depicting such molecules having three polypeptide chains. As provided in

Figures 35A-35F, the trivalent binding molecules of the present invention may have alternative orientations in which the Diabody-Type Binding Domains are N-terminal (Figures 35A, 35C and 35D) or C-terminal (Figures 35B, 35E and 35F) to an Fc Domain. [0197] In certain embodiments, the first polypeptide chain of such trivalent binding molecules of the present invention contains: (i) a VL1-containing Domain, (ii) a VH2-containing Domain, (iii) a Heterodimer-Promoting Domain, and (iv) a Domain containing a CH2-CH3 sequence. The VL1 and VL2 Domains are located N-terminal or C-terminal to the CH2-CH3- containing domain as presented in Table 3 (also see, Figures 35A and 35B). The second polypeptide chain of such embodiments contains: (i) a VL2-containing Domain, (ii) a VH1- containing Domain, and (iii) a Heterodimer-Promoting Domain. The third polypeptide chain of such embodiments contains: (i) a VH3-containing Domain, (ii) a CH1-containing Domain and (iii) a Domain containing a CH2-CH3 sequence. The third polypeptide chain may be the heavy chain of an antibody that contains a VH3 and a heavy chain constant region, or a polypeptide that contains such domains. The fourth polypeptide of such embodiments contains: (i) a VL3-containing Domain and (ii) a CL-containing Domain. The fourth polypeptide chains may be a light chain of an antibody that contains a VL3 complementary to the VH3 of the third polypeptide chain, or a polypeptide that contains such domains. The third or fourth polypeptide chains may be isolated from naturally occurring antibodies.

Alternatively, they may be constructed recombinantly, synthetically or by other means. [0198] The Light Chain Variable Domain of the first and second polypeptide chains are separated from the Heavy Chain Variable Domains of such polypeptide chains by an intervening spacer peptide having a length that is too short to permit their VL1/VH2 (or their VL2/VH1) domains to associate together to form epitope-binding site capable of binding to either the first or second epitope. A preferred intervening peptide linker (Peptide Linker 1) for this purpose has the sequence (SEQ ID NO:508): GGGSGGGG. Other Domains of the trivalent binding molecules may be separated by one or more intervening peptide linkers (Peptide Linkers), optionally comprising a cysteine residue. In particular, as provided above, such Peptide Linkers will typically be incorporated between Variable Domains (i.e., VH or VL) and peptide Heterodimer-Promoting Domains (e.g., an E-coil or K-coil) and between such peptide Heterodimer-Promoting Domains (e.g., an E-coil or K-coil) and CH2-CH3 Domains. Exemplary peptide linkers (e.g., Peptide Linker 2, Peptide Linker 2-C, Peptide Linker 3, Spacer Linker 3, etc.) useful for the generation of trivalent binding molecules are provided above. Such linkers are also provided in PCT Publication Nos. WO 2015/184207 and WO 2015/184203. Thus, the first and second polypeptide chains of such trivalent binding molecules associate together to form a VL1/VH1 binding site capable of binding a first epitope, as well as a VL2/VH2 binding site that is capable of binding to a second epitope. The third and fourth polypeptide chains of such trivalent binding molecules associate together to form a VL3/VH3 binding site that is capable of binding to a third epitope. [0199] As described above, the trivalent binding molecules of the present invention may comprise three polypeptides. Trivalent binding molecules comprising three polypeptide chains may be obtained by linking the domains of the fourth polypeptide N-terminal to the VH3-containing Domain of the third polypeptide (e.g., using an intervening spacer peptide (Peptide Linker 5)). Alternatively, a third polypeptide chain of a trivalent binding molecule of the invention containing the following domains is utilized: (i) a VL3-containing Domain, (ii) a VH3-containing Domain, and (iii) a Domain containing a CH2-CH3 sequence, wherein the VL3 and VH3 are spaced apart from one another by an intervening spacer peptide that is sufficiently long (at least 9 or more amino acid residues) so as to allow the association of these domains to form an epitope-binding site. One preferred intervening spacer peptide for this purpose has the sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 526). [0200] It will be understood that the VL1/VH1, VL2/VH2, and VL3/VH3 Domains of such trivalent binding molecules may be different so as to permit binding that is monospecific, bispecific, or trispecific. In particular, the VL and VH Domains may be selected such that a trivalent binding molecule comprises two binding sites for a first epitope and one binding sites for a second epitope, or one binding site for a first epitope and two binding sites for a second epitope, or one binding site for a first epitope, one binding site for a second epitope and one binding site for a third epitope.

[0201] In one embodiment, these domains are selected so as to bind an epitope of HIV-1 Env, an epitope of second molecule, and an epitope of a third molecule, wherein the second molecule and the third molecule (e.g., CD2, CD3, CD8, CD16, T-Cell Receptor (TCR),

KG2D, etc.) are present on the surface of an effector cell, such as a T lymphocyte, a natural killer (NK) cell or other mononuclear cell.

[0202] The general structure of the polypeptide chains of representative trivalent binding molecules of invention is provided in Figures 35A-35F and in Table 3:

HPD = Heterodimer-Promoting Domain

[0203] One embodiment of the present invention relates to trivalent binding molecules that comprise two epitope-binding sites for HIV-1 Env and one epitope-binding site for a second molecule. The two epitope-binding sites for HIV-1 Env may bind the same epitope or different epitopes. Another embodiment of the present invention relates to trivalent binding molecules that comprise, one epitope-binding site for HIV-1 Env and two epitope-binding sites for a second molecule. The two epitope-binding sites for the second molecule may bind the same epitope or different epitopes of the second molecule. A further embodiment of the present invention relates to trispecific trivalent binding molecules that comprise, one epitope- binding site for HIV-1 Env, one epitope-binding site for a second molecule, and one epitope- binding site for a third molecule. In certain embodiments, the second molecule is a molecule (e.g., CD2, CD3, CD8, CD16, T-Cell Receptor (TCR), NKG2D, etc.) present on the surface of an effector cell, such as a T lymphocyte, a natural killer (NK) cell or other mononuclear cell. In certain embodiments, the second molecule is CD3 and the third molecule is CD8. As provided above, such trivalent binding molecules may comprise three, four, five, or more polypeptide chains. [0204] In one embodiment, these domains are selected so as to bind two epitopes of HIV-1 Env, which may be the same epitopes or different epitopes, and an epitope of second molecule, wherein the second molecule is a molecule (e.g., CD2, CD3, CD8, CD16, T-Cell Receptor (TCR), NKG2D, etc.) present on the surface of an effector cell, such as a T lymphocyte, a natural killer (NK) cell or other mononuclear cell. In a specific embodiment, the two epitopes of HIV-1 Env are the same, and the second molecule is CD3. In an alternative embodiment, the two epitopes of HIV-1 Env are different, and the second molecule is CD3. [0205] The molecules, and fragments thereof, described above can be formulated as a composition (e.g., a pharmaceutical composition). Suitable compositions can comprise the molecules (or fragments thereof) in a pharmaceutically acceptable carrier e.g., dissolved or dispersed in an aqueous medium, or lyophilized. The compositions can be sterile and can be in an injectable form (e.g. but not limited to a form suitable for intravenous injection, or intramuscular injection). The molecules (and fragments thereof) can also be formulated as a composition appropriate for topical administration to the skin or mucosa. Such compositions can take the form of liquids, ointments, creams, gels and pastes. The molecules (and fragments thereof) can also be formulated as a composition appropriate for intranasal administration. The molecules (and fragments thereof) can be formulated so as to be administered as a post-coital douche or with a condom. Standard formulation techniques can be used in preparing suitable compositions.

[0206] In certain embodiments the invention provides multispecific molecules such as but not limited to bispecific and trispecific molecules (e.g., bispecific antibodies, bispecific diabodies, trivalent binding molecules, etc.) comprising the binding domains from human, humanized and/or chimeric antibodies. Methods to construct such antibodies are well known in the art.

[0207] In certain aspects the invention provides use of the multispecific molecules of the invention such as but not limited to bispecific and trispecific molecules (e.g., bispecific antibodies, bispecific diabodies, trivalent binding molecules, etc.), in methods of treating and preventing HIV-1 infection in an individual, comprising administering to the individual a therapeutically effective amount of a composition comprising a multispecific molecule of the invention in a pharmaceutically acceptable form. In certain embodiment, the methods include a composition which includes more than one HIV-1 targeting multispecific molecule. In certain embodiments, the HIV-1 targeting multispecific molecule in such combination bind different epitopes on the HIV-1 envelope. In certain embodiments, such combinations of multispecific molecule targeting more than one HIV-1 epitope provide increased killing of HIV-1 infected cells. In other embodiments, such combinations of multispecific molecule targeting more than one HIV-1 epitope provide increased breadth in recognition of different HIV-1 subtypes.

[0208] The various multispecific molecule described herein have utility, for example, in settings including but not limited to the following:

i) in the setting of anticipated known exposure to HIV-1 infection, the multispecific molecule described herein can be administered prophylactically (e.g., IV, topically or intranasally) as a microbiocide,

ii) in the setting of known or suspected exposure, such as occurs in the setting of rape victims, or commercial sex workers, or in any homosexual or heterosexual transmission without condom protection, the multispecific molecule described herein can be administered as post-exposure prophylaxis, e.g., IV or topically, and

iii) in the setting of Acute HIV-1 infection (AHI), the multispecific molecule described herein can be administered as a treatment for AHI to control the initial viral load, or for the elimination of virus-infected CD4 T cells.

[0209] In accordance with the invention, the multispecific molecules described herein can be administered prior to contact of the subject or the subject's immune system/cells with HIV-1 or within about 48 hours of such contact. Administration within this time frame can maximize inhibition of infection of vulnerable cells of the subject with HIV-1.

[0210] In addition, various forms of the multispecific molecules described herein can be administered to chronically or acutely infected HIV-1 patients and used to kill remaining virus infected cells by virtue of these multispecific molecule binding to the surface of virus infected cells and being able to mediate redirected cell killing of such infected cells.

[0211] In certain embodiments, the multispecific molecules of the invention can be administered in combination with latency activating agents, so as to activate latent reservoir of HIV-1-infected cells. The expectation is that by activating latent proviral HIV-1 DNA in resting cells, once inactive cells will start producing new virus and they will be recognized and eliminated by the immune system. Non-limiting examples of latency activating agents are HDAC inhibitors, e,g, vorinostat, romidepsin, panobinostat, disulfiram, JQ1, bryostatin, PMA, inonomycin, or any combination thereof. See Bullen et al. Nature Medicine 20, 425– 429 (2014).

[0212] In certain embodiments the multispecific molecules of the invention can be

administered in combination with anti-retroviral agents.

[0213] Suitable dose ranges can depend on the multispecific molecule and on the nature of the formulation and route of administration. Optimum doses can be determined by one skilled in the art without undue experimentation. For example, doses of antibodies in the range of 1-50 mg/kg of unlabeled or labeled antibody (with toxins or radioactive moieties) can be used. If antibody fragments, with or without toxins are used or antibodies are used that can be targeted to specific CD4 infected T cells, then less antibody can be used (e.g., from 5 mg/kg to 0.01 mg/kg). If multispecific molecules are used, doses in the range of 0.01 μg/kg to about 30 mg/kg or more of the subject’s body weight can be used. Suitable dose ranges can depend on the antibody (or fragment, or multispecific molecule) and on the nature of the formulation and route of administration. Optimum doses can be determined by one skilled in the art without undue experimentation. For example but not limited, doses of multispecific molecules in the range of 0.01-100 μg/kg, 0.1-50 mg/kg, 1-50 mg/kg, 1-10 mg/kg, 1, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mg/kg of unlabeled or labeled multispecific molecule (with toxins or radioactive moieties) can be used. If antibody fragments, with or without toxins are used or antibodies are used that can be targeted to specific CD4 infected T cells, then less antibody can be used (e.g., from 5 mg/kg to 0.01 mg/kg). In other

embodiments, the molecules of the invention can be administered at a suitable fixed dose, regardless of body size or weight. See Bai et al. Clinical Pharmacokinetics February 2012, Volume 51, Issue 2, pp 119-135. [0214] Multispecific molecules of the invention can be produced recombinantly using nucleic acids comprising nucleotide sequences encoding VH and VL sequences selected from those shown in the figures and examples, or those known in the art.

[0215] In certain embodiments the invention provides multispecific binding molecules comprising antigen binding fragments. Typically, multispecific binding molecules compete with the intact antibody from which they were derived for specific binding to the target including separate heavy chains, light chains Fab, Fab', F(ab').sub.2, F(ab)c, diabodies, Dabs, nanobodies, and Fv. Fragments that can be produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins.

[0216] Nucleic acid sequences encoding polypeptides for the production of multispecific molecules with specificities as described herein can be used to produce plasmids for stable expression of such multispecific molecules. Methods for recombinant expression and purification are known in the art. In certain embodiments of Fc bearing multispecific molecules, the plasmids also comprise any of the changes to the Fc portion described herein.

[0217] In certain embodiments, the nucleic acids are optimized for recombinant expression in a suitable host cell. In certain embodiments, the vector is suitable for gene delivery and expression. There are numerous expression systems available for expression of proteins including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines.

[0218] Any suitable cell line can be used for expression of the polypeptides of the invention, including but not limited to CHO cells, 293T cells. In some aspects, the invention provides nucleic acids encoding these antibodies, expression cassettes and vectors including these nucleic acids, and isolated cells that express the nucleic acids which encode the multispecific molecules of the invention are also provided. The polypeptides of the invention can be purified by any suitable method for purification of polypeptides and/or antibodies.

[0219] Table 4. Summary listing of various sequences listed throughout the specification; starting at SEQ ID NO: 500

[0220] Various antigen binding domains capable of binding to an epitope of HIV-1, CD3, CD16 and CD8 are contemplated by the invention and are disclosed in the specification.

Table 4 discloses non-limiting embodiments of such antigen binding domains which may be incorporated into the multispecific molecules of the invention. Additional, alternative antigen binding domains from other antibodies having specificity for the desired antigens may be utilized. Many such antibodies are known in the art, for example additional anti-CD3 antibodies are described in WO2012/162067 and WO 2014/110601 the contents of each of which are hereby incorporated by reference; additional anti-CD16 antibodies are described in WO 03/101485 the contents of which is hereby incorporated by reference; and additional anti- CD8 antibodies are described in WO 2014/164553 the contents of which is hereby

incorporated by reference. [0221] Various Heterodimer-Promoting Domain (HPD) sequences are contemplated by the invention and disclosed in the specification. Table 4 discloses non-limiting embodiments of various HPDs. In some embodiments the HPD include E/K-coils (SEQ ID NOs: 518, 510) or cysteine engineered E/K-coils (SEQ ID NOs: 519, 521). In some embodiments HPD includes combinations of SEQ ID NOs: 511, 512, 513, and 514 sequences (e.g., SEQ ID NOs: 511 and 513; SEQ ID NOs: 512 and 513; SEQ ID NOs: 511 and 514; SEQ ID NOs: 512 and 514); kappa and lambda light chain constant domains (SEQ ID NOs: 516 and 517). In some embodiments HPD include any suitable sequences with a Cystein residue to permit disulfide bond. In some embodiments HPD includes suitable CH1 and CL domains. [0222] A skilled artisan readily appreciates that the disclosure throughout the application of various design elements such as but not limited to HPDs and sequences, linkers, CH2-CH3 domain and Fc domain and their variants is applicable and could be used in any of the designs of the multispecific molecules of the invention that comprise these design elements.

[0223] HIV-1 Antibodies

[0224] Broadly neutralizing and potent HIV-1 envelope glycoprotein (Env) antibodies are now being developed for both prevention of HIV-1 (Rudicell RS et al. J. Virol 88: 12669,-82, 2014) and for treatment of HIV-1 infected individuals (Barouch DH, et al. Nature 503: 224-8, 2013; Shingai M et al. Nature 503: 277-80, 2013). Thus, human recombinant antibodies either alone or in combinations have great prophylactic and therapeutic potential for the prevention and treatment of HIV-1 infection. Moreover, antibodies that bind with high affinity to Env may be useful in eliminating the latent pool of HIV-1–infected CD4 T cells and curing HIV-1 infection, when either used to sensitize HIV-1 expressing target cells with bispecific bnAbs for NK or CD8 T cell killing or when bnAbs are conjugated with toxins or radionucleotides.

[0225] In certain aspects the invention provides fully human antibodies and fragments that specifically bind to and potently neutralize various isolates of HIV-1. In some embodiments, the antibodies bind to HIV-1 env V3 glycan. In some embodiments, the antibodies of the invention are combined in compositions with antibodies to HIV-1 gp120 Env CD4 binding site.

[0226] In certain aspects the invention provides pharmaceutical compositions including these human antibodies and a pharmaceutically acceptable carrier. In certain aspects the invention provides antibodies for passive immunization against HIV/AIDS. Nucleic acids encoding these antibodies, expression cassettes and vectors including these nucleic acids, and isolated cells that express the nucleic acids which encode the antibodies of the invention are also provided.

[0227] In some embodiments, the invention provides antibodies which are clonal variants. In some embodiments, clonal variants are sequences that differ by one or more nucleotides or amino acids, and have a V region with shared mutations compared to the germline, identical VHDJH or VJH gene usage, identical or similar HCDR3 length, and the same VL and JL usage. The germline sequence (unmutated common ancestor“UCA”) is intended to be the sequence coding for the antibody/immunoglobulin (or of any fragment thereof) deprived of mutations, for example somatic mutations. Antibodies in a clone that are designated as UCA and/or I (for“Intermediate”) are typically not identified from a biological sample, but are derived computationally based on VH and/or VL sequences isolated from subjects infected with HIV-1.

[0228] Compositions including the human antibodies of the invention, including V3 glycan and CD4 binding site antibodies, can be used for any purpose including but not limited to research, diagnostic and therapeutic purposes.

[0229] The neutralization breadth of the inventive antibodies is demonstrated by the diversity of viruses which are neutralized in the TZMbl Env pseudovirus inhibition assay. In certain embodiments, the neutralization breadth and/or binding of the antibodies of the invention can be maintained in the presence of tolerate changes to the epitope. Comparing the sequences of the neutralized viruses, versus viruses that are not neutralized, a skilled artisan can readily determine the % virus changes, including changes in the epitope, which can be tolerated while neutralization and/or binding is maintained.

[0230] Comparing the sequences of the antibodies and their neutralization properties, a skilled artisan can readily determine sequence identity, compare sequence length and determine the % sequence identity and/or changes, including % sequence identity and/or changes in the VH and VL sequences, including % sequence identity and/or changes in the CDRs, as well as the specific positions and types of substitutions which can be tolerated while neutralization potency and breadth is maintained.

[0231] Various algorithms for sequence alignment are known in the art. The similarity between amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods.

[0232] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol.48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444, 1988. Altschul et al., Nature Genet.6:119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations.

[0233] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for

Biotechnology Information (NCBI, Bethesda, Md.) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.

[0234] Homologs and variants of a VL or a VH of an antibody that specifically binds a polypeptide are typically characterized by possession of at least about 75%, for example at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full length alignment with the amino acid sequence of interest.

[0235] As used herein, reference to antibodies, without explicit mention of antibody fragments and antibody-fragment comprising molecules, may encompass antibody fragments and antibody-fragment comprising molecules.

[0236] In certain embodiments, the invention provides antibodies and antibody-fragment comprising molecules, including multispecific molecules, such as, but not limited to bispecific and trispecific molecules (e.g., bispecific antibodies, bispecific diabodies, trivalent binding molecules, etc.) which are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% identical to the VH and VL amino acid sequences of the antibodies described herein and still maintain the neutralization breadth, biding and/or potency. In certain embodiments, the invention provides antibodies and antibody-fragment comprising molecules, including multispecific molecules, such as, but not limited to bispecific and trispecific molecules (e.g., bispecific antibodies, bispecific diabodies, trivalent binding molecules, etc.) which are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% identical to the CDR1, 2, and/or 3 of VH and CDR1, 2, and/or 3 VL amino acid sequences of the antibodies described herein and still maintain the neutralization breadth, biding and/or potency.

[0237] In certain embodiments, the invention provides antibodies and antibody-fragment comprising molecules, including multispecific molecules, such as, but not limited to bispecific and trispecific molecules (e.g., bispecific antibodies, bispecific diabodies, trivalent binding molecules, etc.) which can tolerate a larger percent variation in the sequences outside of the VH and/VL sequences of the antibodies. In certain embodiments, the invention provides antibodies which are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65% identical, wherein the identity is outside of the VH or VL regions, or the CDRs of the VH or VL chains of the antibodies described herein.

[0238] Antibodies of the invention are expected to have the same binding specificity, for example as intact immunoglobulins and antigen binding variants or fragments e.g. as a number of well characterized fragments produced by digestion with various peptidases. For instance and without limitation, Fabs, Fvs, scFvs are fragments which are expected to have the same binding specificities as intact antibodies. Binding specificity can be determined by any suitable assay in the art, for example but not limited competition binding assays, epitope mapping, etc. Assays to determine glycan dependence and glycan specificity binding are also known in the art. A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. Provided are also genetically engineered forms such as chimeric antibodies and heteroconjugate antibodies such as bispecific antibodies. See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby,

Immunology, 3.sup.rd Ed., W.H. Freeman & Co., New York, 1997.

[0239] In certain embodiments the invention provides antibody fragments and molecules comprising antibody fragments, which have the binding specificity and/or properties of the inventive antibodies. Non-limiting examples include: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab').sub.2, the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab').sub.2, a dimer of two Fab' fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. In certain embodiments, the antibody fragments can be produces recombinantly. [0240] In certain embodiments, VH refers to the variable region of an immunoglobulin heavy chain, including but not limited to that of an antibody fragment, such as Fv, scFv, dsFv or Fab. In certain embodiments, VL refers to the variable region of an immunoglobulin light chain, including but not limited to that of an Fv, scFv, dsFv or Fab.

[0241] Any of the nucleic acids encoding any of the antibodies, or fragment thereof can be expressed in a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells. The nucleic acid sequences include any sequence necessary for expression, including but not limited to a promoter, a leader sequence. These antibodies can be expressed as individual VH and/or VL chain, or can be expressed as a fusion protein. In certain embodiments, the antibodies can be expressed by viral vector mediated delivery of genes encoding the antibodies of the invention (See e.g. Yang et al. Viruses 2014, 6, 428-447).

[0242] The present invention also encompasses molecules comprising a hinge domain. The hinge domain be derived from any immunoglobulin isotype or allotype including IgA, IgD, IgG, IgE and IgM. In preferred embodiments, the hinge domain is derived from IgG, wherein the IgG isotype is IgG1, IgG2, IgG3 or IgG4, or an allotype thereof. The hinge domain may be engineered into a polypeptide chain comprising the diabody molecule together with an Fc domain such that the diabody molecule comprises a hinge-Fc domain. In certain

embodiments, the hinge and Fc domain are independently selected from any immunoglobulin isotype known in the art or exemplified herein. In other embodiments the hinge and Fc domain are separated by at least one other domain of the polypeptide chain, e.g., the VL domain.

[0243] Antibody nomenclature and names: UCA4 = DH270.UCA; IA4 = DH270.IA4; IA3 = DH270.IA3; IA2 = DH270.IA2; IA1 = DH270.IA1; DH270 = DH270.1; DH473 = DH270.2; DH391 = DH270.3; DH429 = DH270.4; DH471 = DH270.5; DH542 = DH270.6; DH542-L4 (comprising VH from DH542 and VL from DH429), DH542_QSA.

[0244] It is readily understood that the nucleic acid sequences disclosed in the application are non-limiting embodiments of representative nucleotide sequences encoding the respective amino acid sequences. [0245] The contents of the various publications cited throughout the specification are incorporated by reference in their entirety.

[0246] The following examples are provided to illustrate particular features of certain embodiments, but the scope of the claims should not be limited to those features exemplified. EXAMPLES^

Example^1:^Isolating^antibodies^from^natural^HIV‐1^infected^

individuals^

[0247] Methods to identify and isolate antigen specific reactive antibodies were carried out essentially as described in Liao HX et al. J. Virol. Methods 158: 171-9, 2009, incorporated by reference in its entirety. Specific hooks are designed to identify memory B cells which express antibodies that bind to specific HIV-1 envelope targets/antigens. Using such hooks, with fluorophore labeled streptavidin in two colors, cells are sorted by flow cytometry, into single wells, and the diagonally (that reacted with both colors hooks) reactive memory B cells are picked. B cells enriched from PBMC are sorted, and plated at limiting dilution (as single cell per well). Optionally, these cultures are grown and supernatants are functionally characterized.

[0248] PCR on these cells is carried out according to the protocol in Liao HX et al. J. Virol. Methods 158: 171-9, 2009. PCR amplifications are carried out to amplify rearranged VH and VL fragment pairs from the diagonally sorted memory B cells (Liao et al JVM 158: 171-9, 2009). Overlapping PCR is used to construct full length Ig heavy and Ig light linear genes comprising the rearranged VH and VL fragment pairs. RT-PCR and PCR reactions is carried out essentially as described in Liao HX et al. J. Virol. Methods 158: 171-9, 2009, see for example Figure 1, Section 3.3. Sequence analysis of the VH and VL genes was carried out to determine the VH and VL gene usage, CDR lengths, the % mutation of HCDR3 and LCDR3. Based on this sequence analysis, one to two pairs of linear VH and VL genes are selected and made in linear cassettes (essentially as described in Liao HX et al. J. Virol. Methods 158: 171-9, 2009, see for example Figure 1, Section 3.3) to produce recombinant monoclonal antibodies by transient transfection, e.g. in 293T cells.

[0249] Recombinant antibodies are grown and supernatants and/or purified antibodies are functionally characterized.

[0250] Pairs of VH and VL genes as selected above can also be used to produce plasmids for stable expression of recombinant antibodies.

[0251] In certain embodiments, the plasmids or linear constructs for recombinant antibody expression also comprise AAAA substitution in and around the Fc region of the antibody that has been reported to enhance ADCC via NK cells (AAA mutations) containing the Fc region aa of S298A as well as E333A and K334A (Shields RI et al JBC , 276: 6591-6604, 2001) and the 4^th A (N434A) is to enhance FcR neonatal mediated transport of the IgG to mucosal sites (Shields RI et al. ibid).

[0252] The antibodies of the invention were selected based on a combination of criteria including sequence analyses, and functional analyses including but not limited as

neutralization breadth, and potency.

[0253] In certain embodiments, the antibodies of the invention comprise naturally rearranged VH and VL gene pairs isolated as nucleic acids, wherein the rest of the Ig gene is not naturally occurring with the isolated rearranged VH and VL fragments. In certain

embodiments, the antibodies of the invention are recombinantly produced. Example^2:^Antibodies^from^DH270^lineage^

[0254] Antibodies I1 (DH270IA1), I2, I4, I3 and UCA in Figures 8-11 are not isolated from human subjects but are derived computationally based on VH and VL sequences of other clonal antibodies identified from memory B cells: DH471, DH429, DH473, DH391 and DH270. The VH and VL sequences of DH471, DH429, DH473, DH391 and DH270 are derived from a human subject infected with HIV-1.

[0255] The VH and VL sequences of DH471, DH429 and DH473are derived essentially as described in Example 1, using Consensus C gp120 Env and Consensus C gp120 N332A Env glycopeptides and by sorting cells that bound to Consensus C gp120 Env but not to

Consensus C gp120 N332A Env. DH270 and DH391 were recombinantly produced from VH and VL chains isolated from clonal memory B cell cultures that bound to Consensus C gp120 Env but not to Consensus C gp120 N332A Env using the method previously described (Bonsignori et al J Virol 2011, Gao Bonsignori Liao et al Cell 2014).

[0256] DH542 antibody isolation

[0257] Biotinylated Man₉GlcNac₂ V3 peptides were tetramerized via streptavidin and conjugated with either AF647 or BV421 (Invitrogen) dyes. Peptide tetramer quality following conjugation was assessed by flow cytometry to a panel of well-characterized HIV-1 V3 glycan antibodies (PGT128, and 2G12) and linear V3 antibodies (F39F). The sequence of Man9V3 glycopeptide is EINCTRPNNNTRPGEIIGDIRQAHCNISRA. This is a synthetic glycopeptide which has N-linked glycans (Man9GlcNac2) placed at the Asparagine residues in bold/underlined. The cysteine residues at the N and C terminal form a disulfide linkage under oxidative conditions creating a very stable cyclical conformation that expresses the epitope bound by V3 glycan bnAbs such as PGT128, 125, and 2G12, and now DH542 and other DH270 lineage members.

[0258] Roughly 10 million peripheral blood mononuclear cells (PBMCs) from the HIV-1- infected donor 703-01-084-8 (CH848) collected 234 weeks post infection were stained with AquaVital dye, anti-human IgM (FITC), anti-human IgD (PE), anti-human CD10 (ECD), anti-human CD3 (PE-Cy5), anti-human CD235a (PE-Cy5), anti-human CD27 (PE-Cy7), anti- human CD38 (APC-AF700), anti-human CD19 (APC-Cy7), anti-human CD16 (BV570), anti- human CD14 (BV605), and Man₉GlcNac₂ V3 tetramer in both AF647 and BV421. PBMCs that were AquaVital dye-, CD14-, CD16-, CD3-, CD235a-, IgD-, CD19⁺, CD38⁺, and

Man₉GlcNac₂ V3⁺ were single-cell sorted using a BD FACS Aria II into 96-well plates containing 20 μl of reverse transcriptase buffer (RT). cDNA synthesis was performed as previously described (1). Immunoglobulin (Ig) heavy (VH) chains were PCR amplified using a nested approach. VH genes were amplified in the first round of amplification with primers grouped in Table 9a-9e as previously described (2). Nested amplification of VH genes was performed as in (Liao et al., 2009) with primers grouped in Table 10a. Kappa and lambda were amplified as in (Liao et al., 2009), with primers grouped in Table 10b-10c. PCR products were analyzed on 2% SYBR Safe E-Gels (Invitrogen). PCR-amplified VH and VL genes were purified and sequenced. Sequences were analyzed and VDJ arrangements were inferred using computational methods as previously described (3, 4).

[0259] Table 9a (SEQ ID NOs: 201-211, in order of appearance)

SEQ ID NOs: 212-222, in order of appearance:

Forward Primer Sequence

VH1 Leader A ATGGACTGGACCTGGAGGAT

VH1 Leader B ATGGACTGGACCTGGAGCAT

VH1 Leader C ATGGACTGGACCTGGAGAAT

VH1 Leader D GGTTCCTCTTTGTGGTGGC

VH1 Leader E ATGGACTGGACCTGGAGGGT

VH1 Leader F ATGGACTGGATTTGGAGGAT

VH1 Leader G AGGTTCCTCTTTGTGGTGGCAG VH2 Leader A ATGGACATACTTTGTTCCACGCTC VH2 Leader B ATGGACACACTTTGCTCCACGCT VH2 Leader C ATGGACACACTTTGCTACACACTC Reverse Primer

3’ Cγ CH1 GGAAGGTGTGCACGCCGCTGGTC

[0260] Table 9b (SEQ ID NOs: 223-230, in order of appearance) Forward Primer Sequence

VH3 Leader A TAAAAGGTGTCCAGTGT

VH3 Leader B TAAGAGGTGTCCAGTGT VH3 Leader C TAGAAGGTGTCCAGTGT

VH3 Leader E TACAAGGTGTCCAGTGT

VH3 Leader F TTAAAGGTGTCCAGTGT

VH4 Leader D ATGAAACATCTGTGGTTCTT

VH5 Leader A TTCTCCAAGGAGTCTGT

Reverse Primer

3’ Cγ CH1 GGAAGGTGTGCACGCCGCTGGTC

[0261] Table 9c (SEQ ID NOs: 231-238, in order of appearance) Forward Primer Sequence

VH3 Leader D GCTATTTTTAAAGGTGTCCAGTGT

VH4 Leader A ATGAAACACCTGTGGTTCTTCC

VH4 Leader B ATGAAACACCTGTGGTTCTT

VH4 Leader C ATGAAGCACCTGTGGTTCTT

VH5 Leader B CCTCCACAGTGAGAGTCTG

VH6 Leader A ATGTCTGTCTCCTTCCTCATC

VH7 Leader A GGCAGCAGCAACAGGTGCCCA

Reverse Primer

3’ Cγ CH1 GGAAGGTGTGCACGCCGCTGGTC

[0262] Table 9d (SEQ ID NOs: 239-243, in order of appearance) Forward Primer Sequence

Vκ1,2 Ext GCTCAGCTCCTGGGGCT

Vκ3 Ext GGAARCCCCAGCDCAGC

Vκ4/5 Ext CTSTTSCTYTGGATCTCTG

Vκ6/7 Ext CTSCTGCTCTGGGYTGC

Reverse Primer

CK Ext GAGGCAGTTCCAGATTTCAA

[0263] Table 9e (SEQ ID NOs: 244-254, in order of appearance) Forward Primer Sequence

VL1 Ext CCTGGGCCCAGTCTGTG

VL2 Ext CTCCTCASYCTCCTCACT

VL3 Ext GGCCTCCTATGWGCTGAC

VL3l Ext GTTCTGTGGTTTCTTCTGAGCTG

VL4ab Ext ACAGGGTCTCTCTCCCAG VL4c Ext ACAGGTCTCTGTGCTCTGC

VL5,9 Ext CCCTCTCSCAGSCTGTG

VL6 Ext TCTTGGGCCAATTTTATGC

VL7,8 Ext ATTCYCAGRCTGTGGTGAC

VL10 CAGTGGTCCAGGCAGGG

Reverse Primer

CL Ext AGGCCACTGTCACAGCT

[0264] Table 10a (SEQ ID NOs: 255-261, in order of appearance)

Forward Primer Sequence

VH1 Int

CTGGGTTCCAGGTTCCACTGGTGACCAGGTGCAGCTGGTRCAGTCTGGG VH2 Int

CTGGGTTCCAGGTTCCACTGGTGACCAGRGCACCTTGARGGAGTCTGGTCC VH3 Int

CTGGGTTCCAGGTTCCACTGGTGACGAGGTKCAGCTGGTGGAGTCTGGG VH4 Int

CTGGGTTCCAGGTTCCACTGGTGACCAGGTGCAGCTGCAGGAGTCGG VH5 Int

CTGGGTTCCAGGTTCCACTGGTGACGARGTGCAGCTGGTGCAGTCTGGAG VH6 Int

CTGGGTTCCAGGTTCCACTGGTGACCAGGTACAGCTGCAGCAGTCAGGTCC

Reverse Primer

IgG Int GGGCCGCTGTGCCCCCAGAGGTGCTCTYGGA

[0265] Table 10b (SEQ ID NOs: 262-269, in order of appearance)

Forward Primer Sequence

VK1 Int

CTGGGTTCCAGGTTCCACTGGTGACGACATCCAGWTGACCCAGTCTC VK2 Int

CTGGGTTCCAGGTTCCACTGGTGACGATATTGTGATGACCCAGWCTCCAC VK3 Int

CTGGGTTCCAGGTTCCACTGGTGACGAAATTGTGTTGACRCAGTCTCCA VK4 Int

CTGGGTTCCAGGTTCCACTGGTGACGACATCGTGATGACCCAGTCTC VK5 Int

CTGGGTTCCAGGTTCCACTGGTGACGAAACGACACTCACGCAGTCTC VK6 Int

CTGGGTTCCAGGTTCCACTGGTGACGAAATTGTGCTGACWCAGTCTCCA VK7 Int CTGGGTTCCAGGTTCCACTGGTGACGACATTGTGCTGACCCAGTCT Reverse Primer

CK Int GGGAAGATGAAGACAGATGGT

[0266] Table 10c (SEQ ID NOs: 270-280, in order of appearance)

Forward Primer Sequence

VL1 Int CTGGGTTCCAGGTTCCACTGGTGACCAGTCTGTGYTGACKCAGCC VL2 Int CTGGGTTCCAGGTTCCACTGGTGACCAGTCTGCCCTGACTCAGCC VL3 Int

CTGGGTTCCAGGTTCCACTGGTGACTCYTATGAGCTGACWCAGCCAC VL3l Int

CTGGGTTCCAGGTTCCACTGGTGACTCTTCTGAGCTGACTCAGGACCC VL4ab Int CTGGGTTCCAGGTTCCACTGGTGACCAGCYTGTGCTGACTCAATC VL4c Int CTGGGTTCCAGGTTCCACTGGTGACCTGCCTGTGCTGACTCAGC VL5,9 Int CTGGGTTCCAGGTTCCACTGGTGACCAGSCTGTGCTGACTCAGCC VL6 Int

CTGGGTTCCAGGTTCCACTGGTGACAATTTTATGCTGACTCAGCCCCACT VL7,8 Int CTGGGTTCCAGGTTCCACTGGTGACCAGRCTGTGGTGACYCAGGAG VL10 Int CTGGGTTCCAGGTTCCACTGGTGACCAGGCAGGGCWGACTCAG Reverse Primer

CL Int GGGYGGGAACAGAGTGACC

[0267] Antibody Expression

[0268] Transient and recombinant monoclonal antibody production was performed as previously described (5, 6).

[0269] DH270 N332 dependent V3 glycan bnAb lineage.

[0270] We describe here the co-evolution of a founder virus and a memory B cell lineage of gp120 V3-glycan directed bnAbs (DH270). We sequenced ~1400 HIV quasispecies, isolated natural heavy- and light-chain pairs of 6 lineage DH270 antibodies, and analyzed this lineage by next generation sequencing (NGS) and structural studies. We found two additional TF- induced cooperating B-cell lineages that selected virus escape mutants that stimulated the DH270 lineage to potent neutralization breadth. Within the DH270 lineage we found a single early antibody CDR H2 mutation that was necessary for bnAb B cell lineage initiation. The combination for multiple cooperating lineages plus a rare antibody mutation thus explains the long period of antigenic stimulation required for bnAb induction.

[0271] We studied an African individual (CH848) followed from time of infection to development of plasma neutralization breadth. Abrogation of the N332 glycan near the Env V3 loop by introducing a N332A mutation into consensus C, TRO.11, Q23.17 and DU156 HIV-1 pseudoviruses reduced CH848 plasma neutralization of these viruses, and

demonstrated the presence of plasma N332-sensitive bnAbs (reference Georgiev paper). To isolate them, we probed memory B cells from weeks 205, 232 and 234 post-transmission using clonal memory B cell cultures and antigen-specific memory B cell sorting and isolated 6 naturally paired VH + VL N332-sensitive antibodies, designated DH270.1-6.

Neutralization studies demonstrated that DH270 though containing only 5.5% VH mutations, mediated potent heterologous neutralization breadth (65.2% breadth and median IC50=0.17 ug/ml) (Figure 28A-C).

[0272] We interrogated the depth of the DH270 clonal lineage by sequencing the DH270 variable heavy (V_H) gene using next generation sequencing (NGS) of memory B cell cDNA isolated at 11, 19, 64, 111, 160, 186 and 240 weeks post-transmission. A total of 767 unique DH270 lineage V_H sequences from duplicated NGS experiments were found, with the earliest V_H detected 186 weeks post-transmission (Figure 28A). The DH270 lineage used VH1-2*02 paired with V ^2-23 and had a 20 amino acid-long CDR H3. Clonal lineage intermediate and ancestor antibodies were computed from the naturally-paired sequences (Kepler refs) (Figure 29). V_H mutation frequencies of the isolated antibodies ranged from 5.6% (DH270.1) to 12.9% (DH270.6) (Figure 29). Neutralization of wild-type and N332 mutated HIV-1 strains AC13.8, PVO4, TRO.11, AC10.0.29 and RHPA confirmed DH270 lineage N332 sensitivity of neutralization (Figure 30). In competition binding assays, DH270.1 blocked binding of both V3-glycan bnAbs PGT125 and PGT128 to JRFL gp120 with IC50 = 0.4 ug/ml and DH270.1 binding to gp120 Env was dependent on N332 glycans.

[0273] Ontogeny of DH270 neutralizing B cell lineage

[0274] DH270 lineage antibodies displayed bnAb activity in a panel of 24 heterologous HIV- 1 isolates, with DH270.6 the most broad (17 of 24) and potent (IC50 = 0.21 ug/ml) (Figure 28B). The DH270 unmutated common ancestor antibody (DH270.UCA) did not neutralize heterologous HIV-1, but intermediate antibody 4 (DH270.IA4), which differed from UCA by 4 amino acids in the VH gene segment and one amino acid in VL neutralized 4/24 strains (16.7%) (Figure 28B). Notably, DH270.IA4 acquired neutralizing activity while retaining the unmutated CDR H3 of the UCA.

[0275] As V_H mutations accumulated in the DH270 B cell lineage, neutralization broadend only modestly while potency increased by 2 orders of magnitude. Thus, DH270-lineage heterologous neutralization evolved in two phases: first, early mutations conferred neutralization breadth , and second, further mutations enhanced neutralization potency.

[0276] DH270.1, DH270.5 and DH270.6 neutralization breadth was further evaluated in a large multi-clade panel of 201 HIV-1 heterologous strains (Figure 28C). DH270.1 neutralized 88/201 (43.8%) HIV-1 isolates (median IC50 = 0.39 ug/ml), DH270.5 neutralized 99/201 (49.3%) HIV-1 isolates (median IC50 = 0.14 ug/ml), while DH270.6 was the most broad and potent antibody and neutralized 111/199 viruses (56%) (median IC50 = 0.07 ug/ml).

[0277] Antibody DH270.6 displayed strong clade preference for clade B (78%, n = 41, p = 0.0043) and sensitivity for clade C viruses (68%, n = 55, p = 0.029). It also neutralized 46% of clade A viruses and did not neutralize CRF01 AE viruses. Presence for a glycosylation site in position N332 explained 75% of resistance of heterologous viruses to DH270.6. None of the viruses that shifted N332 glycan to N334 were neutralized by DH270.6 and, within each clade, N332 glycan tracked well with virus sensitivity to DH270.6 (p=0.03, Kendall’s rank correlation). When compared to PGT128 and 10-1074, the neutralization profile of DH270.6 most closely paralleled that of 10-1074 (Figure 31).

[0278] References for Example 2: 1. Wardemann H, Yurasov S, Schaefer A, Young JW, Meffre E, Nussenzweig MC. Predominant autoantibody production by early human B cell precursors. Science (New York, NY).2003;301(5638):1374-7. Epub 2003/08/16. doi: 10.1126/science.1086907. PubMed PMID: 12920303.

2. Scheid JF, Mouquet H, Ueberheide B, Diskin R, Klein F, Oliveira TY, Pietzsch J, Fenyo D, Abadir A, Velinzon K, Hurley A, Myung S, Boulad F, Poignard P, Burton DR, Pereyra F, Ho DD, Walker BD, Seaman MS, Bjorkman PJ, Chait BT, Nussenzweig MC. Sequence and structural convergence of broad and potent HIV antibodies that mimic CD4 binding. Science (New York, NY).2011;333(6049):1633-7. Epub 2011/07/19. doi:

10.1126/science.1207227. PubMed PMID: 21764753; PubMed Central PMCID:

PMCPmc3351836. 3. Kepler TB. Reconstructing a B-cell clonal lineage. I. Statistical inference of unobserved ancestors. F1000Research.2013;2:103. Epub 2014/02/21. doi:

10.12688/f1000research.2-103.v1. PubMed PMID: 24555054; PubMed Central PMCID: PMCPmc3901458.

4. Kepler TB, Munshaw S, Wiehe K, Zhang R, Yu JS, Woods CW, Denny TN, Tomaras GD, Alam SM, Moody MA, Kelsoe G, Liao HX, Haynes BF. Reconstructing a B-Cell Clonal Lineage. II. Mutation, Selection, and Affinity Maturation. Frontiers in immunology.

2014;5:170. Epub 2014/05/06. doi: 10.3389/fimmu.2014.00170. PubMed PMID: 24795717; PubMed Central PMCID: PMCPmc4001017.

5. Liao HX, Chen X, Munshaw S, Zhang R, Marshall DJ, Vandergrift N, Whitesides JF, Lu X, Yu JS, Hwang KK, Gao F, Markowitz M, Heath SL, Bar KJ, Goepfert PA, Montefiori DC, Shaw GC, Alam SM, Margolis DM, Denny TN, Boyd SD, Marshal E, Egholm M, Simen BB, Hanczaruk B, Fire AZ, Voss G, Kelsoe G, Tomaras GD, Moody MA, Kepler TB, Haynes BF. Initial antibodies binding to HIV-1 gp41 in acutely infected subjects are polyreactive and highly mutated. The Journal of experimental medicine.2011;208(11):2237-49. Epub

2011/10/12. doi: 10.1084/jem.20110363. PubMed PMID: 21987658; PubMed Central PMCID: PMCPmc3201211.

6. Liao HX, Levesque MC, Nagel A, Dixon A, Zhang R, Walter E, Parks R, Whitesides J, Marshall DJ, Hwang KK, Yang Y, Chen X, Gao F, Munshaw S, Kepler TB, Denny T, Moody MA, Haynes BF. High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies. Journal of virological methods. 2009;158(1-2):171-9. Epub 2009/05/12. doi: 10.1016/j.jviromet.2009.02.014. PubMed PMID: 19428587; PubMed Central PMCID: PMCPmc2805188.

[0279] DH542 is a V3 glycan bnAb from individual CH848 identified at 234 weeks post infection. This antibody was produced recombinantly from VH and VL sequences amplified by PCR from single cells sorted from PBMCs using Man9V3 glycopeptide tetramer (Figure 1). DH542 is a member of the DH270 V3 glycan BnAb lineage (Figures 8-11).

[0280] Figure 2A shows the gene information of DH542 and Figure 2B shows DH542 sequences. [0281] Neutralization studies demonstrated that DH270 though containing only 5.5% VH mutations, mediated potent heterologous neutralization breadth (65.2% breadth and median IC50=0.17 ug/ml). Example^3:^TZM‐bl^cells^pseudo‐viruses^neutralization^assay^

[0282] TZMbl neutralization assay is a standard way to evaluate antibody breadth and potency. See Montefiori, D. Methods Mol Biol.2009;485:395-405; HIV-1 Env- pseudoviruses infection of TZM-bl cells. Exemplary pseudovirus neutralization assays and panels of HIV-1 pseudovirus are described for example, in Li et al., J Virol 79, 10108-10125, 2005, Seaman et al, J. Virol., 84:1439-1452, 2010; Sarzotti-Kelsoe et al., J. Immunol.

Methods, 409:131-46, 2014; and WO2011/038290, each of which is incorporated by reference herein. Various HIV-1 isolates, both Tier 1 and Tier 2 viruses can be included in this assay.

[0283] The TZMbl assay was conducted to determine neutralization potency and breadth of the various antibodies of the invention on different HIV-1 pseudoviruses. Example^4:^Binding^assays^and^Kd^determination^

[0284] Kd measurements of antibody binding to HIV-1 envelope, e.g. gp120 or any other suitable peptide, will be determined by Surface Plasmon Resonance measurements, for example using Biacore, or any other suitable technology which permits detection of interaction between two molecules in a quantitative way. Example^5:^Assay^for^self‐reactivity^

[0285] Table 11 below summarizes some of the known types of disease associated antibodies.

[0286] Various assays for self-reactivity of human antibodies are known in the art. AtheNA Multi-Lyte ANA Plus Test System is one such assay. This is luminex-based assay, which is also used to screen patient sera

[0287] Figures 6 and 7 show a summary of binding to autoantigen in the AtheNA assay and HEp-2 ell IF staining for DH542. Example^6:^CD4^binding^site^antibodies^^

[0288] CH557 is one example of a CD4 binding site antibody which can be used in combination with the V3 glycan antibodies of the invention. VH and VL sequences of CD4 binding site antibodies are described in Figures 21, 22 and 23. Example^7:^TZM‐bl^neutralization^profiles^of^V3^antibodies^

[0289] TZM-bl neutralization assay was conducted to determine neutralization potency and breadth of different viruses by DH542-L4, DH542, PGT128, PGT121, 10-1074, DH270 and DH471. Figures 13, 14 and 15 show the results of neutralization against a panel of HIV isolates in the TZM-bl pseudovirus neutralization assay. Figures 13, 14 and 15 also show the mean IC50, IC80 and percent of isolates neutralized at an different IC50 or IC80 values. Example^8:^Heavy^and^Light^chain^chimeric^V3^antibodies^

[0290] This example describes chimeric antibodies comprising non-natural VH and VL chain pairs. Recombinantly expressed VH or VL chains from naturally occurring VH:VL pairs are combined in non-natural pairs as described in Figure 25. Lines 2-4 in Figure 25 show antibodies having DH542 VH chain paired with VLs from other antibodies from the DH270 lineage.

[0291] In some instances VH chains (I0848_00001_L1_4A; I0848_00004_L1_4A;

I0848_00005_L1_4A; I0848_00006_L1_4A; I0848_00007_L1_4A) which were identified by Illumina sequences and not as natural VH:VL pair, were paired with VL sequences from a VH:VL pair with the closest VH sequence—lines 5-9 in Figure 25. In other instances, VH chains (I0848_00001_L1_4A; I0848_00004_L1_4A; I0848_00005_L1_4A;

I0848_00006_L1_4A; I0848_00007_L1_4A) were paired with DH542_QSA which is the corrected VL chain of DH542—lines 12-15 in Figure 25.

[0292] For Illumina sequencing RNA was isolated from patient PBMCs using an RNAeasy isolation kit (Qiagen). RNA was reverse transcribed and PCR amplified with primers targeting the IgG VH1 family of Immunoglobulin genes. Illumina adapters were added by PCR using the Nextera sample prep kit (Illumina). Illumina cDNA libraries were quantified using qPCR (Kappa biosciences) and sequenced using Illumina Miseq (2x300bp; Illumina). Analysis of Immunoglobulin sequence genetics were performed using Cloanalyst software (T. Kepler; Boston University).

[0293] Primer Sequences: AGGTGTGCACGCCGCTGGTC IgG-RT (SEQ ID NO: 283);

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCATGGACTGGACCTGGAGG VH1-Ext__P5_1^ST (SEQ ID NO: 284);

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCGATGGGCCCTTGGTGGA HUIGG_Deep_R__P7_1^ST (SEQ ID NO: 285)

[0294] Neutralization by these antibodies was determined in the TZMBl assay using a selection of viruses as shown in Figure 26. Data in Figure 26A and 26B show the

neutralization profiles of these chimeric antibodies. Antibody DH542-L4-4A (also referred to as DH542-L4), comprising VH from DH542 and VL from DH429, shows improved neutralization potency compared to the DH542 antibody---Figures 26A and 27.

[0295] Throughout examples and figures VH sequences are referenced interchangeably as I0848_00001 or I0848_00001 _L1_4A, . I0848_00004 or I0848_00004 _L1_4A I0848_00005 or I0848_00005 _L1_4A, I0848_00006 or I0848_00006 _L1_4A, I0848_00007 or

I0848_00007 _L1_4A.

[0296] Additional chimeric comprising non-natural VH and VL chain pairs are contemplated. In non-limiting examples, these pairings include VH and/or VL chains from antibodies DH542, DH542QSA, DH429, DH471, DH391, and/or DH473. Example^9:^Exemplary^HIVxCD3^Bispecific^Diabodies^with^Fc^Domains^^

[0297] Provided herein are stable, structurally compact, bispecific diabodies that have inter- chain disulfide bonds that may be engineered with an Fc Domain to extend serum half-life. The antigen binding arms of such bispecific diabodies are advantageously selected to co- engage immune effector cells (e.g., T cells, NK cells, etc.) with antigen-expressing target cells (e.g., HIV-1 infected cells) and activate and redirect the cytolytic activity of immune effector cell against the antigen expressing target cells. [0298] An HIVxCD3 bispecific diabody with an Fc Domain designated“CH557xCD3 Fc” was designed and expressed. This molecule comprises three polypeptide chains and includes an HIV-1 binding arm derived from the CH557 antibody described herein, a CD3 effector cell binding arm derived from a humanized anti-CD3ε mAb (hXRCD3), and CH2-CH3 IgG1 Fc Domains. The general structure of the polypeptide chains and a schematic of the assembled chains are shown in Figures 36A-36B. The amino acid sequences and a representative polynucleotide encoding each polypeptide chain are presented in Figures 36C-36D. A corresponding negative control bispecific diabody with an irrelevant binding arm [ ^RSV derived from palivizumab] instead of the CH557 HIV-1 arm (designated“RSVxCD3 Fc,”) was also generated. In addition, a comparator bispecific diabody (designated“A32xCD3 Fc”) having an HIV-1 binding arm derived from the A32 antibody (Protein Data Bank (PBD) ID Code 3TNM) instead of CH557, and additional negative control bispecific diabodies having the A32 HIV-1 arm with an irrelevant binding arm [ ^RSV or αfluorescein] instead of the CD3 arm (designated“A32xRSV Fc,” and“A32x4420,” respectively) were generated. The HIVxCD3 Fc bispecific diabodies are capable of simultaneously binding to HIV-1 and CD3. The control RSVxCD3 Fc bispecific diabody is capable of simultaneously binding to RSV and CD3 and the control A32xRSV Fc bispecific diabody is capable of simultaneously binding to HIV-1 and RSV. Each of the generated bispecific diabodies is a heterotrimer of polypeptide Chains 1, 2 and 3 have the general structure provided in Figure 34A (also see, e.g., Figures 36A-36B). Chains 1 and 2 comprise the VH and VL Domains of CH557, A32, XRCD3, or palivizumab, as detailed above, while Chain 3 is common to each diabody molecule. [0299] Methods for forming bispecific diabodies, including such diabodies comprising an Fc Domain, are provided in WO 2006/113665, WO 2008/157379, WO 2010/080538, WO 2012/018687, WO 2012/162068, WO 2012/162067, WO 2014/159940, WO 2015/021089, WO 2015/026892 and WO 2015/026894. [0300] Table 5 provides the SEQ ID NOs for each of the polypeptide chains present in CH557xCD3 Fc molecule. Table 4 provides SEQ ID NOs for the VL and VH Domains of CH557 (as provided herein), A32 (additional HIV-1 Env antibodies are known in the art, see, e.g., WO 2016/054101), hXRCD3 (additional anti-CD3 antibodies are known in the art, see, e.g., WO 2012/162067), and palivizumab (additional anti-RSV antibodies are known in the art, see, e.g., US 6,818,216), the SEQ ID NOs for the VL and VH Domains of the exemplary anti-CD16 mAb h3G8, anti-CD8 mAb OKT8 and anti-CD8 mAb TRX2 are also provided. In addition, the SEQ ID NOs for representative nucleic acid encoding sequences are provided.

[0301] For additional sequences related to this example see Table 4. Example^10A:^Binding^Properties^of^HIVxCD3^Bispecific^Diabodies^with^Fc^ Domains^

[0302] The binding of CH557xCD3 Fc to recombinant HIV-1 Env and human CD3 was examined by ELISA assay. Briefly, microtiter plates were coated with recombinant proteins (human CD3ε/δ heterodimer, or M.CONS gp140 (Liao HX, et al. A group M consensus envelope glycoprotein induces antibodies that neutralize subsets of subtype B and C HIV-1 primary viruses. Virology.2006;353(2):268–282)) in buffer and blocked. Serial dilutions of the bispecific diabodies (CH557xCD3 Fc, the comparator A32xCD3 Fc, or the controls RSVxCD3 Fc and A32xRSV Fc) were applied followed by sequential addition of biotinylated anti-EK coil antibody and streptavidin-HRP. For bispecific binding assays, the plate was coated with M.CONS gp140, and diabody application was followed by sequential addition of biotinylated CD3ε/δ and streptavidin-HRP. HRP activity was detected with a

chemiluminescent substrate. For bispecific binding assays, the plate was coated with

M.CONS gp140, and diabody application was followed by sequential addition of biotinylated CD3ε/δ and streptavidin-HRP. CH557xCD3 Fc and A32xCD3 Fc both exhibited binding to recombinant human CD3 and HIV-1 Env, individually and simultaneously, while the control molecules, RSVxCD3 Fc and A32xRSV Fc, only exhibited binding to human CD3 or HIV-1 Env, respectively, as shown by ELISA. [0303] Cell surface binding of CH557xCD3 Fc to cells expressing HIV-1 Env (HEK293- D371 cells, expressing HIV-1 CM244 (subtype AE) gp140) and human CD3 (human primary T cells) was examined by flow cytometric analysis. Briefly, Serial dilutions of the bispecific diabodies: CH557xCD3 Fc; the comparator A32xCD3 Fc; or the control RSVxCD3 Fc, were incubated with HEK293-D371 cells or human primary T cells (Pan T cells) in FACS buffer containing a blocking agent (e.g., human albumin serum). After washing, cells were resuspended in buffer containing biotin-conjugated mouse anti-EK antibody (recognizes the E/K heterodimerization region of diabody proteins), mixed with streptavidin-PE and incubated in the dark. Cells were washed, resuspended with FACS buffer, and analyzed by flow cytometry. CH557xCD3 Fc and A32xCD3 Fc exhibited binding to both HEK293-D375 and Pan T cells, while the control, RSVxCD3 Fc, only exhibited binding to Pan T cells. CH557xCD3 Fc exhibited stronger binding to HEK293-D375 cells in these studies. Example^10B:^Cell^Killing^Activity^of^HIVxCD3^Bispecific^Diabodies^with^Fc^ Domains^

[0304] The ability of CH557xCD3 Fc to mediate redirected cell killing of target cells expressing HIV-1 Env was examined using a cytotoxic T lymphocyte (CTL) assay. Briefly, the HIV-1 Env expressing cell line HEK293-D371 was treated with serial dilutions of the bispecific monovalent diabodies: CH557xCD3 Fc; the comparator A32xCD3 Fc; or the control RSVxCD3 Fc, together with effector cells (human PBMCs) from two different donors at an E/T ratio of 30:1 for 24 hours. The percentage cytotoxicity (i.e., cell killing) was determined by measuring the release of lactate dehydrogenase (LDH) into the media by damaged cells as described previously (Moore PA et al. Application of dual affinity retargeting molecules to achieve optimal redirected T-cell killing of B-cell lymphoma. Blood. 2011;117(17):4542–4551). As measured by LDH release assays, CH557xCD3 Fc and A32xCD3 Fc both mediated redirected human immune cells derived from healthy donors to kill the HEK293-D371 cells in a concentration dependent manner at an E:T ratio of 30:1. The average EC50 values were 13.8 ng/mL and 12.3 ng/mL for CH557xCD3 Fc and A32xCD3 Fc, respectively. In contrast, no diabody-mediated redirected T-cell killing occurred with the RSVxCD3 control diabody in which the HIV-1 arm was replaced by an irrelevant one. Example^10C:^Binding^Properties^of^HIVxCD3^Bispecific^Diabodies^with^Fc^ Domains^to^autologous^HIV‐1^CH557^envelope^

Methods.^

[0305] The 491 (control) and 598 (CH505 Transmitted/Founder Envelope- and GFP- transfected cell lines were used to detect the ability of the diabodies of interest to bind specifically to the HIV-1 envelope of the CH505 transmitted/founder isolate. The cell lines were incubated for 24 hours with Doxycycline at concentrations of 0.5, 0.0125, and 0.0031, and 0ug/ml to achieve different levels of expression of the HIV-1 CH505 Envelope as determined in preliminary experiments. The GFP expression was used to determine the level of expression as of the transduced proteins. The A32x4420 and CH557xhXR32 diabodies were tested for staining at the concentration of 10 and 1 µg/ml. Palivizumabx4420

(PAlix4420) and PalivizumabxhXR32 (PalixhXR32) diabodies based on the anti-RSV mAb were used as control for each of the two anti-HIV-1 Env diabodies, respectively, using the same concentrations. In addition the A32 (anti-C1/C2 Env mAb; positive control), Synagis (anti-RSV mAb, negative control), and CH65 (anti-Flu HA mAb; negative control) were used as controls for the specificity of the staining.

[0306] After 24hr. incubation with the different doses of Doxycycline, each cells culture was plated with and without Palix4420, PalixhXR32, A32 x 4420, and CH557 x hXR32 DARTS at 1ug/ml and 10ug/ml. In addition, the same cell culture were plated with and without Synagis, A32, and CH65 human mAbs at 1ug/ml and 10ug/ml.

[0307] All the different cell cultures were incubated with diabodies and human mAbs for 2hrs at 37 degrees Celsius.

[0308] After 2hr incubation cells were washed 2x in PBS and stained with a LIVE/DEAD cell marker (Aqua LIVE/DEAD) for 20 minutes at RT.

[0309] After viability staining, cells were washed 2x in Wash Buffer and appropriate secondary mixes (anti-EK + IgG2b PE for DARTS or IgG PE for human mAbs) were added to wells for 30min at 4 degrees Celsius. The anti-EK and anti-EK+IgG2 conditions were used to stain the cells as additional control conditions.

[0310] Cells were washed 2x in Wash Buffer and fixed in 1% Formalin Solution until acquiring on BD LSR Fortessa. [0311] The analysis of data was performed by gating on the viable (Aqua Live/Dead negative) and GFP positive cells for the 598 cell line and on viable cells for the 491 control cell line. Each frequency reported in the figure refers to the percentage of diabodies or mAb positive cells referred to the Live+/GFP+ cells.

[0312] Results: The levels of GFP expression detected with each Doxycycline concentration in each staining condition was reported for the diabodies and control mAbs, respectively.

[0313] As expected, we observed three different levels of GFP expression ranging from >80% of cells to less than 20%. A minimal constitutive expression of GFP was also observed in absence of Doxycycline.

[0314] The analysis of the staining with the A32x4420 and CH557xhXR32 to the 491 control cell line did not reveal any non specific staining. In contrast, the 598 cell lines were stained proportionally to the level of GFP observed due to the stimulation with Doxycycline, ranging from 82% to 11.7% with the CH557xhXR32 DART, and CH557xhXR32 diabody binding was always better than the A32x4420 diabody binding at each concentration. A minimum background of 10% staining with the CH557xhXR32 was observed when the 598 cell line was used without incubation with Doxycycline. This is due to a minimum constitutive expression of the transduced genes as indicated for GFP expression. The staining with the A32 mAb indicated the expression of HIV-1 Env on the membrane of the cells according to a similar profile and the specificity of the staining of the 598 cell line.

[0315] The analysis of the staining with diabody and mAb controls represented by the anti- EK, anti-EK+IgG2, Palix4420, PalixhXR32, Synagis, and CH65 mAbs combination did not reveal non-specific staining of the 598 cell line.

Example^11A:^Additional^multispecific^molecules^

[0316] Any one of the antibodies from the CH235 lineage or DH270 lineage could be constructed in any one of the multispecific molecule formats described herein. The effector arm could target be any one of the non-limiting examples of CD2, CD3, CD8, CD16, T-Cell Receptor (TCR), NKG2D, etc. epitopes. Non-limiting examples are provided in the Table below. The information in the specification can be readily used for alternative designs of the listed molecules, and for design of other bispecific molecules, for example CH235 lineage members or DH270 lineage members using CDRs, or VH and VL chains from these HIV-1 antibodies. [0317] Table 6 shows a summary of some additional non limiting embodiments of bispecific diabody (with and without Fc Domains) that may be generated comprising alternative HIV-1 and/or alternative effector cell binding specificities. The information in specification can be readily used for alternative design of the listed bispecific diabodies, and for design of other bispecific diabodies, for example bispecific diabodies comprising a CD16 binding specificity instead of CD3 and/or incorporating the HIV-1 binding specificity of CH235 lineage antibodies or DH270 lineage antibodies using CDRs, or VH and VL chains from these antibodies.

Linker 3 523 Linker 3 523

[0318] Wherein SEQ ID NOs: 553, 565, 567, 570, 574, 578, and 582 correspond to the VL chain of antibody CH557, CH556, CH555, CH493, CH492, CH491, and CH490, respectively and as described in Table 4. Wherein SEQ ID NOs: 586, 590, and 594 correspond to the VL chain of antibody DH542, DH542_QSA, and D542_L4, respectively and as described in Table 4. [0319] Wherein SEQ ID NOs: 551, 564, 566, 568, 572, 576, and 580 correspond to the VH chain of antibody CH557, CH556, CH555, CH493, CH492, CH491, and CH490, respectively and as described in Table 4. Wherein SEQ ID NOs: 584, 588, and 592 correspond to the VH chain of antibody DH542, DH542_QSA, and D542_L4, respectively and as described in Table 4. ] Example^11B:^Exemplary Trivalent and Control Molecules with Fc Domains

[0320] As provided herein trivalent binding molecules having three Antigen-Binding Domains provide additional functionality as they can co-engage multiple epitopes present on the surface of an effector cell, such as a T lymphocyte (e.g., CD3 and CD8), or they can bind multiple epitopes of HIV-1 (e.g., epitopes of different HIV-1 isolates or different epitopes of HIV-1 Env). The antigen binding arms of such trivalent binding molecules are advantageously selected to co-engage immune effector cells (e.g., T cells, NK cells, etc.) with antigen-expressing target cells (e.g., HIV-1 infected cells) and activate and redirect the cytolytic activity of immune effector cell against the antigen expressing target cells. [0321] Table 7 shows a summary of some non-limiting embodiments of trivalent binding molecules (having 3 or 4 polypeptide chain) that may be generated. The information in specification can be readily used for alternative design of the listed trivalent binding molecules, and for design of other trivalent binding molecules, for example trivalent binding molecules comprising alternative CD8 binding specificities and/or incorporating the HIV-1 binding specificity of CH235 lineage antibodies or DH270 lineage antibodies using CDRs, or VH and VL chains from these antibodies.

[0322] Wherein SEQ ID NOs: 553, 565, 567, 570, 574, 578, and 582 correspond to the VL chain of antibody CH557, CH556, CH555, CH493, CH492, CH491, and CH490, respectively and as described in Table 4. Wherein SEQ ID NOs: 586, 590, and 594 correspond to the VL chain of antibody DH542, DH542_QSA, and D542_L4, respectively and as described in Table 4. [0323] Wherein SEQ ID NOs: 551, 564, 566, 568, 572, 576, and 580 correspond to the VH chain of antibody CH557, CH556, CH555, CH493, CH492, CH491, and CH490, respectively and as described in Table 4. Wherein SEQ ID NOs: 584, 588, and 592 correspond to the VH chain of antibody DH542, DH542_QSA, and D542_L4, respectively and as described in Table 4. [0324] Table 4 provides the amino acid sequences of one such exemplary trivalent binding molecule having four polypeptide chain which may be generated are provided in SEQ ID NOs: 555, 557, 561, and 562. Chain 1 of this exemplary molecule comprises: an N-terminus, the VL Domain of CH557 (SEQ ID NO: 553), a Peptide Linker 1 (SEQ ID NO: 508), the VH Domain of hXR32 (SEQ ID NO: 500), a Peptide Linker 2 (SEQ ID NO: 510), a cysteine engineered E-coil, a Spacer Linker 3 (SEQ ID NO: 522), a knob bearing CH2-CH3 (SEQ ID NO: 531), and a C-terminus. Chain 2 of this exemplary molecule comprises: an N-terminus, the VL Domain of hXR32 (SEQ ID NO: 503), a Peptide Linker 1 (SEQ ID NO: 508), the VH Domain of CH557 (SEQ ID NO: 551), a Peptide Linker 2 (SEQ ID NO: 510), a cysteine engineered K-coil, a Spacer Linker 3 (SEQ ID NO: 521), and a C-terminus. Chain 3 of this exemplary molecule comprises: an N-terminus, the VH Domain of OKT8 (SEQ ID NO: 543), a CH1-Hinge Domain (SEQ ID NO: 515), a hole bearing CH2-CH3 (SEQ ID NO: 533), and a C-terminus. Chain 4 of this exemplary molecule comprises: an N-terminus, the VL Domain of OKT8 (SEQ ID NO: 545), a CL Kappa Domain (SEQ ID NO: 516), and a C- terminus. It will be noted that polypeptide chains 1 and 2 of such an exemplary trivalent binding molecule are the same as those present in the bispecific Fc bearing diabody

CH557xCD3 Fc described above. [0325] Table 4 provides the amino acid sequences of another such exemplary trivalent binding molecule having three polypeptide chain which may be generated are provided in SEQ ID NOs: 555, 557, and 563. Chain 1 of this exemplary molecule comprises: an N- terminus, the VL Domain of CH557 (SEQ ID NO: 553), a Peptide Linker 1 (SEQ ID NO: 508), the VH Domain of hXR32 (SEQ ID NO: 500), a Peptide Linker 2 (SEQ ID NO: 510), a cysteine engineered E-coil, a Spacer Linker 3 (SEQ ID NO: 522), a knob bearing CH2-CH3 (SEQ ID NO: 531), and a C-terminus. Chain 2 of this exemplary molecule comprises: an N- terminus, the VL Domain of hXR32 (SEQ ID NO: 503), a Peptide Linker 1 (SEQ ID NO: 508), the VH Domain of CH557 (SEQ ID NO: 551), a Peptide Linker 2 (SEQ ID NO: 510), a cysteine engineered K-coil, a Spacer Linker 3 (SEQ ID NO: 521), and a C-terminus. Chain 3 of this exemplary molecule comprises: an N-terminus, the VL Domain of OKT8 (SEQ ID NO: 545), a Peptide Linker 5 (SEQ ID NO: 526), the VH Domain of OKT8 (SEQ ID NO: 543), a CH1-Hinge Domain (SEQ ID NO: 515), a hole bearing CH2-CH3 (SEQ ID NO: 533), and a C-terminus. It will be noted that polypeptide chains 1 and 2 of such an exemplary trivalent binding molecule are the same as those present in the bispecific Fc bearing diabody CH557xCD3 Fc described above.

[0326] Exemplary trivalent binding molecules having three or four polypeptide chains, comprising two HIV-1 Env binding sites and one CD3 binding site are readily generated, for example by replace the VL and VH Domains of the anti-CD antibody in the above described molecules with the VL and VH Domains of an anti-HIV-1 Env antibody (e.g., CH557).

[0327] The ability of such exemplary trivalent binding molecules to bind to HIV-1 Env, CD3, and/or CD8 may be evaluated using the methods describe above, or well known in the art (see, e.g., WO2016/054101). The activity of such trivalent binding molecules to mediate redirected cell killing of target cells expressing HIV-1 Env may be examined using the cytotoxic T lymphocyte (CTL) assay described above, or similar assays known in the art (see, e.g., Sung, JAM et al. Dual-Affinity Re-Targeting proteins direct T cell–mediated cytolysis of latently HIV-1-infected cells. J Clin Invest.2015;125(11):4077-4090;

and WO2016/054101). Example^12:^^Combinations^of^multispecific^anitbodies^

[0328] Multispecific antibodies with different HIV-1 specificity, e.g CD4 binding site and V3 glycan binding, could also be tested in combination.

[0329] Various combinations of HIV-1 multispecific antibodies to mediate redirected cell killing of target cells expressing HIV-1 Env may be examined using the cytotoxic T lymphocyte (CTL) assay described above, or similar assays known in the art (see, e.g., Sung, JAM et al. Dual-Affinity Re-Targeting proteins direct T cell–mediated cytolysis of latently HIV-1-infected cells. J Clin Invest.2015;125(11):4077-4090; and WO2016/054101).

Ccombinations of multispecific molecules, either with different HIV-1 specificity and/or different effector cell specificity, will be tested whether they provide enhanced benefits. Example^13:^Exemplary V3 glycan Specific HIVxCD3 Bispecific Diabody

[0330] As provided herein, the binding specificity of the anti-HIV-1 antibody DH542 differs from the other anti-HIV-1 antibodies provide herein, in that it is specific for the V3 glycan of HIV-1 Env, According, the VH and VL Domains of DH542 are utilized in the generation of HIVxCD3 bispecific diabodies (and/or trivalent binding molecule as provided herein) having binding specificity for the V3 glycan of HIV-1 Env. Exemplary, HIVxCD3 bispecific diabodies, having three polypeptide chains, an HIV-1 binding arm derived from the DH542 antibody described herein, a CD3 effector cell binding arm derived from a humanized anti- CD3ε mAb (hXRCD3), and CH2-CH3 IgG1 Fc Domains may have the general structure shown in Figures 36A-36B. Table 8 provides the SEQ ID NOs for each of the polypeptide chains present in one non-limiting embodiment of such a molecule, designated herein as “DH542xCD3 Fc”. It will be appreciated based on the instant disclosure that the third polypeptide chain of DH542xCD3 Fc may be identical to the HIVxCD3 bivalent diabodies having an Fc Domain provide above.

[0331] Chain 1 of this exemplary molecule comprises: an N-terminus, the VL Domain of DH542 (SEQ ID NO: 586), a Peptide Linker 1 (SEQ ID NO: 508), the VH Domain of hXR32 (SEQ ID NO: 500), a Peptide Linker 2 (SEQ ID NO: 510), a cysteine engineered E-coil, a Spacer Linker 3 (SEQ ID NO: 522), a knob bearing CH2-CH3 (SEQ ID NO: 531), and a C- terminus. Chain 2 of this exemplary molecule comprises: an N-terminus, the VL Domain of hXR32 (SEQ ID NO: 503), a Peptide Linker 1 (SEQ ID NO: 508), the VH Domain of DH542 (SEQ ID NO: 584), a Peptide Linker 2 (SEQ ID NO: 510), a cysteine engineered K-coil, a Spacer Linker 3 (SEQ ID NO: 521), and a C-terminus. Chain 3 of this exemplary molecule comprises: an N-terminus, a Peptide Linker 3 (SEQ ID NO: 523), a hole bearing CH2-CH3 (SEQ ID NO: 533), and a C-terminus. Such bispecific diabodies, may be generated using the methods as provided above. [0332] The ability of such exemplary HIV-1 Env V3 glycan binding bispecific diabodies to bind to HIV-1 Env V3 glycan and CD3 may be evaluated using the methods describe above, or well known in the art (see, e.g., WO2016/054101). The activity of such exemplary bispecific diabodies to mediate redirected cell killing of target cells expressing HIV-1 Env may be examined using the cytotoxic T lymphocyte (CTL) assay described above, or similar assays known in the art (see, e.g., Sung, JAM et al. Dual-Affinity Re-Targeting proteins direct T cell–mediated cytolysis of latently HIV-1-infected cells. J Clin

Invest.2015;125(11):4077-4090; and WO2016/054101). Example^14:^Prophetic^examples^^

[0333] All multispecific molecules described herein could be tested in any other suitable assay. For non-limiting examples of further studies and characterization See Sung et al. J Clin Invest.2015;125(11):4077-4090 ;Sloan DD, Lam C-YK, Irrinki A, Liu L, Tsai A, Pace CS, et al. (2015) Targeting HIV Reservoir in Infected CD4 T Cells by Dual-Affinity Re- targeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells. PLoS Pathog 11(11): e1005233. doi:10.1371/journal.ppat.1005233

[0334] Non-limiting examples of these assays include: determination of binding properties of the multispecific molecules to HIV-1 envelope expressing cells and/or HIV-1 infected cells; determining whether the multispecific molecules of the invention induce redirected T-cell killing of cell lines expressing various Envelopes and concomitant T-cell activation;

determining whether multispecific molecules bind to the surface of HIV-1- infected CD4⁺T cells and redirect CD8⁺ T-cells to kill HIV-1 infected CD4⁺ cells using lymphocytes from HIV-1 seronegative donors; determining whether multispecific molecules redirect CD8⁺ T- cells to clear HIV-1 (e.g. JR-CSF, or any other suitable HIV-1 type)-superinfected CD4⁺ cells using lymphocytes from patients on suppressive ART; determining whether multispecific molecules redirect T cells from HIV-1-infected individuals on suppressive ART to clear virus from resting CD4⁺ T cells following induction of latent virus expression.

[0335] Methods and Reagents

[0336] Generation of Infectious Molecular Clones (IMCs). HIV-1 IMCs for any subtype B BaL, subtype AE CM235 and subtype C 1086.C were generated with the backbone derived from NHL4-3 isolate as previously described See Edmonds TG et al. Replication competent molecular clones of HIV-1 expressing Renilla luciferase facilitate the analysis of antibody inhibition in PBMC. Virology.2010;408(1):1–13; Adachi A et al. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol.1986;59(2):284–291 ). All IMCs expressed the Renilla luciferase reporter gene and preserved all nine viral open reading frames. The Renilla luciferase reporter gene was expressed under the control of the HIV-1 Tat gene. Upon HIV-1 infection of CD4+ T cells, expression of Tat during HIV-1 replication will induce luciferase expression, which allows quantitation of infected cells by measuring relative luminescence units (RLU).

[0337] Cell Lines. Jurkat-522 F/Y GF cells, which constitutively express a fusion protein of Copepod Green Fluorescent Protein (copGFP) and Firefly Luciferase (System Biosciences), were generated at Macrogenics from Jurkat-522 F/Y cells by transduction and clone selection. HEK293-D371 cells, which have doxycycline-inducible expression of HIV-1 CM244 (subtype AE) gp140 or CH505 T/F envelope, were obtained from Dr. John Kappes

(University of Alabama at Birmingham).

[0338] Flow Cytometric Analysis of diabody or mAb Binding to Cells. Diabodies at a desired concentration, e.g.4 µg/mL are incubated with 10⁵ cells in 200 µL FACS buffer containing 10% human AB serum for 30 minutes at room temperature. After washing, cells are resuspended in 100 µL of 1 µg/mL biotin-conjugated mouse anti-EK antibody (recognizes the E/K heterodimerization region of diabody proteins and/or trivalent binding molecules), are mixed with 1:500 diluted streptavidin-PE and incubated in the dark for 45 minutes at 2-8 ^C. Cells are washed, resuspended with FACS buffer, and analyzed with a BD Calibur flow cytometer and FlowJo software (TreeStar, Ashland OR). Binding to IMC-infected CD4⁺ T cells from normal human donors is conducted as previously described (54) for the A32 and 7B2 mAbs, and with biotin-conjugated mouse anti-EK antibody and 1:500 diluted

streptavidin-PE for the HIVx4420 control diabodies.

[0339] The ability of CH557xCD3 Fc (or any of the bispecific diabodies and/or trivalent binding molecules) to mediate redirected cell killing of HIV-1 IMC-Infected CD4⁺ Cells may be examined using assays well known in the art (see, e.g., Sung, JAM et al. Dual-Affinity Re-Targeting proteins direct T cell–mediated cytolysis of latently HIV-infected cells. J Clin Invest.2015;125(11):4077-4090; and WO2016/054101). Briefly, resting PBMC from normal healthy HIV-1 seronegative donors are activated (e.g., with anti–human CD3 and anti–human CD28 antibodies). A CD4+ enriched cell population is obtained by depletion of CD8+ T cells (e.g., using commercially available magnetic beads) and is spinoculated in the presence of the luciferase-expressing IMC-representing an HIV-1 subtype (e.g., AE (CM235); B (BaL); or C (1086.C)) and cultured for ~72 hours. The CD4+-infected target cells are then incubated with resting CD8+ effector cells (which may be isolated by negative selection from autologous PBMC, using a commercial CD8+ T cell isolation kit) at different E/T ratios (e.g., 33:1, 11:1, 3:1, and 0:1) in the absence or presence of bispecific diabodies for 6–48 hours at

concentrations ranging from 0.0001–1,000 ng/ml. Uninfected and infected target cells alone are included as additional controls. Each condition is tested in duplicate. After incubation, the percentage of specific lysis (%SL) of target cells is determined, for example by adding ViviRen Live Cell Substrate (Promega) and measuring the RLU on a luminometer. The %SL of target cells may be determined as described previously (Pollara J et al. HIV-1 Vaccine- Induced C1 and V2 Env-Specific Antibodies Synergize for Increased Antiviral Activities. J Virol.2014;88(14):7715–7726). [0340] Redirected T-Cell Cytotoxicity Assay Against HIV-1 Env-expressing Cell Lines and Assessment of T-Cell Activation. Pan T cells are isolated from healthy human PBMCs with the Dynabeads® Untouched™ Human T Cells Kit (Invitrogen). HIV-1 Env expressing cell lines (1-4 x10⁵ cells/mL) are treated with serial dilutions of diabodies (or trivalent binding molecules), together with human T cells at an effector:target (E:T) ratio = 10:1, or otherwise at varying E:T ratios as indicated, and incubated at 37 ^C, 5% CO₂ overnight. Cytotoxicity is measured by lactate dehydrogenase (LDH) release (CytoTox 96® Non-Radioactive

Cytotoxicity Assay, Promega) as described previously (32). With the Jurkat-522 F/Y GF cell line, cytotoxicity is also measured by luminescence using Luciferase-Glo substrate

(Promega). Specific lysis is calculated from luminiscence counts (RLU): cytotoxicity (%) = 100 x (1-(RLU of Sample ^ RLU of Control)), where Control = average RLU of target cells incubated with effector cells in the absence of DART. Data are fit to a sigmoidal dose- response function to obtain 50% effective concentration (EC₅₀) and percent maximum specific lysis values. T-cell activation is measured by FACS analysis after cells in the assay plate are labeled with CD8-FITC, CD4-APC, and CD25-PE antibodies (BD Biosciences), followed by cell collection by FACS Calibur flow cytometer equipped with acquisition software CellQuest Pro Version 5.2.1 (BD Biosciences). Data analysis is performed using FlowJo software (Treestar, Inc).

[0341] Redirected T-Cell Cytotoxicity Assay Against HIV-1 IMC-Infected CD4⁺ Cells.

Cryopreserved resting PBMC from normal healthy HIV-1 seronegative donors are activated for 72 hours with anti-human CD3 (clone OKT3; eBioscience) and anti-human CD28 (clone CD28.2; BD Pharmingen). Subsequently, a CD4⁺ enriched cell population (purity >92.3%; average±standard deviation 95.73±2.6%) is obtained by depletion of CD8⁺ T cells using magnetic beads (Miltenyi Biosciences), spinoculated in presence of the luciferase-expressing IMC representing HIV-1 subtype AE (CM235), B (BaL) or C (1086.C) and cultured for 72 hours. CD4⁺ infected target cells are incubated with resting CD8⁺ effector cells (isolated by negative selection from autologous PBMC, CD8⁺ T cell Isolation Kit, Miltenyi Biosciences) at 33:1, 11:1, 3:1, and 0:1 E:T ratios in the absence or presence of diabodies (and/or trivalent binding molecules) for 6-48 hours at concentration ranging from 1,000 to 0.0001 ng/mL. Uninfected and infected target cells alone are included as additional controls. Each condition is tested in duplicate. After incubation, ViviRen™ Live Cell Substrate (Promega) is added and RLU measured on a luminometer; percentage specific lysis (%SL) of target cells is determined as described previously. SeePollara J et al. HIV-1 Vaccine-Induced C1 and V2 Env-Specific Antibodies Synergize for Increased Antiviral Activities. J Virol.

2014;88(14):7715–7726.

[0342] T-Cell Degranulation (CD107) Assay. As described for the cytotoxicity assay with HIV-1 IMC-infected cells as targets, activated CD4⁺ cells infected with HIV-1 BaL IMC are plated with resting CD8⁺ effector cells at a 33:1 E:T ratio in the absence or presence of 1ng/mL diabodies and incubated for 6 hour. For the CD4 T cell degranulation, activated CD4⁺ T cells are either infected with JR-CSF and labeled with the viability (NFL1) and target specific (TFL4) markers utilized in an ADCC assay or added to targets as effectors at a 10:1 ratio prior to addition of diabodies. Each condition is tested in duplicate. CD107 PE-Cy5 (clone H4A3; eBioscience) is titered and added during the last six hours of the incubation along with Monensin solution (BD GolgiStop). A panel of antibodies consisting of

LIVE/DEAD Aqua stain, anti-CD3 APC-H7 (clone SK7; BD Pharmingen), anti-CD4 BV605 (clone OKT4; Biolegend), anti-CD8 BV650 (clone RPA-T8; Biolegend) are used to detect CD107⁺ CD8⁺ T cells. After washing and fixation, samples are acquired on a custom made LSRII (BD Bioscience, San Jose, CA) within the next 24 hours. A minimum of 300,000 total viable events is acquired for each test. The analysis of the data is performed using the Flow-Jo software (Treestar, Ashland, OR).

[0343] T-Cell Viability and Activation Assays. CD8⁺ T cells and CD8 depleted PBMCs obtained from HIV infected ART suppressed patients are plated at 5x10⁴ cells per well in 96 well plates with 100ng/mL of the indicated DART. Cells are cultured in 0.2mL of cIMDM media supplemented with 10% FBS, 1% Penicillin/Streptomycin and 5U/mL IL-2 for 7 days, and then are stained with the following antibodies: HLA-DR-PerCP (clone L243), CD25-PE (clone M-A251), CD8-FITC (clone HIT8a), CD8-PE (clone HIT8a), CD4-FITC (clone RPA- T4), and Annexin V-PE and 7-AAD (all BD biosciences, San Jose, CA).

[0344] Redirected T-Cell Viral Clearance Assay. CD8⁺ T-cells are isolated from PBMCs by positive selection (EasySep human CD8⁺ Selection Kit, Stem Cell). CD8-depleted PBMCs are first activated with 2μg/mL of PHA (Remel, Lenexa, KS) and 60U/mL of IL-2, and then infected by spinoculation at 1200xg for 90 minutes with either JR-CSF or autologous reservoir virus (AR) at an MOI of 0.01). AR virus is obtained from pooled supernatants of replicate wells from outgrowth assays of resting CD4+ T-cells for each patient performed. Fifty-thousand (5x10⁴) targets/well are co-cultured with CD8⁺ T cells in triplicate at the indicated E:T ratio in the absence or presence of 100 ng/mL of diabody in 0.2m of cIMDM media supplemented with 10% FBS, 1% Penicillin/Streptomycin and 5 U/mL IL-2. For experiments performed in the presence of antiretrovirals (ARVs), 24 hours after spinoculation cells are washed and 1μM of raltegravir and 4μM of abacavir are added, and then diabodies and CD8⁺ T-cells are added to cultures. Supernatant is assayed on day 7 by p24 ELISA (ABL, Rockville, MD). Results are calculated as the log (p24 of infected target cells only control divided by p24 of the test condition).

[0345] Latency Clearance Assay (LCA). The reduction of virus recovery from CD4⁺ infected cells is assessed by a standard quantitative viral outgrowth assay using the resting CD4⁺ T cells of aviremic, ART-treated patients, following the addition of antiviral effector cells and/or molecules, as previously described. See Sung JA et al. Expanded Cytotoxic T-Cell Lymphocytes Target the Latent Hiv Reservoir. J Infect Dis.2015;:1–15.. In this case the LCA is used to model the ability of diabodies to clear virus emerging from the latent reservoir under clinically and pharmacologically relevant conditions. Resting CD4⁺ T-cells are isolated from a leukapheresis product as previously described (72) and exposed to PHA (4μg/mL) and IL-2 (60U/mL) for 24 hours or vorinostat (VOR) (335nM, 6 hours) (Merck Research

Laboratories), and plated at 0.5 to 1 x 10⁶ cells/well in 12 to 36 replicate wells depending on the size of the reservoir. The VOR is then washed off and CD8s added at an E:T of 1:10 as well as 100 ng/mL of the indicated DART. Cells are co-cultured for 24 hours (unless specified otherwise) following which the diabody (and/or trivalent binding molecule) proteins are washed off and allogeneic CD8-depleted PBMCs from an HIV negative donor are added to amplify residual virus. Supernatant is assayed for the presence of p24 antigen on day 15 for each well. Results are calculated as % viral recovery [(# of positive wells/total number plated)x100], normalized to a control in which no CD8⁺ T cells are added.

[0346] Additional animal studies will be conducted to evaluate toxicity, safety, PK and PD profiles, and efficacy in preventing or treating HIV infection of the multispecific molecules of the invention.

[0347] All documents and other information sources cited herein are hereby incorporated in their entirety by reference.

Claims

1. A bispecific molecule comprising a first polypeptide chain and a second polypeptide chain, covalently bonded to one another, wherein:

(I) the first polypeptide chain comprises in the N- to C-terminal direction:

(i) a domain (A) comprising a binding region of the light chain variable domain of a first immunoglobulin (VL1) having the binding specificity of a V3 glycan HIV-1 antibody (1);

(iii) a domain (C) comprising a heterodimer promoting domain;

(II) the second polypeptide chain comprises in the N- to C-terminal direction:

(ii) a domain (E) comprising a binding region of a heavy chain variable domain of the first immunoglobulin (VH1) having the binding specificity of a V3 glycan HIV-1 antibody (1); and

(iii) a domain (F) comprising a heterodimer promoting domain; and wherein:

the domains (A) and (E) associate to form a binding site that binds the HIV-1 envelope like a V3 glycan HIV-1 antibody (1); and the domains (B) and (D) associate to form a binding site that binds the epitope (2).

2. The bispecific molecule of claim 1, wherein:

(i) domains (A) and (B) are separated by a peptide linker 1;

(ii) domains (C) and (B) are separated by a peptide linker 2;

(iii) domains (D) and (E) are separated by a peptide linker 1; and

(iv) domains (F) and (E) are separated by a peptide linker 2.

3. A bispecific molecule comprising a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first and second polypeptide chains are covalently bonded and the second and third polypeptide chains are covalently bonded, and wherein:

(I) the first polypeptide chain comprises in the N- to C-terminal direction:

(iii) a domain (C) comprising a heterodimer promoting domain; and

(iv) a CH2-CH3 domain;

(II) the second polypeptide chain comprises in the N- to C-terminal direction:

(iii) a domain (F) comprising a heterodimer promoting domain;

(III) the third polypeptide chain comprises in the N- to C-terminal direction:

(i) a CH2-CH3 domain, and wherein:

the domains (A) and (E) associate to form a binding site that binds the HIV-1 envelope like a V3 glycanHIV-1 antibody (1);

4. The bispecific molecule of claim 3, wherein:

(i) the third polypeptide chain further comprises a peptide linker 3 N-terminal to the CH2-CH3 domain; (ii) domains (A) and (B) are separated by a peptide linker 1;

(iii) domains (C) and (B) are separated by a peptide linker 2;

(v) domains (D) and (E) are separated by a peptide linker 1; and

(vi) domains (F) and (E) are separated by a peptide linker 2.

5. A bispecific molecule comprising a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, wherein the first and second polypeptide chains are covalently bonded and the second and third polypeptide chains are covalently bonded, and wherein:

(I) the first polypeptide chain comprises in the N- to C-terminal direction:

(i) a CH2-CH3 domain;

(ii) a domain (A) comprising a binding region of the light chain variable domain of a first immunoglobulin (VL1) having the binding specificity of a V3 glycan HIV-1 antibody (1);

(iii) a domain (B) comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for an epitope (2); and

(iv) a domain (C) comprising a heterodimer promoting domain;

(II) the second polypeptide chain comprises in the N- to C-terminal direction:

(iii) a domain (F) comprising a heterodimer promoting domain;

(III) the third polypeptide chain comprises in the N- to C-terminal direction:

(i) a CH2-CH3 domain, and wherein:

the domains (D) and (E) are linked so that they do not associate with one another to form an epitope binding site; the domains (A) and (E) associate to form a binding site that binds the HIV-1 envelope like a V3 glycanHIV-1 antibody (1);

6. The bispecific molecule of claim 5, wherein:

(i) the CH2-CH3 domain and domain (A) are separated by a peptide linker 4; (ii) domains (A) and (B) are separated by a peptide linker 1;

(iii) domains (C) and (B) are separated by a peptide linker 2;

(iv) domains (D) and (E) are separated by a peptide linker 1;

(v) domains (F) and (E) are separated by a peptide linker 2;

(vi) the first polypeptide chain further comprises a peptide linker 3 N-terminal to the CH2-CH3 domain; and

(vii) the third polypeptide chain further comprises a peptide linker 3 N-terminal to the CH2-CH3 domain.

7. A bispecific molecule comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first and second polypeptide chains are covalently bonded and the first and third polypeptide chains are covalently bonded, and wherein:

(I) the first and the third polypeptide chains each comprise in the N- to C-terminal direction:

(iii) a domain (C) comprising a heterodimer promoting domain; and

(iv) a CH2-CH3 domain;

(i) a domain (D) comprising a binding region of a light chain variable domain of the second immunoglobulin (VL2) specific for the epitope (2); (ii) a domain (E) comprising a binding region of a heavy chain variable domain of the first immunoglobulin (VH1) having the binding specificity of a V3 glycanHIV-1 antibody (1);

(iii) a domain (F) comprising a heterodimer promoting domain; and wherein the domains (A) and (B) are linked so that they do not associate with one another to form an epitope binding site;

8. The bispecific molecule of claim 7, wherein:

(i) domains (A) and (B) are separated by a peptide linker 1;

(ii) domains (C) and (B) are separated by a peptide linker 2;

(iv) domains (D) and (E) are separated by a peptide linker 1; and

(v) domains (F) and (E) are separated by a peptide linker 2.

9. A trivalent binding molecule comprising a first, second, third and fourth polypeptide chain wherein:

(iii) a domain (C) comprising a heterodimer promoting domain; and

(iv) a CH2-CH3 Domain;

(i) a domain (D) comprising a binding region of a light chain variable domain of the first immunoglobulin (VL1) specific for the epitope (2); (ii) a domain (E) comprising a binding region of a heavy chain variable domain of the second immunoglobulin (VH1) specific for the epitope (1);

(iii) a domain (F) comprising a heterodimer promoting domain; and

(ii) a CH1-Hinge Domain, and a CH2-CH3 Domain; and

(ii) CL Kappa Domain or a CL Lambda Domain; and wherein

at least one of epitope (1), epitope (2), and epitope (3) is an epitope bound by an V3 glycan HIV-1antibody, and at least one of epitope (1), epitope (2), and epitope (3) is an epitope of CD3, CD8, or CD16;

the CH2-CH3 domains of the first and third polypeptide form an Fc Domain;

said first and second polypeptide chains are covalently bonded to one another;

said third and fourth polypeptide chains are covalently bonded to one another.

10. The trivalent binding molecule of claim 9, wherein:

(i) domains (A) and (B) are separated by a peptide linker 1;

(ii) domains (C) and (B) are separated by a peptide linker 2 or a peptide linker 2-C; (iii) the CH2-CH3 domain and domain (C) are separated by a peptide linker 3 or a spacer linker 3;

(iv) domains (D) and (E) are separated by a peptide linker 1; and

(v) domains (F) and (E) are separated by a peptide linker 2 or a peptide linker 2-C.

11. A trivalent binding molecule comprising a first, second, and third polypeptide chain wherein:

(iii) a domain (C) comprising a heterodimer promoting domain; and

(iv) a CH2-CH3 Domain;

(iii) a domain (F) comprising a heterodimer promoting domain; and

(iii) a CH2-CH3 Domain; and wherein

at least one of epitope (1), epitope (2), and epitope (3) is an epitope bound by a V3 glycan HIV-1 antibody, and at least one of epitope (1), epitope (2), and epitope (3) is and epitope of CD3, CD8, or CD16;

the CH2-CH3 domains of the first and third polypeptide form an Fc Domain; said first and second polypeptide chains are covalently bonded to one another; said first and third polypeptide chains are covalently bonded to one another; and

said third and fourth polypeptide chains are covalently bonded to one another.

12. The trivalent binding molecule of claim 11, wherein:

(i) domains (A) and (B) are separated by a peptide linker 1;

(iv) domains (D) and (E) are separated by a peptide linker 1;

(v) domains (F) and (E) are separated by a peptide linker 2 or a peptide linker 2-C; (vi) domains (H) and (G) are separated by a peptide linker 5; and

(vii) the CH2-CH3 domain and domain (G) are separated by a peptide linker 3.

13. The trivalent binding molecule of claims 9-12, wherein one of epitope (1), epitope (2), or epitope (3) is an epitope of CD8.

14. The molecules of claims 3-8 and 9-12, wherein the CH2-CH3 domain of the first

polypeptide chain is the of the“knob” design (SEQ ID NOs: 531 or 532) and the CH2- CH3 domain of the third polypeptide chain is of the“hole” design (SEQ ID NOs: 533 or 534).

15. The molecules of claim 14, wherein the CH2-CH3 domain of the first polypeptide

comprises SEQ ID NO: 531 and the CH2-CH3 domain of the third polypeptide chain comprises SEQ ID NO: 533.

16. The molecules of claims 3-8 and 9-12, wherein the CH2-CH3 domain of the third

polypeptide chain is the of the“knob” design (SEQ ID NOs: 531 or 532) and the CH2- CH3 domain of the first polypeptide chain is of the“hole” design (SEQ ID NOs: 533 or 534).

17. The molecules of claim 16, wherein the CH2-CH3 domain of the third polypeptide

comprises SEQ ID NO: 531 and the CH2-CH3 domain of the first polypeptide chain comprises SEQ ID NO: 533.

18. The molecules of claims 1-12 wherein the epitope (2) is a CD3 epitope, CD8 epitope, or a CD16 epitope.

19. The molecules of claims 1-12, wherein the V3 glycanHIV-1 antibody is DH542,

DH542_QSA, DH542_L4 or any of the antibodies of the DH542 lineage.

20. The molecules of claims 1-12, wherein the molecule binds HIV-1 envelope with the specificity of DH542, DH542_QSA, DH542_L4 antibody and binds CD3, CD8, or CD16.

21. The molecules of claims 1-12, wherein domain (A) comprises the CDR1, CDR2, and CDR3 of the light chain variable domain of immunoglobulin DH542, DH542_QSA, or DH542_L4.

22. The molecules of claims 1-12, wherein domain (E) comprises the CDR1, CDR2, and CDR3 of the heavy chain variable domain of immunoglobulin DH542, DH542_QSA, or DH542_L4.

23. The molecules of claims 1-12, wherein domain (A) comprises the light chain variable domain of immunoglobulin DH542, DH542_QSA, or DH542_L4.

24. The molecules of claims 1-12, wherein domain (E) comprises the heavy chain variable domain of immunoglobulin DH542, DH542_QSA, or DH542_L4.

25. The molecules of claims 1-12, wherein domain (B) comprises the heavy chain variable domain of an anti-CD3 antibody.

26. The molecules of claims 1-12, wherein the domain (D) comprises the light chain variable domain of an anti-CD3 antibody.

27. The trivalent binding molecule of claims 9-12, wherein domain (G) comprises heavy chain variable domain of an anti-CD8 antibody.

28. The trivalent binding molecule of claims 9-12, wherein domain (H) comprises light chain variable domain of an anti-CD8 antibody.

29. The bispecific molecule of claim 3, wherein the first polypeptide comprises SEQ ID NO: 555, the second polypeptide comprises SEQ ID NO: 557, and the third polypeptide comprises SEQ ID NO: 559.

30. The trivalent binding molecule of claim 9, wherein the first polypeptide comprises SEQ ID NO: 596, the second polypeptide comprises SEQ ID NO: 597, the third polypeptide comprises SEQ ID NO: 561, and the fourth polypeptide comprises SEQ ID NO: 562.

31. The trivalent binding molecule of claim 11, wherein the first polypeptide comprises SEQ ID NO: 596, the second polypeptide comprises SEQ ID NO: 597, and the third

polypeptide comprises SEQ ID NO: 563.

32. A composition comprising any one of the molecules of claims 1-31or any combination thereof and a carrier.

33. A composition comprising a bispecific molecule or trivalent molecule which comprises at least one arm with the binding specificity of HIV-1 antibody DH542, DH542_QSA, or DH542_L4, and a second arm targeting CD3, CD8 or CD16.

34. The composition of claim 33, further comprising a second bispecific molecule or trivalent molecule comprising a first arm with the binding specificity of HIV-1 antibody different from the binding specificity of the first bispecific molecule or trivalent molecule and a second arm targeting CD3, CD8 or CD16, wherein the first and secondmolecules are different.

35. A method to treat or prevent HIV-1 infection in a subject in need thereof comprising

administering to the subject a composition comprising any one of the molecules of claim 1-31 or a combination of any one of these molecules in a therapeutically effective amount.

36. The method of claim 35, further comprising administering a latency activating agent.

37. The method of claim 36, wherein the latency activating agent is vorinostat, romidepsin, panobinostat, disulfiram, JQ1, bryostatin, PMA, inonomycin, or any combination thereof.

38. A vector comprising nucleic acids comprising nucleotides encoding the molecules of any one of claim 1-31.

39. A composition comprising a vector comprising a nucleic acid encoding the molecules of any one of claim 1-31.