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WO2025229018A1 - Neutralizing antibody constructs against hiv - Google Patents

Neutralizing antibody constructs against hiv

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Publication number
WO2025229018A1
WO2025229018A1 PCT/EP2025/061759 EP2025061759W WO2025229018A1 WO 2025229018 A1 WO2025229018 A1 WO 2025229018A1 EP 2025061759 W EP2025061759 W EP 2025061759W WO 2025229018 A1 WO2025229018 A1 WO 2025229018A1
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WO
WIPO (PCT)
Prior art keywords
seq
domain
binding protein
hiv
linker
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/061759
Other languages
French (fr)
Inventor
Tiancen HU
James SCHAWALDER
Eric Arnoult
Alan P. Lewis
Mark R. Krystal
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ViiV Healthcare UK No 5 Ltd
Original Assignee
ViiV Healthcare UK No 5 Ltd
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Application filed by ViiV Healthcare UK No 5 Ltd filed Critical ViiV Healthcare UK No 5 Ltd
Publication of WO2025229018A1 publication Critical patent/WO2025229018A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C07K16/1145—
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70514—CD4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention is directed to a multimeric antigen binding protein that binds to the Human Immunodeficiency Virus (HIV) envelope spike complex and its use in treating or preventing HIV infection.
  • HIV Human Immunodeficiency Virus
  • the antigen binding proteins of the invention bind to the MPER region of glycoprotein 41 (gp41) as well as having a CD4 domain that binds to the CD4bs of gpl20.
  • HIV the virus that over time may result in Acquired Immunodeficiency Syndrome (AIDS)
  • AIDS Acquired Immunodeficiency Syndrome
  • HIV attacks the body's immune system, targeting CD4-positive white blood cells, and leaves those infected vulnerable to opportunistic infections such as tuberculosis and fungal infections, severe bacterial infections and some cancers.
  • WHO Key Facts HIV, July 2022).
  • ART antiretroviral therapy
  • NRTIs nucleoside reverse transcriptase inhibitors
  • NRTIs non-nucleoside reverse transcriptase inhibitors
  • protease inhibitors entry inhibitors and integrase inhibitors
  • bNAbs Broadly neutralizing antibodies
  • individual bNAbs have only had limited success in previous studies. This is in part because antibody-resistant virus either already existed in the patient or emerged soon after treatment began (NIH Research Matters, 14 June 2022). Combinations of bNAbs are currently being investigated in the presence or absence of ART (Nature, 606, 368-374, 2022).
  • a multimeric anti-HIV envelope spike complexbinding protein comprising: i. a broadly neutralizing anti-MPER antibody, comprising at least one heavy chain or light chain, wherein the antibody binds to the MPER region of a gp41 protein; and ii. at least one CD4 domain which binds to gpl20, wherein the CD4 domain is attached directly or by a linker to the N-terminus or C-terminus of at least one of the heavy chains or light chains of the broadly neutralizing antibody.
  • compositions comprising anti-HIV envelope spike complex-binding proteins of the invention, methods of preventing HIV infection and methods of treating HIV infection with anti-HIV envelope spike complex-binding proteins of the invention, uses of anti-HIV envelope spike complex-binding proteins of the invention, methods of manufacturing anti-HIV envelope spike complex-binding proteins of the invention and kits comprising anti-HIV envelope spike complex-binding proteins of the invention are also provided.
  • FIG. 1 shows schematic designs of example bispecific binding proteins of the invention.
  • Human CD4 domains or variants thereof are fused, either directly or via linkers, to the N-termini the heavy chains (A), the light chains (B) or to the N-termini of heavy chains (C) or light chains (D) of bNAbs.
  • Such designs facilitate concomitant binding of the human CD4 domain of the bispecific molecule and gpl60 binding domain (or in other words, the envelope spike complex broadly neutralizing antibody or fragment) of the bispecific molecule to HIV-1 gpl60 (the envelope spike complex) and prevent HIV-1 virions from binding to the cell surface receptors and fusing with the cell membrane (E).
  • FIG. 2 shows IC50 values (nM) of soluble CD4 domains (SEQ ID NOs:4-15) against a panel of HIV-1 envelopes in a PSV assay (ACTOne), together with the Tm for each soluble CD4 domain.
  • the horizontal bars indicate geometric mean IC50.
  • FIG. 3 shows that linker length between the CD4 domain and a similar model bNAb to the invention (V3-bNAbl) heavy chain N-terminus does not particularly affect anti-viral activity in a PSV assay (ACTOne) (A) but does change the PK of the resultant bispecific molecules in a humanized mouse model (Tg32-hFcRn strain) (B). Thermal stability of the CD4 domain also affects the PK of the bispecific molecules (B).
  • FIG. 4 shows IC50 (nM) and MPV (%) values for anti-viral activity of MPER-bNAb34-derived bispecifics according to the invention (targeting MPER). These include bispecific 34-1, bispecific 34- 2, bispecific 34-3 and bispecific 34-4 detailed in Table 1 below.
  • bispecific 34-1 contains a 4x GS linker (SEQ ID NO: 33) and a DlD2wt CD4 domain (SEQ ID NO: 1)
  • the HC of bispecific 34-2 contains a 4XGS linker (SEQ ID NO: 33) and a D1.22D2 CD4 domain (SEQ ID NO: 2)
  • the HC of bispecific 34-3 contains a 4XGS linker (SEQ ID NO: 33)and a DI.22 CD4 domain (SEQ ID NO: 4)
  • the HC of bispecific 34-4 contains a 2XG4S linker (SEQ ID NO: 31) and a DI.22 CD4 domain (SEQ ID NO: 4).
  • FIG. 5 shows IC50 (nM), IC90 (nM) and MPV (%) values for anti-viral activity of MPER- bNAb35-derived bispecifics according to the invention (targeting MPER as detailed in Table 2 below.
  • all seventeen of these bispecific molecules contain the same LC (SEQ ID NO: 200), but vary in their HC sequences as different linkers and CD4 domains were used.
  • the HC of bispecific 35-6 contains a 6x GS linker (SEQ ID NO: 35) and a DI.22 CD4 domain (SEQ ID NO: 4)
  • the HC of bispecific 35-5 contains a 5x GS linker (SEQ ID NO: 34) and a DI.22 CD4 domain (SEQ ID NO: 4)
  • the HC of bispecific 35-4 contains a 4x GS linker (SEQ ID NO: 33) and a DI.22 CD4 domain (SEQ ID NO: 4)
  • the HC of bispecific 35-3 contains a 3x GS linker (SEQ ID NO: 32) and a DI.22 CD4 domain (SEQ ID NO: 4)
  • the HC of bispecific 35-2 contains a 2x GS linker (SEQ ID NO: 31) and a DI.22 CD4 domain (SEQ ID NO: 4)
  • the HC of bispecific 35-1 contains a lx GS linker (SEQ ID NO: 30) and a DI.22 CD4
  • CD4D1 SEQ ID NO:3
  • CD4 ECD/D1D2 SEQ ID NO:1
  • A-C bNAb35-derived bispecifics containing CD4 domain 1 and different linkers
  • D-F bNAb35-derived bispecifics containing CD4 domains 1 and 2 and different linkers.
  • A) and (D) show IC50
  • B) and (E) show IC90
  • C) and (F) show MPI.
  • FIG. 6 shows IC50 (nM), IC90 (nM) and MPV (%) values for anti-viral activity of (A-C) a selected MPER-bNAb35-derived bispecific of the invention (specifically bispecifics 35-4 and 35-4-LS as per Figure 5 and Table 2 above) and control molecules against a panel of HIV-1 envelopes in PSV assay (ACTOne cells), said selected bNAb35-derived bispecific further including an "LS" mutation against (D-F) the same panel as well as (G-I) a further panel including further envelopes insensitive to MPER bNAbs described herein.
  • A-C a selected MPER-bNAb35-derived bispecific of the invention (specifically bispecifics 35-4 and 35-4-LS as per Figure 5 and Table 2 above) and control molecules against a panel of HIV-1 envelopes in PSV assay (ACTOne cells), said selected bNAb35-derived bispecific further including an "LS" mutation against (D-F) the same panel as well as (G-I
  • FIG. 7 shows (A) IC50 (nM), (B) IC90 (nM) and (C) MPV (%) values for anti-viral activity of
  • MPER- bNAb36-derived bispecifics according to the invention (targeting MPER, containing a HC with a linker + CD4 domain as per SEQ ID NO: 59 and a LC according to SEQ ID NO: 76) and control molecules against a panel of HIV-1 envelopes in PSV assay (ACTOne cells)
  • FIG. 8 shows (A) IC50 (nM), (B) IC90 (nM) and (C) MPV (%) values for anti-viral activity of MPER-bNAb38-derived bispecifics according to the invention (targeting MPER, containing a HC with a linker + CD4 domain as per SEQ ID NO: 60 and a LC according to SEQ ID NO: 87) and control molecules against a panel of HIV-1 envelopes in PSV assay (ACTOne cells)
  • FIG. 9 shows (A) IC50 (nM), (B) IC90 (nM) and (C) MPV (%) values for anti-viral activity of MPER-bNAb39-derived bispecifics (targeting MPER, containing a HC with a linker + CD4 domain as per SEQ ID NO: 61 and a LC according to SEQ ID NO: 98) and control molecules against a panel of HIV-1 envelopes in PSV assay (ACTOne cells)
  • FIG. 10 shows (A) IC50 (nM), (B) IC90 (nM) and (C) MPV (%) values for anti-viral activity of MPER-bNAb40-derived bispecifics (targeting MPER, containing a HC with a linker + CD4 domain as per SEQ ID NO: 62 and a LC according to SEQ ID NO: 109) and control molecules against a panel of HIV-1 envelopes in PSV assay (ACTOne cells)
  • FIG. 11 shows (A) IC50 (nM), (B) IC90 (nM) and (C) MPV (%) values for anti-viral activity of MPER-bNAb41-derived bispecifics (targeting MPER, containing a HC with a linker + CD4 domain as per SEQ ID NO: 63 and a LC according to SEQ ID NO: 120) and control molecules against a panel of HIV-1 envelopes in PSV assay (ACTOne cells)
  • FIG. 12 shows (A) IC50 (nM), (B) IC90 (nM) and (C) MPV (%) values for anti-viral activity of MPER-bNAb42-derived bispecifics (targeting MPER, containing a HC with a linker + CD4 domain as per SEQ ID NO: 64 and a LC according to SEQ ID NO: 131) and control molecules against a panel of HIV-1 envelopes in PSV assay (ACTOne cells)
  • FIG. 13 shows (A) IC50 (nM), (B) IC90 (nM) and (C) MPV (%) values for anti-viral activity of MPER-bNAb43-derived bispecifics (targeting MPER, containing a HC with a linker + CD4 domain as per SEQ ID NO: 65 and a LC according to SEQ ID NO: 142) and control molecules against a panel of HIV-1 envelopes in PSV assay (ACTOne cells).
  • Binding affinity is the strength of binding at a single interaction site, i.e., of one molecule, e.g., an antigen binding protein, to another molecule, e.g., its target antigen, at a single binding site.
  • the binding affinity of an antigen binding protein to its target may be determined by equilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE analysis).
  • “Alternative antibody formats” include alternative scaffolds in which one or more CDRs of the antigen binding protein can be arranged onto a suitable non-immunoglobulin protein scaffold 5 or skeleton, such as an affibody, a SpA scaffold, an LDL receptor class A domain, an avimer (see, e.g., U.S. Patent Application Publication Nos. 2005/0053973, 2005/0089932, 2005/0164301) or an EGF domain.
  • a suitable non-immunoglobulin protein scaffold 5 or skeleton such as an affibody, a SpA scaffold, an LDL receptor class A domain, an avimer (see, e.g., U.S. Patent Application Publication Nos. 2005/0053973, 2005/0089932, 2005/0164301) or an EGF domain.
  • Antibody is used herein to refer to a heterotetra meric glycoprotein with an approximate molecular weight of 150,000 daltons.
  • An intact antibody is composed of two identical heavy chains (HCs) and two identical light chains (LCs) linked by covalent disulphide bonds. This H2L2 structure folds to form a 'Y' shape with three functional domains comprising two antigen-binding fragments, known as 'Fab' fragments (the 'top' of the ), and a fragment crystallisable 'Fc' (the 'bottom' of the "YQ.
  • the Fab fragment is composed of the variable domain at the amino-terminus, variable heavy (VH) or variable light (VL), and the constant domain at the carboxyl terminus, CHI (heavy) and CL (light).
  • the Fc fragment is composed of two domains formed by dimerization of paired CH2 and CH3 regions. The Fc may elicit effector functions by binding to receptors on immune cells or by binding Clq, the first component of the classical complement pathway.
  • the five classes of antibodies IgM, IgA, IgG, IgE and IgD are defined by distinct heavy chain amino acid sequences, which are called p, a, y, £ and 5 respectively; each heavy chain can pair with either a K or A light chain.
  • an anti-CD4bs antibody refers to an antibody that binds to a CD4 binding site
  • Antigen binding antibody fragments or “antigen binding fragments” or “antibody fragments” as used herein include Fab, F(abQ2, Fv, disulphide linked Fv, single chain Fv (scFv), disulphide-linked scFv, diabodies, TANDABS, etc. and modified versions of any of the foregoing (for a summary of alternative “antibody” formats see Holliger and Hudson, Nature Biotechnology, 23(9), 1126-1136, 2005).
  • Antigen binding protein of the invention and "anti-HIV envelope spike complex binding protein” are used interchangeably herein and refer to antibodies and fragments thereof, alternative antibody formats, and other protein constructs, such as domains, that are capable of binding to the HIV envelope spike complex comprised of gpl20 and gp41, or in other words are capable of binding to either gpl20 or gp41.
  • the HIV env gene encodes a gene product of around 850 amino acids.
  • the primary env product is the protein gpl60 (or envelope glycoprotein gpl60) which is gets cleaved into gpl20 (about 480 amino acids) and gp41 (about 345 amino acids) in the endoplasmic reticulum by the cellular protease furin.
  • gpl20 (or gpl20) is a 120 kDa glycoprotein that is part of the outer layer of HIV. It presents itself as viral membrane spikes consisting of three molecules of gpl20 linked together and anchored to the membrane by gp41 protein. Gpl20 is essential for viral infection as it facilitates HIV entry into the host cell through its interaction with cell surface receptors.
  • Gp41 is a transmembrane protein that contains several sites within its ectodomain that are required for infection of host cells.
  • the amino acid sequence of an exemplary gpl60 from HIV clone WITO is provided below (SEQ ID NO: 55):
  • amino acid of an exemplary gpl20 is provided below (SEQ ID NO: 56):
  • amino acid of an exemplary gp41 is provided below (SEQ ID NO: 57; the MPER region is boldened, and shown separately as SEQ ID NO: 58):
  • Antigen binding site and “paratope” are used interchangeably herein and refer to a particular site on an antigen binding protein that makes contact with and is capable of specifically binding to a site (i.e., epitope) on an antigen, e.g., HIV gpl20 or gp41.
  • the antigen binding site may be formed by a single variable domain, or paired VH/VL domains as can be found on a standard antibody.
  • Single-chain Fv (ScFv) domains can also provide antigen binding sites.
  • “Avidity” also referred to as functional affinity, is the cumulative strength of binding at multiple interaction sites, e.g., the sum total of the strength of binding of two molecules (or more) to one another at multiple sites, e.g., taking into account the valency of the interaction.
  • a “bispecific molecule” or “bispecific antigen binding protein” as used herein is an antigen binding protein that is capable of binding to two different epitopes on the same antigen, i.e., HIV gp41 protein.
  • one epitope comprises part of or the whole of MPER region of gpl41 and the other epitope comprises part of or the whole of the CD4 binding site of gpl20.
  • BNAb Broadly neutralizing antibody
  • a broadly neutralizing antibody may neutralize at least 2, 3, 4, 5, 6, 7, 8, 9 or more different strains of HIV-1, the strains belonging to the same or different clades.
  • CD4 binding site or “CD4-binding site” or “CD4bs” refers to a site on the HIV envelope protein gpl20 that binds to CD4. (Cluster of differentiation factor 4).
  • CD4 is a T-cell surface protein that serves as the primary receptor site for HIV during HIV infection.
  • the CD4 binding site on gpl20 is a highly conserved, discontinuous and conformational that comprises residues on either side of the HIV V4 loop (Curr HIV/AIDS Rep, 9(1): 52-63, 2021) that binds to CD4.
  • a “CD4 domain” as used herein is a soluble recombinant form of human CD4 (Cluster of differentiation factor 4, a transmembrane glycoprotein found on T-cells ), or a fragment thereof, that mimics the activity of native membrane-anchored human CD4 in its binding interactions with the HIV envelope protein.
  • a CD4 domain of the present invention binds to the CD4-binding site of HIV gpl20 and may block the ability of HIV gpl20 to bind membrane-anchored CD4, e.g., on CD4+ T cells.
  • a CD4 domain of the invention may induce a structural rearrangement in gpl20 upon binding, including a structural rearrangement of part or all of the MPER of gp41 (PMID: 32601441).
  • CD4 comprises four domains that are exposed on the extracellular surface of the cell, DI, D2, D3 and D4; a transmembrane domain; and a cytoplasmic tail domain. DI and D3 resemble Ig variable domains and D2 and D4 resemble Ig constant domains.
  • CD4 domains of the invention include one or more of domains DI to D4 of CD4, or variants thereof.
  • CD4 domains of the invention include wild-type DI (SEQ ID NO:3); "mD1.22”/Dlm (SEQ ID NO:4), which is a variant of DI of CD4 (Chen et al, JVI 88(2): 1125-39, 2014); wild-type D1D2 (SEQ ID NO:1); "mD1.22-D2" (SEQ ID NO:2), which is a variant of D1D2 (Fetzer et al., Journal of Virology, 92(12), 2018); and further variants of mD1.22 (SEQ ID NOs:5- 21).
  • CDRs are defined as the complementarity determining region amino acid sequences of an antigen binding protein.
  • the hypervariable regions of immunoglobulin heavy and light chains There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin.
  • the CDRs are defined based on the Kabat definition.
  • the CDRs are defined based on the Chothia definition.
  • the Chothia definition is from Discovery Studio which uses the definitions fromChothia and Lesk, JMol Biol. 196(4):901-17 (1987) and Morea et al, Methods, 20:267-279 (2000).
  • the Chothia definition is based on the Chothia from Abysis definition.
  • CDRs are defined based on the IMGT definition. In another embodiment, the CDRs are defined based on the Honegger definition. In another embodiment, the CDRs are defined based on the contact definition.
  • CDRs refers to all three heavy chain CDRs, all three light chain CDRs, all heavy and light chain CDRs, or at least two CDRs.
  • Domain refers to a folded polypeptide structure that retains its tertiary structure independent of the rest of the polypeptide. Generally, domains are responsible for discrete functional properties of polypeptides and in many cases may be added, removed or transferred to other polypeptides without loss of function of the remainder of the protein and/or of the domain.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • DCP complement-dependent cell- mediated phagocytosis
  • ADCML antibody dependent complement-mediated cell lysis
  • ADCP Fc-mediated phagocytosis or antibody-dependent cellular phagocytosis
  • Epitope refers to the portion of an antigen (e.g., gpl20) that makes contact with and is capable of specifically binding to a particular site (paratope) on an antigen binding protein.
  • An epitope may be linear or conformational/discontinuous.
  • a conformational/ discontinuous epitope comprises amino acid residues that are separated by other sequences, i.e., it does not comprise a continuous sequence in the antigen's primary amino acid sequence, but instead relies on the tertiary folding of the polypeptide.
  • residues within a confirmational/ discontinuous epitope may be from different regions of the polypeptide chain, they are in close proximity in the three-dimensional structure of the antigen.
  • a conformational or discontinuous epitope may include residues from different polypeptide chains. Particular residues comprised within an epitope can be determined through computer modelling programs or via three-dimensional structures obtained through methods known in the art, such as X-ray crystallography. Epitope mapping can be carried out using various techniques known to persons skilled in the art as described in publications such as Methods in Molecular Biology 'Epitope Mapping Protocols', by Mike Schutkowski and Ulrich Reineke (volume 524, 2009) and Johan Rockberg and Johan Nilvebrant (volume 1785, 2018).
  • Exemplary methods include peptide-based approaches such as pepscan whereby a series of overlapping peptides are screened for binding using techniques such as ELISA or by in vitro display of large libraries of peptides or protein mutants, e.g., on phage.
  • Detailed epitope information can be determined by structural techniques including X-ray crystallography, solution nuclear magnetic resonance (NMR) spectroscopy and cryogenic-electron microscopy (cryo- EM). Mutagenesis, such as alanine scanning, is an effective approach whereby loss of binding analysis is used for epitope mapping.
  • Another method is hydrogen/deuterium exchange (HDX) combined with proteolysis and liquid-chromatography mass spectrometry (LC-MS) analysis to characterize discontinuous or conformational epitopes.
  • HDX hydrogen/deuterium exchange
  • LC-MS liquid-chromatography mass spectrometry
  • “Half-life” or “tl/2” refers to the time required for the serum concentration of an antigen binding protein to reach half of its original value.
  • the serum half-life of proteins can be measured by pharmacokinetic studies according to the method described by Kim et al., 1994, Eur. J. of Immuno. 24: 542-548. According to this method, radio-labelled protein is injected intravenously into mice and its plasma concentration is periodically measured as a function of time, for example, at about 3 minutes to about 72 hours after the injection. Other methods for pharmacokinetic analysis and determination of the half-life of a molecule will be familiar to those skilled in the art.
  • HIV envelope protein or "ENV” or “spike complex” refers to a trimeric viral membrane- associated glycoprotein (gp) or 'spike' comprised of non-covalently linked heterodimers of surface gpl20 and transmembrane gp41. It is found on both the viral membrane and the cell membrane of infected host cells and is encoded by the Env gene. The env gene encodes the gpl60 polypeptide which forms a homotrimer and is cleaved into gpl20 and gp41 polypeptides.
  • Gpl20 is a surface (SU) glycoprotein responsible for binding to receptor molecules and the transmembrane (TM) glycoprotein, gp41, mediates fusion of the viral membrane with the plasma cell membrane.
  • TM transmembrane
  • Over half of the mass of the trimeric envelope 'spike' is an N-linked glycan shield that hides most amino acid-based epitopes on gpl20.
  • Binding of the cell surface receptor CD4 to HIV gpl20 induces a structural rearrangement creating a high affinity binding site for a chemokine coreceptor (CXCR4 and/or CCR5), on gpl20.
  • CXCR4 and/or CCR5 chemokine coreceptor
  • HIV Human immunodeficiency virus
  • HIV-1 is more virulent and more infective than HIV-2 and is the cause of the majority of HIV infections globally, whereas HIV-2 is limited to a much smaller number of people, mostly in West Africa (Gilbert et al., Statistics in Medicine 22(4): 573-593).
  • HIV may mean HIV-1 and HIV-2, or just “HIV-1”.
  • HIV virions are spherical with viral glycoprotein "spikes", the HIV envelope protein, protruding outwards.
  • a conical capsid exists within the virion, enclosing a ribonucleoprotein complex comprising two copies of positive-sense single stranded RNA tightly bound to nucleocapsid proteins and enzymes needed for viral replication.
  • Symptoms of HIV include raised temperature (fever), sore throat, body rash, tiredness, joint pain, muscle pain, swollen glands, weight loss, chronic diarrhoea, night sweats, skin problems, recurrent infections, headaches.
  • a “linker” is a suitable structure that can be used to join together the CD4 domain and antibody or fragment of the invention.
  • a linker may be a chemical linker such as PEG, or an amino acid sequence that links one domain in a polypeptide to another domain in a polypeptide.
  • a linker within the meaning of the invention includes an amino acid sequence that joins a CD4 domain to a bNAb heavy chain or a bNAb light chain.
  • the linker is not cleavable under intracellular conditions.
  • Multimeric antigen binding protein or “multispecific antigen binding protein” refers to an antigen binding protein that comprises at least two different polypeptide chains having antigen binding sites. Each of these antigen-binding sites is capable of binding to a different epitope, which may be present on the same antigen or different antigens.
  • the multi-specific antigen binding proteins of the invention are bispecific molecules capable of binding to two different epitopes on the HIV envelope protein.
  • one epitope may comprise part of or the whole of the MPER region of gp41 and the other epitope may comprise part of or the whole of the CD4 binding site of gpl20 (through binding of the CD4 domain portion of the molecule).
  • Symmetric formats of MSABPs combine multiple binding specificities in a single polypeptide chain or single HL pair including Fc-fusion proteins of fragment-based formats and formats whereby antibody fragments are fused to regular antibody molecules.
  • Examples of symmetric formats may include DVD-Ig, TVD-Ig, CODV-Ig, (scFv)4-Fc, IgG-(scFv)2, Tetravalent DART-Fc, F(ab)4CrossMab, IgG-HC-scFv, IgG-LC-scFv, mAb-dAb etc.
  • a multimeric antigen binding protein described herein for example, a bispecific antigen binding protein having a broadly neutralizing anti-HIV envelope spike complex antibody and a CD4 domain, may be encoded by one or more isolated nucleic acid sequences.
  • Production of a multimeric antigen binding protein, such as a bispecific antigen binding protein may be achieved in a cell or living organism by delivering exogenous isolated nucleic acids encoding the multimeric antigen binding protein, for example, a heavy chain and a light chain of a broadly neutralizing anti-HIV envelope spike complex antibody and a CD4 domain.
  • Production of a multimeric antigen binding protein may be achieved in a cell in vitro or in vivo by delivering exogenous isolated nucleic acids encoding the multimeric antigen binding protein, for example, a heavy chain and a light chain of a broadly neutralizing anti-HIV envelope spike complex antibody and a CD4 domain.
  • a subject in need may be delivered one or more nucleic acids encoding an multimeric antigen binding protein provided herein, such as a heavy chain and a light chain of a broadly neutralizing anti-HIV envelope spike complex antibody and a CD4 domain.
  • the heavy chain and the light chain of the antibody may be delivered by the same or separate nucleic acids.
  • the nucleic acids may be DNA or RIMA.
  • the nucleic acids encoding the multimeric antigen binding protein may be delivered to the subject naked (i.e. without an encapsulating particle) or packaged (i.e. encapsulated in liposomes or polymer-based vehicles).
  • the nucleic acids encoding the multimeric antigen binding protein may be delivered without a delivery vehicle (i.e., "naked") or delivered with a viral or non-viral delivery vehicle (i.e., as a viral vector, adsorbed to or encapsulated in liposomes or polymer-based vehicles, and the like).
  • the nucleic acid may include elements such as a poly-A tail, a 5' and/or 3' untranslated region (UTR).
  • the nucleic acids may be mRNA.
  • the mRNA may include a cap structure.
  • the mRNA may be self-replicating RNA.
  • the nucleic acid coding for the multimeric antigen binding proteins may be modified or unmodified.
  • the nucleic acids coding for the multimeric antigen binding proteins may comprise at least one chemical modification.
  • Nucleic acids e.g., mRNAs
  • Such chemical modifications include, but are not limited to, a modified nucleotide, a modified sugar backbone, and the like.
  • a method of producing a multimeric antigen binding protein in a cell, tissue, or organism comprising contacting said cell, tissue, or organism with a composition comprising an isolated nucleic acid comprising at least one chemical modification and which encodes the multimeric antigen binding protein.
  • Also provided herein is a method of producing a multimeric antigen binding protein in a cell, tissue or organism comprising contacting said cell, tissue or organism with a composition comprising a polynucleotide comprising at least one chemical modification and which encodes a multimeric antigen binding protein. Also provided herein is a method of producing a multimeric antigen binding protein in a cell, in vitro or in vivo, comprising contacting said cell with a composition comprising a nucleic acid comprising at least one chemical modification and which encodes a multimeric antigen binding protein.
  • Neutralizes as used throughout the present specification means that the biological activity of HIV is reduced in the presence of an antigen binding protein as described herein in comparison to the biological activity of HIV in the absence of the antigen binding protein, in vitro or in vivo.
  • a neutralizing antigen binding protein of the invention may inhibit HIV entry into a target cell and reduce viral load in a patient infected with HIV.
  • Percent identity or “% identity” between a query amino acid sequence and a subject amino acid sequence is the "Identities” value, expressed as a percentage, that is calculated using a suitable algorithm (e.g., BLASTP, FASTA, Needleman-Wunsch, Smith-Waterman, LALIGN, or GenePAST/KERR) or software (e.g., DNASTAR Lasergene, GenomeQuest, EMBOSS needle or EMBOSS infoalign), over the entire length of the query sequence after a pair-wise global sequence alignment has been performed using a suitable algorithm (e.g., Needleman-Wunsch or GenePAST/KERR) or software (e.g. DNASTAR Lasergene or GenePAST/KERR).
  • a query amino acid sequence may be described by an amino acid sequence disclosed herein, in particular in one or more of the claims.
  • the query sequence may be 100% identical to the subject sequence, or it may include up to a certain integer number of amino acid alterations as compared to the subject sequence such that the % identity is less than 100%.
  • the query sequence is at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the subject sequence.
  • amino acid sequences such alterations include at least one amino acid residue deletion, substitution (including conservative and non-conservative substitutions), or insertion, wherein said alterations may occur at the amino- or carboxy-terminal positions of the query sequence or anywhere between those terminal positions, interspersed either individually among the amino acid residues in the query sequence or in one or more contiguous groups within the query sequence.
  • the % identity may be determined across the entire length of the query sequence, including the CDRs.
  • the % identity may exclude one or more or all of the CDRs, for example all of the CDRs are 100% identical to the subject sequence and the % identity variation is in the remaining portion of the query sequence, e.g., the framework sequence, so that the CDR sequences are fixed and intact.
  • Protein scaffold as used herein includes, but is not limited to, an immunoglobulin (Ig) scaffold, for example an IgG scaffold, which may be a four chain or two chain antibody, or which may comprise only the Fc region of an antibody, or which may comprise one or more constant regions from an antibody, which constant regions may be of human origin.
  • Ig immunoglobulin
  • the protein scaffold may be an Ig scaffold, for example an IgG, or IgA scaffold.
  • the IgG scaffold may comprise some or all the domains of an intact antibody (i.e., CHI, CH2, CH3, VH, VL).
  • the antigen binding protein may comprise an IgG scaffold selected from IgGl, IgG2, IgG3, IgG4 or IgG4PE.
  • the scaffold may be IgGl.
  • the scaffold may consist of, or comprise, the Fc region of an antibody, or is a part thereof.
  • the protein scaffold may be a non-Ig scaffold.
  • the protein scaffold may be a derivative of a scaffold selected from one or more of CTLA-4, lipocalin, Protein A derived molecules such as Z- domain of Protein A (Affibody, SpA), A-domain (Avimer/Maxibody); heat shock proteins such as GroEl and GroES; transferrin (trans-body); ankyrin repeat protein (DARPin); peptide aptamer; Ctype lectin domain (Tetranectin); human y-crystallin and human ubiquitin (affilins); PDZ domains; scorpion toxin kunitz type domains of human protease inhibitors; and fibronectin/adnectin; which has been subjected to protein engineering in order to obtain binding to an antigen, such as gp41.
  • Protein A derived molecules such as Z- domain of Protein A (Affibody, SpA), A-domain (Avimer/Maxibody); heat shock proteins such as
  • Single variable domain refers to a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes complete antibody variable domains such as VH, VHH and VL and modified antibody variable domains, for example, in which one or more loops have been replaced by sequences that are not characteristic of antibody variable domains, or antibody variable domains that have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains that retain at least the binding activity and specificity of the full-length domain.
  • a single variable domain as defined herein is capable of binding an antigen or epitope independently of a different variable region or domain.
  • a "domain antibody” or “DAB” may be considered the same as a human "single variable domain”.
  • a single variable domain may be a human single variable domain, but also includes single variable domains from other species such as rodent (for example, as disclosed in WO 00/29004), nurse shark and Camelid VHHs
  • Camelid VHHs are immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain only antibodies naturally devoid of light chains.
  • Such VHH domains may be humanised according to standard techniques available in the art, and such domains are considered to be "single variable domains".
  • Stabilizing mutation refers to a change of an amino acid residue in a polypeptide sequence that increases the thermal thermostability of said polypeptide. Increased thermostability may be reflected in a melting temperature (Tm) increase of, for example, between 1 and 50 °C.
  • Tm melting temperature
  • CD4 domains with stabilizing mutations include SEQ ID NOs:5-21.
  • the VH or VL (or HC or LC) sequence may be a variant sequence with up to 10 amino acid substitutions, additions or deletions.
  • the variant sequence may have up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitution(s), addition(s) or deletion(s).
  • the sequence variation may exclude one or more or all of the CDRs, for example the CDRs are the same as the VH or VL (or HC or LC) sequence and the variation is in the remaining portion of the VH or VL (or HC or LC) sequence, so that the CDR sequences are fixed and intact.
  • V3 loop region refers to the third variable region (V3) of HIV gpl20. Comparison of predicted amino acid sequences from several different isolates has shown that sequence heterogeneity of gpl20 is clustered in five variable regions (designated VI, V2, V3, V4, and V5.) The V3 region contains post-translational modifications, such as glycosylation, and is essential for viral infectivity. The V3 region, although only 35 amino acids long, exhibits considerable sequence variability. Additionally, variability in potential N-linked glycosylation sites allow for further variability in the variable regions of gpl20.
  • V3 region and the N- linked glycosylation sites within and adjacent to the region are understood to comprise the "V3 loop region," "V3/glycan” or V3" as used herein.
  • one site of glycosylation e.g., 30 oligomannose such as Man-5 to Man-9
  • N332 of gpl20 amino acid residue N332 of gpl20.
  • Other sites of potential N-linked glycosylation within and adjacent to the V3 loop region include K295, N301, N386, N392 of gpl20.
  • the V3 loop is generally considered to be in the region between cysteine residues C296 and C331 of gpl20, while some N-linked glycosylation sites are located directly adjacent to the V3 loop.
  • the V3 loop comprises a highly conserved tetrapeptide sequence, GPGR (residues 312 to 315) (Ivanhoff et al., Virology, 187(2) 1992). HIV-1 cellular entry depends on the interaction of the V3 loop region with an HIV co-receptor, commonly CCR5 or CXCR4.
  • the V3 loop comprises: (i) the base (residues 296-299), (ii) the stem (residues 300-303 and 321-326), and (iii) the crown (residues 304-320) (Friedrich et al., Nature Communications 12, 6705 (2021)).
  • a consensus sequence of the V3 region of gpl20 (Milich et al., J Virol., 67(9):5623-5634 (1993)) is provided below:
  • V3-bNAb or "anti-V3 bNAb” is a bNAb that binds within the V3 loop region.
  • a V3- bNAb may also be referred to herein as an anti-V3 antibody.
  • a V3-bNAb may bind the N332 glycan in the V3 loop region and/or other N-linked glycosylation sites within and adjacent to the V3 loop region.
  • MPER Membrane Proximal External Region
  • MPER region refers to a functional segment of HIV gp41.
  • the MPER region is located near the viral membrane and plays a crucial role in the fusion of the viral envelope with the host cell membrane during the process of viral entry.
  • a consensus sequence of the MPER region of gp41 is provided below:
  • consensus sequence describes the highest frequency of residues emerging on each position of this region across multiple subtypes, but that this region of a particular strain may exhibit sequence variability.
  • MPER-bNAb or "anti-MPER bNAb” is a bNAb that binds within the MPER region of gp41.
  • An MPER-bNAb may also be referred to herein as an anti-MPER antibody.
  • a multimeric antigen binding protein of the invention binds to to the MPER region of gp41 as well as having a CD4 domain that binds to the CD4bs of gpl20 have been shown to effectively neutralize HIV and exhibit significantly better anti-viral activity than monospecific molecules that only bind to the MPER region of gp41 or the CD4bs of gpl20, and mixtures of these monospecific molecules.
  • a multimeric anti-HIV envelope spike complex -binding protein comprising: i. a means for binding to the MPER domain of a gp41 protein and neutralizing at least 2 different strains of HIV-1; and ii. at least one CD4 domain which binds to gpl20, wherein the CD4 domain is attached directly or by a linker to the N-terminus or C-terminus of said means for binding.
  • the means for binding to the MPER region of a gp41 protein and neutralizing at least 2 different strains of HIV-1 is a broadly neutralizing antibody or fragment thereof.
  • the means for binding to binding to the MPER region of a gp41 protein and neutralizing at least 2 different strains of HIV-1 may be any of the bispecific binding proteins exemplified in Example 6 and Figures 4-13.
  • the means for binding to binding to the MPER region of a gp41 protein and neutralizing at least 2 different strains of HIV-1 may comprise CDR sequences according to any row of Table 3 and/or heavy and light variable regions or heavy and light chain sequences according to any row of Table 4.
  • a multimeric anti-HIV envelope spike complex-binding protein comprising: i. a broadly neutralizing anti-MPER antibody, comprising at least one heavy chain or light chain, wherein the antibody binds to the MPER domain of a gp41 protein; and ii. at least one CD4 domain which binds to gpl20, wherein the CD4 domain is attached directly or by a linker to the N-terminus or C-terminus of one of the heavy chains or light chains.
  • the binding protein is a bispecific binding protein.
  • CD4 domains of the invention include SEQ ID NOs: 1-21.
  • the CD4 domain is a CD4 DI domain.
  • the CD4 domain is a human CD4 domain.
  • CD4 DI domains include human wild-type DI (SEQ ID NO:3), mD1.22 (SEQ ID NO:4) also known as Dim, and further variants of mD1.22 (SEQ ID NOs: 5-21).
  • the CD4 domain has a sequence that is at least 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 3 or 4.
  • the CD4 domain is a CD4 D1D2 domain.
  • the CD4 domain is a human CD4 D1D2 domain.
  • CD4 D1D2 domains include human wild-type D1D2 (SEQ ID NO:1) and mD1.22-D2 (SEQ ID NO:2).
  • the CD4 domain has a sequence that is at least 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO:1 or
  • a stabilized CD4 domain is provided.
  • a stabilized CD4 DI domain is provided.
  • the CD4 domain is thermally stable, i.e., thermostable.
  • the CD4 domain is a thermostable CD4 DI domain.
  • the CD4 domain comprises one or more stabilizing mutations compared to the wild type sequence.
  • the stabilizing mutations are in the CD4 DI domain.
  • the CD4 DI domain comprises at least one mutation at position 5, 23, 55, 79, 96 and/or 98 of SEQ ID NO: 1 or 3.
  • the CD4 DI domain comprises one or more mutations at positions 8, 11, 13, 21, 25, 27, 38, 52, 58, 61, 65, 70, 72, 87, 91 and or 99 of SEQ ID NO: 1 or 3.
  • the CD4 DI domain comprises a mutation at position 8 or SEQ ID NO: 1 or 3.
  • the CD4 DI domain comprises a mutation at positions 11 and 72 of SEQ ID NO: 1 or 3.
  • the CD4 DI domain comprises mutation at positions 8 and 99.
  • the CD4 DI domain comprises at least one mutation selected from: L5Y, S23N, A55V, I79P, L96V and/or F98V mutations
  • the CD4 DI domain comprises at least one mutation selected from: K8C, K8I, K8V, T11C, E13C, K21C, Q25E, H27C, H27D, G38C, N52W, R58N, R58T, R58V, L61M, G65C, I70C, K72C, E87G, E91H, E91Q, and/or G99C mutations.
  • the CD4 DI domain comprises a K8I mutation.
  • the CD4 DI domain comprises a K8V mutation.
  • the CD4 DI domain comprises a TIC and a K72C mutation. In an embodiment, the CD4 DI domain comprises a K8C and a G99C mutation. In all of these embodiments, these mutations may be compared to a wild type sequence as per SEQ ID NO: 1 or 3.
  • CD4 domains of the invention comprising novel and inventive stabilizing mutations include SEQ ID NOs:5-21.
  • the CD4 domain is a SEQ ID NOs: 5-15.
  • the CD4 domain has a sequence that is at least 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 5-21.
  • the CD4 domain has a sequence that is at least 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 5-15.
  • Tm melting temperature
  • the Tm increase is determined by measuring the Tm of the CD4 domain(s) comprising one or more stabilizing mutations and subtracting the Tm of the corresponding CD4 domain(s) without said mutation(s). For example, measuring the Tm of a stabilized CD4 DI domain and subtracting the Tm of the wild-type CD4 DI domain.
  • the Tm increase is about 8 °C.
  • the Tm increase is about 9 °C.
  • the Tm increase is about 12 °C.
  • the Tm increase is about 21 °C.
  • the Tm of the CD4 domain is above 70 °C. In an embodiment, the Tm of the CD4 domain is between 70 °C and 95 °C. In an embodiment, the Tm of the CD4 domain is between 75 °C and 95 °C. In an embodiment, the Tm of the CD4 domain is between 75 °C and 91 °C.
  • the Tm of the CD4 domain is about 76 °C, about 77 °C, about 78 °C, about 79 °C, about 80 °C, about 81 °C, about 82 °C, about 83 °C, about 84 °C, about 85 °C, about 86 °C, about 87 °C, about 88 °C, about 89 °C, or about 90 °C.
  • the Tm of the CD4 domain is about 90 °C.
  • the Tm of the CD4 domain is about 89 °C.
  • Tm may be determined by routine methods known in the art or as set out in the Examples. In an embodiment, Tm is determined using the Prometheus System (NanoTemper, Munchen Germany).
  • a CD4 domain as described above may be attached directly to a broadly neutralizing antibody, by "directly” is meant that a CD4 domain is covalently bonded to a broadly neutralizing antibody without the use of an additional linking chemical or peptide sequence e.g. OxGS.
  • the CD4 domain as described above may be attached by a linker (for example a peptide linker such as those shown in SEQ ID: 30-35).
  • Anti-HIV envelope spike complex broadly neutralizing antibodies
  • An antigen binding protein of the invention may comprise a broadly neutralizing antibody or fragment thereof having heavy chain CDRs (CDRH1, CDRH2, and CDRH3) as set out in any row of Table 3.
  • An antigen binding protein of the invention may comprise a broadly neutralizing antibody or fragment thereof having light chain CDRs (CDRL1, CDRL2, and CDRL3) as set out in any row of Table 3.
  • An antigen binding protein of the invention may comprise a broadly neutralizing antibody or fragment thereof having a set of six CDRs (CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3) as set out in any row of Table 3.
  • An antigen binding protein of the invention may comprise a broadly neutralizing antibody or fragment thereof having a VH domain as set out in Table 4.
  • An antigen binding protein of the invention may comprise a broadly neutralizing antibody or fragment thereof having a VL domain as set out in Table 4.
  • An antigen binding protein of the invention may comprise a broadly neutralizing antibody or fragment thereof having a pair of variable domains (a VH and a VL) as set out in any row of Table 4.
  • Table 4 SEO ID NOs for the variable heavy regions (VH) and variable light regions (VL) variable domain pairs'), and the full heavy chains (with and without LS mutations') and light chains (LC) of broadly neutralizing antibodies.
  • an antigen binding protein of the invention may comprise an anti-MPER bNAb or a fragment thereof (i.e. a broadly neutralizing antibody or fragment binds to the MPER region of a gp41 protein).
  • a fragment may include Fab, F(abQ2, Fv, disulphide linked Fv, single chain Fv (scFv), disulphide-linked scFv, diabodies, TANDABS, etc. and modified versions of any of the foregoing.
  • scFv variants of anti-MPER bNAbs have been described in van Dorsten et al.
  • An anti-MPER bNAb or fragment thereof includes an antibody comprising a set of CDRs
  • An anti-MPER bNAb or fragment thereof also includes an antibody comprising a pair of variable domains (a VH and VL) set out each row of Table 4.
  • An anti-MPER bNAb may be an antibody comprising a heavy chain (HC), with or without M428L/N434S (EU numbering) 'LS' mutations, and a light chain (LC) as set out in each row of Table 4.
  • the HC comprises LS.
  • the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 168, a CDRH2 of SEQ ID NO: 169, a CDRH3 of SEQ ID NO: 170, a CDRL1 of SEQ ID NO: 171, a CDRL2 of SEQ ID NO: 172 and a CDRL3 of SEQ ID NO: 173.
  • the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 179, a CDRH2 of SEQ ID NO: 180, a CDRH3 of SEQ ID NO: 181, a CDRL1 of SEQ ID NO: 182, a CDRL2 of SEQ ID NO: 183 and a CDRL3 of SEQ ID NO: 184.
  • the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 190, a CDRH2 of SEQ ID NO: 191, a CDRH3 of SEQ ID NO: 192, a CDRL1 of SEQ ID NO: 193, a CDRL2 of SEQ ID NO: 194 and a CDRL3 of SEQ ID NO: 195.
  • the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 66, a CDRH2 of SEQ ID NO: 67, a CDRH3 of SEQ ID NO: 68, a CDRL1 of SEQ ID NO: 69, a CDRL2 of SEQ ID NO: 70 and a CDRL3 of SEQ ID NO: 71.
  • the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 77, a CDRH2 of SEQ ID NO: 78, a CDRH3 of SEQ ID NO: 79, a CDRL1 of SEQ ID NO: 80, a CDRL2 of SEQ ID NO: 81 and a CDRL3 of SEQ ID NO: 82.
  • the anti- MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 88, a CDRH2 of SEQ ID NO: 89, a CDRH3 of SEQ ID NO: 90, a CDRL1 of SEQ ID NO: 91, a CDRL2 of SEQ ID NO: 92 and a CDRL3 of SEQ ID NO: 93.
  • the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 99, a CDRH2 of SEQ ID NO: 100, a CDRH3 of SEQ ID NO: 101, a CDRL1 of SEQ ID NO: 102, a CDRL2 of SEQ ID NO: 103 and a CDRL3 of SEQ ID NO: 104.
  • the anti- MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 110, a CDRH2 of SEQ ID NO: 111, a CDRH3 of SEQ ID NO: 112, a CDRL1 of SEQ ID NO: 113, a CDRL2 of SEQ ID NO: 114 and a CDRL3 of SEQ ID NO: 115.
  • the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 121, a CDRH2 of SEQ ID NO: 122, a CDRH3 of SEQ ID NO: 123, a CDRL1 of SEQ ID NO: 124, a CDRL2 of SEQ ID NO: 125 and a CDRL3 of SEQ ID NO: 126.
  • the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 132, a CDRH2 of SEQ ID NO: 133, a CDRH3 of SEQ ID NO: 134, a CDRL1 of SEQ ID NO: 135, a CDRL2 of SEQ ID NO: 136 and a CDRL3 of SEQ ID NO: 137.
  • the anti-MPER bNAb or fragment thereof comprises a VH domain having at least 95% sequence identity to SEQ ID NO: 174 and a VL domain having at least 95% sequence identity to SEQ ID NO: 175, optionally wherein the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 168, a CDRH2 of SEQ ID NO: 169, a CDRH3 of SEQ ID NO: 170, a CDRL1 of SEQ ID NO: 171, a CDRL2 of SEQ ID NO: 172 and a CDRL3 of SEQ ID NO: 173.
  • the anti-MPER bNAb or fragment thereof comprises a VH domain having at least 95% sequence identity to SEQ ID NO: 185 and a VL domain having at least 95% sequence identity to SEQ ID NO: 186, optionally wherein the the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 179, a CDRH2 of SEQ ID NO: 180, a CDRH3 of SEQ ID NO: 181, a CDRL1 of SEQ ID NO: 182, a CDRL2 of SEQ ID NO: 183 and a CDRL3 of SEQ ID NO: 184.
  • the anti-MPER bNAb or fragment thereof comprises a VH domain having at least 95% sequence identity to SEQ ID NO: 196 and a VL domain having at least 95% sequence identity to SEQ ID NO: 197, optionally wherein the the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 190, a CDRH2 of SEQ ID NO: 191, a CDRH3 of SEQ ID NO: 192, a CDRL1 of SEQ ID NO: 193, a CDRL2 of SEQ ID NO: 194 and a CDRL3 of SEQ ID NO: 195.
  • the anti-MPER bNAb or fragment thereof comprises a VH domain having at least 95% sequence identity to SEQ ID NO: 72 and a VL domain having at least 95% sequence identity to SEQ ID NO: 73, optionally wherein the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 66, a CDRH2 of SEQ ID NO: 67, a CDRH3 of SEQ ID NO: 68, a CDRL1 of SEQ ID NO: 69, a CDRL2 of SEQ ID NO: 70 and a CDRL3 of SEQ ID NO: 71.
  • the anti-MPER bNAb or fragment thereof comprises a VH domain having at least 95% sequence identity to SEQ ID NO: 83 and a VL domain having at least 95% sequence identity to SEQ ID NO: 84, optionally wherein the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 77, a CDRH2 of SEQ ID NO: 78, a CDRH3 of SEQ ID NO: 79, a CDRL1 of SEQ ID NO: 80, a CDRL2 of SEQ ID NO: 81 and a CDRL3 of SEQ ID NO: 82.
  • the anti-MPER bNAb or fragment thereof comprises a VH domain having at least 95% sequence identity to SEQ ID NO: 94 and a VL domain having at least 95% sequence identity to SEQ ID NO: 95, optionally wherein the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 88, a CDRH2 of SEQ ID NO: 89, a CDRH3 of SEQ ID NO: 90, a CDRL1 of SEQ ID NO: 91, a CDRL2 of SEQ ID NO: 92 and a CDRL3 of SEQ ID NO: 93.
  • the anti-MPER bNAb or fragment thereof comprises a VH domain having at least 95% sequence identity to SEQ ID NO: 105 and a VL domain having at least 95% sequence identity to SEQ ID NO: 106, optionally wherein the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 99, a CDRH2 of SEQ ID NO: 100, a CDRH3 of SEQ ID NO: 101, a CDRL1 of SEQ ID NO: 102, a CDRL2 of SEQ ID NO: 103 and a CDRL3 of SEQ ID NO: 104.
  • the anti-MPER bNAb or fragment thereof comprises a VH domain having at least 95% sequence identity to SEQ ID NO: 116 and a VL domain having at least 95% sequence identity to SEQ ID NO: 117, optionally wherein the anti-MPER bNAb or fragment thereof comprises a a CDRH1 of SEQ ID NO: 110, a CDRH2 of SEQ ID NO: 111, a CDRH3 of SEQ ID NO: 112, a CDRL1 of SEQ ID NO: 113, a CDRL2 of SEQ ID NO: 114 and a CDRL3 of SEQ ID NO: 115.
  • the anti-MPER bNAb or fragment thereof comprises a VH domain having at least 95% sequence identity to SEQ ID NO: 116 and a VL domain having at least 95% sequence identity to SEQ ID NO: 117, optionally wherein the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 121, a CDRH2 of SEQ ID NO: 122, a CDRH3 of SEQ ID NO: 123, a CDRL1 of SEQ ID NO: 124, a CDRL2 of SEQ ID NO: 125 and a CDRL3 of SEQ ID NO: 126.
  • the anti-MPER bNAb or fragment thereof comprises a VH domain having at least 95% sequence identity to SEQ ID NO: 127 and a VL domain having at least 95% sequence identity to SEQ ID NO: 128, optionally wherein the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 121, a CDRH2 of SEQ ID NO: 122, a CDRH3 of SEQ ID NO: 123, a CDRL1 of SEQ ID NO: 124, a CDRL2 of SEQ ID NO: 125 and a CDRL3 of SEQ ID NO: 126.
  • the anti-MPER bNAb or fragment thereof comprises a VH domain having at least 95% sequence identity to SEQ ID NO: 138 and a VL domain having at least 95% sequence identity to SEQ ID NO: 139, optionally wherein the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 132, a CDRH2 of SEQ ID NO: 133, a CDRH3 of SEQ ID NO: 134, a CDRL1 of SEQ ID NO: 135, a CDRL2 of SEQ ID NO: 136 and a CDRL3 of SEQ ID NO: 137.
  • the anti-MPER bNAb or fragment thereof comprises a VH domain of SEQ ID NO: 174 and a VL domain of SEQ ID NO: 175. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a VH domain of SEQ ID NO: 185 and a VL domain of SEQ ID NO: 186. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a VH domain of SEQ ID NO: 196 and a VL domain of SEQ ID NO: 197. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a VH domain of SEQ ID NO: 72 and a VL domain of SEQ ID NO: 73.
  • the anti-MPER bNAb or fragment thereof comprises a VH domain of SEQ ID NO: 83 and a VL domain of SEQ ID NO: 84. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a VH domain of SEQ ID NO: 94 and a VL domain of SEQ ID NO: 95. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a VH domain of SEQ ID NO: 105 and a VL domain of SEQ ID NO: 106. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a VH domain of SEQ ID NO: 116 and a VL domain of SEQ ID NO: 117.
  • the anti- MPER bNAb or fragment thereof comprises a VH domain of SEQ ID NO: 127 and a VL domain of SEQ ID NO: 128. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a VH domain of SEQ ID NO: 138 and a VL domain of SEQ ID NO: 139. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a heavy chain (HC) having at least 95% sequence identity to SEQ ID NO: 176 or 177 and a light chain (LC) having at least 95% sequence identity to SEQ ID NO: 178.
  • HC heavy chain
  • LC light chain
  • the anti-MPER bNAb or fragment thereof comprises a heavy chain (HC) having at least 95% sequence identity to SEQ ID NO: 187 or 188 and a light chain (LC) having at least 95% sequence identity to SEQ ID NO: 189.
  • the anti-MPER bNAb or fragment thereof comprises a heavy chain (HC) having at least 95% sequence identity to SEQ ID NO: 198 or 199 and a light chain (LC) having at least 95% sequence identity to SEQ ID NO: 200.
  • the anti-MPER bNAb or fragment thereof comprises a heavy chain (HC) having at least 95% sequence identity to SEQ ID NO: 74 or 75 and a light chain (LC) having at least 95% sequence identity to SEQ ID NO: 76.
  • the anti-MPER bNAb or fragment thereof comprises a HC having at least 95% sequence identity to SEQ ID NO: 85 or 86 and a LC having at least 95% sequence identity to SEQ ID NO: 87.
  • the anti-MPER bNAb or fragment thereof comprises a HC having at least 95% sequence identity to SEQ ID NO: 96 or 97 and a LC having at least 95% sequence identity to SEQ ID NO: 98.
  • the anti-MPER bNAb or fragment thereof comprises a HC having at least 95% sequence identity to SEQ ID NO: 107 or 108 and a LC having at least 95% sequence identity to SEQ ID NO: 109.
  • the anti-MPER bNAb or fragment thereof comprises a HC having at least 95% sequence identity to SEQ ID NO: 118 or 119 and a LC having at least 95% sequence identity to SEQ ID NO: 120.
  • the anti-MPER bNAb or fragment thereof comprises a HC having at least 95% sequence identity to SEQ ID NO: 129 or 130 and a LC having at least 95% sequence identity to SEQ ID NO: 131.
  • the anti-MPER bNAb or fragment thereof comprises a HC having at least 95% sequence identity to SEQ ID NO: 140 or 141 and a LC having at least 95% sequence identity to SEQ ID NO: 142.
  • the anti-MPER bNAb or fragment thereof comprises a heavy chain (HC) of SEQ ID NO: 176 or 177 and a light chain (LC) of SEQ ID NO: 178.
  • the anti- MPER bNAb or fragment thereof comprises a heavy chain (HC) of SEQ ID NO: 187 or 188 and a light chain (LC) of SEQ ID NO: 189.
  • the anti-MPER bNAb or fragment thereof comprises a heavy chain (HC) of SEQ ID NO: 198 or 199 and a light chain (LC) of SEQ ID NO: 200.
  • the anti-MPER bNAb or fragment thereof comprises a heavy chain (HC) of SEQ ID NO: 74 or 75 and a light chain (LC) of SEQ ID NO: 76.
  • the anti-MPER bNAb or fragment thereof comprises a HC of SEQ ID NO: 85 or 86 and a LC of SEQ ID NO: 87.
  • the anti-MPER bNAb or fragment thereof comprises a HC of SEQ ID NO: 96 or 97 and a LC of SEQ ID NO: 98.
  • the anti-MPER bNAb or fragment thereof comprises a HC of SEQ ID NO: 107 or 108 and a LC of SEQ ID NO: 109.
  • the anti-MPER bNAb or fragment thereof comprises a HC of SEQ ID NO: 118 or 119 and a LC of SEQ ID NO: 120. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a HC of SEQ ID NO: 129 or 130 and a LC of SEQ ID NO: 131. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a HC of SEQ ID NO: 140 or 141 and a LC of SEQ ID NO: 142.
  • suitable linkers include amino acid sequences that are from 1 amino acid to 150 amino acids in length.
  • the linker is an amino acid sequence from 5 to 30 amino acids in length.
  • the linker is an amino acid sequence as set forth in any one of SEQ ID NOs: 30 to 35. In an embodiment, the linker is an amino acid sequence as set forth in SEQ ID NO: 30. In an embodiment, the linker is a multimer of the amino acid sequence as set forth in SEQ ID NO: 30. In an embodiment, the linker is [SEQ ID NO:30] n , wherein n is an integer from 1 to 6. In an embodiment, the linker is an amino acid sequence as set forth in SEQ ID NO:31. In an embodiment, the linker is an amino acid sequence as set forth in SEQ ID NO:32. In an embodiment, the linker is an amino acid sequence as set forth in SEQ ID NO:33.
  • the linker is an amino acid sequence as set forth in SEQ ID NO: 34. In an embodiment, the linker is an amino acid sequence as set forth in SEQ ID NO: 35. In another embodiment, the linker is a helical amino acid linker, for example that set forth in SEQ ID NO: 201.
  • any of the aforementioned linkers may be incorporated into an antigen binding protein of the invention.
  • any of the aforementioned linkers may be used to join a domain within the antigen binding protein to another domain within the antigen binding protein.
  • any of the aforementioned linkers may be used to join a CD4 domain within the antigen binding protein that to a broadly neutralizing antibody or fragment thereof that binds to the MPER region of gp41.
  • any of the aforementioned linkers may be used to join a CD4 domain as disclosed herein to a bNAb as disclosed herein.
  • the linker is an amino acid sequence as set forth in any one of SEQ ID NOs: 30 to 35.
  • the linker is an amino acid sequence as set forth in SEQ ID NO: 30. In another embodiment, the linker is a helical amino acid linker, for example that set forth in SEQ ID NO: 201. In an embodiment, a linker is used to join the C-terminus of a CD4 domain to the N- terminus of a bNAb heavy chain variable domain. In an embodiment, a linker is used to join the C- terminus of a CD4 domain to the N-terminus of a bNAb light chain variable domain.
  • a linker is used to join the C-terminus of a CD4 domain to the N-terminus of a bNAb heavy chain variable domain and a linker is used to join the C-terminus of a CD4 domain to the N- terminus of a bNAb light chain variable domain.
  • a linker is used to join the C- terminus of a CD4 domain to the N-terminus of a bNAb heavy chain variable domain and an identical linker is used to join the C-terminus of a CD4 domain to the N-terminus of a bNAb light chain variable domain.
  • the linker is an amino acid sequence as set forth in any one of SEQ ID NOs: 30 to 35. In an embodiment, the linker is an amino acid sequence as set forth in SEQ ID NO: 30.
  • a linker is used to join the N-terminus of a CD4 domain to the C- terminus of a bNAb heavy chain. In an embodiment, a linker is used to join the N-terminus of a CD4 domain to the C-terminus of a bNAb heavy chain variable domain. In an embodiment, a linker is used to join the N-terminus of a CD4 domain to the C-terminus of a bNAb light chain. In an embodiment, a linker is used to join the N-terminus of a CD4 domain to the C-terminus of a bNAb light chain variable domain.
  • a linker is used to join the N-terminus of a CD4 domain to the C-terminus of an Fc domain.
  • the linker is an amino acid sequence as set forth in any one of SEQ ID NOs: 30 to 35.
  • the linker is an amino acid sequence as set forth in SEQ ID NO: 30.
  • any of the aforementioned linkers may be used to join a VH and VL pair as disclosed herein to form a scFv.
  • the linker between the VH domain and the VL domain of the scFv is selected from the one of SEQ ID NOs: 30-35.
  • the linker between the VH domain and the VL domain of the scFv is SEQ ID NO: 33.
  • any of the aforementioned linkers may be used to join a scFv as disclosed herein to an Fc domain.
  • the scFv is fused to a human Fc via a linker selected from one of SEQ ID NO: 30-35.
  • the scFv is fused to a human Fc via a linker of SEQ ID NO: 31.
  • the multimeric binding protein of the invention is a bispecific binding protein.
  • bispecific binding proteins are embodiments as disclosed in the below Table 5.
  • Table 5 Discloses bispecific proteins under the scope of the invention with reference to the sequences provided in Tables 3 and 4.
  • the cell containing an "X" in the table includes a bispecific binding protein of the invention comprising a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a VH domain having at least 95% sequence identity to SEQ ID NO: 72 and a VL domain having at least 95% sequence identity of SEQ ID NO: 73 (fourth row of Table 4) and CDRs having a sequence according to SEQ ID NOs: 66-71 (fourth row of Table 3).
  • a bispecific binding protein of the invention comprising a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a VH domain having at least 95% sequence identity to SEQ ID NO: 72 and a VL domain having at least 95% sequence identity of SEQ ID NO: 73 (fourth row of Table 4) and CDRs having a sequence according to SEQ ID NOs: 66-71 (fourth row of Table 3).
  • the bispecific binding protein comprises a CD4 domain and a broadly neutralizing antibody comprising a pair of variable domains (a VH and a VL) as set out in any row of Table 4.
  • the bispecific binding protein comprises a CD4 domain having a sequence of any one of SEQ ID NOs: 1-21 and a broadly neutralizing antibody comprising a pair of variable domains (a VH and a VL) as set out in any row of Table 4.
  • the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and a broadly neutralizing antibody comprising a pair of variable domains (a VH and a VL) as set out in any row of Table 4.
  • the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a VH domain of SEQ ID NO: 174 and a VL domain of SEQ ID NO: 175.
  • the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a VH domain of SEQ ID NO: 185 and a VL domain of SEQ ID NO: 186.
  • the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a VH domain of SEQ ID NO: 196 and a VL domain of SEQ ID NO: 197.
  • the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a VH domain of SEQ ID NO: 72 and a VL domain of SEQ ID NO: 73.
  • the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a VH domain of SEQ ID NO: 83 and a VL domain of SEQ ID NO: 84.
  • the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a VH domain of SEQ ID NO: 94 and a VL domain of SEQ ID NO: 95.
  • the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a VH domain of SEQ ID NO: 105 and a VL domain of SEQ ID NO: 106. In an embodiment, the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a VH domain of SEQ ID NO: 116 and a VL domain of SEQ ID NO: 117.
  • the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a VH domain of SEQ ID NO: 127 and a VL domain of SEQ ID NO: 128.
  • the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a VH domain of SEQ ID NO: 138 and a VL domain of SEQ ID NO: 139.
  • the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a HC of SEQ ID NO: 176 or 177 and a LC of SEQ ID NO: 178.
  • the CD4 domain is linked to the N-terminus of the HC. In some embodiments, the CD4 domain is linked to the C-terminus of the HC.
  • the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a HC of SEQ ID NO: 187 or 188 and a LC of SEQ ID NO: 189.
  • the CD4 domain is linked to the N-terminus of the HC. In some embodiments, the CD4 domain is linked to the C-terminus of the HC.
  • the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a HC of SEQ ID NO: 198 or 199 and a LC of SEQ ID NO: 200.
  • the CD4 domain is linked to the N-terminus of the HC. In some embodiments, the CD4 domain is linked to the C-terminus of the HC.
  • the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a HC of SEQ ID NO: 74 or 75 and a LC of SEQ ID NO: 76.
  • the CD4 domain is linked to the N-terminus of the HC. In some embodiments, the CD4 domain is linked to the C-terminus of the HC.
  • the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a HC of SEQ ID NO: 85 or 86 and a LC of SEQ ID NO: 87.
  • the CD4 domain is linked to the N-terminus of the HC. In some embodiments, the CD4 domain is linked to the C-terminus of the HC.
  • the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a HC of SEQ ID NO: 96 or 97 and a LC of SEQ ID NO: 98.
  • the CD4 domain is linked to the N-terminus of the HC.
  • the CD4 domain is linked to the C-terminus of the HC.
  • the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a HC of SEQ ID NO: 107 or 108 and a LC of SEQ ID NO: 109.
  • the CD4 domain is linked to the N-terminus of the HC.
  • the CD4 domain is linked to the C-terminus of the HC.
  • the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a HC of SEQ ID NO: 118 or 119 and a LC of SEQ ID NO: 120.
  • the CD4 domain is linked to the N-terminus of the HC. In some embodiments, the CD4 domain is linked to the C-terminus of the HC.
  • the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a HC of SEQ ID NO: 129 or 130 and a LC of SEQ ID NO: 131.
  • the CD4 domain is linked to the N-terminus of the HC. In some embodiments, the CD4 domain is linked to the C-terminus of the HC.
  • the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a HC of SEQ ID NO: 140 or 141 and a LC of SEQ ID NO: 142.
  • the CD4 domain is linked to the N-terminus of the HC. In some embodiments, the CD4 domain is linked to the C-terminus of the HC.
  • the bispecific binding protein comprises a sequence according to SEQ ID NO: 147-150. In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 147-150 and a sequence according to SEQ ID NO: 189.
  • the bispecific binding protein comprises a sequence according to SEQ ID NO: 59. In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 59 and a sequence according to SEQ ID NO: 76.
  • the bispecific binding protein comprises a sequence according to SEQ ID NO: 60. In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 60 and a sequence according to SEQ ID NO: 87.
  • the bispecific binding protein comprises a sequence according to SEQ ID NO: 61. In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 61 and a sequence according to SEQ ID NO: 98. In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 62. In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 62 and a sequence according to SEQ ID NO: 109.
  • the bispecific binding protein comprises a sequence according to SEQ ID NO: 63. In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 63 and a sequence according to SEQ ID NO: 120.
  • the bispecific binding protein comprises a sequence according to SEQ ID NO: 64. In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 64 and a sequence according to SEQ ID NO: 131.
  • the bispecific binding protein comprises a sequence according to SEQ ID NO: 65. In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 65 and a sequence according to SEQ ID NO: 142.
  • Antigen binding proteins may be prepared by any of a number of conventional techniques. For example, antigen binding proteins may be purified from cells that naturally express them (e.g., an antibody can be purified from a hybridoma that produces it), or produced in recombinant expression systems.
  • a number of different expression systems and purification regimes can be used to generate the antigen binding proteins of the invention.
  • host cells are transformed with a recombinant expression vector encoding the desired antigen binding protein.
  • the expression vector may be maintained by the host as a separate genetic element or integrated into the host chromosome depending on the expression system.
  • a wide range of host cells can be employed, including Prokaryotes (including Gram-negative or Gram-positive bacteria, for example Escherichia coii, Bacilli sp., Pseudomonas sp., Corynebacterium sp.), Eukaryotes including yeast (for example Saccharomyces cerevisiae, Pichia pastoris), fungi (for example Aspergiius sp.), or higher Eukaryotes including insect cells and cell lines of mammalian origin (for example, CHO, NSO, PER.C6, HEK293, HeLa).
  • Prokaryotes including Gram-negative or Gram-positive bacteria, for example Escherichia coii, Bacilli sp., Pseudomonas sp., Corynebacterium sp.
  • Eukaryotes including yeast (for example Saccharomyces cerevisiae, Pichia pastoris), fungi (for example Aspergi
  • the host cell may be an isolated host cell.
  • the host cell is usually not part of a multicellular organism (e.g., plant or animal).
  • the host cell may be a non-human host cell.
  • the host cell may be a microorganism cell.
  • Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian host cells are known in the art.
  • the cells can be cultured under conditions that promote expression of the antigen binding protein using a variety of equipment such as shake flasks, spinner flasks, and bioreactors.
  • the polypeptide(s) is(are) recovered by conventional protein purification procedures. Protein purification procedures typically consist of a series of unit operations comprised of various filtration and chromatographic processes developed to selectively concentrate and isolate the antigen binding protein.
  • the purified antigen binding protein may be formulated in a pharmaceutically acceptable composition.
  • Fc engineering methods can be applied to modify the functional or pharmacokinetics properties of an antigen binding protein, in particular an antibody. Effector function may be altered by making mutations in the Fc region that increase or decrease binding to Clq or Fey receptors and modify CDC or ADCC activity respectively. Modifications to the glycosylation pattern of an antibody can also be made to change the effector function.
  • FcR Fc receptors
  • FcR Fc receptors
  • FcR Fc receptors
  • FcR FcyRI
  • FcyRII CD32
  • FcyRIII CD16
  • FcRn FcRn
  • Clq type II Fc receptors
  • significant biological effects can be a consequence of effector functionality.
  • the ability to mediate effector function requires binding of the antigen binding protein or antibody to an antigen and not all antigen binding proteins or antibodies will mediate every effector function.
  • Effector function can be assessed in a number of ways including, for example, evaluating ADCC effector function of antibody coated to target cells mediated by Natural Killer (NK) cells via FcyRIII, or monocytes/macrophages via FcyRI, or evaluating CDC effector function of antibody coated to target cells mediated by complement cascade via Clq.
  • an antigen binding protein of the present invention can be assessed for ADCC effector function in a Natural Killer cell assay. Examples of such assays can be found in Shields etal, 2001, The Journal of Biological Chemistry, Vol. 276, p. 6591-6604; Chappel etal, 1993, The Journal of Biological Chemistry, Vol 268, p. 25124-25131; Lazar et al, 2006, PNAS, 103; 4005-4010.
  • Examples of assays to determine CDC function include those described in J Imm Meth, 1995, 184: 29-38.
  • the long half-life of IgG antibodies is reported to be dependent on their binding to FcRn. Therefore, substitutions that increase the binding affinity of IgG to FcRn at pH 6.0 while maintaining the pH dependence of the interaction with target, by engineering the constant region, have been extensively studied (Ghetie etai., Nature Biotech. 15: 637-640, 1997; Hinton etai., JBC 279: 6213- 6216, 2004; Dall'Acqua eta!., 10 J Immunol 117: 1129-1138, 2006).
  • the in-vivo half-life of antigen binding proteins of the present invention may be altered by modification of a heavy chain constant domain or an FcRn binding domain therein.
  • FcRn plays a key role in maintaining serum antibody levels by acting as a protective receptor that binds and salvages antibodies of the IgG isotype from degradation.
  • IgG molecules are endocytosed by endothelial cells and, if they bind to FcRn, are recycled out of the cells back into circulation. In contrast, IgG molecules that enter the cells and do not bind to FcRn and are targeted to the lysosomal pathway where they are degraded.
  • FcRn is believed to be involved in both antibody clearance and the transcytosis across tissues (see Junghans R.P (1997) Immunol. Res 16. 29-57 and Ghetie et a/ (2000) Annu. Rev. Immunol. 18, 739-766).
  • Human IgGl residues determined to interact directly with human FcRn include Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 and His435. Mutations at any of these positions may enable increased serum half-life and/or altered effector properties of antigen binding proteins of the invention.
  • Antigen binding proteins of the present invention may have amino acid modifications that increase the affinity of the constant domain or fragment thereof for FcRn. Increasing the half-life (Ze., serum half-life) of therapeutic and diagnostic IgG antibodies and other bioactive molecules has many benefits including reducing the amount and/or frequency of dosing of these molecules.
  • an antigen binding protein of the invention comprises all or a portion (an FcRn binding portion) of an IgG constant domain having one or more of the following amino acid modifications.
  • an antigen binding protein of the invention comprises an Fc domain with the LS mutations.
  • an antigen binding protein of the invention comprises a bNAb in which the LS mutations are present in both of the heavy chain Fc domains.
  • Half-life and FcRn binding can also be extended by introducing H433K and N434F mutations (commonly referred to as "HN” or “NHance” mutations) (with reference to IgGl) (W02006/130834).
  • an antigen binding protein such as a bispecific molecule of the invention in a host cell
  • post-translational modifications may occur. For example, this may include the cleavage of certain leader sequences, the addition of various sugar moieties in various glycosylation patterns, non-enzymatic glycation, deamidation, oxidation, disulfide bond scrambling and other cysteine variants such as free sulfhydryls, racemized disulfides, thioethers and trisulfide bonds, isomerisation, C-terminal lysine clipping, and N-terminal glutamine cyclisation.
  • an antigen binding protein of the invention includes an "antigen binding protein" as defined earlier that has undergone a post-translational modification such as described herein.
  • Glycation is a post-translational non-enzymatic chemical reaction between a reducing sugar, such as glucose, and a free amine group in the protein, and is typically observed at the epsilon amine of lysine side chains or at the N-Terminus of the protein. Glycation can occur during production and storage only in the presence of reducing sugars.
  • a reducing sugar such as glucose
  • Deamidation can occur during production and storage, is an enzymatic reaction primarily converting asparagine (N) to iso-aspartic acid (iso-aspartate) and aspartic acid (aspartate) (D) at approximately 3:1 ratio. This deamidation reaction is therefore related to isomerization of aspartate (D) to iso-aspartate.
  • the deamidation of asparagine and the isomerisation of aspartate both involve the intermediate succinimide.
  • deamidation can occur with glutamine residues in a similar manner. Deamidation can occur in a CDR, in a Fab (non-CDR region), or in the Fc region.
  • Oxidation can occur during production and storage (Ze., in the presence of oxidizing conditions) and results in a covalent modification of a protein, induced either directly by reactive oxygen species or indirectly by reaction with secondary by-products of oxidative stress. Oxidation happens primarily with methionine residues, but may occur at tryptophan and free cysteine residues. Oxidation can occur in a CDR, in a Fab (non-CDR) region, or in the Fc region.
  • Disulfide bond scrambling can occur during production and basic storage conditions. Under certain circumstances, disulfide bonds can break or form incorrectly, resulting in unpaired cysteine residues (-SH). These free (unpaired) sulfhydryls (-SH) can promote shuffling.
  • thioether and racemization of a disulphide bond can occur under basic conditions, in production or storage, through a beta elimination of disulphide bridges back to cysteine residues via a dehydroalanine and persulfide intermediate. Subsequent crosslinking of dehydroalanine and cysteine results in the formation of a thioether bond or the free cysteine residues can reform a disulphide bond with a mixture of D- and L-cysteine.
  • Trisulfides result from insertion of a sulfur atom into a disulphide bond (Cys-S-S-S-Cys) and are formed due to the presence of hydrogen sulphide in production cell culture.
  • N-terminal glutamine (Q) and glutamate (glutamic acid) (E) in the heavy chain and/or light chain is likely to form pyroglutamate (pGlu) via cyclization.
  • pGlu pyroglutamate
  • C-terminal lysine clipping is an enzymatic reaction catalyzed by carboxypeptidases, and is commonly observed in recombinant and natural human antibodies. Variants of this process include removal of lysine from one or both heavy chains due to cellular enzymes from the recombinant host cell. Upon administration to the human subject/patient is likely to result in the removal of any remaining C-terminal lysines.
  • Antigen binding proteins as described herein may be incorporated into pharmaceutical compositions for use in the treatment or prevention of HIV infection.
  • the pharmaceutical composition comprises an antigen binding protein in combination with one or more pharmaceutically acceptable carriers and/or excipients.
  • compositions comprise a pharmaceutically acceptable carrier as known and called for by acceptable pharmaceutical practice.
  • compositions may be administered by injection or continuous infusion (examples include, but are not limited to, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intraocular, and intraportal). In one embodiment, the composition is suitable for intravenous administration. In one embodiment, the composition is suitable for subcutaneous administration. Pharmaceutical compositions may be suitable for topical administration (which includes, but is not limited to, epicutaneous, inhaled, intranasal or ocular administration) or enteral administration (which includes, but is not limited to, oral, vaginal, or rectal administration).
  • the pharmaceutical composition may be included in a kit containing the antigen binding protein together with other medicaments, for example dolutegravir or cabotegravir, and/or with instructions for use.
  • the kit may comprise the reagents in predetermined amounts with instructions for use.
  • the kit may also include devices used for administration of the pharmaceutical composition.
  • subject is a human.
  • the antigen binding proteins described herein may be used in methods of treatment or prevention of HIV infection and AIDs.
  • the antigen binding proteins described herein may be used in the manufacture of medicaments for the treatment or prevention of HIV infection and AIDs.
  • the antigen binding proteins described may be used in an effective amount for therapeutic, prophylactic or preventative treatment.
  • a therapeutically effective amount of the antigen binding protein described herein is an amount effective to ameliorate or reduce one or more symptoms of HIV infection.
  • a prophylactically effective amount of the antigen binding protein described herein is an amount effective to prevent one or more symptoms of HIV infection.
  • Antigen binding proteins of the present invention may be employed alone or in combination with other therapeutic agents, or a prodrug thereof.
  • Combination therapies according to the present invention thus comprise the administration of an antigen binding protein and the administration of at least one other agent which may be useful in the treatment or prevention of HIV infection and/or AIDS.
  • An antigen binding protein of the present invention and the other therapeutic agent may be formulated and administered together in a single pharmaceutical composition or may be formulated and administered separately. When formulated and administered separately, administration may occur simultaneously or sequentially in any order.
  • Antigen binding proteins as described herein may be combined with, for example, one or more of an antiretroviral agent, an anti-infective agent, an immunomodulator, and other HIV entry inhibitors.
  • NRTIs may include, but are not limited to: abacavir, adefovir, adefovir dipivoxil, alovudine, amdoxovir, apricitabine, calanolide A, censavudine, didanosine, elvucitabine, emtricitabine, fozivudine, lamivudine, racivir, stampidine, stavudine, tenofovir disoproxil fumerate, tenofovir alafenamide, todoxil, zalcitabine, and zidovudine.
  • NNRTIs may include, but are not limited to, HBY 097 (Hoechst/Bayer), capravirine, delaviridine, doravirine, efavirenz, etravirine, immunocal, lersivirine, loviride, nevirapine, oltipraz, and rilpivirine.
  • NRTTIs include, but are not limited to, islatravir.
  • Pls may include, but are not limited to, amprenavir, atazanavir, brecanavir, cobicistat, darunavir, fosamprenavir, indinavir, lasinavir, lopinavir, palinavir, nelfinavir, ritonavir, saquinavir, and tipranavir.
  • the antigen binding proteins of the invention can be utilized in combination with attachment inhibitors, fusion inhibitors, and chemokine receptor antagonists aimed at either the CCR5 or CXCR4 coreceptor.
  • HIV attachment inhibitors are also set forth in US 7,354,924 and US 7,745,625.
  • Els may include, but are not limited to, cenicriviroc, enfuvirtide, fostemsavir, ibalizumab, leronlimab, maraviroc, vicriviroc and VIR-576.
  • INSTIs may include, but are not limited to, bictegravir, cabotegravir, dolutegravir, elvitegravir, and raltegravir, .
  • the INSTI is dolutegravir or cabotegravir.
  • the INSTI is cabotegravir.
  • Maturation inhibitors may include, but are not limited to, bevirimat, BMS-955176, GSK3640254, GSK3739937, PA-344 and PA-457. It will be understood that GSK3640254 is a compound as described in Dicker I, Jeffrey JL, Protack T, eta/., Antimicrob Agents Chemother. 2022;66(l). GSK3739937, also known as VH3739937, is the compound of clinical trial NCT04493684.
  • Capsid inhibitors may include, but are not limited to, GSK4004280, GSK4011499, and lencapavir.
  • Anti -infective agents include, but are not limited to, clindamycin with primaquine, daunorubicin, fluconazole, intraconazole, nystatin pastille, ornidyl eflornithine, megestrol acetate, pentamidine isethionate, piritrexim, trimethoprim, trimetrexate, recombinant human erythropoietin, recombinant human growth hormone, spiramycin, testosterone and total enteral nutrition,
  • Immunomodulators include, but are not limited to, acemannan, alpha-2-interferon, AS-101, bropirimine, CL246,738, FP-21399, gamma interferon, granulocyte macrophage colony stimulating factor, HIV core particle immunostimulant, interleukin-2, immune globulin, IMREG-1, IMREG-2, imuthiol diethyl dithio carbamate, methionine enkephalin, MTP-PE muramyl tripeptide, remune, recombinant soluble human CD4, rCD4-IgG hybrids, SK&F106528, thymopentin, and tumour necrosis factor (TNF).
  • acemannan alpha-2-interferon
  • AS-101 bropirimine
  • CL246,738, FP-21399 gamma interferon
  • granulocyte macrophage colony stimulating factor HIV core particle immunostimulant
  • interleukin-2 immune
  • the antigen binding proteins of the present invention may also be used in combination with agents that induce HIV expression, such as latency reversing agents.
  • latency reversing agents include, but are not limited to, the following: histone deacetylase inhibitors e.g, vorinostat, panobinostat, romidepin), histone crotonyl transferase inhibitors (sodium corotonate), protein kinase C agonists e.g., bryostatin, ingenol B), disulfiram, TLR7 agonists e.g., GS-9620), and bromodomain inhibitors (e.g., JQ1, iBET151).
  • histone deacetylase inhibitors e.g, vorinostat, panobinostat, romidepin
  • histone crotonyl transferase inhibitors sodium corotonate
  • protein kinase C agonists e.g., bryostatin, in
  • the antigen binding proteins of the present invention may also be used in combination with other agents that induce HIV expression, such as agents for clearance therapy.
  • agents for clearance therapy or of immunological combinations for clearance, include, but are not limited to, the following: bNAbs, CD4-Ig, eCD4-Ig, and dual-affinity re-targeting (DART) proteins.
  • Antigen binding proteins of the invention may be used in combination with broadly neutralizing HIV-1 antibodies, including 1NC9, 1B2530, 2F5, 2G12, 3NBC60, 3BNC117, 4E10, 8ANC131, 8ANC134, 10-1074, 10-1074LS, 10E8, 12A12, 12A21, bl2, CAP206-CH12, CH01-04, CH103-106, elipovimab (formerly known as GS-9722), HJ16, M66.6, N6LS (also known as VRC- HIVMAB091-00-AB and the compound of clinical trial NCT03538626), NIH45-46, PG9, PG16, PGT121-123, PGT125-131, PCT135-137, PGT141-145, PGT121.414.LS, PGT151 2G12, QA013.2, VRC01-03, VRC-PG04, VRC-PG04b, VRC-CH30-34.
  • agents that may be combined with antigen binding proteins of the invention include BIT225, GSK4000422/VH4000422, and S-648414 (the compound of clinical trial NCT04147715).
  • a subject is administered DNA or RNA encoding an multimeric antigen binding protein of the invention to provide in vivo antibody production, for example using the cellular machinery of the subject.
  • Administration of nucleic acid constructs is known in the art and taught, for example, in U.S. Patent No. 5,643,578, U.S. Patent. No. 5,593,972 and U.S. Patent No. 5,817,637.
  • U.S. Patent. No. 5,880,103 describes several methods of delivery of nucleic acids encoding proteins to an organism.
  • One approach to administration of nucleic acids is direct administration with plasmid DNA. such as with a mammalian expression plasmid.
  • the nucleotide sequence encoding the disclosed antigen binding protein can be placed under the control of a promoter to increase expression.
  • the methods include liposomal delivery of the nucleic acids. Such methods can be applied to the production of an antigen binding protein of the invention.
  • a multimeric antigen binding protein of the invention is expressed in a subject using the pVRC8400 vector (described in Barouch er u/., J. Virol., 79(14), 8828-8834, 2005).
  • a subject (such as a human subject at risk of HIV infection) can be administered an effective amount of an adeno-associated virus (AAV) viral vector that includes one or more nucleic acid molecules encoding an multimeric antigen binding protein of the invention.
  • the AAV viral vector is designed for expression of the nucleic acid molecules encoding a disclosed antigen binding protein, and administration of an effective amount of the AAV viral vector to the subject leads to expression of an effective amount of the antigen binding protein in the subject.
  • AAV viral vectors that can be used to express a disclosed antigen binding protein in a subject include those provided in Johnson et al., Nut. Med., 15(8):901-906, 2009 and Gardner et al., Nature, 519(7541):87-91, 2015.
  • a multimeric anti-HIV envelope spike complex -binding protein comprising: i. a broadly neutralizing anti-MPER antibody, comprising at least one heavy chain or light chain, wherein the antibody binds to the MPER domain of a gp41 protein; and ii. at least one CD4 domain which binds to gpl20, wherein the CD4 domain is attached directly or by a linker to the N-terminus or C-terminus of one of the heavy chains or light chains of the broadly neutralizing antibody.
  • binding protein of any preceding clause wherein the CD4 domain is attached via a linker to the N-terminus or C-terminus of at least one heavy or light chain of the broadly neutralizing antibody.
  • binding protein of any preceding clause wherein the binding protein comprises at least four CD4 domains.
  • a multimeric anti-HIV envelope spike complex-binding protein comprising: i. A broadly neutralizing anti-MPER antibody or an antigen-binding Fab' or F(abQ2 fragment thereof comprising at least one heavy chain variable region or light chain variable region, wherein said antibody or fragment binds to the MPER region of gp41; and ii. at least one CD4 domain, wherein the C-terminus of the CD4 domain is attached directly or by a linker to the N-terminus of the heavy chain variable region or light chain variable region.
  • the binding protein of clause 8 comprising at least two CD4 domains, wherein the fragment is an antigen-binding Fab' fragment, wherein the first CD4 domain is attached via a linker to the heavy chain variable region of the Fab' fragment and the second CD4 domain is attached via a linker to the light chain variable region of the Fab' fragment.
  • binding protein of clause 8 comprising at least two CD4 domains, wherein the fragment is an antigen-binding F(abQ2 fragment, and wherein each of the CD4 domains is attached via a linker to each of the heavy chain variable regions of the F(abQ2 fragment.
  • the binding protein of clause 8 comprising at least two CD4 domains, wherein the fragment is an antigen-binding F(abQ2 fragment, and wherein each of the CD4 domains is attached via a linker to each of the light chain variable regions of the F(abQ2 fragment.
  • binding protein of clause 8 comprising at least four CD4 domains, wherein the fragment is an antigen-binding F(abQ2 fragment, and wherein each of the CD4 domains is attached via a linker to each of the heavy chain variable regions and each of the light chain variable regions of the F(abQ2 fragment.
  • binding protein of any preceding clause wherein the binding protein is a bispecific binding protein.
  • linker is a multimeter of the amino sequence as set forth in SEQ ID NO: 30.
  • linker is selected from SEQ ID NOs: 30 to 35
  • CD4 domain is a human CD4 DI domain or CD4 D1D2 domain.
  • CD4 domain comprises at least one stabilizing mutation compared to a wild type CD4 domain.
  • CD4 domain comprises at least one mutation selected from: L5Y, S23N, A55V, I79P, L96V and/or F98V.
  • CD4 domain comprises L5Y, S23N, A55V, I79P, L96V and F98V mutations; and at least one further mutations selected from: E91Q, E91H, E87G, N52W, K8V, K8I, K8C, G99C, T11C, K72C, E13C, I70C, H27C, G38C, K21C, G65C, Q25E, H27D, R58V, R58N, R58Y, and/or L61M.
  • CD4 domain comprises or consists of any one of SEQ ID NO: 4 - 21.
  • CD4 domain comprises or consists of any one of SEQ ID NO: 5 - 21.
  • CD4 domain comprises or consists of any one of SEQ ID NO: 5 - 15.
  • CD4 domain comprises or consists of SEQ ID NO: 11.
  • binding protein of any preceding clause, wherein the broadly neutralizing antibody comprises a mutation that increases the half-life of the binding protein compared to the same binding protein without said mutation.
  • binding protein of clause 32 wherein the binding protein comprises an Fc domain and the half-life increasing mutation is a mutation in said Fc domain.
  • the broadly neutralizing antibody comprises a heavy chain variable domain and a light chain variable domain pair that is at least 95% identical to any of the variable domain pairs set out in any one row of Table 4.
  • binding protein of any preceding clause, wherein the broadly neutralizing antibody comprises a heavy chain and a light chain pair that is at least 95% identical to any of the chain pairs set out in any one row of Table 4.
  • binding protein of any preceding clause, wherein the broadly neutralizing antibody comprises a heavy chain variable domain and a light chain variable domain pair that is at least 95% identical to: i. SEQ ID NOs: 174 and 175, or ii. SEQ ID NOs: 185 and 186, or iii. SEQ ID NOs: 196 and 197, or iv. SEQ ID NOs: 72 and 73, or
  • binding protein of any preceding clause wherein the binding protein can reduce the IC50 (mean/specific envelope) in a PSV assay compared to the IC50 reduction caused by the broadly neutralizing antibody and CD4 domain alone or in combination.
  • binding protein of any preceding clause wherein the binding protein is linked to an effector molecule or detectable marker, optionally wherein the detectable marker is a fluorescent, enzymatic or radioactive marker.
  • a method of detecting an HIV infection in a human subject comprising contacting a biological sample from the human subject with the binding protein according to any one of clauses 1 to 45 under conditions sufficient to form an immune complex; and detecting the presence of the immune complex in the sample, wherein the presence of the immune complex in the sample indicates that the subject has a HIV infection.
  • a pharmaceutical composition comprising the binding protein as defined in any one of the preceding clauses and a pharmaceutically acceptable excipient.
  • a method of treating or preventing an HIV infection in a human comprising administering to the human an anti-HIV binding protein according to any one of clauses 1 to 45, or a pharmaceutical composition according to clause 47.
  • kits comprising in separate containers: an anti-HIV binding protein according to any one of clauses 1 to 45 and at least one anti-viral drug that inhibits cellular entry, replication, or transcription of HIV in a human.
  • the antiviral drug is selected from: Nucleoside Reverse Transcriptase Inhibitors (NRTIs), Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs), Protease Inhibitors (Pls), Entry Inhibitors, Integrase Strand Transfer Inhibitors (INSTI), Maturation Inhibitors (Mis), Capsid Inhibitors (Cis) and Nucleoside Reverse Transcriptase Translocation Inhibitors (NRTTIs).
  • a recombinant host cell that comprises the nucleic acid sequence of clause 56 or the expression vector of clause 57.
  • a method of producing an anti-HIV binding protein comprising culturing the host cell as defined in clause 58 under conditions suitable for expression of said nucleic acid sequence or vector, whereby an anti-HIV binding protein is produced.
  • Plasmids encoding the antigen binding proteins of the invention were expressed in EXPI293 or FREESTYLE 293-F cells using the manufacturer's standard protocol (ThermoFisher Scientific, Waltham, MA). The expressed medium was harvested by centrifugation (4000 rpm for 10 min) and the antigen binding proteins were purified by filtration through a 0.22 pm filter (Millipore Sigma, Burlington, MA) and fast protein liquid chromatography (FPLC) (AKTATM Pure, Cytiva, Marlborough MA).
  • FPLC fast protein liquid chromatography
  • the medium was then passed through a Mabselect SuRe column (Cytiva, Marlborough MA) to capture the antigen binding proteins and the column was washed sequentially with phosphate- buffered saline (PBS) before elution.
  • Mabselect SuRe column Cytiva, Marlborough MA
  • the antigen binding proteins were then exchanged into a final buffer by using dialysis, a desalting column and preparative size exclusion column (SEC).
  • SEC preparative size exclusion column
  • the purity of the antigen binding proteins was evaluated by using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS- PAGE) and on a size exclusion column on a high-performance liquid chromatography system (SEC- HPLC).
  • Antigen binding protein concentrations were determined by measuring absorbance at 280nm wavelength (A280) on a NanoDrop machine (ThermoFisher Scientific, Waltham, MA), and their molecular mass was measured by using liquid chromatography - mass spectrometry (LC-MS) to confirm their identity.
  • A280 absorbance at 280nm wavelength
  • LC-MS liquid chromatography - mass spectrometry
  • the endotoxin level in the final purified products was measured on an ENDOSAFE system (Charles River Labs, Wilmington MA) to make sure it was sufficiently low (usually ⁇ 1 EU (Endotoxin Unit) / mg of protein) for downstream anti-viral studies.
  • Pseudotyped HIV-1 virus contains deletions in the genome that make it unable to produce infectious virions, but it can be used to measure the activity of cell entry inhibitors (i.e., molecules that prevent the binding of HIV-1 virions to the target cell membrane and/or prevent entry of HIV-1 into target cells), which include the antigen binding proteins of the invention.
  • PSV was produced in HEK-293T cells (ATCC, Manassas VA) by co-transfecting expression plasmids encoding the HIV-1 gpl60 envelope gene and an HIV-1 backbone plasmid using TRANSIT- 2020 transfection reagent (Mirus Bio, Madison WI).
  • a panel of HIV-1 PSVs expressing different gpl60 envelope trimers was generated to evaluate the effectiveness of the antigen binding proteins of the invention against a wide spectrum of HIV-1 strains.
  • the complex N- glycans on selected HIV-1 envelopes were removed by adding Kifunensine during PSV production.
  • Kifunensine inhibits mannosidase I, prevents processing of high mannose N-glycans into complex N- glycans in the cells and consequently produces high mannose glycoproteins.
  • Kifunensine was added to a final concentration of 100 pM, the cells were incubated for another 48 hours, and the supernatant which contains the PSV was harvested and aliquoted. i. ACTOne cells
  • the genome of PSV used in this assay contains a luciferase gene that is expressed once the virus enters cells. Accordingly, the luminescence signal (after adding a substrate of luciferase) can be used to determine the level of viral infection.
  • TCID tissue culture infectious dose
  • ACTOne cells were detached by treating the cell culture flask with trypsin (trypsin ization) and resuspended in growth medium containing 2% of DMSO to a density of 2.5 x 10 5 cells/ml.
  • trypsin ization trypsin ization
  • growth medium containing 2% of DMSO growth medium containing 2% of DMSO to a density of 2.5 x 10 5 cells/ml.
  • One hundred pl of such cells was added to 10 pl of antigen binding protein pre-loaded in a 96-well plate.
  • Ninety pl of PSV was then added to each well.
  • the assay plates were incubated at 37°C in a humidified incubator at 5% CO2 level.
  • the PSV assay was carried out using a luciferase-based assay in a TZM.bl cell line.
  • the TZM-bl cell line is derived from a HeLa cell clone that was engineered to express CD4, CCR5 and CXCR4 and to contain integrated reporter genes for firefly luciferase and E. coli 0-galactosidase under the control of an HIV-1 long terminal repeat (Wei et al., Antimicrobial agents and chemotherapy 46:1896-905(2002)) permitting sensitive and accurate measurements of infection.
  • the assay measures the reduction in luciferase reporter gene expression in TZM.bl cells following a single round of virus infection.
  • TZM.bl cells were then added (lxl0 4 /well in a 100-pl volume) in 10% D-MEM growth medium containing DEAE-dextran (Sigma, St. Louis, MO) at a final concentration of 11 pg/ml.
  • Assay controls included TZM.bl cells alone (cell control) and TZM.bl cells with virus (virus control).
  • the anti-viral activity of the antigen binding proteins was also measured in a replicating virus assay against NL4-3 strain and its variants in MT2 cells.
  • the proviral clone of NL4-3 (obtained from NIH) was used to make the replicating reporter virus NLRepRIuc, in which a section of the nef gene from the proviral clone of NL4-3 was replaced with the Renilla luciferase gene (Techniques in HIV Research. Tech HIV Res. Published online 1990. doi: 10.1007/978-1-349-11888-5).
  • Virus was produced through transfection of HEK293T cells using Lipofectamine Plus (Invitrogen, Carlsbad CA), according to the manufacturer's instructions.
  • the replication -com petent virus was harvested 3 days after transfection of HEK 293T cells with the modified pNLRepRIuc proviral clone and titrated in MT-2 cells using luciferase activity as a biomarker.
  • the MPER-bNAb insensitive strains are engineered based on this proviral clone NL4-3 by introducing point mutations into its envelope gene, including W680R/K683Q and W672L/F673L.
  • MT-2 cells were obtained from the American Type Culture Collection (ATCC) and were propagated in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml of penicillin G, 100 pg/ml of streptomycin, 10 mM HEPES buffer pH 7.55 and 2-mM L-glutamine.
  • FBS heat-inactivated fetal bovine serum
  • HEK293T cells were obtained from the ATCC and propagated in DMEM media supplemented with 10% heat-inactivated FBS.
  • the NLRepRIuc was used to infect MT-2 cells at a multiplicity of 0.01 for 1 hour before adding the proteins to the 96-well plates.
  • the antigen binding proteins were serially diluted four-fold and 11 concentrations were plated in triplicate. After 4 days of incubation, cells were processed and quantitated for virus growth by the amount of expressed luciferase. Luciferase was quantitated using the ENDUREN substrate from Promega (Madison, WI) according to the manufacturer's instructions. Luciferase activity was measured immediately on an ENVISION multilabel plate reader (PerkinElmer, Waltham MA).
  • IC50 half maximal inhibitory concentration
  • IC50 is a measure of the potency of a substance in inhibiting a specific biological or biochemical function (in this case, viral replication).
  • IC50 is a quantitative measure that indicates how much of a substance (e.g. drug) is needed to inhibit, in vitro, a given biological process or biological component by 50%.
  • IC80 or IC90 are the amount of the particular inhibitory substance needed to inhibit, in vitro, a given biological process or biological component by 80% or 90% respectively.
  • soluble human CD4 domains tested contain a set of "base” mutations in human CD4 domain 1 (DI) over the wild-type sequence (SEQ ID NO:3) that enable the folding of human CD4 DI on its own.
  • Soluble CD4 DI with this set of mutations is known as mD1.22 (Chen et al., J Virol. 2014 Jan;88(2): 1125-39) and the mutations therein consist of: L5Y, S23N, A55V, I76P, L96V, and F98V (SEQ ID N0:4, also referred to as Dim herein).
  • mD1.22 SEQ ID NO:4
  • the additional stabilizing mutations were designed based on several methodologies: 1) computational simulation by using Free Energy Perturbation (FEP+, Schrodinger, New York, NY USA); 2) computational simulation by using disulfide-bond scan in Molecular Operating Environment program (MOE, Chemical Computing Group, Montreal Canada); and 3) panning a library of human CD4 DI with each residue mutated, one by one, to the other 19 types of amino acids (site saturation mutagenesis, TWIST BioScience, San Francisco, CA USA) using phage display under thermally challenging conditions (i.e., incubating the phage at room temperature, 70 °C, and 80 °C, then selecting the CD4 domain variants that can still bind to recombinant HIV-1 gpl20 (CN54 strain, Aero Biosystems, Beijing China)).
  • FEP+ Free Energy Perturbation
  • MOE Chemical Computing Group, Montreal Canada
  • SEQ ID NOs: 5-21 The best performing variants (SEQ ID NOs: 5-21) were fused with 6xHis tag at their C- termini, expressed and purified from mammalian cells using methods as described in Example 1, except that purification was via a Ni-NTA resin (Cytiva, Marlborough MA) instead of Mabselect SuRe column, with standard protocol from the vendor.
  • the fusion position of the CD4 domain in the broadly neutralizing mAbs was tested in some examples V3-bNAbs (another class of bNAbs that bind to a distinct target called the V3 loop).
  • the shorter-linker bispecific (Dlm_lxG4S_bNAbl, SEQ ID NOs: 36 and 23) showed much better PK (longer half-life and lower clearance rate) than the longer-linker bispecific (Dlm_4xG4S_bNAbl, SEQ ID NOs: 37 and 23)) in a humanized mouse model (Tg32 strain where human neonatal Fc receptor (hFcRn) replaced the corresponding mouse gene (mFcRn), The Jackson Laboratory, Bar Harbor, Maine USA).
  • hFcRn human neonatal Fc receptor
  • Table 7 IC50 (nM) of different V3-bNAbl-derived bispecific formats and control molecules against a panel of HIV-1 envelopes in a PSV assay (ACTOne cells)
  • Table 8 IC50 (nM) of different V3-bNAb2-, bNAb3- and bNAb4-derived bispecific formats and control molecules against a panel of HIV-1 envelopes in PSV assay (ACTOne cells)
  • Thermal stabilization of CD4 DI further enhanced the PK of the bispecific molecules (Dlm-K8C-G99C_lxG4S_bNAbl, SEQ ID NOs:44 and 23; Dlm-TllC- K72C_lxG4S_bNAbl, SEQ ID NOs:45 and 23; Dlm-K8I_lxG4S_bNAbl, SEQ ID NOs:42 and 23; and Dlm-K8V_lxG4S_bNAbl, SEQ ID NOs:43 and 23) as shown in Table 9 below.
  • the bispecific molecules Dlm-K8C-G99C_lxG4S_bNAbl, SEQ ID NOs:44 and 23; Dlm-TllC- K72C_lxG4S_bNAbl, SEQ ID NOs:45 and 23; Dlm-K8I_lxG4S_bNAbl, SEQ ID NOs:42 and 23; and Dl
  • Table 9 The effect of (1) linker length between the CD4 domain and V3-bNAbl heavy chain, and (2) thermal stability of CD4 DI, on the PK of bispecific molecules in hFcRn mice (Tq32)
  • the best molecules for further development contain shorter linker lengths between the CD4 domain and bNAb (lxG4S) and contain a thermally stable CD4 domain(s).
  • CD4 domains was fused to different broadly neutralizing antibodies (bNAbs) that target the membrane proximal external region (MPER) of the HIV-1 envelope protein gpl60. These bispecifics were tested against a panel of HIV-1 envelopes in PSV assay, along with control molecules including CD4 domains alone, bNAbs alone, their mixtures at a 1:1 molar ratio (combo), and CD4 domains fused to an anti-RSV control antibody (Synagis - bispecific32). Several such bispecifics showed much higher anti-viral activity than the control molecules, indicating strong anti-viral synergy between the CD4 domain and the fused bNAbs ( Figures 4-13).
  • This synergy might be a combined outcome from 1) the CD4 domain and the linked bNAb bind to a same gpl60 envelope trimer; 2) CD4-induced conformational change of gpl60 exposes the MPER epitope for the bNAb (Prasad et al., Cell. 2022 Feb 17;185(4):641-653.el7.; Li et al., Nat Struct Mol Biol. 2020 Aug;27(8):726-734), which might be inaccessible in some resistant envelopes; and 3) the bNAb is sequestered at a high local concentration near the CD4 domain, providing advantage of binding kinetics.
  • SEQ ID NO: 48 (D1m-K8C-G99C 4xG4S bNAb2-HC)
  • SEQ ID NO: 70 (CDRL2 of bNAb36)
  • SEQ ID NO: 72 VH of bNAb36
  • SEQ ID NO: 75 (HO (LS) of bNAb36)
  • SEQ ID NO: 76 (LC of bNAb36)
  • SEQ ID NO: 80 (CDRL1 of bNAb38)
  • SEQ ID NO: 82 (CDRL3 of bNAb38)
  • SEQ ID NO: 84 VL of bNAb38
  • SEQ ID NO: 87 (LC of bNAb38)
  • SEQ ID NO: 90 CDRH3 of bNAb39
  • RASQSVIRYLA SEQ ID NO: 92 CDRL2 of bNAb39
  • SCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 108 (HC (LS) of bNAb40)
  • SEQ ID NO: 110 CDRH1 of bNAb41
  • SEQ ID NO: 120 (LC of bNAb41)
  • GIIPLLGLPNYAPKFQG SEQ ID NO: 123 (CDRH3 of bNAb42)
  • SEQ ID NO: 128 VL of bNAb42
  • SEQ ID NO: 142 (LC of bNAb44)

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Abstract

Antigen binding proteins of the invention bind to Human Immunodeficiency Virus (HIV) envelope protein and are useful in treating and preventing HIV infection. In particular, the antigen binding proteins of the MPER region of gp140 as well as having a CD4 domain that binds to the CD4bs of gp120.

Description

NEUTRALIZING ANTIBODY CONSTRUCTS AGAINST HIV
FIELD OF THE INVENTION
The invention is directed to a multimeric antigen binding protein that binds to the Human Immunodeficiency Virus (HIV) envelope spike complex and its use in treating or preventing HIV infection. The antigen binding proteins of the invention bind to the MPER region of glycoprotein 41 (gp41) as well as having a CD4 domain that binds to the CD4bs of gpl20.
BACKGROUND TO THE INVENTION
HIV, the virus that over time may result in Acquired Immunodeficiency Syndrome (AIDS), continues to be a serious public health challenge and has claimed 40.1 million lives so far. HIV attacks the body's immune system, targeting CD4-positive white blood cells, and leaves those infected vulnerable to opportunistic infections such as tuberculosis and fungal infections, severe bacterial infections and some cancers. Globally, 38.4 million people were living with HIV at the end of 2021, with 1.5 million people becoming newly infected (WHO, Key Facts HIV, July 2022).
Whilst there is currently no cure for HIV infection, it can be treated with antiretroviral therapy (ART), which includes a number of different types of drugs that prevent the virus from multiplying (nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors, entry inhibitors and integrase inhibitors), allowing the body's immune system to recover sufficiently for the infected patient to be asymptomatic. 75% of people living with HIV in 2021 received some form of ART. However, ART often requires taking medication every day for life and has the risk of serious and debilitating side effects. Further, increased use of ART has also been accompanied by the emergence of drug resistance, the levels of which have steadily increased in recent years.
Broadly neutralizing antibodies (bNAbs) could potentially provide longer-term HIV suppression, but individual bNAbs have only had limited success in previous studies. This is in part because antibody-resistant virus either already existed in the patient or emerged soon after treatment began (NIH Research Matters, 14 June 2022). Combinations of bNAbs are currently being investigated in the presence or absence of ART (Nature, 606, 368-374, 2022).
Further treatment options are needed for HIV infection, in particular drugs that are long- acting and effective against a wide spectrum of HIV strains so that patients taking them are less susceptible to drug resistance. SUMMARY OF THE INVENTION
In a first aspect of the invention, there is provided a multimeric anti-HIV envelope spike complexbinding protein comprising: i. a broadly neutralizing anti-MPER antibody, comprising at least one heavy chain or light chain, wherein the antibody binds to the MPER region of a gp41 protein; and ii. at least one CD4 domain which binds to gpl20, wherein the CD4 domain is attached directly or by a linker to the N-terminus or C-terminus of at least one of the heavy chains or light chains of the broadly neutralizing antibody.
In further aspects of the invention, pharmaceutical compositions comprising anti-HIV envelope spike complex-binding proteins of the invention, methods of preventing HIV infection and methods of treating HIV infection with anti-HIV envelope spike complex-binding proteins of the invention, uses of anti-HIV envelope spike complex-binding proteins of the invention, methods of manufacturing anti-HIV envelope spike complex-binding proteins of the invention and kits comprising anti-HIV envelope spike complex-binding proteins of the invention are also provided
DESCRIPTION OF DRAWINGS/ FIGURES
FIG. 1 shows schematic designs of example bispecific binding proteins of the invention. Human CD4 domains or variants thereof are fused, either directly or via linkers, to the N-termini the heavy chains (A), the light chains (B) or to the N-termini of heavy chains (C) or light chains (D) of bNAbs. Such designs facilitate concomitant binding of the human CD4 domain of the bispecific molecule and gpl60 binding domain (or in other words, the envelope spike complex broadly neutralizing antibody or fragment) of the bispecific molecule to HIV-1 gpl60 (the envelope spike complex) and prevent HIV-1 virions from binding to the cell surface receptors and fusing with the cell membrane (E).
FIG. 2 shows IC50 values (nM) of soluble CD4 domains (SEQ ID NOs:4-15) against a panel of HIV-1 envelopes in a PSV assay (ACTOne), together with the Tm for each soluble CD4 domain. The horizontal bars indicate geometric mean IC50.
FIG. 3 shows that linker length between the CD4 domain and a similar model bNAb to the invention (V3-bNAbl) heavy chain N-terminus does not particularly affect anti-viral activity in a PSV assay (ACTOne) (A) but does change the PK of the resultant bispecific molecules in a humanized mouse model (Tg32-hFcRn strain) (B). Thermal stability of the CD4 domain also affects the PK of the bispecific molecules (B). FIG. 4 shows IC50 (nM) and MPV (%) values for anti-viral activity of MPER-bNAb34-derived bispecifics according to the invention (targeting MPER). These include bispecific 34-1, bispecific 34- 2, bispecific 34-3 and bispecific 34-4 detailed in Table 1 below. In short, all four of these bispecifics contain the same LC (SEQ ID NO: 189), but vary in their HC sequences as different CD4 domains and/or linkers were used. In particular the HC of bispecific 34-1 contains a 4x GS linker (SEQ ID NO: 33) and a DlD2wt CD4 domain (SEQ ID NO: 1), the HC of bispecific 34-2 contains a 4XGS linker (SEQ ID NO: 33) and a D1.22D2 CD4 domain (SEQ ID NO: 2), the HC of bispecific 34-3 contains a 4XGS linker (SEQ ID NO: 33)and a DI.22 CD4 domain (SEQ ID NO: 4), and the HC of bispecific 34-4 contains a 2XG4S linker (SEQ ID NO: 31) and a DI.22 CD4 domain (SEQ ID NO: 4).
These experiments tested these MPER-bNAb34-derived bispecifics as well as control molecules against (A) a panel of HIV-1 envelopes in PSV assay (ACTOne cells) shown as IC50, and (B-C) replicating virus (MT2 cells) shown in both IC50 (B) and MPI (C).
Table 1- Figure 4 molecule key (SEQ ID NOs:)
FIG. 5 shows IC50 (nM), IC90 (nM) and MPV (%) values for anti-viral activity of MPER- bNAb35-derived bispecifics according to the invention (targeting MPER as detailed in Table 2 below. In short, all seventeen of these bispecific molecules contain the same LC (SEQ ID NO: 200), but vary in their HC sequences as different linkers and CD4 domains were used. In particular the HC of bispecific 35-6 contains a 6x GS linker (SEQ ID NO: 35) and a DI.22 CD4 domain (SEQ ID NO: 4), the HC of bispecific 35-5 contains a 5x GS linker (SEQ ID NO: 34) and a DI.22 CD4 domain (SEQ ID NO: 4), the HC of bispecific 35-4 contains a 4x GS linker (SEQ ID NO: 33) and a DI.22 CD4 domain (SEQ ID NO: 4), the HC of bispecific 35-3 contains a 3x GS linker (SEQ ID NO: 32) and a DI.22 CD4 domain (SEQ ID NO: 4), the HC of bispecific 35-2 contains a 2x GS linker (SEQ ID NO: 31) and a DI.22 CD4 domain (SEQ ID NO: 4), the HC of bispecific 35-1 contains a lx GS linker (SEQ ID NO: 30) and a DI.22 CD4 domain (SEQ ID NO: 4), the HC of bispecific 35-6 contains a Ox GS linker (no linker) and a DI.22 CD4 domain (SEQ ID NO: 4), and the HC of bispecific 35-EP contains an EP linker (SEQ ID NO: 201) and a DI.22 CD4 domain (SEQ ID NO: 4), the HC of bispecific 35-6-D2 contains a 6x GS linker (SEQ ID NO: 35) and a D1.22D2 CD4 domain (SEQ ID NO: 2), the HC of bispecific 35-5-D2 contains a 5x GS linker (SEQ ID NO: 34) and a D1.22D2 CD4 domain (SEQ ID NO: 2), the HC of bispecific 35-4-D2 contains a 4x GS linker (SEQ ID NO: 33) and a D1.22D2 CD4 domain (SEQ ID NO: 2), the HC of bispecific 35-3-D2 contains a 3x GS linker (SEQ ID NO: 32) and a D1.22D2 CD4 domain (SEQ ID NO: 2), the HC of bispecific 35-2-D2 contains a 2x GS linker (SEQ ID NO: 31) and a D1.22D2 CD4 domain (SEQ ID NO: 2), the HC of bispecific 35-1-D2 contains a lx GS linker (SEQ ID NO: 30) and a D1.22D2 CD4 domain (SEQ ID NO: 2), the HC of bispecific 35-0-D2 contains a Ox GS linker (no linker) ) and a D1.22D2 CD4 domain (SEQ ID NO: 2), and the HC of bispecific 35-EP-D2 contains an EP linker (SEQ ID NO: 201) and a D1.22D2 CD4 domain (SEQ ID NO: 2), and lastly the HC of bispecific 35-4-LS contains a 4xGS linker (SEQ ID NO: 33), an LS mutation and a DI.22 CD4 domain (SEQ ID NO: 2).
These experiments tested these MPER-bNAb34-derived bispecifics as well as control molecules (e.g. CD4D1= SEQ ID NO:3 and CD4 ECD/D1D2 = SEQ ID NO:1) against a panel of HIV-1 envelopes in PSV assay (ACTOne cells) for (A-C) bNAb35-derived bispecifics containing CD4 domain 1 and different linkers and (D-F) bNAb35-derived bispecifics containing CD4 domains 1 and 2 and different linkers. (A) and (D) show IC50, (B) and (E) show IC90, and (C) and (F) show MPI.
Table 2- Figures 5 and 6 molecule key (SEQ ID NOs:)
FIG. 6 shows IC50 (nM), IC90 (nM) and MPV (%) values for anti-viral activity of (A-C) a selected MPER-bNAb35-derived bispecific of the invention (specifically bispecifics 35-4 and 35-4-LS as per Figure 5 and Table 2 above) and control molecules against a panel of HIV-1 envelopes in PSV assay (ACTOne cells), said selected bNAb35-derived bispecific further including an "LS" mutation against (D-F) the same panel as well as (G-I) a further panel including further envelopes insensitive to MPER bNAbs described herein. (A), (D), and (G) show IC50; (B), (E), and (H) show IC90; (C), (F), and (I) show MPI. FIG. 7 shows (A) IC50 (nM), (B) IC90 (nM) and (C) MPV (%) values for anti-viral activity of
MPER- bNAb36-derived bispecifics according to the invention (targeting MPER, containing a HC with a linker + CD4 domain as per SEQ ID NO: 59 and a LC according to SEQ ID NO: 76) and control molecules against a panel of HIV-1 envelopes in PSV assay (ACTOne cells)
FIG. 8 shows (A) IC50 (nM), (B) IC90 (nM) and (C) MPV (%) values for anti-viral activity of MPER-bNAb38-derived bispecifics according to the invention (targeting MPER, containing a HC with a linker + CD4 domain as per SEQ ID NO: 60 and a LC according to SEQ ID NO: 87) and control molecules against a panel of HIV-1 envelopes in PSV assay (ACTOne cells)
FIG. 9 shows (A) IC50 (nM), (B) IC90 (nM) and (C) MPV (%) values for anti-viral activity of MPER-bNAb39-derived bispecifics (targeting MPER, containing a HC with a linker + CD4 domain as per SEQ ID NO: 61 and a LC according to SEQ ID NO: 98) and control molecules against a panel of HIV-1 envelopes in PSV assay (ACTOne cells)
FIG. 10 shows (A) IC50 (nM), (B) IC90 (nM) and (C) MPV (%) values for anti-viral activity of MPER-bNAb40-derived bispecifics (targeting MPER, containing a HC with a linker + CD4 domain as per SEQ ID NO: 62 and a LC according to SEQ ID NO: 109) and control molecules against a panel of HIV-1 envelopes in PSV assay (ACTOne cells)
FIG. 11 shows (A) IC50 (nM), (B) IC90 (nM) and (C) MPV (%) values for anti-viral activity of MPER-bNAb41-derived bispecifics (targeting MPER, containing a HC with a linker + CD4 domain as per SEQ ID NO: 63 and a LC according to SEQ ID NO: 120) and control molecules against a panel of HIV-1 envelopes in PSV assay (ACTOne cells)
FIG. 12 shows (A) IC50 (nM), (B) IC90 (nM) and (C) MPV (%) values for anti-viral activity of MPER-bNAb42-derived bispecifics (targeting MPER, containing a HC with a linker + CD4 domain as per SEQ ID NO: 64 and a LC according to SEQ ID NO: 131) and control molecules against a panel of HIV-1 envelopes in PSV assay (ACTOne cells)
FIG. 13 shows (A) IC50 (nM), (B) IC90 (nM) and (C) MPV (%) values for anti-viral activity of MPER-bNAb43-derived bispecifics (targeting MPER, containing a HC with a linker + CD4 domain as per SEQ ID NO: 65 and a LC according to SEQ ID NO: 142) and control molecules against a panel of HIV-1 envelopes in PSV assay (ACTOne cells).
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
"Affinity", also referred to as "binding affinity", is the strength of binding at a single interaction site, i.e., of one molecule, e.g., an antigen binding protein, to another molecule, e.g., its target antigen, at a single binding site. The binding affinity of an antigen binding protein to its target may be determined by equilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE analysis).
"Alternative antibody formats" include alternative scaffolds in which one or more CDRs of the antigen binding protein can be arranged onto a suitable non-immunoglobulin protein scaffold 5 or skeleton, such as an affibody, a SpA scaffold, an LDL receptor class A domain, an avimer (see, e.g., U.S. Patent Application Publication Nos. 2005/0053973, 2005/0089932, 2005/0164301) or an EGF domain.
"Antibody" is used herein to refer to a heterotetra meric glycoprotein with an approximate molecular weight of 150,000 daltons. An intact antibody is composed of two identical heavy chains (HCs) and two identical light chains (LCs) linked by covalent disulphide bonds. This H2L2 structure folds to form a 'Y' shape with three functional domains comprising two antigen-binding fragments, known as 'Fab' fragments (the 'top' of the ), and a fragment crystallisable 'Fc' (the 'bottom' of the "YQ. The Fab fragment is composed of the variable domain at the amino-terminus, variable heavy (VH) or variable light (VL), and the constant domain at the carboxyl terminus, CHI (heavy) and CL (light). The Fc fragment is composed of two domains formed by dimerization of paired CH2 and CH3 regions. The Fc may elicit effector functions by binding to receptors on immune cells or by binding Clq, the first component of the classical complement pathway. The five classes of antibodies IgM, IgA, IgG, IgE and IgD are defined by distinct heavy chain amino acid sequences, which are called p, a, y, £ and 5 respectively; each heavy chain can pair with either a K or A light chain. The majority of antibodies in the serum belong to the IgG class, there are four isotypes of human IgG (IgGl, IgG2, IgG3 and IgG4), the sequences of which differ mainly in their hinge region. In an embodiment, an anti-CD4bs antibody, as used herein, refers to an antibody that binds to a CD4 binding site
"Antigen binding antibody fragments" or "antigen binding fragments" or "antibody fragments" as used herein include Fab, F(abQ2, Fv, disulphide linked Fv, single chain Fv (scFv), disulphide-linked scFv, diabodies, TANDABS, etc. and modified versions of any of the foregoing (for a summary of alternative "antibody" formats see Holliger and Hudson, Nature Biotechnology, 23(9), 1126-1136, 2005).
"Antigen binding protein of the invention" and "anti-HIV envelope spike complex binding protein" are used interchangeably herein and refer to antibodies and fragments thereof, alternative antibody formats, and other protein constructs, such as domains, that are capable of binding to the HIV envelope spike complex comprised of gpl20 and gp41, or in other words are capable of binding to either gpl20 or gp41. The HIV env gene encodes a gene product of around 850 amino acids. The primary env product is the protein gpl60 (or envelope glycoprotein gpl60) which is gets cleaved into gpl20 (about 480 amino acids) and gp41 (about 345 amino acids) in the endoplasmic reticulum by the cellular protease furin. gpl20 (or gpl20) is a 120 kDa glycoprotein that is part of the outer layer of HIV. It presents itself as viral membrane spikes consisting of three molecules of gpl20 linked together and anchored to the membrane by gp41 protein. Gpl20 is essential for viral infection as it facilitates HIV entry into the host cell through its interaction with cell surface receptors. Gp41 is a transmembrane protein that contains several sites within its ectodomain that are required for infection of host cells. The amino acid sequence of an exemplary gpl60 from HIV clone WITO is provided below (SEQ ID NO: 55):
MKVMGTKKNYQHLWRWGIMLLGMLMMSSAAEQLWVTVYYGVPVWREANTTLFCASDAKAYDTEVHNVWAT HACVPTDPNPQEWMGNVTEDFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNVTISSTNGSTA NVTMREEMKNCSFNTTTVIRDKIQKEYALFYKLDIVPIEGKNTNTSYRLINCNTSVITQACPKVSFEPIPIHYCAPA GFAILKCNNKTFNGKGPCRNVSTVQCTHGIKPWSTQLLLNGSLAEEDIIIRSENFTNNGKNIIVQLKEPVKINCT RPGNNTRRSINIGPGRAFYATGAIIGDIRKAHCNISTEQWNNTLTQIVDKLREQFGNKTIIFNQSSGGDP
EWMHTFNCGGEFFYCNSTQLFNSTWFNNGTSTWNSTADNITLPCRIKQVINMWQEVGKAMYAPPIRGQIDCS SNITGLILTRDGGSNSSQNETFRPGGGNMKDNWRSELYKYKWKIEPLGIAPTRAKRRWQREKRAVTLGAVFLG FLGAAGSTMGAASLTLTVQARLLLSGIVQQQSNLLRAIEAQQHMLQLTVWGIKQLQARVLAIERYLKDQQLLGI WGCSGKLICTTTVPWNTSWSNKSYDYIWNNMTWMQWEREIDNYTGFIYTLIEESQNQQEKNELELLELDKWA SLWNWFNITNWLWYIKLFIMIIGGLVGLRIVCAVLSIVNRVRQGYSPLSFQTRLPNPRGPDRPEETEGEGGERDR DRSARLVNGFLAIIWDDLRSLCLFSYHRLRDLLLIVARWEILGRRGWEILKYWWNLLKYWSQELKNSAVSLLNV TAIAVAEGTDRVIEIVQRAVRAILHIPTRIRQGFERALL
The amino acid of an exemplary gpl20 is provided below (SEQ ID NO: 56):
AEQLWVIVYYGVPVWREANTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEWMGNVTEDFNMWKNNMV EQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNVTISSTNGSTANVTMREEMKNCSFNTTTVIRDKIQKEYALFYK LDIVPIEGKNTNTSYRLINCNTSVrTQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGKGPCRNVSTVQCTHGIKP WSTQLLLNGSLAEEDIIIRSENFTNNGKNIIVQLKEPVKINCTRPGNNTRRSINIGPGRAFYATGAIIGDIR KAHCNISTEQWNNTLTQIVDKLREQFGNKTIIFNQSSGGDPEWMHTFNCGGEFFYCNSTQLFNSTWFNNGTS TWNSTADNITLPCRIKQVINMWQEVGKAMYAPPIRGQIDCSSNITGLILTRDGGSNSSQNETFRPGGGNMKDN WRSELYKYKWKIEPLGIAPTRAKRRWQREKR
The amino acid of an exemplary gp41 is provided below (SEQ ID NO: 57; the MPER region is boldened, and shown separately as SEQ ID NO: 58):
AVGIGALFLGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARI LAVER YLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNHTTWMEWDREINNYTSLIHSLIEESQNQQEKNE QELLELDKWASLWNWFNrTNWLWYIKLFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGPD RPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYWWNLLQYW SQELKNSAVSLLNATAIAVAEGTDRVIEWQGACRAIRHIPRRIRQGLERILL
"Antigen binding site" and "paratope" are used interchangeably herein and refer to a particular site on an antigen binding protein that makes contact with and is capable of specifically binding to a site (i.e., epitope) on an antigen, e.g., HIV gpl20 or gp41. The antigen binding site may be formed by a single variable domain, or paired VH/VL domains as can be found on a standard antibody. Single-chain Fv (ScFv) domains can also provide antigen binding sites. "Avidity" also referred to as functional affinity, is the cumulative strength of binding at multiple interaction sites, e.g., the sum total of the strength of binding of two molecules (or more) to one another at multiple sites, e.g., taking into account the valency of the interaction.
A "bispecific molecule" or "bispecific antigen binding protein" as used herein is an antigen binding protein that is capable of binding to two different epitopes on the same antigen, i.e., HIV gp41 protein. In particular, one epitope comprises part of or the whole of MPER region of gpl41 and the other epitope comprises part of or the whole of the CD4 binding site of gpl20.
"Broadly neutralizing antibody" or "bNAb" as used herein, is meant an antibody that neutralizes more than one HIV-1 virus species (from diverse clades and different strains within a clade) in a neutralization assay. A broadly neutralizing antibody may neutralize at least 2, 3, 4, 5, 6, 7, 8, 9 or more different strains of HIV-1, the strains belonging to the same or different clades.
"CD4 binding site" or "CD4-binding site" or "CD4bs" refers to a site on the HIV envelope protein gpl20 that binds to CD4. (Cluster of differentiation factor 4). CD4 is a T-cell surface protein that serves as the primary receptor site for HIV during HIV infection. The CD4 binding site on gpl20 is a highly conserved, discontinuous and conformational that comprises residues on either side of the HIV V4 loop (Curr HIV/AIDS Rep, 9(1): 52-63, 2021) that binds to CD4.
A "CD4 domain" as used herein is a soluble recombinant form of human CD4 (Cluster of differentiation factor 4, a transmembrane glycoprotein found on T-cells ), or a fragment thereof, that mimics the activity of native membrane-anchored human CD4 in its binding interactions with the HIV envelope protein. A CD4 domain of the present invention binds to the CD4-binding site of HIV gpl20 and may block the ability of HIV gpl20 to bind membrane-anchored CD4, e.g., on CD4+ T cells. A CD4 domain of the invention may induce a structural rearrangement in gpl20 upon binding, including a structural rearrangement of part or all of the MPER of gp41 (PMID: 32601441). This structural rearrangement in gpl20 results in a high affinity binding site for a chemokine coreceptor (CXCR4 and/or CCR5) being exposed. Native CD4 comprises four domains that are exposed on the extracellular surface of the cell, DI, D2, D3 and D4; a transmembrane domain; and a cytoplasmic tail domain. DI and D3 resemble Ig variable domains and D2 and D4 resemble Ig constant domains. CD4 domains of the invention include one or more of domains DI to D4 of CD4, or variants thereof. Examples of CD4 domains of the invention include wild-type DI (SEQ ID NO:3); "mD1.22"/Dlm (SEQ ID NO:4), which is a variant of DI of CD4 (Chen et al, JVI 88(2): 1125-39, 2014); wild-type D1D2 (SEQ ID NO:1); "mD1.22-D2" (SEQ ID NO:2), which is a variant of D1D2 (Fetzer et al., Journal of Virology, 92(12), 2018); and further variants of mD1.22 (SEQ ID NOs:5- 21). "CDRs" are defined as the complementarity determining region amino acid sequences of an antigen binding protein. These are the hypervariable regions of immunoglobulin heavy and light chains. There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. In one embodiment, the CDRs are defined based on the Kabat definition. In another embodiment, the CDRs are defined based on the Chothia definition. In a further embodiment, the Chothia definition is from Discovery Studio which uses the definitions fromChothia and Lesk, JMol Biol. 196(4):901-17 (1987) and Morea et al, Methods, 20:267-279 (2000). In another embodiment, the Chothia definition is based on the Chothia from Abysis definition. In a further embodiment, the CDRs are defined based on the IMGT definition. In another embodiment, the CDRs are defined based on the Honegger definition. In another embodiment, the CDRs are defined based on the contact definition. Thus, "CDRs" as used herein refers to all three heavy chain CDRs, all three light chain CDRs, all heavy and light chain CDRs, or at least two CDRs.
"Domain" refers to a folded polypeptide structure that retains its tertiary structure independent of the rest of the polypeptide. Generally, domains are responsible for discrete functional properties of polypeptides and in many cases may be added, removed or transferred to other polypeptides without loss of function of the remainder of the protein and/or of the domain.
"Effector Function" as used herein refers to one or more of antibody-mediated effects including antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-mediated complement activation including complement-dependent cytotoxicity (CDC), complement-dependent cell- mediated phagocytosis (CDCP), antibody dependent complement-mediated cell lysis (ADCML), and Fc-mediated phagocytosis or antibody-dependent cellular phagocytosis (ADCP).
"Epitope" as used herein refers to the portion of an antigen (e.g., gpl20) that makes contact with and is capable of specifically binding to a particular site (paratope) on an antigen binding protein. An epitope may be linear or conformational/discontinuous. A conformational/ discontinuous epitope comprises amino acid residues that are separated by other sequences, i.e., it does not comprise a continuous sequence in the antigen's primary amino acid sequence, but instead relies on the tertiary folding of the polypeptide. Although the residues within a confirmational/ discontinuous epitope may be from different regions of the polypeptide chain, they are in close proximity in the three-dimensional structure of the antigen.
In the case of multimeric antigens, a conformational or discontinuous epitope may include residues from different polypeptide chains. Particular residues comprised within an epitope can be determined through computer modelling programs or via three-dimensional structures obtained through methods known in the art, such as X-ray crystallography. Epitope mapping can be carried out using various techniques known to persons skilled in the art as described in publications such as Methods in Molecular Biology 'Epitope Mapping Protocols', by Mike Schutkowski and Ulrich Reineke (volume 524, 2009) and Johan Rockberg and Johan Nilvebrant (volume 1785, 2018). Exemplary methods include peptide-based approaches such as pepscan whereby a series of overlapping peptides are screened for binding using techniques such as ELISA or by in vitro display of large libraries of peptides or protein mutants, e.g., on phage. Detailed epitope information can be determined by structural techniques including X-ray crystallography, solution nuclear magnetic resonance (NMR) spectroscopy and cryogenic-electron microscopy (cryo- EM). Mutagenesis, such as alanine scanning, is an effective approach whereby loss of binding analysis is used for epitope mapping. Another method is hydrogen/deuterium exchange (HDX) combined with proteolysis and liquid-chromatography mass spectrometry (LC-MS) analysis to characterize discontinuous or conformational epitopes.
"Half-life" or "tl/2" refers to the time required for the serum concentration of an antigen binding protein to reach half of its original value. The serum half-life of proteins can be measured by pharmacokinetic studies according to the method described by Kim et al., 1994, Eur. J. of Immuno. 24: 542-548. According to this method, radio-labelled protein is injected intravenously into mice and its plasma concentration is periodically measured as a function of time, for example, at about 3 minutes to about 72 hours after the injection. Other methods for pharmacokinetic analysis and determination of the half-life of a molecule will be familiar to those skilled in the art.
"HIV envelope protein" or "ENV" or "spike complex" refers to a trimeric viral membrane- associated glycoprotein (gp) or 'spike' comprised of non-covalently linked heterodimers of surface gpl20 and transmembrane gp41. It is found on both the viral membrane and the cell membrane of infected host cells and is encoded by the Env gene. The env gene encodes the gpl60 polypeptide which forms a homotrimer and is cleaved into gpl20 and gp41 polypeptides. Gpl20 is a surface (SU) glycoprotein responsible for binding to receptor molecules and the transmembrane (TM) glycoprotein, gp41, mediates fusion of the viral membrane with the plasma cell membrane. Over half of the mass of the trimeric envelope 'spike' is an N-linked glycan shield that hides most amino acid-based epitopes on gpl20. Binding of the cell surface receptor CD4 to HIV gpl20 induces a structural rearrangement creating a high affinity binding site for a chemokine coreceptor (CXCR4 and/or CCR5), on gpl20. Following gpl20 binding to CXCR4 or CCR5 further conformational changes are triggered which results in gpl20 disengaging from gp41, allowing for the fusion peptide of gp41 to be inserted into the cell membrane, which in turn triggers a sequence of structural changes resulting in membrane fusion (Dimitrov et al., Biochemistry 44(37): 12471-12479, 2005). "Human immunodeficiency virus (HIV)" has been characterized into two types: HIV-1 and HIV-2. HIV-1 is more virulent and more infective than HIV-2 and is the cause of the majority of HIV infections globally, whereas HIV-2 is limited to a much smaller number of people, mostly in West Africa (Gilbert et al., Statistics in Medicine 22(4): 573-593). Thus "HIV" may mean HIV-1 and HIV-2, or just "HIV-1". HIV virions are spherical with viral glycoprotein "spikes", the HIV envelope protein, protruding outwards. A conical capsid exists within the virion, enclosing a ribonucleoprotein complex comprising two copies of positive-sense single stranded RNA tightly bound to nucleocapsid proteins and enzymes needed for viral replication. "Symptoms" of HIV include raised temperature (fever), sore throat, body rash, tiredness, joint pain, muscle pain, swollen glands, weight loss, chronic diarrhoea, night sweats, skin problems, recurrent infections, headaches.
A "linker" is a suitable structure that can be used to join together the CD4 domain and antibody or fragment of the invention. A linker may be a chemical linker such as PEG, or an amino acid sequence that links one domain in a polypeptide to another domain in a polypeptide. For example, a linker within the meaning of the invention includes an amino acid sequence that joins a CD4 domain to a bNAb heavy chain or a bNAb light chain. In an embodiment, the linker is not cleavable under intracellular conditions.
"Multimeric antigen binding protein" or "multispecific antigen binding protein" refers to an antigen binding protein that comprises at least two different polypeptide chains having antigen binding sites. Each of these antigen-binding sites is capable of binding to a different epitope, which may be present on the same antigen or different antigens.
In an embodiment, the multi-specific antigen binding proteins of the invention are bispecific molecules capable of binding to two different epitopes on the HIV envelope protein. In particular, one epitope may comprise part of or the whole of the MPER region of gp41 and the other epitope may comprise part of or the whole of the CD4 binding site of gpl20 (through binding of the CD4 domain portion of the molecule).
Symmetric formats of MSABPs combine multiple binding specificities in a single polypeptide chain or single HL pair including Fc-fusion proteins of fragment-based formats and formats whereby antibody fragments are fused to regular antibody molecules. Examples of symmetric formats may include DVD-Ig, TVD-Ig, CODV-Ig, (scFv)4-Fc, IgG-(scFv)2, Tetravalent DART-Fc, F(ab)4CrossMab, IgG-HC-scFv, IgG-LC-scFv, mAb-dAb etc.
A multimeric antigen binding protein described herein, for example, a bispecific antigen binding protein having a broadly neutralizing anti-HIV envelope spike complex antibody and a CD4 domain, may be encoded by one or more isolated nucleic acid sequences. Production of a multimeric antigen binding protein, such as a bispecific antigen binding protein, may be achieved in a cell or living organism by delivering exogenous isolated nucleic acids encoding the multimeric antigen binding protein, for example, a heavy chain and a light chain of a broadly neutralizing anti-HIV envelope spike complex antibody and a CD4 domain. Production of a multimeric antigen binding protein, such as a bispecific antigen binding protein, may be achieved in a cell in vitro or in vivo by delivering exogenous isolated nucleic acids encoding the multimeric antigen binding protein, for example, a heavy chain and a light chain of a broadly neutralizing anti-HIV envelope spike complex antibody and a CD4 domain. A subject in need may be delivered one or more nucleic acids encoding an multimeric antigen binding protein provided herein, such as a heavy chain and a light chain of a broadly neutralizing anti-HIV envelope spike complex antibody and a CD4 domain. The heavy chain and the light chain of the antibody may be delivered by the same or separate nucleic acids. The nucleic acids may be DNA or RIMA. The nucleic acids encoding the multimeric antigen binding protein may be delivered to the subject naked (i.e. without an encapsulating particle) or packaged (i.e. encapsulated in liposomes or polymer-based vehicles). The nucleic acids encoding the multimeric antigen binding protein may be delivered without a delivery vehicle (i.e., "naked") or delivered with a viral or non-viral delivery vehicle (i.e., as a viral vector, adsorbed to or encapsulated in liposomes or polymer-based vehicles, and the like). The nucleic acid may include elements such as a poly-A tail, a 5' and/or 3' untranslated region (UTR). The nucleic acids may be mRNA. The mRNA may include a cap structure. The mRNA may be self-replicating RNA.
The nucleic acid coding for the multimeric antigen binding proteins may be modified or unmodified. The nucleic acids coding for the multimeric antigen binding proteins may comprise at least one chemical modification. Nucleic acids (e.g., mRNAs) can be modified to enhance stability by including one or more chemical modifications. Such chemical modifications include, but are not limited to, a modified nucleotide, a modified sugar backbone, and the like. Also provided herein is a method of producing a multimeric antigen binding protein in a cell, tissue, or organism comprising contacting said cell, tissue, or organism with a composition comprising an isolated nucleic acid comprising at least one chemical modification and which encodes the multimeric antigen binding protein. Also provided herein is a method of producing a multimeric antigen binding protein in a cell, tissue or organism comprising contacting said cell, tissue or organism with a composition comprising a polynucleotide comprising at least one chemical modification and which encodes a multimeric antigen binding protein. Also provided herein is a method of producing a multimeric antigen binding protein in a cell, in vitro or in vivo, comprising contacting said cell with a composition comprising a nucleic acid comprising at least one chemical modification and which encodes a multimeric antigen binding protein. "Neutralizes" as used throughout the present specification means that the biological activity of HIV is reduced in the presence of an antigen binding protein as described herein in comparison to the biological activity of HIV in the absence of the antigen binding protein, in vitro or in vivo. For example, a neutralizing antigen binding protein of the invention may inhibit HIV entry into a target cell and reduce viral load in a patient infected with HIV.
"Percent identity" or "% identity" between a query amino acid sequence and a subject amino acid sequence is the "Identities" value, expressed as a percentage, that is calculated using a suitable algorithm (e.g., BLASTP, FASTA, Needleman-Wunsch, Smith-Waterman, LALIGN, or GenePAST/KERR) or software (e.g., DNASTAR Lasergene, GenomeQuest, EMBOSS needle or EMBOSS infoalign), over the entire length of the query sequence after a pair-wise global sequence alignment has been performed using a suitable algorithm (e.g., Needleman-Wunsch or GenePAST/KERR) or software (e.g. DNASTAR Lasergene or GenePAST/KERR). Importantly, a query amino acid sequence may be described by an amino acid sequence disclosed herein, in particular in one or more of the claims.
The query sequence may be 100% identical to the subject sequence, or it may include up to a certain integer number of amino acid alterations as compared to the subject sequence such that the % identity is less than 100%. For example, the query sequence is at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the subject sequence. In the case of amino acid sequences such alterations include at least one amino acid residue deletion, substitution (including conservative and non-conservative substitutions), or insertion, wherein said alterations may occur at the amino- or carboxy-terminal positions of the query sequence or anywhere between those terminal positions, interspersed either individually among the amino acid residues in the query sequence or in one or more contiguous groups within the query sequence.
For antibody sequences, the % identity may be determined across the entire length of the query sequence, including the CDRs. Alternatively, the % identity may exclude one or more or all of the CDRs, for example all of the CDRs are 100% identical to the subject sequence and the % identity variation is in the remaining portion of the query sequence, e.g., the framework sequence, so that the CDR sequences are fixed and intact.
"Protein scaffold" as used herein includes, but is not limited to, an immunoglobulin (Ig) scaffold, for example an IgG scaffold, which may be a four chain or two chain antibody, or which may comprise only the Fc region of an antibody, or which may comprise one or more constant regions from an antibody, which constant regions may be of human origin.
The protein scaffold may be an Ig scaffold, for example an IgG, or IgA scaffold. The IgG scaffold may comprise some or all the domains of an intact antibody (i.e., CHI, CH2, CH3, VH, VL). The antigen binding protein may comprise an IgG scaffold selected from IgGl, IgG2, IgG3, IgG4 or IgG4PE. For example, the scaffold may be IgGl. The scaffold may consist of, or comprise, the Fc region of an antibody, or is a part thereof.
The protein scaffold may be a non-Ig scaffold. The protein scaffold may be a derivative of a scaffold selected from one or more of CTLA-4, lipocalin, Protein A derived molecules such as Z- domain of Protein A (Affibody, SpA), A-domain (Avimer/Maxibody); heat shock proteins such as GroEl and GroES; transferrin (trans-body); ankyrin repeat protein (DARPin); peptide aptamer; Ctype lectin domain (Tetranectin); human y-crystallin and human ubiquitin (affilins); PDZ domains; scorpion toxin kunitz type domains of human protease inhibitors; and fibronectin/adnectin; which has been subjected to protein engineering in order to obtain binding to an antigen, such as gp41.
"Single variable domain" refers to a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes complete antibody variable domains such as VH, VHH and VL and modified antibody variable domains, for example, in which one or more loops have been replaced by sequences that are not characteristic of antibody variable domains, or antibody variable domains that have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains that retain at least the binding activity and specificity of the full-length domain. A single variable domain as defined herein is capable of binding an antigen or epitope independently of a different variable region or domain. A "domain antibody" or "DAB" may be considered the same as a human "single variable domain". A single variable domain may be a human single variable domain, but also includes single variable domains from other species such as rodent (for example, as disclosed in WO 00/29004), nurse shark and Camelid VHHs Camelid VHHs are immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain only antibodies naturally devoid of light chains. Such VHH domains may be humanised according to standard techniques available in the art, and such domains are considered to be "single variable domains".
"Stabilizing mutation" refers to a change of an amino acid residue in a polypeptide sequence that increases the thermal thermostability of said polypeptide. Increased thermostability may be reflected in a melting temperature (Tm) increase of, for example, between 1 and 50 °C. CD4 domains with stabilizing mutations include SEQ ID NOs:5-21.
A "variant sequence" substantially retains the biological characteristics of the unmodified protein. In the case of an antibody sequence disclosed herein, the VH or VL (or HC or LC) sequence may be a variant sequence with up to 10 amino acid substitutions, additions or deletions. For example, the variant sequence may have up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitution(s), addition(s) or deletion(s). The sequence variation may exclude one or more or all of the CDRs, for example the CDRs are the same as the VH or VL (or HC or LC) sequence and the variation is in the remaining portion of the VH or VL (or HC or LC) sequence, so that the CDR sequences are fixed and intact.
"V3 loop region", "V3/glycan" or "V3" as used herein refers to the third variable region (V3) of HIV gpl20. Comparison of predicted amino acid sequences from several different isolates has shown that sequence heterogeneity of gpl20 is clustered in five variable regions (designated VI, V2, V3, V4, and V5.) The V3 region contains post-translational modifications, such as glycosylation, and is essential for viral infectivity. The V3 region, although only 35 amino acids long, exhibits considerable sequence variability. Additionally, variability in potential N-linked glycosylation sites allow for further variability in the variable regions of gpl20. Together, the V3 region and the N- linked glycosylation sites within and adjacent to the region are understood to comprise the "V3 loop region," "V3/glycan" or V3" as used herein. For example, one site of glycosylation (e.g., 30 oligomannose such as Man-5 to Man-9) is centered on amino acid residue N332 of gpl20. Other sites of potential N-linked glycosylation within and adjacent to the V3 loop region include K295, N301, N386, N392 of gpl20. The V3 loop is generally considered to be in the region between cysteine residues C296 and C331 of gpl20, while some N-linked glycosylation sites are located directly adjacent to the V3 loop. The V3 loop comprises a highly conserved tetrapeptide sequence, GPGR (residues 312 to 315) (Ivanhoff et al., Virology, 187(2) 1992). HIV-1 cellular entry depends on the interaction of the V3 loop region with an HIV co-receptor, commonly CCR5 or CXCR4. The V3 loop comprises: (i) the base (residues 296-299), (ii) the stem (residues 300-303 and 321-326), and (iii) the crown (residues 304-320) (Friedrich et al., Nature Communications 12, 6705 (2021)). A consensus sequence of the V3 region of gpl20 (Milich et al., J Virol., 67(9):5623-5634 (1993)) is provided below:
CTRPNNNTRKSIHIGPGRAFYTTGEIIGDIRQAHC (SEQ ID NO: 54)
It is understood that the consensus sequence describes the highest frequency of residues emerging on each position of this region across multiple subtypes, but that the V3 loop region of a particular strain may exhibit sequence variability.
A "V3-bNAb" or "anti-V3 bNAb" is a bNAb that binds within the V3 loop region. A V3- bNAb may also be referred to herein as an anti-V3 antibody. A V3-bNAb may bind the N332 glycan in the V3 loop region and/or other N-linked glycosylation sites within and adjacent to the V3 loop region.
"Membrane Proximal External Region" or "MPER" or "MPER region" or "MPER domain" as used herein refers to a functional segment of HIV gp41. The MPER region is located near the viral membrane and plays a crucial role in the fusion of the viral envelope with the host cell membrane during the process of viral entry. A consensus sequence of the MPER region of gp41 is provided below:
LDKWASLWNWFNITNWLWYIK (SEQ ID NO: 58)
It is understood that the consensus sequence describes the highest frequency of residues emerging on each position of this region across multiple subtypes, but that this region of a particular strain may exhibit sequence variability.
An "MPER-bNAb" or "anti-MPER bNAb" is a bNAb that binds within the MPER region of gp41. An MPER-bNAb may also be referred to herein as an anti-MPER antibody.
STATEMENT OF THE INVENTION
A multimeric antigen binding protein of the invention binds to to the MPER region of gp41 as well as having a CD4 domain that binds to the CD4bs of gpl20 have been shown to effectively neutralize HIV and exhibit significantly better anti-viral activity than monospecific molecules that only bind to the MPER region of gp41 or the CD4bs of gpl20, and mixtures of these monospecific molecules. Without being bound by any particular theory, we postulate that the bispecific molecules of the invention bind the two different epitopes in the same or neighboring HIV envelope protein trimers at the same time, such that the bispecific molecules achieve stronger binding (increased avidity) to the HIV envelope proteins. This may be as a result of the high local concentration of the bispecific molecules' binding sites (paratopes) being "pre-positioned" around their target binding sites (epitopes) on the HIV envelope compared to their monospecific counterparts, which in turn leads to stronger anti-viral activity. In addition, we believe that CD4-induced conformational change of the envelope spike complex (also referred to as gpl60) exposes new epitopes for the bNAb portion of the binding proteins, which overcomes the low susceptibility of insensitive envelopes
Accordingly, in one aspect of the invention there is provided a multimeric anti-HIV envelope spike complex -binding protein comprising: i. a means for binding to the MPER domain of a gp41 protein and neutralizing at least 2 different strains of HIV-1; and ii. at least one CD4 domain which binds to gpl20, wherein the CD4 domain is attached directly or by a linker to the N-terminus or C-terminus of said means for binding.
In some embodiments, the means for binding to the MPER region of a gp41 protein and neutralizing at least 2 different strains of HIV-1 is a broadly neutralizing antibody or fragment thereof. For example, the means for binding to binding to the MPER region of a gp41 protein and neutralizing at least 2 different strains of HIV-1 may be any of the bispecific binding proteins exemplified in Example 6 and Figures 4-13. The means for binding to binding to the MPER region of a gp41 protein and neutralizing at least 2 different strains of HIV-1 may comprise CDR sequences according to any row of Table 3 and/or heavy and light variable regions or heavy and light chain sequences according to any row of Table 4.
Accordingly, in one aspect of the invention there is provided a multimeric anti-HIV envelope spike complex-binding protein comprising: i. a broadly neutralizing anti-MPER antibody, comprising at least one heavy chain or light chain, wherein the antibody binds to the MPER domain of a gp41 protein; and ii. at least one CD4 domain which binds to gpl20, wherein the CD4 domain is attached directly or by a linker to the N-terminus or C-terminus of one of the heavy chains or light chains.
In some embodiments, the binding protein is a bispecific binding protein.
CD4 Domains
CD4 domains of the invention include SEQ ID NOs: 1-21.
In an embodiment of the invention, the CD4 domain is a CD4 DI domain. In an embodiment, the CD4 domain is a human CD4 domain. CD4 DI domains include human wild-type DI (SEQ ID NO:3), mD1.22 (SEQ ID NO:4) also known as Dim, and further variants of mD1.22 (SEQ ID NOs: 5-21). In some embodiments, the CD4 domain has a sequence that is at least 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 3 or 4.
In an embodiment of the invention, the CD4 domain is a CD4 D1D2 domain. In an embodiment, the CD4 domain is a human CD4 D1D2 domain. CD4 D1D2 domains include human wild-type D1D2 (SEQ ID NO:1) and mD1.22-D2 (SEQ ID NO:2). In some embodiments, the CD4 domain has a sequence that is at least 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO:1 or
2.
In an aspect of the invention, a stabilized CD4 domain is provided. In an embodiment of the invention, a stabilized CD4 DI domain is provided. In an embodiment, the CD4 domain is thermally stable, i.e., thermostable. In an embodiment, the CD4 domain is a thermostable CD4 DI domain.
In an embodiment of the invention, the CD4 domain comprises one or more stabilizing mutations compared to the wild type sequence. In an embodiment, the stabilizing mutations are in the CD4 DI domain. In an embodiment, the CD4 DI domain comprises at least one mutation at position 5, 23, 55, 79, 96 and/or 98 of SEQ ID NO: 1 or 3. In an embodiment, the CD4 DI domain comprises one or more mutations at positions 8, 11, 13, 21, 25, 27, 38, 52, 58, 61, 65, 70, 72, 87, 91 and or 99 of SEQ ID NO: 1 or 3. In an embodiment, the CD4 DI domain comprises a mutation at position 8 or SEQ ID NO: 1 or 3. In an embodiment, the CD4 DI domain comprises a mutation at positions 11 and 72 of SEQ ID NO: 1 or 3. In an embodiment, the CD4 DI domain comprises mutation at positions 8 and 99.
In an embodiment, the CD4 DI domain comprises at least one mutation selected from: L5Y, S23N, A55V, I79P, L96V and/or F98V mutations In an embodiment, the CD4 DI domain comprises at least one mutation selected from: K8C, K8I, K8V, T11C, E13C, K21C, Q25E, H27C, H27D, G38C, N52W, R58N, R58T, R58V, L61M, G65C, I70C, K72C, E87G, E91H, E91Q, and/or G99C mutations. In an embodiment, the CD4 DI domain comprises a K8I mutation. In an embodiment, the CD4 DI domain comprises a K8V mutation. In an embodiment, the CD4 DI domain comprises a TIC and a K72C mutation. In an embodiment, the CD4 DI domain comprises a K8C and a G99C mutation. In all of these embodiments, these mutations may be compared to a wild type sequence as per SEQ ID NO: 1 or 3.
CD4 domains of the invention comprising novel and inventive stabilizing mutations include SEQ ID NOs:5-21. In some embodiments, the CD4 domain is a SEQ ID NOs: 5-15. In some embodiments, the CD4 domain has a sequence that is at least 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 5-21. In some embodiments, the CD4 domain has a sequence that is at least 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO: 5-15.
Increased thermostability may be reflected in a melting temperature (Tm) increase of, for example, between 1 and 50 °C; in particular between 1 and 30 °C; in particular between 1 and 25 °C, in particular between 1 and 21 °C, more particularly between 5 and 21 °C. The Tm increase is determined by measuring the Tm of the CD4 domain(s) comprising one or more stabilizing mutations and subtracting the Tm of the corresponding CD4 domain(s) without said mutation(s). For example, measuring the Tm of a stabilized CD4 DI domain and subtracting the Tm of the wild-type CD4 DI domain. In an embodiment, the Tm increase is about 8 °C. In an embodiment, the Tm increase is about 9 °C. In an embodiment, the Tm increase is about 12 °C. In an embodiment, the Tm increase is about 21 °C.
In an embodiment, the Tm of the CD4 domain is above 70 °C. In an embodiment, the Tm of the CD4 domain is between 70 °C and 95 °C. In an embodiment, the Tm of the CD4 domain is between 75 °C and 95 °C. In an embodiment, the Tm of the CD4 domain is between 75 °C and 91 °C. In an embodiment, the Tm of the CD4 domain is about 76 °C, about 77 °C, about 78 °C, about 79 °C, about 80 °C, about 81 °C, about 82 °C, about 83 °C, about 84 °C, about 85 °C, about 86 °C, about 87 °C, about 88 °C, about 89 °C, or about 90 °C. In an embodiment, the Tm of the CD4 domain is about 90 °C. In an embodiment, the Tm of the CD4 domain is about 89 °C. Tm may be determined by routine methods known in the art or as set out in the Examples. In an embodiment, Tm is determined using the Prometheus System (NanoTemper, Munchen Germany).
In some embodiments of the invention, a CD4 domain as described above may be attached directly to a broadly neutralizing antibody, by "directly" is meant that a CD4 domain is covalently bonded to a broadly neutralizing antibody without the use of an additional linking chemical or peptide sequence e.g. OxGS. In other embodiments, the CD4 domain as described above may be attached by a linker (for example a peptide linker such as those shown in SEQ ID: 30-35).
Anti-HIV envelope spike complex broadly neutralizing antibodies
An antigen binding protein of the invention may comprise a broadly neutralizing antibody or fragment thereof having heavy chain CDRs (CDRH1, CDRH2, and CDRH3) as set out in any row of Table 3. An antigen binding protein of the invention may comprise a broadly neutralizing antibody or fragment thereof having light chain CDRs (CDRL1, CDRL2, and CDRL3) as set out in any row of Table 3. An antigen binding protein of the invention may comprise a broadly neutralizing antibody or fragment thereof having a set of six CDRs (CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3) as set out in any row of Table 3.
An antigen binding protein of the invention may comprise a broadly neutralizing antibody or fragment thereof having a VH domain as set out in Table 4. An antigen binding protein of the invention may comprise a broadly neutralizing antibody or fragment thereof having a VL domain as set out in Table 4. An antigen binding protein of the invention may comprise a broadly neutralizing antibody or fragment thereof having a pair of variable domains (a VH and a VL) as set out in any row of Table 4.
Table 3: SEO ID NOs for the complementarity determining regions (CDRs) of broadly neutralizing antibodies
Table 4: SEO ID NOs for the variable heavy regions (VH) and variable light regions (VL) variable domain pairs'), and the full heavy chains (with and without LS mutations') and light chains (LC) of broadly neutralizing antibodies.
Anti-MPER bNAbs
In particular, an antigen binding protein of the invention may comprise an anti-MPER bNAb or a fragment thereof (i.e. a broadly neutralizing antibody or fragment binds to the MPER region of a gp41 protein). A fragment may include Fab, F(abQ2, Fv, disulphide linked Fv, single chain Fv (scFv), disulphide-linked scFv, diabodies, TANDABS, etc. and modified versions of any of the foregoing. For example, scFv variants of anti-MPER bNAbs have been described in van Dorsten et al. 2021 (PMID: 34935437) and Fab variants of anti-MPER bNAbs have been described in Garcia-Porras et al. 2024 (PMID: 38339834). An anti-MPER bNAb or fragment thereof includes an antibody comprising a set of CDRs
(CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3) as set out each row of Table 3. An anti-MPER bNAb or fragment thereof also includes an antibody comprising a pair of variable domains (a VH and VL) set out each row of Table 4.
An anti-MPER bNAb may be an antibody comprising a heavy chain (HC), with or without M428L/N434S (EU numbering) 'LS' mutations, and a light chain (LC) as set out in each row of Table 4. In an embodiment, the HC comprises LS.
In an embodiment, the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 168, a CDRH2 of SEQ ID NO: 169, a CDRH3 of SEQ ID NO: 170, a CDRL1 of SEQ ID NO: 171, a CDRL2 of SEQ ID NO: 172 and a CDRL3 of SEQ ID NO: 173. In an embodiment, the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 179, a CDRH2 of SEQ ID NO: 180, a CDRH3 of SEQ ID NO: 181, a CDRL1 of SEQ ID NO: 182, a CDRL2 of SEQ ID NO: 183 and a CDRL3 of SEQ ID NO: 184. In an embodiment, the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 190, a CDRH2 of SEQ ID NO: 191, a CDRH3 of SEQ ID NO: 192, a CDRL1 of SEQ ID NO: 193, a CDRL2 of SEQ ID NO: 194 and a CDRL3 of SEQ ID NO: 195. In an embodiment, the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 66, a CDRH2 of SEQ ID NO: 67, a CDRH3 of SEQ ID NO: 68, a CDRL1 of SEQ ID NO: 69, a CDRL2 of SEQ ID NO: 70 and a CDRL3 of SEQ ID NO: 71. In an embodiment, the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 77, a CDRH2 of SEQ ID NO: 78, a CDRH3 of SEQ ID NO: 79, a CDRL1 of SEQ ID NO: 80, a CDRL2 of SEQ ID NO: 81 and a CDRL3 of SEQ ID NO: 82. In an embodiment, the anti- MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 88, a CDRH2 of SEQ ID NO: 89, a CDRH3 of SEQ ID NO: 90, a CDRL1 of SEQ ID NO: 91, a CDRL2 of SEQ ID NO: 92 and a CDRL3 of SEQ ID NO: 93. In an embodiment, the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 99, a CDRH2 of SEQ ID NO: 100, a CDRH3 of SEQ ID NO: 101, a CDRL1 of SEQ ID NO: 102, a CDRL2 of SEQ ID NO: 103 and a CDRL3 of SEQ ID NO: 104. In an embodiment, the anti- MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 110, a CDRH2 of SEQ ID NO: 111, a CDRH3 of SEQ ID NO: 112, a CDRL1 of SEQ ID NO: 113, a CDRL2 of SEQ ID NO: 114 and a CDRL3 of SEQ ID NO: 115. In an embodiment, the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 121, a CDRH2 of SEQ ID NO: 122, a CDRH3 of SEQ ID NO: 123, a CDRL1 of SEQ ID NO: 124, a CDRL2 of SEQ ID NO: 125 and a CDRL3 of SEQ ID NO: 126. In an embodiment, the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 132, a CDRH2 of SEQ ID NO: 133, a CDRH3 of SEQ ID NO: 134, a CDRL1 of SEQ ID NO: 135, a CDRL2 of SEQ ID NO: 136 and a CDRL3 of SEQ ID NO: 137.
In an embodiment, the anti-MPER bNAb or fragment thereof comprises a VH domain having at least 95% sequence identity to SEQ ID NO: 174 and a VL domain having at least 95% sequence identity to SEQ ID NO: 175, optionally wherein the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 168, a CDRH2 of SEQ ID NO: 169, a CDRH3 of SEQ ID NO: 170, a CDRL1 of SEQ ID NO: 171, a CDRL2 of SEQ ID NO: 172 and a CDRL3 of SEQ ID NO: 173.
In an embodiment, the anti-MPER bNAb or fragment thereof comprises a VH domain having at least 95% sequence identity to SEQ ID NO: 185 and a VL domain having at least 95% sequence identity to SEQ ID NO: 186, optionally wherein the the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 179, a CDRH2 of SEQ ID NO: 180, a CDRH3 of SEQ ID NO: 181, a CDRL1 of SEQ ID NO: 182, a CDRL2 of SEQ ID NO: 183 and a CDRL3 of SEQ ID NO: 184.
In an embodiment, the anti-MPER bNAb or fragment thereof comprises a VH domain having at least 95% sequence identity to SEQ ID NO: 196 and a VL domain having at least 95% sequence identity to SEQ ID NO: 197, optionally wherein the the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 190, a CDRH2 of SEQ ID NO: 191, a CDRH3 of SEQ ID NO: 192, a CDRL1 of SEQ ID NO: 193, a CDRL2 of SEQ ID NO: 194 and a CDRL3 of SEQ ID NO: 195.
In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a VH domain having at least 95% sequence identity to SEQ ID NO: 72 and a VL domain having at least 95% sequence identity to SEQ ID NO: 73, optionally wherein the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 66, a CDRH2 of SEQ ID NO: 67, a CDRH3 of SEQ ID NO: 68, a CDRL1 of SEQ ID NO: 69, a CDRL2 of SEQ ID NO: 70 and a CDRL3 of SEQ ID NO: 71.
In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a VH domain having at least 95% sequence identity to SEQ ID NO: 83 and a VL domain having at least 95% sequence identity to SEQ ID NO: 84, optionally wherein the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 77, a CDRH2 of SEQ ID NO: 78, a CDRH3 of SEQ ID NO: 79, a CDRL1 of SEQ ID NO: 80, a CDRL2 of SEQ ID NO: 81 and a CDRL3 of SEQ ID NO: 82.
In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a VH domain having at least 95% sequence identity to SEQ ID NO: 94 and a VL domain having at least 95% sequence identity to SEQ ID NO: 95, optionally wherein the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 88, a CDRH2 of SEQ ID NO: 89, a CDRH3 of SEQ ID NO: 90, a CDRL1 of SEQ ID NO: 91, a CDRL2 of SEQ ID NO: 92 and a CDRL3 of SEQ ID NO: 93.
In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a VH domain having at least 95% sequence identity to SEQ ID NO: 105 and a VL domain having at least 95% sequence identity to SEQ ID NO: 106, optionally wherein the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 99, a CDRH2 of SEQ ID NO: 100, a CDRH3 of SEQ ID NO: 101, a CDRL1 of SEQ ID NO: 102, a CDRL2 of SEQ ID NO: 103 and a CDRL3 of SEQ ID NO: 104.
In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a VH domain having at least 95% sequence identity to SEQ ID NO: 116 and a VL domain having at least 95% sequence identity to SEQ ID NO: 117, optionally wherein the anti-MPER bNAb or fragment thereof comprises a a CDRH1 of SEQ ID NO: 110, a CDRH2 of SEQ ID NO: 111, a CDRH3 of SEQ ID NO: 112, a CDRL1 of SEQ ID NO: 113, a CDRL2 of SEQ ID NO: 114 and a CDRL3 of SEQ ID NO: 115.
In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a VH domain having at least 95% sequence identity to SEQ ID NO: 116 and a VL domain having at least 95% sequence identity to SEQ ID NO: 117, optionally wherein the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 121, a CDRH2 of SEQ ID NO: 122, a CDRH3 of SEQ ID NO: 123, a CDRL1 of SEQ ID NO: 124, a CDRL2 of SEQ ID NO: 125 and a CDRL3 of SEQ ID NO: 126.
In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a VH domain having at least 95% sequence identity to SEQ ID NO: 127 and a VL domain having at least 95% sequence identity to SEQ ID NO: 128, optionally wherein the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 121, a CDRH2 of SEQ ID NO: 122, a CDRH3 of SEQ ID NO: 123, a CDRL1 of SEQ ID NO: 124, a CDRL2 of SEQ ID NO: 125 and a CDRL3 of SEQ ID NO: 126.
In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a VH domain having at least 95% sequence identity to SEQ ID NO: 138 and a VL domain having at least 95% sequence identity to SEQ ID NO: 139, optionally wherein the anti-MPER bNAb or fragment thereof comprises a CDRH1 of SEQ ID NO: 132, a CDRH2 of SEQ ID NO: 133, a CDRH3 of SEQ ID NO: 134, a CDRL1 of SEQ ID NO: 135, a CDRL2 of SEQ ID NO: 136 and a CDRL3 of SEQ ID NO: 137.
In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a VH domain of SEQ ID NO: 174 and a VL domain of SEQ ID NO: 175. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a VH domain of SEQ ID NO: 185 and a VL domain of SEQ ID NO: 186. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a VH domain of SEQ ID NO: 196 and a VL domain of SEQ ID NO: 197. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a VH domain of SEQ ID NO: 72 and a VL domain of SEQ ID NO: 73. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a VH domain of SEQ ID NO: 83 and a VL domain of SEQ ID NO: 84. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a VH domain of SEQ ID NO: 94 and a VL domain of SEQ ID NO: 95. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a VH domain of SEQ ID NO: 105 and a VL domain of SEQ ID NO: 106. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a VH domain of SEQ ID NO: 116 and a VL domain of SEQ ID NO: 117. In an embodiment, the anti- MPER bNAb or fragment thereof, comprises a VH domain of SEQ ID NO: 127 and a VL domain of SEQ ID NO: 128. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a VH domain of SEQ ID NO: 138 and a VL domain of SEQ ID NO: 139. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a heavy chain (HC) having at least 95% sequence identity to SEQ ID NO: 176 or 177 and a light chain (LC) having at least 95% sequence identity to SEQ ID NO: 178. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a heavy chain (HC) having at least 95% sequence identity to SEQ ID NO: 187 or 188 and a light chain (LC) having at least 95% sequence identity to SEQ ID NO: 189. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a heavy chain (HC) having at least 95% sequence identity to SEQ ID NO: 198 or 199 and a light chain (LC) having at least 95% sequence identity to SEQ ID NO: 200. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a heavy chain (HC) having at least 95% sequence identity to SEQ ID NO: 74 or 75 and a light chain (LC) having at least 95% sequence identity to SEQ ID NO: 76. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a HC having at least 95% sequence identity to SEQ ID NO: 85 or 86 and a LC having at least 95% sequence identity to SEQ ID NO: 87. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a HC having at least 95% sequence identity to SEQ ID NO: 96 or 97 and a LC having at least 95% sequence identity to SEQ ID NO: 98. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a HC having at least 95% sequence identity to SEQ ID NO: 107 or 108 and a LC having at least 95% sequence identity to SEQ ID NO: 109. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a HC having at least 95% sequence identity to SEQ ID NO: 118 or 119 and a LC having at least 95% sequence identity to SEQ ID NO: 120. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a HC having at least 95% sequence identity to SEQ ID NO: 129 or 130 and a LC having at least 95% sequence identity to SEQ ID NO: 131. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a HC having at least 95% sequence identity to SEQ ID NO: 140 or 141 and a LC having at least 95% sequence identity to SEQ ID NO: 142.
In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a heavy chain (HC) of SEQ ID NO: 176 or 177 and a light chain (LC) of SEQ ID NO: 178. In an embodiment, the anti- MPER bNAb or fragment thereof, comprises a heavy chain (HC) of SEQ ID NO: 187 or 188 and a light chain (LC) of SEQ ID NO: 189. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a heavy chain (HC) of SEQ ID NO: 198 or 199 and a light chain (LC) of SEQ ID NO: 200. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a heavy chain (HC) of SEQ ID NO: 74 or 75 and a light chain (LC) of SEQ ID NO: 76. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a HC of SEQ ID NO: 85 or 86 and a LC of SEQ ID NO: 87. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a HC of SEQ ID NO: 96 or 97 and a LC of SEQ ID NO: 98. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a HC of SEQ ID NO: 107 or 108 and a LC of SEQ ID NO: 109. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a HC of SEQ ID NO: 118 or 119 and a LC of SEQ ID NO: 120. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a HC of SEQ ID NO: 129 or 130 and a LC of SEQ ID NO: 131. In an embodiment, the anti-MPER bNAb or fragment thereof, comprises a HC of SEQ ID NO: 140 or 141 and a LC of SEQ ID NO: 142.
Linkers
Examples of suitable linkers include amino acid sequences that are from 1 amino acid to 150 amino acids in length. In particular, from 1 to 140 amino acids, from 1 to 130 amino acids, from 1 to 120 amino acids, from 1 to 110 amino acids, from 1 to 100 amino acids, from 1 to 90 amino acids, from 1 to 80 amino acids, from 1 to 70 amino acids, from 1 to 60 amino acids, from 1 to 50 amino acids, from 1 to 40 amino acids, from 1 to 30 amino acids, from 1 to 20 amino acids, from 1 to 10 amino acids, from 5 to 30 amino acids. In an embodiment, the linker is an amino acid sequence from 5 to 30 amino acids in length.
In an embodiment, the linker is an amino acid sequence as set forth in any one of SEQ ID NOs: 30 to 35. In an embodiment, the linker is an amino acid sequence as set forth in SEQ ID NO: 30. In an embodiment, the linker is a multimer of the amino acid sequence as set forth in SEQ ID NO: 30. In an embodiment, the linker is [SEQ ID NO:30]n, wherein n is an integer from 1 to 6. In an embodiment, the linker is an amino acid sequence as set forth in SEQ ID NO:31. In an embodiment, the linker is an amino acid sequence as set forth in SEQ ID NO:32. In an embodiment, the linker is an amino acid sequence as set forth in SEQ ID NO:33. In an embodiment, the linker is an amino acid sequence as set forth in SEQ ID NO: 34. In an embodiment, the linker is an amino acid sequence as set forth in SEQ ID NO: 35. In another embodiment, the linker is a helical amino acid linker, for example that set forth in SEQ ID NO: 201.
Any of the aforementioned linkers may be incorporated into an antigen binding protein of the invention. In particular, any of the aforementioned linkers may be used to join a domain within the antigen binding protein to another domain within the antigen binding protein. In particular, any of the aforementioned linkers may be used to join a CD4 domain within the antigen binding protein that to a broadly neutralizing antibody or fragment thereof that binds to the MPER region of gp41. Further, any of the aforementioned linkers may be used to join a CD4 domain as disclosed herein to a bNAb as disclosed herein. In an embodiment, the linker is an amino acid sequence as set forth in any one of SEQ ID NOs: 30 to 35. In an embodiment, the linker is an amino acid sequence as set forth in SEQ ID NO: 30. In another embodiment, the linker is a helical amino acid linker, for example that set forth in SEQ ID NO: 201. In an embodiment, a linker is used to join the C-terminus of a CD4 domain to the N- terminus of a bNAb heavy chain variable domain. In an embodiment, a linker is used to join the C- terminus of a CD4 domain to the N-terminus of a bNAb light chain variable domain. In an embodiment, a linker is used to join the C-terminus of a CD4 domain to the N-terminus of a bNAb heavy chain variable domain and a linker is used to join the C-terminus of a CD4 domain to the N- terminus of a bNAb light chain variable domain. In an embodiment, a linker is used to join the C- terminus of a CD4 domain to the N-terminus of a bNAb heavy chain variable domain and an identical linker is used to join the C-terminus of a CD4 domain to the N-terminus of a bNAb light chain variable domain. In an embodiment, the linker is an amino acid sequence as set forth in any one of SEQ ID NOs: 30 to 35. In an embodiment, the linker is an amino acid sequence as set forth in SEQ ID NO: 30.
In an embodiment, a linker is used to join the N-terminus of a CD4 domain to the C- terminus of a bNAb heavy chain. In an embodiment, a linker is used to join the N-terminus of a CD4 domain to the C-terminus of a bNAb heavy chain variable domain. In an embodiment, a linker is used to join the N-terminus of a CD4 domain to the C-terminus of a bNAb light chain. In an embodiment, a linker is used to join the N-terminus of a CD4 domain to the C-terminus of a bNAb light chain variable domain. In an embodiment, a linker is used to join the N-terminus of a CD4 domain to the C-terminus of an Fc domain. In an embodiment, the linker is an amino acid sequence as set forth in any one of SEQ ID NOs: 30 to 35. In an embodiment, the linker is an amino acid sequence as set forth in SEQ ID NO: 30.
Any of the aforementioned linkers may be used to join a VH and VL pair as disclosed herein to form a scFv. In an embodiment, the linker between the VH domain and the VL domain of the scFv is selected from the one of SEQ ID NOs: 30-35. In a particular embodiment, the linker between the VH domain and the VL domain of the scFv is SEQ ID NO: 33.
Any of the aforementioned linkers may be used to join a scFv as disclosed herein to an Fc domain. In an embodiment, the scFv is fused to a human Fc via a linker selected from one of SEQ ID NO: 30-35. In an embodiment, the scFv is fused to a human Fc via a linker of SEQ ID NO: 31.
Bispecific proteins
In an embodiment, the multimeric binding protein of the invention is a bispecific binding protein. Examples of such bispecific binding proteins are embodiments as disclosed in the below Table 5. Table 5: Discloses bispecific proteins under the scope of the invention with reference to the sequences provided in Tables 3 and 4. For example, the cell containing an "X" in the table includes a bispecific binding protein of the invention comprising a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a VH domain having at least 95% sequence identity to SEQ ID NO: 72 and a VL domain having at least 95% sequence identity of SEQ ID NO: 73 (fourth row of Table 4) and CDRs having a sequence according to SEQ ID NOs: 66-71 (fourth row of Table 3).
In an embodiment, the bispecific binding protein comprises a CD4 domain and a broadly neutralizing antibody comprising a pair of variable domains (a VH and a VL) as set out in any row of Table 4.
In an embodiment, the bispecific binding protein comprises a CD4 domain having a sequence of any one of SEQ ID NOs: 1-21 and a broadly neutralizing antibody comprising a pair of variable domains (a VH and a VL) as set out in any row of Table 4.
In an embodiment, the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and a broadly neutralizing antibody comprising a pair of variable domains (a VH and a VL) as set out in any row of Table 4.
In an embodiment, the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a VH domain of SEQ ID NO: 174 and a VL domain of SEQ ID NO: 175.
In an embodiment, the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a VH domain of SEQ ID NO: 185 and a VL domain of SEQ ID NO: 186.
In an embodiment, the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a VH domain of SEQ ID NO: 196 and a VL domain of SEQ ID NO: 197.
In an embodiment, the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a VH domain of SEQ ID NO: 72 and a VL domain of SEQ ID NO: 73.
In an embodiment, the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a VH domain of SEQ ID NO: 83 and a VL domain of SEQ ID NO: 84.
In an embodiment, the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a VH domain of SEQ ID NO: 94 and a VL domain of SEQ ID NO: 95.
In an embodiment, the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a VH domain of SEQ ID NO: 105 and a VL domain of SEQ ID NO: 106. In an embodiment, the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a VH domain of SEQ ID NO: 116 and a VL domain of SEQ ID NO: 117.
In an embodiment, the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a VH domain of SEQ ID NO: 127 and a VL domain of SEQ ID NO: 128.
In an embodiment, the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a VH domain of SEQ ID NO: 138 and a VL domain of SEQ ID NO: 139.
In an embodiment, the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a HC of SEQ ID NO: 176 or 177 and a LC of SEQ ID NO: 178. In some embodiments, the CD4 domain is linked to the N-terminus of the HC. In some embodiments, the CD4 domain is linked to the C-terminus of the HC.
In an embodiment, the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a HC of SEQ ID NO: 187 or 188 and a LC of SEQ ID NO: 189. In some embodiments, the CD4 domain is linked to the N-terminus of the HC. In some embodiments, the CD4 domain is linked to the C-terminus of the HC.
In an embodiment, the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a HC of SEQ ID NO: 198 or 199 and a LC of SEQ ID NO: 200. In some embodiments, the CD4 domain is linked to the N-terminus of the HC. In some embodiments, the CD4 domain is linked to the C-terminus of the HC.
In an embodiment, the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a HC of SEQ ID NO: 74 or 75 and a LC of SEQ ID NO: 76. In some embodiments, the CD4 domain is linked to the N-terminus of the HC. In some embodiments, the CD4 domain is linked to the C-terminus of the HC.
In an embodiment, the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a HC of SEQ ID NO: 85 or 86 and a LC of SEQ ID NO: 87. In some embodiments, the CD4 domain is linked to the N-terminus of the HC. In some embodiments, the CD4 domain is linked to the C-terminus of the HC.
In an embodiment, the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a HC of SEQ ID NO: 96 or 97 and a LC of SEQ ID NO: 98. In some embodiments, the CD4 domain is linked to the N-terminus of the HC. In some embodiments, the CD4 domain is linked to the C-terminus of the HC. In an embodiment, the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a HC of SEQ ID NO: 107 or 108 and a LC of SEQ ID NO: 109. In some embodiments, the CD4 domain is linked to the N-terminus of the HC. In some embodiments, the CD4 domain is linked to the C-terminus of the HC.
In an embodiment, the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a HC of SEQ ID NO: 118 or 119 and a LC of SEQ ID NO: 120. In some embodiments, the CD4 domain is linked to the N-terminus of the HC. In some embodiments, the CD4 domain is linked to the C-terminus of the HC.
In an embodiment, the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a HC of SEQ ID NO: 129 or 130 and a LC of SEQ ID NO: 131. In some embodiments, the CD4 domain is linked to the N-terminus of the HC. In some embodiments, the CD4 domain is linked to the C-terminus of the HC.
In an embodiment, the bispecific binding protein comprises a CD4 domain having a sequence of SEQ ID NO: 11 and an anti-MPER bNAb comprising a HC of SEQ ID NO: 140 or 141 and a LC of SEQ ID NO: 142. In some embodiments, the CD4 domain is linked to the N-terminus of the HC. In some embodiments, the CD4 domain is linked to the C-terminus of the HC.
In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 147-150. In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 147-150 and a sequence according to SEQ ID NO: 189.
In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 151-167. In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 151-167 and a sequence according to SEQ ID NO: 200.
In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 59. In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 59 and a sequence according to SEQ ID NO: 76.
In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 60. In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 60 and a sequence according to SEQ ID NO: 87.
In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 61. In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 61 and a sequence according to SEQ ID NO: 98. In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 62. In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 62 and a sequence according to SEQ ID NO: 109.
In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 63. In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 63 and a sequence according to SEQ ID NO: 120.
In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 64. In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 64 and a sequence according to SEQ ID NO: 131.
In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 65. In an embodiment, the bispecific binding protein comprises a sequence according to SEQ ID NO: 65 and a sequence according to SEQ ID NO: 142.
Production methods
Antigen binding proteins may be prepared by any of a number of conventional techniques. For example, antigen binding proteins may be purified from cells that naturally express them (e.g., an antibody can be purified from a hybridoma that produces it), or produced in recombinant expression systems.
A number of different expression systems and purification regimes can be used to generate the antigen binding proteins of the invention. Generally, host cells are transformed with a recombinant expression vector encoding the desired antigen binding protein. The expression vector may be maintained by the host as a separate genetic element or integrated into the host chromosome depending on the expression system. A wide range of host cells can be employed, including Prokaryotes (including Gram-negative or Gram-positive bacteria, for example Escherichia coii, Bacilli sp., Pseudomonas sp., Corynebacterium sp.), Eukaryotes including yeast (for example Saccharomyces cerevisiae, Pichia pastoris), fungi (for example Aspergiius sp.), or higher Eukaryotes including insect cells and cell lines of mammalian origin (for example, CHO, NSO, PER.C6, HEK293, HeLa).
The host cell may be an isolated host cell. The host cell is usually not part of a multicellular organism (e.g., plant or animal). The host cell may be a non-human host cell. The host cell may be a microorganism cell.
Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian host cells are known in the art. The cells can be cultured under conditions that promote expression of the antigen binding protein using a variety of equipment such as shake flasks, spinner flasks, and bioreactors. The polypeptide(s) is(are) recovered by conventional protein purification procedures. Protein purification procedures typically consist of a series of unit operations comprised of various filtration and chromatographic processes developed to selectively concentrate and isolate the antigen binding protein. The purified antigen binding protein may be formulated in a pharmaceutically acceptable composition.
Fc modifications
Fc engineering methods can be applied to modify the functional or pharmacokinetics properties of an antigen binding protein, in particular an antibody. Effector function may be altered by making mutations in the Fc region that increase or decrease binding to Clq or Fey receptors and modify CDC or ADCC activity respectively. Modifications to the glycosylation pattern of an antibody can also be made to change the effector function.
The interaction between the Fc region of an antigen binding protein or antibody and various Fc receptors (FcR), including FcyRI (CD64), FcyRII (CD32), FcyRIII (CD16), FcRn, Clq, and type II Fc receptors is believed to mediate the effector functions of the antigen binding protein or antibody. Significant biological effects can be a consequence of effector functionality. Usually, the ability to mediate effector function requires binding of the antigen binding protein or antibody to an antigen and not all antigen binding proteins or antibodies will mediate every effector function.
Effector function can be assessed in a number of ways including, for example, evaluating ADCC effector function of antibody coated to target cells mediated by Natural Killer (NK) cells via FcyRIII, or monocytes/macrophages via FcyRI, or evaluating CDC effector function of antibody coated to target cells mediated by complement cascade via Clq. For example, an antigen binding protein of the present invention can be assessed for ADCC effector function in a Natural Killer cell assay. Examples of such assays can be found in Shields etal, 2001, The Journal of Biological Chemistry, Vol. 276, p. 6591-6604; Chappel etal, 1993, The Journal of Biological Chemistry, Vol 268, p. 25124-25131; Lazar et al, 2006, PNAS, 103; 4005-4010.
Examples of assays to determine CDC function include those described in J Imm Meth, 1995, 184: 29-38.
The effects of mutations on effector functions e.g., FcRn binding, FcyRs and Clq binding, CDC, ADCML, ADCC, ADCP) can be assessed, e.g., as described in Grevys et al., J Immunol. 2015 Jun 1; 194(11): 5497-5508, or Tam etal., Antibodies 2017, 6(3); Monnet et al., 2014 mAbs, 6:2, 422-436. Throughout this specification, amino acid residues in Fc regions, in antibody sequences or full-length antigen binding protein sequences, are numbered according to the EU index numbering convention.
The long half-life of IgG antibodies is reported to be dependent on their binding to FcRn. Therefore, substitutions that increase the binding affinity of IgG to FcRn at pH 6.0 while maintaining the pH dependence of the interaction with target, by engineering the constant region, have been extensively studied (Ghetie etai., Nature Biotech. 15: 637-640, 1997; Hinton etai., JBC 279: 6213- 6216, 2004; Dall'Acqua eta!., 10 J Immunol 117: 1129-1138, 2006). The in-vivo half-life of antigen binding proteins of the present invention may be altered by modification of a heavy chain constant domain or an FcRn binding domain therein.
In adult mammals, FcRn, plays a key role in maintaining serum antibody levels by acting as a protective receptor that binds and salvages antibodies of the IgG isotype from degradation. IgG molecules are endocytosed by endothelial cells and, if they bind to FcRn, are recycled out of the cells back into circulation. In contrast, IgG molecules that enter the cells and do not bind to FcRn and are targeted to the lysosomal pathway where they are degraded.
FcRn is believed to be involved in both antibody clearance and the transcytosis across tissues (see Junghans R.P (1997) Immunol. Res 16. 29-57 and Ghetie et a/ (2000) Annu. Rev. Immunol. 18, 739-766). Human IgGl residues determined to interact directly with human FcRn include Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 and His435. Mutations at any of these positions may enable increased serum half-life and/or altered effector properties of antigen binding proteins of the invention.
Antigen binding proteins of the present invention may have amino acid modifications that increase the affinity of the constant domain or fragment thereof for FcRn. Increasing the half-life (Ze., serum half-life) of therapeutic and diagnostic IgG antibodies and other bioactive molecules has many benefits including reducing the amount and/or frequency of dosing of these molecules. In one embodiment, an antigen binding protein of the invention comprises all or a portion (an FcRn binding portion) of an IgG constant domain having one or more of the following amino acid modifications.
For example, with reference to IgGl, M252Y/S254T/T256E (commonly referred to as "YTE" mutations) and M428L/N434S (commonly referred to as "LS" mutations) increase FcRn binding at pH 6.0 (Wang etai. 2018). In an embodiment, an antigen binding protein of the invention comprises an Fc domain with the LS mutations. In an embodiment, an antigen binding protein of the invention comprises a bNAb in which the LS mutations are present in both of the heavy chain Fc domains. Half-life and FcRn binding can also be extended by introducing H433K and N434F mutations (commonly referred to as "HN" or "NHance" mutations) (with reference to IgGl) (W02006/130834).
Additionally, various publications describe methods for obtaining physiologically active molecules with modified half-lives, either by introducing an FcRn-binding polypeptide into the molecules (WO97/43316, US5869046, US5747035, WO96/32478 and WO91/14438) or by fusing the molecules with antibodies whose FcRn-binding affinities are preserved, but affinities for other Fc receptors have been greatly reduced (WO99/43713), or fusing with FcRn binding domains of antibodies (WO00/09560, US4703039).
Post-translational modifications
The skilled person will appreciate that, upon production of an antigen binding protein, such as a bispecific molecule of the invention in a host cell, post-translational modifications may occur. For example, this may include the cleavage of certain leader sequences, the addition of various sugar moieties in various glycosylation patterns, non-enzymatic glycation, deamidation, oxidation, disulfide bond scrambling and other cysteine variants such as free sulfhydryls, racemized disulfides, thioethers and trisulfide bonds, isomerisation, C-terminal lysine clipping, and N-terminal glutamine cyclisation. The present invention encompasses the use of antigen binding proteins that have been subjected to, or have undergone, one or more post-translational modifications. Thus an antigen binding protein of the invention includes an "antigen binding protein" as defined earlier that has undergone a post-translational modification such as described herein.
Glycation is a post-translational non-enzymatic chemical reaction between a reducing sugar, such as glucose, and a free amine group in the protein, and is typically observed at the epsilon amine of lysine side chains or at the N-Terminus of the protein. Glycation can occur during production and storage only in the presence of reducing sugars.
Deamidation can occur during production and storage, is an enzymatic reaction primarily converting asparagine (N) to iso-aspartic acid (iso-aspartate) and aspartic acid (aspartate) (D) at approximately 3:1 ratio. This deamidation reaction is therefore related to isomerization of aspartate (D) to iso-aspartate. The deamidation of asparagine and the isomerisation of aspartate, both involve the intermediate succinimide. To a much lesser degree, deamidation can occur with glutamine residues in a similar manner. Deamidation can occur in a CDR, in a Fab (non-CDR region), or in the Fc region.
Oxidation can occur during production and storage (Ze., in the presence of oxidizing conditions) and results in a covalent modification of a protein, induced either directly by reactive oxygen species or indirectly by reaction with secondary by-products of oxidative stress. Oxidation happens primarily with methionine residues, but may occur at tryptophan and free cysteine residues. Oxidation can occur in a CDR, in a Fab (non-CDR) region, or in the Fc region.
Disulfide bond scrambling can occur during production and basic storage conditions. Under certain circumstances, disulfide bonds can break or form incorrectly, resulting in unpaired cysteine residues (-SH). These free (unpaired) sulfhydryls (-SH) can promote shuffling.
The formation of a thioether and racemization of a disulphide bond can occur under basic conditions, in production or storage, through a beta elimination of disulphide bridges back to cysteine residues via a dehydroalanine and persulfide intermediate. Subsequent crosslinking of dehydroalanine and cysteine results in the formation of a thioether bond or the free cysteine residues can reform a disulphide bond with a mixture of D- and L-cysteine.
Trisulfides result from insertion of a sulfur atom into a disulphide bond (Cys-S-S-S-Cys) and are formed due to the presence of hydrogen sulphide in production cell culture.
N-terminal glutamine (Q) and glutamate (glutamic acid) (E) in the heavy chain and/or light chain is likely to form pyroglutamate (pGlu) via cyclization. Most pGlu formation happens in the production bioreactor, but it can be formed non-enzymatically, depending on pH and temperature of processing and storage conditions. Cyclization of N-terminal Q or E is commonly observed in natural human antibodies.
C-terminal lysine clipping is an enzymatic reaction catalyzed by carboxypeptidases, and is commonly observed in recombinant and natural human antibodies. Variants of this process include removal of lysine from one or both heavy chains due to cellular enzymes from the recombinant host cell. Upon administration to the human subject/patient is likely to result in the removal of any remaining C-terminal lysines.
Pharmaceutical compositions
Antigen binding proteins as described herein may be incorporated into pharmaceutical compositions for use in the treatment or prevention of HIV infection. In one embodiment, the pharmaceutical composition comprises an antigen binding protein in combination with one or more pharmaceutically acceptable carriers and/or excipients.
Such compositions comprise a pharmaceutically acceptable carrier as known and called for by acceptable pharmaceutical practice.
Pharmaceutical compositions may be administered by injection or continuous infusion (examples include, but are not limited to, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intraocular, and intraportal). In one embodiment, the composition is suitable for intravenous administration. In one embodiment, the composition is suitable for subcutaneous administration. Pharmaceutical compositions may be suitable for topical administration (which includes, but is not limited to, epicutaneous, inhaled, intranasal or ocular administration) or enteral administration (which includes, but is not limited to, oral, vaginal, or rectal administration).
The pharmaceutical composition may be included in a kit containing the antigen binding protein together with other medicaments, for example dolutegravir or cabotegravir, and/or with instructions for use. For convenience, the kit may comprise the reagents in predetermined amounts with instructions for use. The kit may also include devices used for administration of the pharmaceutical composition.
The terms "individual", "subject" and "patient" are used herein interchangeably. In one embodiment the subject is a human.
The antigen binding proteins described herein may be used in methods of treatment or prevention of HIV infection and AIDs. The antigen binding proteins described herein may be used in the manufacture of medicaments for the treatment or prevention of HIV infection and AIDs. The antigen binding proteins described may be used in an effective amount for therapeutic, prophylactic or preventative treatment. A therapeutically effective amount of the antigen binding protein described herein is an amount effective to ameliorate or reduce one or more symptoms of HIV infection. A prophylactically effective amount of the antigen binding protein described herein is an amount effective to prevent one or more symptoms of HIV infection.
Combinations
Antigen binding proteins of the present invention may be employed alone or in combination with other therapeutic agents, or a prodrug thereof. Combination therapies according to the present invention thus comprise the administration of an antigen binding protein and the administration of at least one other agent which may be useful in the treatment or prevention of HIV infection and/or AIDS. An antigen binding protein of the present invention and the other therapeutic agent may be formulated and administered together in a single pharmaceutical composition or may be formulated and administered separately. When formulated and administered separately, administration may occur simultaneously or sequentially in any order.
Antigen binding proteins as described herein may be combined with, for example, one or more of an antiretroviral agent, an anti-infective agent, an immunomodulator, and other HIV entry inhibitors.
Antiretroviral agents include Nucleoside Reverse Transcriptase Inhibitors (NRTIs), Non- Nucleoside Reverse Transcriptase Inhibitors (NNRTIs), Nucleoside Reverse Transcriptase Translocation Inhibitors (NRTTIs), Protease Inhibitors (Pls), Entry Inhibitors (El), Integrase Strand Transfer Inhibitors (INSTI), Maturation Inhibitors (Mis), and Capsid Inhibitors (Cis).
NRTIs may include, but are not limited to: abacavir, adefovir, adefovir dipivoxil, alovudine, amdoxovir, apricitabine, calanolide A, censavudine, didanosine, elvucitabine, emtricitabine, fozivudine, lamivudine, racivir, stampidine, stavudine, tenofovir disoproxil fumerate, tenofovir alafenamide, todoxil, zalcitabine, and zidovudine.
NNRTIs may include, but are not limited to, HBY 097 (Hoechst/Bayer), capravirine, delaviridine, doravirine, efavirenz, etravirine, immunocal, lersivirine, loviride, nevirapine, oltipraz, and rilpivirine.
NRTTIs include, but are not limited to, islatravir.
Pls may include, but are not limited to, amprenavir, atazanavir, brecanavir, cobicistat, darunavir, fosamprenavir, indinavir, lasinavir, lopinavir, palinavir, nelfinavir, ritonavir, saquinavir, and tipranavir.
Els are discussed in DRUGS OF THE FUTURE 1999, 24(12), 1355-1362; CELL, Vol. 9, 243- 246, Oct. 29, 1999; and DRUG DISCOVERY TODAY, Vol. 5, No. 5, May 2000, pp. 183-194; and Meanwell eta/., Current Opinion in Drug Discovery 8i Development (2003), 6(4), 451-461. In particular, the antigen binding proteins of the invention can be utilized in combination with attachment inhibitors, fusion inhibitors, and chemokine receptor antagonists aimed at either the CCR5 or CXCR4 coreceptor. HIV attachment inhibitors are also set forth in US 7,354,924 and US 7,745,625. Els may include, but are not limited to, cenicriviroc, enfuvirtide, fostemsavir, ibalizumab, leronlimab, maraviroc, vicriviroc and VIR-576.
INSTIs may include, but are not limited to, bictegravir, cabotegravir, dolutegravir, elvitegravir, and raltegravir, . In an embodiment, the INSTI is dolutegravir or cabotegravir. In an embodiment, the INSTI is cabotegravir.
Maturation inhibitors may include, but are not limited to, bevirimat, BMS-955176, GSK3640254, GSK3739937, PA-344 and PA-457. It will be understood that GSK3640254 is a compound as described in Dicker I, Jeffrey JL, Protack T, eta/., Antimicrob Agents Chemother. 2022;66(l). GSK3739937, also known as VH3739937, is the compound of clinical trial NCT04493684.
Capsid inhibitors may include, but are not limited to, GSK4004280, GSK4011499, and lencapavir.
Anti -infective agents include, but are not limited to, clindamycin with primaquine, daunorubicin, fluconazole, intraconazole, nystatin pastille, ornidyl eflornithine, megestrol acetate, pentamidine isethionate, piritrexim, trimethoprim, trimetrexate, recombinant human erythropoietin, recombinant human growth hormone, spiramycin, testosterone and total enteral nutrition,
Immunomodulators include, but are not limited to, acemannan, alpha-2-interferon, AS-101, bropirimine, CL246,738, FP-21399, gamma interferon, granulocyte macrophage colony stimulating factor, HIV core particle immunostimulant, interleukin-2, immune globulin, IMREG-1, IMREG-2, imuthiol diethyl dithio carbamate, methionine enkephalin, MTP-PE muramyl tripeptide, remune, recombinant soluble human CD4, rCD4-IgG hybrids, SK&F106528, thymopentin, and tumour necrosis factor (TNF).
The antigen binding proteins of the present invention may also be used in combination with agents that induce HIV expression, such as latency reversing agents. Several latency reversing agents include, but are not limited to, the following: histone deacetylase inhibitors e.g, vorinostat, panobinostat, romidepin), histone crotonyl transferase inhibitors (sodium corotonate), protein kinase C agonists e.g., bryostatin, ingenol B), disulfiram, TLR7 agonists e.g., GS-9620), and bromodomain inhibitors (e.g., JQ1, iBET151).
The antigen binding proteins of the present invention may also be used in combination with other agents that induce HIV expression, such as agents for clearance therapy. Several examples of agents for clearance therapy, or of immunological combinations for clearance, include, but are not limited to, the following: bNAbs, CD4-Ig, eCD4-Ig, and dual-affinity re-targeting (DART) proteins.
Antigen binding proteins of the invention may be used in combination with broadly neutralizing HIV-1 antibodies, including 1NC9, 1B2530, 2F5, 2G12, 3NBC60, 3BNC117, 4E10, 8ANC131, 8ANC134, 10-1074, 10-1074LS, 10E8, 12A12, 12A21, bl2, CAP206-CH12, CH01-04, CH103-106, elipovimab (formerly known as GS-9722), HJ16, M66.6, N6LS (also known as VRC- HIVMAB091-00-AB and the compound of clinical trial NCT03538626), NIH45-46, PG9, PG16, PGT121-123, PGT125-131, PCT135-137, PGT141-145, PGT121.414.LS, PGT151 2G12, QA013.2, VRC01-03, VRC-PG04, VRC-PG04b, VRC-CH30-34.
Other agents that may be combined with antigen binding proteins of the invention include BIT225, GSK4000422/VH4000422, and S-648414 (the compound of clinical trial NCT04147715).
The scope of combinations of compounds of this invention with HIV agents is not limited to those mentioned above, but includes in principle any combination with any pharmaceutical composition useful for the treatment and/or prevention of HIV infection and/or AIDS.
Nucleic acid
In some embodiments, a subject is administered DNA or RNA encoding an multimeric antigen binding protein of the invention to provide in vivo antibody production, for example using the cellular machinery of the subject. Administration of nucleic acid constructs is known in the art and taught, for example, in U.S. Patent No. 5,643,578, U.S. Patent. No. 5,593,972 and U.S. Patent No. 5,817,637. U.S. Patent. No. 5,880,103 describes several methods of delivery of nucleic acids encoding proteins to an organism. One approach to administration of nucleic acids is direct administration with plasmid DNA. such as with a mammalian expression plasmid. The nucleotide sequence encoding the disclosed antigen binding protein can be placed under the control of a promoter to increase expression. The methods include liposomal delivery of the nucleic acids. Such methods can be applied to the production of an antigen binding protein of the invention. In some embodiments, a multimeric antigen binding protein of the invention is expressed in a subject using the pVRC8400 vector (described in Barouch er u/., J. Virol., 79(14), 8828-8834, 2005).
In some embodiments, a subject (such as a human subject at risk of HIV infection) can be administered an effective amount of an adeno-associated virus (AAV) viral vector that includes one or more nucleic acid molecules encoding an multimeric antigen binding protein of the invention. The AAV viral vector is designed for expression of the nucleic acid molecules encoding a disclosed antigen binding protein, and administration of an effective amount of the AAV viral vector to the subject leads to expression of an effective amount of the antigen binding protein in the subject. Non-limiting examples of AAV viral vectors that can be used to express a disclosed antigen binding protein in a subject include those provided in Johnson et al., Nut. Med., 15(8):901-906, 2009 and Gardner et al., Nature, 519(7541):87-91, 2015.
The invention is illustrated by the following clauses:
1. A multimeric anti-HIV envelope spike complex -binding protein comprising: i. a broadly neutralizing anti-MPER antibody, comprising at least one heavy chain or light chain, wherein the antibody binds to the MPER domain of a gp41 protein; and ii. at least one CD4 domain which binds to gpl20, wherein the CD4 domain is attached directly or by a linker to the N-terminus or C-terminus of one of the heavy chains or light chains of the broadly neutralizing antibody.
2. The binding protein of any preceding clause, wherein the CD4 domain is attached via a linker to the N-terminus or C-terminus of at least one heavy or light chain of the broadly neutralizing antibody. 3. The binding protein of any preceding clause, wherein the CD4 domain is attached via a linker to the N-terminus at least one heavy chain or light chain of the broadly neutralizing antibody.
4. The binding protein of any preceding clause, wherein the CD4 domain is attached via a linker to the N-terminus at least one heavy chain of the broadly neutralizing antibody.
5. The binding protein of any preceding clause, wherein the CD4 domain is attached via a linker to the N-terminus or C-terminus of at least one heavy or light chain of the broadly neutralizing antibody.
6. The binding protein of any preceding clause, wherein the binding protein comprises at least four CD4 domains.
7. The binding protein of clause 6, wherein broadly neutralizing antibody has two heavy chains and two light chains; and the C-terminus of a first CD4 domain is attached by a linker to the N-terminus of a first heavy chain, a second CD4 domain is attached by a linker to the N- terminus of a second heavy chain, a third CD4 domain is attached by a linker to the N- terminus of a first light chain, an a fourth CD4 domain is attached by a linker to the N- terminus of a second light chain.
8. A multimeric anti-HIV envelope spike complex-binding protein comprising: i. A broadly neutralizing anti-MPER antibody or an antigen-binding Fab' or F(abQ2 fragment thereof comprising at least one heavy chain variable region or light chain variable region, wherein said antibody or fragment binds to the MPER region of gp41; and ii. at least one CD4 domain, wherein the C-terminus of the CD4 domain is attached directly or by a linker to the N-terminus of the heavy chain variable region or light chain variable region.
9. The binding protein of clause 8, wherein the fragment is an antigen-binding Fab' fragment, and wherein the CD4 domain is attached via a linker to the heavy chain variable region of the Fab' fragment. 10. The binding protein of clause 8, wherein the fragment is an antigen-binding Fab' fragment, and wherein the CD4 domain is attached via a linker to the light chain variable region of the Fab' fragment.
11. The binding protein of clause 8, comprising at least two CD4 domains, wherein the fragment is an antigen-binding Fab' fragment, wherein the first CD4 domain is attached via a linker to the heavy chain variable region of the Fab' fragment and the second CD4 domain is attached via a linker to the light chain variable region of the Fab' fragment.
12. The binding protein of clause 8, comprising at least two CD4 domains, wherein the fragment is an antigen-binding F(abQ2 fragment, and wherein each of the CD4 domains is attached via a linker to each of the heavy chain variable regions of the F(abQ2 fragment.
13. The binding protein of clause 8, comprising at least two CD4 domains, wherein the fragment is an antigen-binding F(abQ2 fragment, and wherein each of the CD4 domains is attached via a linker to each of the light chain variable regions of the F(abQ2 fragment.
14. The binding protein of clause 8, comprising at least four CD4 domains, wherein the fragment is an antigen-binding F(abQ2 fragment, and wherein each of the CD4 domains is attached via a linker to each of the heavy chain variable regions and each of the light chain variable regions of the F(abQ2 fragment.
15. The binding protein of clauses 8 to 14, wherein the C-terminus of the CD4 domains are each attached via a linker to the N-terminus of the heavy chain variable region or light chain variable region of the Fab or F(abQ2 fragment.
16. The binding protein of any preceding clause, wherein the binding protein is a bispecific binding protein.
17. The binding protein of any preceding clause, wherein the linker is a peptide linker.
18. The binding protein of any clause 17, wherein the peptide linker is between 5 and 30 amino acids in length.
19. The binding protein of any preceding clause, wherein the linker is a multimeter of the amino sequence as set forth in SEQ ID NO: 30. 20. The binding protein of any preceding clause, wherein the linker is selected from SEQ ID NOs: 30 to 35
21. The binding protein of any preceding clause, wherein the CD4 domain is a human CD4 DI domain or CD4 D1D2 domain.
22. The binding protein of any preceding clause, wherein the CD4 domain comprises at least one stabilizing mutation compared to a wild type CD4 domain.
23. The binding protein of any preceding clause, wherein the CD4 domain comprises at least one mutation selected from: L5Y, S23N, A55V, I79P, L96V and/or F98V.
24. The binding protein of any preceding clause, wherein the CD4 domain comprises L5Y, S23N, A55V, I79P, L96V and F98V mutations; and at least one further mutations selected from: E91Q, E91H, E87G, N52W, K8V, K8I, K8C, G99C, T11C, K72C, E13C, I70C, H27C, G38C, K21C, G65C, Q25E, H27D, R58V, R58N, R58Y, and/or L61M.
25. The binding protein of any preceding clause, wherein the CD4 domain comprises or consists of any one of SEQ ID NO: 4 - 21.
26. The binding protein of any preceding clause, wherein the CD4 domain comprises or consists of any one of SEQ ID NO: 5 - 21.
27. The binding protein of any preceding clause, wherein the CD4 domain comprises or consists of any one of SEQ ID NO: 5 - 15.
28. The binding protein of any preceding clause, wherein the CD4 domain comprises or consists of SEQ ID NO: 11.
29. The binding protein of any preceding clause, wherein the CD4 domain has a Tm above about 70°C.
30. The binding protein of any preceding clause, wherein the CD4 domain has a Tm between about 70°C and 95°C. 31. The binding protein of any preceding clause, wherein the CD4 domain has a Tm of about 90°C.
32. The binding protein of any preceding clause, wherein the broadly neutralizing antibody comprises a mutation that increases the half-life of the binding protein compared to the same binding protein without said mutation.
33. The binding protein of clause 32, wherein the binding protein comprises an Fc domain and the half-life increasing mutation is a mutation in said Fc domain.
34. The binding protein of clause 33, wherein the Fc domain comprises at least one of the following sets of mutations (EU numbering):
M428L and N434S (LS);
L309D, Q311H and N434S (DHS);
M252Y, S254T and T256E (YTE); and
H433K and N434F (HN).
35. The binding protein of clause 33, wherein the Fc domain comprises M428L and N434S (LS) mutations (EU numbering).
36. The binding protein of any preceding clause, wherein the broadly neutralizing antibody comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDL3 as set out in any one row of Table 3.
37. The binding protein of any preceding clause, wherein the broadly neutralizing antibody comprises a heavy chain variable domain and a light chain variable domain pair that is at least 95% identical to any of the variable domain pairs set out in any one row of Table 4.
38. The binding protein of any preceding clause, wherein the broadly neutralizing antibody comprises a heavy chain and a light chain pair that is at least 95% identical to any of the chain pairs set out in any one row of Table 4.
39. The binding protein of any preceding clause, wherein the broadly neutralizing antibody comprises a heavy chain variable domain and a light chain variable domain pair that is at least 95% identical to: i. SEQ ID NOs: 174 and 175, or ii. SEQ ID NOs: 185 and 186, or iii. SEQ ID NOs: 196 and 197, or iv. SEQ ID NOs: 72 and 73, or
V. SEQ ID NOs: 83 and 84. The binding protein of any preceding clause, wherein the broadly neutralizing antibody comprises a heavy chain and a light chain pair that is at least 95% identical to: i. SEQ ID NOs: 176 or 177, and 178, or ii. SEQ ID NOs: 187 or 188, and 189, or iii. SEQ ID NOs: 198 or 199, and 200, or iv. SEQ ID NOs: 74 or 75, and 76, or
V. SEQ ID NOs: 85 or 86, and 87. The binding protein of any preceding clause, wherein the broadly neutralizing antibody comprises a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3 according to: i. SEQ ID NOs: 168 - 173, or ii. SEQ ID NOs: 179 - 184, or iii. SEQ ID NOs: 190 - 195, or iv. SEQ ID NOs: 66 - 71, or v. SEQ ID NOs: 77 - 82. The binding protein of any preceding clause, wherein the broadly neutralizing antibody comprises a heavy chain variable domain and a light chain variable domain pair according to: i. SEQ ID NOs: 174 and 175, or ii. SEQ ID NOs: 185 and 186, or iii. SEQ ID NOs: 196 and 197, or iv. SEQ ID NOs: 72 and 73, or
V. SEQ ID NOs: 83 and 84. The binding protein of any preceding clause, wherein the broadly neutralizing antibody comprises a heavy chain and a light chain pair according to: i. SEQ ID NOs: 176 or 177, and 178, or ii. SEQ ID NOs: 187 or 188, and 189, or iii. SEQ ID NOs: 198 or 199, and 200, or iv. SEQ ID NOs: 74 or 75, and 76, or
V. SEQ ID NOs: 85 or 86, and 87.
44. The binding protein of any preceding clause, wherein the binding protein can reduce the IC50 (mean/specific envelope) in a PSV assay compared to the IC50 reduction caused by the broadly neutralizing antibody and CD4 domain alone or in combination.
45. The binding protein of any preceding clause, wherein the binding protein is linked to an effector molecule or detectable marker, optionally wherein the detectable marker is a fluorescent, enzymatic or radioactive marker.
46. A method of detecting an HIV infection in a human subject comprising contacting a biological sample from the human subject with the binding protein according to any one of clauses 1 to 45 under conditions sufficient to form an immune complex; and detecting the presence of the immune complex in the sample, wherein the presence of the immune complex in the sample indicates that the subject has a HIV infection.
47. A pharmaceutical composition comprising the binding protein as defined in any one of the preceding clauses and a pharmaceutically acceptable excipient.
48. A method of treating or preventing an HIV infection in a human comprising administering to the human an anti-HIV binding protein according to any one of clauses 1 to 45, or a pharmaceutical composition according to clause 47.
49. An anti-HIV binding protein according to any one of clauses 1 to 45, or a pharmaceutical composition according to clause 47, for use in treating or preventing an HIV infection in a human.
50. Use of an anti-HIV binding protein according to any one of clauses 1 to 45, or a pharmaceutical composition according to clause 47, in the manufacture of a medicament for treating or preventing an HIV infection in a human. 51. The method of clause 48, the protein or composition for use according to clause 49, or the use according to clause 50, whereby viral load in the human is decreased.
52. A kit comprising in separate containers: an anti-HIV binding protein according to any one of clauses 1 to 45 and at least one anti-viral drug that inhibits cellular entry, replication, or transcription of HIV in a human.
53. The kit of clause 52, wherein the antiviral drug is selected from: Nucleoside Reverse Transcriptase Inhibitors (NRTIs), Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs), Protease Inhibitors (Pls), Entry Inhibitors, Integrase Strand Transfer Inhibitors (INSTI), Maturation Inhibitors (Mis), Capsid Inhibitors (Cis) and Nucleoside Reverse Transcriptase Translocation Inhibitors (NRTTIs).
54. The kit of clause 53, wherein the antiviral drug is an INSTI.
55. The kit of clause 54, wherein the INSTI is dolutegravir or cabotegravir.
56. A nucleic acid sequence that encodes an anti-HIV binding protein according to any one of clauses 1 to 45.
57. An expression vector that comprises the nucleic acid sequence of clause 56.
58. A recombinant host cell that comprises the nucleic acid sequence of clause 56 or the expression vector of clause 57.
59. A method of producing an anti-HIV binding protein, comprising culturing the host cell as defined in clause 58 under conditions suitable for expression of said nucleic acid sequence or vector, whereby an anti-HIV binding protein is produced.
60. The anti-HIV binding protein produced by the method of clause 59.
EXAMPLES
Example 1 - Antigen Binding Protein Production
Plasmids encoding the antigen binding proteins of the invention were expressed in EXPI293 or FREESTYLE 293-F cells using the manufacturer's standard protocol (ThermoFisher Scientific, Waltham, MA). The expressed medium was harvested by centrifugation (4000 rpm for 10 min) and the antigen binding proteins were purified by filtration through a 0.22 pm filter (Millipore Sigma, Burlington, MA) and fast protein liquid chromatography (FPLC) (AKTATM Pure, Cytiva, Marlborough MA). The medium was then passed through a Mabselect SuRe column (Cytiva, Marlborough MA) to capture the antigen binding proteins and the column was washed sequentially with phosphate- buffered saline (PBS) before elution.
The antigen binding proteins were then exchanged into a final buffer by using dialysis, a desalting column and preparative size exclusion column (SEC). The purity of the antigen binding proteins was evaluated by using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS- PAGE) and on a size exclusion column on a high-performance liquid chromatography system (SEC- HPLC).
Antigen binding protein concentrations were determined by measuring absorbance at 280nm wavelength (A280) on a NanoDrop machine (ThermoFisher Scientific, Waltham, MA), and their molecular mass was measured by using liquid chromatography - mass spectrometry (LC-MS) to confirm their identity.
The endotoxin level in the final purified products was measured on an ENDOSAFE system (Charles River Labs, Wilmington MA) to make sure it was sufficiently low (usually < 1 EU (Endotoxin Unit) / mg of protein) for downstream anti-viral studies.
Example 2 - Anti-Viral Activity Using Pseudotyped Virus Assay
The anti-viral activity of the antigen binding proteins was measured in a pseudotyped virus (PSV) assay. Pseudotyped HIV-1 virus (PSV) contains deletions in the genome that make it unable to produce infectious virions, but it can be used to measure the activity of cell entry inhibitors (i.e., molecules that prevent the binding of HIV-1 virions to the target cell membrane and/or prevent entry of HIV-1 into target cells), which include the antigen binding proteins of the invention.
PSV was produced in HEK-293T cells (ATCC, Manassas VA) by co-transfecting expression plasmids encoding the HIV-1 gpl60 envelope gene and an HIV-1 backbone plasmid using TRANSIT- 2020 transfection reagent (Mirus Bio, Madison WI). A panel of HIV-1 PSVs expressing different gpl60 envelope trimers was generated to evaluate the effectiveness of the antigen binding proteins of the invention against a wide spectrum of HIV-1 strains.
Meanwhile, to facilitate mechanistic studies of the antigen binding proteins, the complex N- glycans on selected HIV-1 envelopes were removed by adding Kifunensine during PSV production. Kifunensine inhibits mannosidase I, prevents processing of high mannose N-glycans into complex N- glycans in the cells and consequently produces high mannose glycoproteins. In brief, after 6 hours of transfection, Kifunensine was added to a final concentration of 100 pM, the cells were incubated for another 48 hours, and the supernatant which contains the PSV was harvested and aliquoted. i. ACTOne cells
The genome of PSV used in this assay contains a luciferase gene that is expressed once the virus enters cells. Accordingly, the luminescence signal (after adding a substrate of luciferase) can be used to determine the level of viral infection.
The 50% tissue culture infectious dose (TCID) of a single thawed aliquot of each batch of PSV was determined in ACTOne cells. The ACTOne cell-line used in this assay was derived in-house from a genetically engineered 293T cell clone that expresses CD4, CXCR4, and CCR5. Cells were maintained in growth medium composed of Dulbecco's modified Eagle's medium (DMEM, Life Technologies) at 37 °C in a humidified 5% COz-95% air environment. Cell monolayers were split by treatment with Trypsin-EDTA (0.05%).
To run the anti-viral assay, ACTOne cells were detached by treating the cell culture flask with trypsin (trypsin ization) and resuspended in growth medium containing 2% of DMSO to a density of 2.5 x 105 cells/ml. One hundred pl of such cells was added to 10 pl of antigen binding protein pre-loaded in a 96-well plate. Ninety pl of PSV was then added to each well. The assay plates were incubated at 37°C in a humidified incubator at 5% CO2 level. Plates were developed after 72 hours of incubation by adding 50 pl of BRIGHTGLO luciferase reagent (Promega, Madison WI) to each well, and transferring the plates to an ENVISION multilabel plate reader (PerkinElmer, Waltham MA) to measure the luminescence and determine the level of virus that had infected the cells. The higher the luminescence signal, the higher the level of infection.
Raw data were analyzed using an in-house template in an IDBS system to calculate half- maximal inhibitory concentration (IC50) values which reflects the activity of the antigen binding proteins of the invention at inhibiting viral entry (the smaller the number is, the more active the molecule is). Maximal percent inhibition values were calculated as MPI = 100 - (mean signals at the 2 highest concentrations of compound/mean signal of 2 no-drug control wells) x 100 (PMID: 34780263). ii. TMZ. bl cells
Alternatively, the PSV assay was carried out using a luciferase-based assay in a TZM.bl cell line. The TZM-bl cell line is derived from a HeLa cell clone that was engineered to express CD4, CCR5 and CXCR4 and to contain integrated reporter genes for firefly luciferase and E. coli 0-galactosidase under the control of an HIV-1 long terminal repeat (Wei et al., Antimicrobial agents and chemotherapy 46:1896-905(2002)) permitting sensitive and accurate measurements of infection.
The detailed materials and methodology have been described elsewhere (Mentefiori, Curr. Protoc. Immunol., 2005, Chapter 12; Seaman et al., Journal of Virology, Feb. 2010, 84(3), p. 1439- 1452). In brief, the assay measures the reduction in luciferase reporter gene expression in TZM.bl cells following a single round of virus infection.
Five-fold serial dilutions of the antigen binding proteins of the invention, from 50 pg/ml to 3.2 ng/ml, were performed in duplicate in 10% DMEM growth medium (100 p/well). An amount of 200 TCID50 (50% tissue culture infectious dose) of virus was added to each well in a volume of pl, and the plates were incubated for 1 h at 37°C.
TZM.bl cells were then added (lxl04/well in a 100-pl volume) in 10% D-MEM growth medium containing DEAE-dextran (Sigma, St. Louis, MO) at a final concentration of 11 pg/ml. Assay controls included TZM.bl cells alone (cell control) and TZM.bl cells with virus (virus control).
Following a 48-hour incubation at 37°C, 150 pl of assay medium was removed from each well and 100 pl of BRIGHTGLO luciferase reagent (Promega, Madison, WI) was added. The cells were allowed to lyse for 2 min, and then 150 pl of the cell lysate was transferred to a 96-well black solid plate, and luminescence was measured using a Victor 3 luminometer (Perkin Elmer).
The 50% and 80% inhibitory concentration (IC50 and IC80) values were calculated as the serum dilution that caused a 50% and 80% reduction respectively, in relative luminescence units (RLU) compared to the level in the virus control wells after subtraction of cell control RLU. All data were analyzed with 5-parameter curve fitting using neutralizing antibody analysis software provided by the CAVD Vaccine Immunology Statistical Center. Maximal percent inhibition values were calculated as MPI = 100 - (mean signals at the 2 highest concentrations of compound/mean signal of 2 no-drug control wells) x 100 (PMID: 34780263).
Example 3 - Anti-Viral Activity Using Pseudotyped Virus Assay
The anti-viral activity of the antigen binding proteins was also measured in a replicating virus assay against NL4-3 strain and its variants in MT2 cells.
The proviral clone of NL4-3 (obtained from NIH) was used to make the replicating reporter virus NLRepRIuc, in which a section of the nef gene from the proviral clone of NL4-3 was replaced with the Renilla luciferase gene (Techniques in HIV Research. Tech HIV Res. Published online 1990. doi: 10.1007/978-1-349-11888-5). Virus was produced through transfection of HEK293T cells using Lipofectamine Plus (Invitrogen, Carlsbad CA), according to the manufacturer's instructions. The replication -com petent virus was harvested 3 days after transfection of HEK 293T cells with the modified pNLRepRIuc proviral clone and titrated in MT-2 cells using luciferase activity as a biomarker. The MPER-bNAb insensitive strains are engineered based on this proviral clone NL4-3 by introducing point mutations into its envelope gene, including W680R/K683Q and W672L/F673L. MT-2 cells were obtained from the American Type Culture Collection (ATCC) and were propagated in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml of penicillin G, 100 pg/ml of streptomycin, 10 mM HEPES buffer pH 7.55 and 2-mM L-glutamine. HEK293T cells were obtained from the ATCC and propagated in DMEM media supplemented with 10% heat-inactivated FBS.
The NLRepRIuc was used to infect MT-2 cells at a multiplicity of 0.01 for 1 hour before adding the proteins to the 96-well plates. The antigen binding proteins were serially diluted four-fold and 11 concentrations were plated in triplicate. After 4 days of incubation, cells were processed and quantitated for virus growth by the amount of expressed luciferase. Luciferase was quantitated using the ENDUREN substrate from Promega (Madison, WI) according to the manufacturer's instructions. Luciferase activity was measured immediately on an ENVISION multilabel plate reader (PerkinElmer, Waltham MA).
ECso values were calculated by comparing the amount of luciferase produced in the presence of antigen binding protein compared to wells where no antigen binding protein (DMSO control) was added. A 5-parameter sigmoidal equation was used to fit the resulting signal vs. concentration curves, and the concentration of each antigen binding protein that produced 50% maximal inhibition (ECso) was determined. Maximal percent inhibition values were calculated as MPI = 100 - (mean signals at the 2 highest concentrations of compound/mean signal of 2 no-drug control wells) x 100 (PMID: 34780263).
Those in the field will know that half maximal inhibitory concentration (IC50) is a measure of the potency of a substance in inhibiting a specific biological or biochemical function (in this case, viral replication). IC50 is a quantitative measure that indicates how much of a substance (e.g. drug) is needed to inhibit, in vitro, a given biological process or biological component by 50%. Similarly, IC80 or IC90 are the amount of the particular inhibitory substance needed to inhibit, in vitro, a given biological process or biological component by 80% or 90% respectively.
Example 4 - Stability of soluble CD4 domains
All soluble human CD4 domains tested contain a set of "base" mutations in human CD4 domain 1 (DI) over the wild-type sequence (SEQ ID NO:3) that enable the folding of human CD4 DI on its own. Soluble CD4 DI with this set of mutations is known as mD1.22 (Chen et al., J Virol. 2014 Jan;88(2): 1125-39) and the mutations therein consist of: L5Y, S23N, A55V, I76P, L96V, and F98V (SEQ ID N0:4, also referred to as Dim herein).
To achieve better developability and pharmacokinetics, further mutations were introduced into mD1.22 (SEQ ID NO:4) to enhance its thermal stability. The additional stabilizing mutations were designed based on several methodologies: 1) computational simulation by using Free Energy Perturbation (FEP+, Schrodinger, New York, NY USA); 2) computational simulation by using disulfide-bond scan in Molecular Operating Environment program (MOE, Chemical Computing Group, Montreal Canada); and 3) panning a library of human CD4 DI with each residue mutated, one by one, to the other 19 types of amino acids (site saturation mutagenesis, TWIST BioScience, San Francisco, CA USA) using phage display under thermally challenging conditions (i.e., incubating the phage at room temperature, 70 °C, and 80 °C, then selecting the CD4 domain variants that can still bind to recombinant HIV-1 gpl20 (CN54 strain, Aero Biosystems, Beijing China)).
The best performing variants (SEQ ID NOs: 5-21) were fused with 6xHis tag at their C- termini, expressed and purified from mammalian cells using methods as described in Example 1, except that purification was via a Ni-NTA resin (Cytiva, Marlborough MA) instead of Mabselect SuRe column, with standard protocol from the vendor.
These purified CD4 DI variants (with C-terminal 6xHis tag) were then evaluated to determine their melting temperature (Tm, using Prometheus System, NanoTemper, Munchen Germany), which indicates thermal stability, as well as their anti-viral activity against HIV-1 pseudotyped virus (see Example 2 above for methods using ACTOne cells).
As shown in Table 6 below and in Figure 2, several CD4 DI variants (SEQ ID NO:5-15) showed dramatically improved thermal stability over the "baseline" or "control" CD4 DI (Dim, SEQ 5 ID NO:4), while maintaining similar anti-viral activity.
Table 6 - Melting temperature of soluble CD4 domains
Example 5 - Antigen Binding Protein Format and Linker Length
The fusion position of the CD4 domain in the broadly neutralizing mAbs (e.g., whether to fuse the CD4 domain to the light chain or heavy chain or both, whether to fuse the CD4 domain to the N- terminus or C-terminus of these chains, or whether to fuse the CD4 domain in the middle of the heavy chain (in between CHI and CH2 domains)) was tested in some examples V3-bNAbs (another class of bNAbs that bind to a distinct target called the V3 loop).
We observed that the most potent bispecific molecule resulted from fusing CD4 DI to the N- terminus of the heavy chain of V3-bNAbl (molecule 1 in Table 7 [SEQ ID NOs: 44+23] which neutralized 6 envelopes with IC50 < 160 pM and 1 envelope with IC50 about 3 nM in PSV assay). In this bispecific format, the linker length between the CD4 domain and V3-bNAbl heavy chain N- terminus does not particularly affect anti-viral activity (Figure 3A), but changes the pharmacokinetics (PK) of the resultant bispecific molecules dramatically (Figure 3B).
As shown in Figure 3B, the shorter-linker bispecific (Dlm_lxG4S_bNAbl, SEQ ID NOs: 36 and 23) showed much better PK (longer half-life and lower clearance rate) than the longer-linker bispecific (Dlm_4xG4S_bNAbl, SEQ ID NOs: 37 and 23)) in a humanized mouse model (Tg32 strain where human neonatal Fc receptor (hFcRn) replaced the corresponding mouse gene (mFcRn), The Jackson Laboratory, Bar Harbor, Maine USA).
Table 7: IC50 (nM) of different V3-bNAbl-derived bispecific formats and control molecules against a panel of HIV-1 envelopes in a PSV assay (ACTOne cells)
1 = Dlm-K8C-G99C_lxG4S_bNAbl (SEQ ID NOs: 44+23) 7 = Dlm_His (SEQ ID NO:4*) + bNAbl (SEQ ID NOs:22+23)(combo)
2 = Dlm-K8C-G99C_lxG4S_bNAbl-LC (SEQ ID NOs:22+143) 8 = Dlm-K8C-G99C_Fc (SEQ ID NO:53) + bNAbl (SEQ ID NOs:22+23) (combo)
3 = Dlm_lxG4S_bNAbl-BothChains (SEQ ID NOs:36+38) 9 = Dlm_His (SEQ ID NO:4*)
4 = bNAbl-mid_lxG4S_Dlm-K8C-G99C (SEQ ID NOs:39+23) 10 = Dlm-K8C-G99C_Fc (SEQ ID NO:53)
5 = bNAbl-HC_lxG4S_Dlm-K8C-G99C (SEQ ID NOs:40+23) 11 = bNAbl (SEQ ID NOs:22+23)
6 = bNAbl-LC_lxG4S_Dlm-K8C-G99C (SEQ ID NOs:22+41)
* plus a 6xHis tag (six C-terminal histidine residues)
Table 8: IC50 (nM) of different V3-bNAb2-, bNAb3- and bNAb4-derived bispecific formats and control molecules against a panel of HIV-1 envelopes in PSV assay (ACTOne cells)
* not tested
Table 8 Molecule Key:
1 = Dlm-K8C-G99C_2xG4S_bNAb2 (SEQ ID NOs:46+25)
2 = Dlm-K8C-G99C_3xG4S_bNAb2 (SEQ ID NOs:47+25)
3 = Dlm-K8C-G99C_4xG4S_bNAb2 (SEQ ID NOs:48+25)
4 = bNAb2-LC_lxG4S_Dlm-K8C-G99C (SEQ ID NOs:24+49)
5 = bNAb2 (SEQ ID NOs:24+25) + DlmJHis (SEQ ID NO:4*) (combo)
6 = bNAb2 (SEQ ID NOs:24+25) + Dlm-K8C-G99C_Fc (SEQ ID NO:53) (combo)
7 = bNAb2 (SEQ ID NOs:24+25)
8 = Dlm-K8C-G99C_lxG4S_bNAb3 (SEQ ID NOs:50+27)
9 = Dlm-K8C-G99C_4xG4S_bNAb3 (SEQ ID NOs:51+27)
10 = bNAb3 (SEQ ID NOs:26+27) + DlmJHis (SEQ ID NO:4*) (combo)
11 = bNAb3 (SEQ ID NOs:26+27) + Dlm-K8C-G99C_Fc (SEQ ID NO:53) (combo)
12 = bNAb3 (SEQ ID NOs:26+27)
13 = Dlm-K8C-G99C_4xG4S_bNAb4 (SEQ ID NOs:52+29)
14 = bNAb4 (SEQ ID NOs:28+29) + DlmJHis (SEQ ID NO:4*) (combo)
15 = bNAb4 (SEQ ID NOs:28+29) + Dlm-K8C-G99C_Fc (SEQ ID NO:53) (combo)
16 = bNAb4 (SEQ ID NOs:28+29)
17 = Dlm_His (SEQ ID NO:4*)
18 = Dlm-K8C-G99C_Fc (SEQ ID NO:53)
* plus a 6xHis tag (six C-terminal histidine residues)
Thermal stabilization of CD4 DI (see Example 3 above) further enhanced the PK of the bispecific molecules (Dlm-K8C-G99C_lxG4S_bNAbl, SEQ ID NOs:44 and 23; Dlm-TllC- K72C_lxG4S_bNAbl, SEQ ID NOs:45 and 23; Dlm-K8I_lxG4S_bNAbl, SEQ ID NOs:42 and 23; and Dlm-K8V_lxG4S_bNAbl, SEQ ID NOs:43 and 23) as shown in Table 9 below.
Table 9: The effect of (1) linker length between the CD4 domain and V3-bNAbl heavy chain, and (2) thermal stability of CD4 DI, on the PK of bispecific molecules in hFcRn mice (Tq32)
Accordingly, the best molecules for further development contain shorter linker lengths between the CD4 domain and bNAb (lxG4S) and contain a thermally stable CD4 domain(s).
Example 6 - Bisoecifics based on MPER binders
To generate "CD4-MPER" bispecifics, CD4 domains was fused to different broadly neutralizing antibodies (bNAbs) that target the membrane proximal external region (MPER) of the HIV-1 envelope protein gpl60. These bispecifics were tested against a panel of HIV-1 envelopes in PSV assay, along with control molecules including CD4 domains alone, bNAbs alone, their mixtures at a 1:1 molar ratio (combo), and CD4 domains fused to an anti-RSV control antibody (Synagis - bispecific32). Several such bispecifics showed much higher anti-viral activity than the control molecules, indicating strong anti-viral synergy between the CD4 domain and the fused bNAbs (Figures 4-13). For example, bispecifics based on bNAb34 and bNAb35 showed much higher potency than the individual bNAb34 or bNAb35, the CD4 domains, and the combinations of bNAb34 or bNAb35 with the CD4 domains (Figures 4-7). This clearly indicates that the fusion of CD4 with the bNAb into a single molecule gained significant anti-viral advantage over simply combining the two reagents.
This synergy might be a combined outcome from 1) the CD4 domain and the linked bNAb bind to a same gpl60 envelope trimer; 2) CD4-induced conformational change of gpl60 exposes the MPER epitope for the bNAb (Prasad et al., Cell. 2022 Feb 17;185(4):641-653.el7.; Li et al., Nat Struct Mol Biol. 2020 Aug;27(8):726-734), which might be inaccessible in some resistant envelopes; and 3) the bNAb is sequestered at a high local concentration near the CD4 domain, providing advantage of binding kinetics. Indeed, we observed in the case of a class of MPER-bispecifics (based on bNAb33-bNAb36), the synergy is particularly obvious when the CD4 domain is attached to the C- terminus of the heavy chain (Figures 4-7). Based on structural modeling, this heavy chain C-terminal fusion can accommodate the cobinding of the CD4 domain and the linked bNAb on a same gpl60 trimer, which is hard to achieve by other fusion positions. SEQUENCE LISTING
SEQ ID NO: 1 (D1 D2)
KKWLGKKGDTVELTCTASQKKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRADSRRSLWD
QGN FPLIIKNLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRS
PRG KNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIWLA
SEQ ID NO:2 (D1mD2)
KKWLGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD
QGN FPLIIKNLKIEDSDTYICEVEDQKEEVQLWFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRS
PR GKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAF
KKWYGKKGDCVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD
QG NFPLIICNLKPEDSDTYICEVEDQKEEVQLWVG
SEQ ID NO:13 (D1m-E13C-l70C)
KKWYGKKGDTVCLTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD
QG NFPLCIKNLKPEDSDTYICEVEDQKEEVQLWVG
SEQ ID NO:14 (D1 m-H27C-G38C)
KKWYGKKGDTVELTCTASQKKNIQFCWKNSNQIKILCNQGSFLTKGPSKLNDRVDSRRSLWD
QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVG
SEQ ID NO:15 (D1 m-K21C-G65C)
KKWYGKKGDTVELTCTASQCKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD
QCNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVG
SEQ ID NO:16 (D1 m-Q25E)
KKWYGKKGDTVELTCTASQKKNIEFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD
QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVG
SEQ ID NO:17 (D1 m-H27D)
KKWYGKKGDTVELTCTASQKKNIQFDWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD
QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVG
SEQ ID NO:18 (D1 m-R58V)
KKWYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSVRSLWD
QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVG
SEQ ID NO:19 (D1 m-R58N)
KKWYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSNRSLWD
QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVG
SEQ ID NQ:20 (D1 m-R58T)
KKWYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSTRSLWD
QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVG
SEQ ID NO:21 (D1m-L61M)
KKWYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSMW
DQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVG SEQ ID NO:22 (V3-bNAb1 HC+LS)
QPQLQESGPTLVEASETLSLTCAVSGDSTAACNSFWGWVRQPPGKGLEWVGSLSHCASYWN
RGWTYHNPSLKSRLTLALDTPKNLVFLKLNSVTAADTATYYCARFGGEVLRYTDWPKPA WDL
WGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVLHEALHSHYTQKSLSLSPGK
SEQ ID NO:23 (V3-bNAb1 LC)
QSALTQPPSASGSPGQSITISCTGTSNNFVSWYQQHAGKAPKLVIYDVNKRPSGVPDRFSGSK
SGNTASLTVSGLQTDDEAVYYCGSLVGNWDVIFGGGTKLTVLGQPKAAPSVTLFPPSSEELQA
NKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRS
YSCQVTHEGSTVEKTVAPTECS
SEQ ID NO:24 (V3-bNAb2 HC+LS)
QMQLQESGPGLVKPSETLSLTCSVSGASISDSYWSWIRRSPGKGLEWIGYVHKSGDTNYSPSL
KSRVNLSLDTSKNQVSLSLVAATAADSGKYYCARTLHGRRIYGIVAFNEWFTYFYMDVWGNGT
QVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS
LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
VLHEALHSHYTQKSLSLSPGK
SEQ ID NO:25 (V3-bNAb2 LC)
SDISVAPGETARISCGEKSLGSRAVQWYQHRAGQAPSLIIYNNQDRPSGIPERFSGSPDSPFGT
TATLTITSVEAGDEADYYCHIWDSRVPTKWVFGGGTTLTVLGQPKAAPSVTLFPPSSEELQANK
ATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYS
CQVTHEGSTVEKTVAPTECS
SEQ ID NO:26 (V3-bNAb3 HC+LS)
QVQLRESGPGLVKPSETLSLSCTVSNDSRPSDHSWTWVRQSPGKALEWIGDIHYNGATTYNP
SLRSRVRIELDQSIPRFSLKMTSMTAADTGMYYCARNAIRIYGWALGEWFHYGMDVWGQGTA
VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGP
SVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
WSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
LHEALHSHYTQKSLSLSPGK
SEQ ID NO:27 (V3-bNAb3 LC)
SSELTQPPSVSVSPGQTARITCSGAPLTSRFTYWYRQKPGQAPVLIISRSSQRSSGWSGRFSA
SWSGTTVTLTIRGVQADDEADYYCQSSDTSDSYKMFGGGTKLTVLGQPAAAPSVTLFPPSSEE
LQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKS
HKSYSCQVTHEGSTVEKTVAPTEC
SEQ ID NO:28 (V3-bNAb4 HC+LS)
QVQLQESGPGLVKPSETLSVTCSVSGDSMNNYYWTWIRQSPGKGLEWIGYISDRESATYNPS
LNSRWISRDTSKNQLSLKLNSVTPADTAVYYCATARRGQRIYGWSFGEFFYYYSMDVWGKG
TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLG
GPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVLHEALHSHYTQKSLSLSPGK
SEQ ID NO:29 (V3-bNAb4 LC)
SYVRPLSVALGETARISCGRQALGSRAVQWYQHRPGQAPILLIYNNQDRPSGIPERFSGTPDIN
FGTRATLTISGVEAGDEADYYCHMWDSRSGFSWSFGGATRLTVLGQPKAAPSVTLFPPSSEE
LQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKS
HRSYSCQVTHEGSTVEKTVAPTECS
SEQ ID NQ:30 (1XG4S)
GGGGS
SEQ ID NO: 31 (2XG4S)
GGGGSGGGGS
SEQ ID NO: 32 (3XG4S)
GGGGSGGGGSGGGGS SEQ ID NO: 33 (4XG4S)
GGGGSGGGGSGGGGSGGGGS
SEQ ID NO: 34 (5XG4S)
GGGGSGGGGSGGGGSGGGGSGGGGS
SEQ ID NO: 35 (6XG4S)
GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS
SEQ ID NO: 36 (Di m 1xG4S bNAb1-HC)
KKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD
QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSQPQLQESGPTLVEASETLSLTCA
VSGDSTAACNSFWGWVRQPPGKGLEWVGSLSHCASYWNRGWTYHNPSLKSRLTLALDTPK
NLVFLKLNSVTAADTATYYCARFGGEVLRYTDWPKPAWVDLWGRGTLVTVSSASTKGPSVFP
LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS
SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLS LSPGK
SEQ ID NO: 37 (Di m 4xG4S bNAb2-HC)
KKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD
QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVGGGGGSGGGGSGGGGSGGGGSQPQLQ
ESGPTLVEASETLSLTCAVSGDSTAACNSFWGWVRQPPGKGLEWVGSLSHCASYWNRGWT
YHNPSLKSRLTLALDTPKNLVFLKLNSVTAADTATYYCARFGGEVLRYTDWPKPAWVDLWGR
GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG
GPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVLHEALHSHYTQKSLSLSPGK
SEQ ID NO: 38 (Di m 1xG4S bNAb1-LC)
KKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD
QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVGGGGGSQSALTQPPSASGSPGQSITISCT
GTS N N F VS WYQQ HAG KAP KLVI YD VN KR PSG VP DR FSGS KSG NTAS LTVSG LQTDD EAVYYC GSLVGNWDVIFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKA
DSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
SEQ ID NO: 39 (bNAb1-HC-mid 1xG4S D1 m-K8C-G99C)
QPQLQESGPTLVEASETLSLTCAVSGDSTAACNSFWGWVRQPPGKGLEWVGSLSHCASYWN
RGWTYHNPSLKSRLTLALDTPKNLVFLKLNSVTAADTATYYCARFGGEVLRYTDWPKPA WDL
WGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGGGGSKKVVYG
KCGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPL
IIKNLKPEDSDTYICEVEDQKEEVQLWVCGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKD
TLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD
IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQ
KSLSLSPGK
SEQ ID NO: 40 (bNAb1-HC 1xG4S D1 m-K8C-G99C)
QPQLQESGPTLVEASETLSLTCAVSGDSTAACNSFWGWVRQPPGKGLEWVGSLSHCASYWN
RGWTYHNPSLKSRLTLALDTPKNLVFLKLNSVTAADTATYYCARFGGEVLRYTDWPKPA WDL
WGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVLHEALHSHYTQKSLSLSPGGGGGSKKVVYGKCGDTVELTCTASQKKNIQFHWKNSN
QIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLW
VC
SEQ ID NO: 41 (bNAb1-LC 1xG4S D1 m-K8C-G99C)
QSALTQPPSASGSPGQSITISCTGTSNNFVSWYQQHAGKAPKLVIYDVNKRPSGVPDRFSGSK
SGNTASLTVSGLQTDDEAVYYCGSLVGNWDVIFGGGTKLTVLGQPKAAPSVTLFPPSSEELQA
NKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRS
YSCQVTHEGSTVEKTVAPTECSGGGGSKKVVYGKCGDTVELTCTASQKKNIQFHWKNSNQIKI
LGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVWC
SEQ ID NO: 42 (D1m-K8l 1xG4S bNAb1-HC)
KKWYGKIGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVGGGGGSQPQLQESGPTLVEASETLSLTCA
VSGDSTAACNSFWG WRQPPGKGLEVWGSLSHCASYWNRGWTYHNPSLKSRLTLALDTPK
NLVFLKLNSVTAADTATYYCARFGGEVLRYTDWPKPAWVDLWGRGTLVTVSSASTKGPSVFP
LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS
SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLS LSPGK
SEQ ID NO: 43 (D1m-K8V 1xG4S bNAb1-HC)
KKWYGKVGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD
QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVGGGGGSQPQLQESGPTLVEASETLSLTCA
VSGDSTAACNSFWG WRQPPGKGLEVWGSLSHCASYWNRGWTYHNPSLKSRLTLALDTPK
NLVFLKLNSVTAADTATYYCARFGGEVLRYTDWPKPAWVDLWGRGTLVTVSSASTKGPSVFP
LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS
SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLS LSPGK
SEQ ID NO: 44 (D1m-K8C-G99C 1xG4S bNAb1-HC)
KKWYGKCGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD
QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVCGGGGSQPQLQESGPTLVEASETLSLTCA
VSGDSTAACNSFWGWVRQPPGKGLEWVGSLSHCASYWNRGWTYHNPSLKSRLTLALDTPK
NLVFLKLNSVTAADTATYYCARFGGEVLRYTDWPKPAWVDLWGRGTLVTVSSASTKGPSVFP
LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS
SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS
RTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLS LSPGK
SEQ ID NO: 45 (D1m-T11C-K72C 1xG4S bNAb1-HC)
KKWYGKKGDCVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD QGNFPLIICNLKPEDSDTYICEVEDQKEEVQLWVGGGGGSQPQLQESGPTLVEASETLSLTCA
VSGDSTAACNSFWG WRQPPGKGLEVWGSLSHCASYWNRGWTYHNPSLKSRLTLALDTPK
NLVFLKLNSVTAADTATYYCARFGGEVLRYTDWPKPAWVDLWGRGTLVTVSSASTKGPSVFP
LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS
SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLS LSPGK
SEQ ID NO: 46 (D1m-K8C-G99C 2xG4S bNAb2-HC)
KKWYGKCGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD
QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVCGGGGSGGGGSQMQLQESGPGLVKPSE
TLSLTCSVSGASISDSYWSWIRRSPGKGLEWIGYVHKSGDTNYSPSLKSRVNLSLDTSKNQVS
LSLVAATAADSGKYYCARTLHGRRIYGIVAFNEWFTYFYMDVWGNGTQVTVSSASTKGPSVFP
LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS
SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLS LSPGK
SEQ ID NO: 47 (D1m-K8C-G99C 3xG4S bNAb2-HC)
KKWYGKCGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD
QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVCGGGGSGGGGSGGGGSQMQLQESGPGL
VKPSETLSLTCSVSGASISDSYWSWIRRSPGKGLEWIGYVHKSGDTNYSPSLKSRVNLSLDTS
KNQVSLSLVAATAADSGKYYCARTLHGRRIYGIVAFNEWFTYFYMDVWGNGTQVTVSSASTK
GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYT QKSLSLSPGK
SEQ ID NO: 48 (D1m-K8C-G99C 4xG4S bNAb2-HC)
KKWYGKCGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVCGGGGSGGGGSGGGGSGGGGSQMQLQ
ESGPGLVKPSETLSLTCSVSGASISDSYWSWIRRSPGKGLEWIGYVHKSGDTNYSPSLKSRVN
LSLDTSKNQVSLSLVAATAADSGKYYCARTLHGRRIYGIVAFNEWFTYFYMDVWGNGTQVTVS
SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEAL HSHYTQKSLSLSPGK
SEQ ID NO: 49 (bNAb2-LC 1xG4S D1 m-K8C-G99C)
SDISVAPGETARISCGEKSLGSRAVQWYQHRAGQAPSLIIYNNQDRPSGIPERFSGSPDSPFGT
TATLTITSVEAGDEADYYCHIWDSRVPTKWVFGGGTTLTVLGQPKAAPSVTLFPPSSEELQANK
ATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYS
CQVTHEGSTVEKTVAPTECSGGGGSKKWYGKCGDTVELTCTASQKKNIQFHWKNSNQIKILG NQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVC
SEQ ID NO: 50 (D1m-K8C-G99C 1xG4S bNAb3-HC)
KKWYGKCGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD
QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVWCGGGGSQVQLRESGPGLVKPSETLSLSCT
VSNDSRPSDHSWTWVRQSPGKALEWIGDIHYNGATTYNPSLRSRVRIELDQSIPRFSLKMTSM
TAADTGMYYCARNAIRIYGWALGEWFHYGMDVWGQGTAVTVSSASTKGPSVFPLAPSSKST
SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYI
CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV
WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
SEQ ID NO: 51 (D1m-K8C-G99C 4xG4S bNAb3-HC)
KKWYGKCGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD
QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVCGGGGSGGGGSGGGGSGGGGSQVQLRE
SGPGLVKPSETLSLSCTVSNDSRPSDHSWTWVRQSPGKALEWIGDIHYNGATTYNPSLRSRV
RIELDQSIPRFSLKMTSMTAADTGMYYCARNAIRIYGWALGEWFHYGMDVWGQGTAVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHS HYTQKSLSLSPGK
SEQ ID NO: 52 (D1m-K8C-G99C 4xG4S bNAb4-HC)
KKWYGKCGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD
QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVCGGGGSGGGGSGGGGSGGGGSQVQLQ
ESGPGLVKPSETLSVTCSVSGDSMNNYYWTWIRQSPGKGLEWIGYISDRESATYNPSLNSRW
ISRDTSKNQLSLKLNSVTPADTAVYYCATARRGQRIYGWSFGEFFYYYSMDVWGKGTTVTVS
SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEAL HSHYTQKSLSLSPGK
SEQ ID NO: 53 (D1m-K8C-G99C Fc)
KKWYGKCGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD
QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVCGGGGSGGGGSGGGGSGGGGSDKTHTC
PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
SEQ ID NO: 54 (V3 loop)
CTRPNNNTRKSIHIGPGRAFYTTGEIIGDIRQAHC
SEQ ID NO: 55 (Exemplary qp160)
MKVMGTKKNYQHLWRWGIMLLGMLMMSSAAEQLWVTVYYGVPVWREANTTLFCASDAKAY
DTEVHNVWATHACVPTDPNPQEWMGNVTEDFNMWKNNMVEQMHEDIISLWDQSLKPCVKL
TPLCVTLHCTNVTISSTNGSTANVTMREEMKNCSFNTTTVIRDKIQKEYALFYKLDIVPIEGKNTN
TSYRLINCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGKGPCRNVSTVQCTHGIKPV
VSTQLLLNGSLAEEDIIIRSENFTNNGKNIIVQLKEPVKINCTRPGNNTRRSINIGPGRAFYATGAII
GDIRKAHCNISTEQWNNTLTQIVDKLREQFGNKTIIFNQSSGGDPEWMHTFNCGGEFFYCNST
QLFNSTWFNNGTSTWNSTADNITLPCRIKQVINMWQEVGKAMYAPPIRGQIDCSSNITGLILTR
DGGSNSSQNETFRPGGGNMKDNWRSELYKYKWKIEPLGIAPTRAKRRWQREKRAVTLGAV FLGFLGAAGSTMGAASLTLTVQARLLLSGIVQQQSNLLRAIEAQQHMLQLTVWGIKQLQARVLA
IERYLKDQQLLGIWGCSGKLICTTTVPWNTSWSNKSYDYIWNNMTWMQWEREIDNYTGFIYTLI
EESQNQQEKNELELLELDKWASLWNWFNITNWLWYIKLFIMIIGGLVGLRIVCAVLSIVNRVRQG
YSPLSFQTRLPNPRGPDRPEETEGEGGERDRDRSARLVNGFLAIIWDDLRSLCLFSYHRLRDL
LLIVARWEILGRRGWEILKYWWNLLKYWSQELKNSAVSLLNVTAIAVAEGTDRVIEIVQRAVRAI LHIPTRIRQGFERALL
SEQ ID NO: 56 (Exemplary gp120)
AEQLWVIVYYGVPVWREANTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEWMGNVTED
FNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNVTISSTNGSTANVTMREEMKNC
SFNTTTVIRDKIQKEYALFYKLDIVPIEGKNTNTSYRLINCNTSVITQACPKVSFEPIPIHYCAPAGF
AILKCNNKTFNGKGPCRNVSTVQCTHGIKPWSTQLLLNGSLAEEDIIIRSENFTNNGKNIIVQLK
EPVKINCTRPGNNTRRSINIGPGRAFYATGAIIGDIRKAHCNISTEQWNNTLTQIVDKLREQFGN
KTIIFNQSSGGDPEWMHTFNCGGEFFYCNSTQLFNSTWFNNGTSTWNSTADNITLPCRIKQVI
NMWQEVGKAMYAPPIRGQIDCSSNITGLILTRDGGSNSSQNETFRPGGGNMKDNWRSELYKY
KWKIEPLGIAPTRAKRRWQREKR
SEQ ID NO: 57 (Exemplary gp41)
AVGIGALFLGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQ
LQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNHTTWMEWDREINNY
TSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSI
VNRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFS
YHRLRDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIE
VVQGACRAIRHIPRRIRQGLERILL
SEQ ID NO: 58 (MPER)
LDKWASLWNWFNITNWLWYIK
SEQ ID NO: 59 (MPER-bispecific36)
KKWYGKCGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD
QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVCGGGGSEVQLVESGGGLVKPGGSLRLSC
SASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNTLYL
EMNNVRTEDTGYYYCARTGKYYDFWSGYPPGEEYFQDWGQGTLVIVSSASTKGPSVFPLAPS
SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG
TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE
VTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPG K
SEQ ID NO: 60 (MPER-bispecific38)
KKWYGKCGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD
QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVCGGGGSQVQLVQSGAEVKKPGASVRVSC
KASGYTFTGYYMNWVRQAPGQGLEWMGWINPNRGGINYAQKFQGRVTMTRDTSITTAYMEL
SRLTSDDTAVYYCARGKNSDYNWDFQHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA
LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNH
KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA
PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
SEQ ID NO: 61 (MPER-bispecific39)
KKWYGKCGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD
QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVCGGGGSQVQLVQSGAEVKKPGASVKVSC
KASGYTFTGHYIHWVRQAPGQGLEWMGWINPNGGGTNYAHKFQGRVAMTRDTSISTAYMEL
SRLRSDDTAVYYCARGKNSDYNWDFQHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
SEQ ID NO: 62 (MPER-bispecific40)
KKWYGKCGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD
QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVCGGGGSQVQLVQSGGGLVKPGGSLTLSC
SASGFFFDNSWMGWVRQAPGKGLEWVGRIRRLKDGATGEYGAAVKDRFTISRDDSRNMLYL
HMRTLKTEDSGTYYCTMDEGTPVTRFLEWGYFYYYMAVWGRGTTVIVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSP GK
SEQ ID NO: 63 (MPER-bispecific41)
KKWYGKCGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD
QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVCGGGGSKVQLVQSGAELKKPWSSVRVSC
KASGGSFSSYAFNWVRQAPGQRLEWLGGIVPLVSSTNYAQRFRGRVTISADRSTSTVYLEMT
GLTSADTAVYFCAREGEGWFGRPLRAFEFWGQGTLVTVSTASTKGPSVFPLAPSSKSTSGGT
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN
HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
SEQ ID NO: 64 (MPER- bispecific42)
KKWYGKCGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD
QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVCGGGGSQVQLVQSGPEVKKPGSSLKVSC
KASGGSFSTYTLSWVRQTPGQGLEWMGGIIPLLGLPNYAPKFQGRVTFSADTSTNTAYMEMS
RLRFEDTAVYFCAREGAGWFGKPVGAMGYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGG
TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNV
NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL
PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
SEQ ID NO: 65 (MPER-bispecific43)
KKWYGKCGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD
QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVCGGGGSEVQLVESGPGLVQPWGTLSLTC
RVSGDSVSNDNYYWAWIRQTPGRELQVIGTIYYSGTTYYNPSLRNRVTISLDKSVNWSLRLGS
VSAADTAQYYCVRMPSHGFWSTSFSYWYFDLWGRGHFVAVSWASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV
WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK SEQ ID NO: 66 (CDRH1 of bNAb36)
NAWMT
SEQ ID NO: 67 (CDRH2 of bNAb36)
RITGPGEGWSVDYAESVKG
SEQ ID NO: 68 (CDRH3 of bNAb36)
TGKYYDFWSGYPPGEEYFQD
SEQ ID NO: 69 (CDRL1 of bNAb36)
QGDSLRSHYAS
SEQ ID NO: 70 (CDRL2 of bNAb36)
GKNNRPS
SEQ ID NO: 71 (CDRL3 of bNAb36)
SSRDKSGSRLSV
SEQ ID NO: 72 (VH of bNAb36)
EVQLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYYCARTGKYYDFWSGYPPGEEYFQDWG
QGTLVIVSS
SEQ ID NO: 73 ( VL of bNAb36)
SELTQDPAVSVALKQTVTITCQGDSLRSHYASWYQQKPGQAPVLLFYGKNNRPSGIPDRFSGS
ASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVL
SEQ ID NO: 74 (HO (no LS) of bNAb36)
EVQLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYYCARTGKYYDFWSGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SOS VM H EALH N H YTQ KS LS LS PG K
SEQ ID NO: 75 (HO (LS) of bNAb36)
EVQLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYYCARTGKYYDFWSGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVLHEALHSHYTQKSLSLSPGK
SEQ ID NO: 76 (LC of bNAb36)
SELTQDPAVSVALKQTVTITCQGDSLRSHYASWYQQKPGQAPVLLFYGKNNRPSGIPDRFSGS
ASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVLSQPKAAPSVTLFPPSSEE
LQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKS
HRSYSCQVTHEGSTVEKTVAPTECS
SEQ ID NO: 77 (CDRH1 of bNAb38)
GYYMN
SEQ ID NO: 78 (CDRH2 of bNAb38)
WINPNRGGINYAQKFQG
SEQ ID NO: 79 (CDRH3 of bNAb38)
GKNSDYNWDFQH
SEQ ID NO: 80 (CDRL1 of bNAb38)
RASQSVSRYLA
SEQ ID NO: 81 (CDRL2 of bNAb38)
DAS N RAT
SEQ ID NO: 82 (CDRL3 of bNAb38)
QQYEF
SEQ ID NO: 83 (VH of bNAb38)
QVQLVQSGAEVKKPGASVRVSCKASG YTFTG YYM N WVRQAPGQG LEWMG Wl N PN RGG I N Y
AQKFQGRVTMTRDTSITTAYMELSRLTSDDTAVYYCARGKNSDYNWDFQHWGQGTLVTVS
SEQ ID NO: 84 (VL of bNAb38)
EIVLTQSPATLSLSPGERATLSCRASQSVSRYLAWYQQKPGQAPRLLIYDASNRATGIPASFSG
SGSGTDFTLTISSLEPEDFAVYYCQQYEFFGQGTKVEIK SEQ ID NO: 85 (HC (no LS) of bNAb38)
QVQLVQSGAEVKKPGASVRVSCKASG YTFTG YYM N WVRQAPGQG LEWMG Wl N PN RGG I N Y
AQKFQGRVTMTRDTSITTAYMELSRLTSDDTAVYYCARGKNSDYNWDFQHWGQGTLVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTV
LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK
SEQ ID NO: 86 (HC (LS) of bNAb38)
QVQLVQSGAEVKKPGASVRVSCKASG YTFTG YYM N WVRQAPGQG LEWMG Wl N PN RGG I N Y
AQKFQGRVTMTRDTSITTAYMELSRLTSDDTAVYYCARGKNSDYNWDFQHWGQGTLVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV
LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHS HYTQKSLSLSPGK
SEQ ID NO: 87 (LC of bNAb38)
EIVLTQSPATLSLSPGERATLSCRASQSVSRYLAWYQQKPGQAPRLLIYDASNRATGIPASFSG
SGSGTDFTLTISSLEPEDFAVYYCQQYEFFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASW
CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC
SEQ ID NO: 88 (CDRH1 of bNAb39)
GHYIH
SEQ ID NO: 89 (CDRH2 of bNAb39)
WINPNGGGTNYAHKFQG
SEQ ID NO: 90 (CDRH3 of bNAb39)
GKNSDYNWDFQH
SEQ ID NO: 91 (CDRL1 of bNAb39)
RASQSVIRYLA SEQ ID NO: 92 (CDRL2 of bNAb39)
DAS N RAT
SEQ ID NO: 93 (CDRL3 of bNAb39)
QQYEF
SEQ ID NO: 94 (VH of bNAb39)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGHYIHWVRQAPGQGLEWMGWINPNGGGTNYA
HKFQGRVAMTRDTSISTAYMELSRLRSDDTAVYYCARGKNSDYNWDFQHWGQGTLVTVSS
SEQ ID NO: 95 (VL of bNAb39)
EIVLTQSPATLSLSPGERATLSCRASQSVIRYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGS
GSGTDFTLTISSLEPEDFAVYYCQQYEFFGQGTKLEIK
SEQ ID NO: 96 (HO (no LS) of bNAb39)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGHYIHWVRQAPGQGLEWMGWINPNGGGTNYA
HKFQGRVAMTRDTSISTAYMELSRLRSDDTAVYYCARGKNSDYNWDFQHWGQGTLVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTV
LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK
SEQ ID NO: 97 (HO (LS) of bNAb39)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGHYIHWVRQAPGQGLEWMGWINPNGGGTNYA
HKFQGRVAMTRDTSISTAYMELSRLRSDDTAVYYCARGKNSDYNWDFQHWGQGTLVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTV
LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHS HYTQKSLSLSPGK
SEQ ID NO: 98 (LC of bNAb39)
EIVLTQSPATLSLSPGERATLSCRASQSVIRYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGS
GSGTDFTLTISSLEPEDFAVYYCQQYEFFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
HQGLSSPVTKSFNRGEC
SEQ ID NO: 99 ( CDRH1 of bNAb40)
NSWMG
SEQ ID NO: 100 (CDRH2 of bNAb40)
RIRRLKDGATGEYGAAVKD
SEQ ID NO: 101 (CDRH3 of bNAb40)
DEGTPVTRFLEWGYFYYYMAV
SEQ ID NO: 102 (CDRL1 of bNAb40)
RASQNIRDYLN
SEQ ID NO: 103 (CDRL2 of bNAb40)
AASTLQT
SEQ ID NO: 104 (CDRL3 of bNAb40)
QENYNTIPSLS
SEQ ID NO: 105 (VH of bNAb40)
QVQLVQSGGGLVKPGGSLTLSCSASGFFFDNSWMGWVRQAPGKGLEWVGRIRRLKDGATG
EYGAAVKDRFTISRDDSRNMLYLHMRTLKTEDSGTYYCTMDEGTPVTRFLEWGYFYYYMAVW
GRGTTVIVSS
SEQ ID NO: 106 (VL of bNAb40)
DIVMTQSPSSVSASVGDRVTITCRASQNIRDYLNWYQHKPGGSPRLLIYAASTLQTGVPSRFSG
SGSGNLFTLTITNLQPEDFATYYCQENYNTIPSLSFGQGTKVDIR
SEQ ID NO: 107 (HO (no LS) of bNAb40)
QVQLVQSGGGLVKPGGSLTLSCSASGFFFDNSWMGWVRQAPGKGLEWVGRIRRLKDGATG
EYGAAVKDRFTISRDDSRNMLYLHMRTLKTEDSGTYYCTMDEGTPVTRFLEWGYFYYYMAVW
GRGTTVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 108 (HC (LS) of bNAb40)
QVQLVQSGGGLVKPGGSLTLSCSASGFFFDNSWMGWVRQAPGKGLEWVGRIRRLKDGATG
EYGAAVKDRFTISRDDSRNMLYLHMRTLKTEDSGTYYCTMDEGTPVTRFLEWGYFYYYMAVW
GRGTTVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVLHEALHSHYTQKSLSLSPGK
SEQ ID NO: 109 ( LC of bNAb40)
DIVMTQSPSSVSASVGDRVTITCRASQNIRDYLNWYQHKPGGSPRLLIYAASTLQTGVPSRFSG
SGSGNLFTLTITNLQPEDFATYYCQENYNTIPSLSFGQGTKVDIRRTVAAPSVFIFPPSDEQLKS
GTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
KVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 110 (CDRH1 of bNAb41)
SYAFN
SEQ ID NO: 111 (CDRH2 of bNAb41)
GIVPLVSSTNYAQRFRG
SEQ ID NO: 112 (CDRH3 of bNAb41)
EGEGWFGRPLRAFEF
SEQ ID NO: 113 (CDRL1 of bNAb41)
RASQSVSGGALA
SEQ ID NO: 114 (CDRL2 of bNAb41)
GTSGRAT
SEQ ID NO: 115 ( CDRL3 of bNAb41)
QQYGTSQST
SEQ ID NO: 116 (VH of bNAb41)
KVQLVQSGAELKKPWSSVRVSCKASGGSFSSYAFNWVRQAPGQRLEWLGGIVPLVSSTNYA
QRFRGRVTISADRSTSTVYLEMTGLTSADTAVYFCAREGEGWFGRPLRAFEFWGQGTLVTVS T SEQ ID NO: 117 (VL of bNAb41)
EIVLTQSPGTFALSPGERATLSCRASQSVSGGALAWYQQKAGQAPRLLIYGTSGRATGVPGRF
SGSGSETDFSLTISRLEPEDFAVYYCQQYGTSQSTFGQGTRLETR
SEQ ID NO: 118 (HC (no LS) of bNAb41)
KVQLVQSGAELKKPWSSVRVSCKASGGSFSSYAFNWVRQAPGQRLEWLGGIVPLVSSTNYA
QRFRGRVTISADRSTSTVYLEMTGLTSADTAVYFCAREGEGWFGRPLRAFEFWGQGTLVTVS
TASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK
SEQ ID NO: 119 (HC (LS) of bNAb41)
KVQLVQSGAELKKPWSSVRVSCKASGGSFSSYAFNWVRQAPGQRLEWLGGIVPLVSSTNYA
QRFRGRVTISADRSTSTVYLEMTGLTSADTAVYFCAREGEGWFGRPLRAFEFWGQGTLVTVS
TASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEAL HSHYTQKSLSLSPGK
SEQ ID NO: 120 (LC of bNAb41)
EIVLTQSPGTFALSPGERATLSCRASQSVSGGALAWYQQKAGQAPRLLIYGTSGRATGVPGRF
SGSGSETDFSLTISRLEPEDFAVYYCQQYGTSQSTFGQGTRLETRRTVAAPSVFIFPPSDEQLK
SGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 121 ( CDRH1 of bNAb42)
TYTLS
SEQ ID NO: 122 (CDRH2 of bNAb42)
GIIPLLGLPNYAPKFQG SEQ ID NO: 123 (CDRH3 of bNAb42)
EGAGWFGKPVGAMGY
SEQ ID NO: 124 (CDRL1 of bNAb42)
RASQNVRNSNLA
SEQ ID NO: 125 (CDRL2 of bNAb42)
GASSRAS
SEQ ID NO: 126 (CDRL3 of bNAb42)
QQYGGSFGT
SEQ ID NO: 127 (VH of bNAb42)
QVQLVQSGPEVKKPGSSLKVSCKASGGSFSTYTLSWVRQTPGQGLEWMGGIIPLLGLPNYAP
KFQGRVTFSADTSTNTAYMEMSRLRFEDTAVYFCAREGAGWFGKPVGAMGYWGQGTTVTVS S
SEQ ID NO: 128 (VL of bNAb42)
EIVLTQSPDTLSLSPGERASLSCRASQNVRNSNLAWYQHKPGQPPRLLIYGASSRASGIPGRFS
GSGSGTDFTLTISRLEPEDFAVYYCQQYGGSFGTFGQGTKVEFR
SEQ ID NO: 129 (HO (no LS) of bNAb42)
QVQLVQSGPEVKKPGSSLKVSCKASGGSFSTYTLSWVRQTPGQGLEWMGGIIPLLGLPNYAP
KFQGRVTFSADTSTNTAYMEMSRLRFEDTAVYFCAREGAGWFGKPVGAMGYWGQGTTVTVS
SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK
SEQ ID NO: 130 (HO (LS) of bNAb42)
QVQLVQSGPEVKKPGSSLKVSCKASGGSFSTYTLSWVRQTPGQGLEWMGGIIPLLGLPNYAP
KFQGRVTFSADTSTNTAYMEMSRLRFEDTAVYFCAREGAGWFGKPVGAMGYWGQGTTVTVS
SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEAL HSHYTQKSLSLSPGK
SEQ ID NO: 131 (LC of bNAb42)
EIVLTQSPDTLSLSPGERASLSCRASQNVRNSNLAWYQHKPGQPPRLLIYGASSRASGIPGRFS
GSGSGTDFTLTISRLEPEDFAVYYCQQYGGSFGTFGQGTKVEFRRTVAAPSVFIFPPSDEQLK
SGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 132 (CDRH1 of bNAb44)
NDNYYWA
SEQ ID NO: 133 (CDRH2 of bNAb44)
TIYYSGTTYYNPSLRN
SEQ ID NO: 134 (CDRH3 of bNAb44)
MPSHGFWSTSFSYWYFDL
SEQ ID NO: 135 ( CDRL1 of bNAb44)
RASQSVTKYLN
SEQ ID NO: 136 (CDRL2 of bNAb44)
GTYTLLS
SEQ ID NO: 137 (CDRL3 of bNAb44)
QQAHSTPWT
SEQ ID NO: 138 (VH of bNAb44)
EVQLVESGPGLVQPWGTLSLTCRVSGDSVSNDNYYWAWIRQTPGRELQVIGTIYYSGTTYYNP
SLRNRVTISLDKSVNWSLRLGSVSAADTAQYYCVRMPSHGFWSTSFSYWYFDLWGRGHFVA VSW
SEQ ID NO: 139 (VL of bNAb44)
DIQMTQSPSSLSASVGDKVTITCRASQSVTKYLNWYQFKTGQAPRILIYGTYTLLSGVSPRFSG
AGSGSLYTLTITNIQPEDFATYYCQQAHSTPWTFGQGTHVAAN
SEQ ID NO: 140 (HO (no LS) of bNAb44)
EVQLVESGPGLVQPWGTLSLTCRVSGDSVSNDNYYWAWIRQTPGRELQVIGTIYYSGTTYYNP
SLRNRVTISLDKSVNWSLRLGSVSAADTAQYYCVRMPSHGFWSTSFSYWYFDLWGRGHFVA
VSWASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGK
SEQ ID NO: 141 (HC (LS) of bNAb44)
EVQLVESGPGLVQPWGTLSLTCRVSGDSVSNDNYYWAWIRQTPGRELQVIGTIYYSGTTYYNP
SLRNRVTISLDKSVNWSLRLGSVSAADTAQYYCVRMPSHGFWSTSFSYWYFDLWGRGHFVA
VSWASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHE ALHSHYTQKSLSLSPGK
SEQ ID NO: 142 (LC of bNAb44)
DIQMTQSPSSLSASVGDKVTITCRASQSVTKYLNWYQFKTGQAPRILIYGTYTLLSGVSPRFSG AGSGSLYTLTITNIQPEDFATYYCQQAHSTPWTFGQGTHVAANRTVAAPSVFIFPPSDEQLKSG
TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 143 Di m K8C G99C_1xG4S_bNAb1 LC
KKWYGKCGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWD QGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVWCGGGGSQSALTQPPSASGSPGQSITISCT
GTS N N F VS WYQQ HAG KAP KLVI YD VN KR PSG VP DR FSGS KSG NTAS LTVSG LQTDD EAVYYC GSLVGNWDVIFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKA DSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
SEQ ID NO: 144 Bispecific 32-1
QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDDKKDYN PSLKSRLTISKDTSKNQVVLKVTNMDPADTATYYCARSMITNWYFDVWGAGTTVTVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYT
QKSLSLSPGKGGGGSGGGGSGGGGSGGGGSKKWLGKKGDTVELTCTASQKKSIQFHWKN
SNQIKILGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQL
LVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWT
CTVLQNQKKVEFKIDIWLAF
SEQ ID NO: 145 Bispecific 32-2
QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDDKKDYN
PSLKSRLTISKDTSKNQVVLKVTNMDPADTATYYCARSMITNWYFDVWGAGTTVTVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD
TLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYT
QKSLSLSPGKGGGGSGGGGSGGGGSGGGGSKKVVLGKKGDTVELTCTASQKKNIQFHWKN
SNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQL
VVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWT
CTVLQNQKKVEFKIDIWLAF
SEQ ID NO: 146 Bispecific 32-3
QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDDKKDYN
PSLKSRLTISKDTSKNQVVLKVTNMDPADTATYYCARSMITNWYFDVWGAGTTVTVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD
TLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYT
QKSLSLSPGKGGGGSGGGGSGGGGSGGGGSKKVVYGKKGDTVELTCTASQKKNIQFHWKN
SNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQL
VWG
SEQ ID NO: 147 Bispecific 34-1 EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWFGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVLHEALHSHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSKKVVLGKKGDTVELTCT
ASQKKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTY
ICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSV
SQLELQDSGTWTCTVLQNQKKVEFKIDIWLAF
SEQ ID NO: 148 Bispecific 34-2
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWFGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVLHEALHSHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSKKVVLGKKGDTVELTCT
ASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKIEDSDTY
ICEVEDQKEEVQLWFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSV
SQLELQDSGTWTCTVLQNQKKVEFKIDIWLAF
SEQ ID NO: 149 Bispecific 34-3
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWFGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVLHEALHSHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSKKVVYGKKGDTVELTCT ASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDT
YICEVEDQKEEVQLVVVG
SEQ ID NO: 150 Bispecific 34-4
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWFGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVLHEALHSHYTQKSLSLSPGKGGGGSGGGGSKKVVYGKKGDTVELTCTASQKKNIQFHW KNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEE
VQLVVVG
SEQ ID NO: 151 Bispecific 35-6
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWG QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSKKWY GKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFP
LIIKNLKPEDSDTYICEVEDQKEEVQLWVG
SEQ ID NO: 152 Bispecific 35-5 EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSKKVVYGKKGD
TVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNL
KPEDSDTYICEVEDQKEEVQLVVVG
SEQ ID NO: 153 Bispecific 35-4
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSKKVVYGKKGDTVELTC
TASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSD TYICEVEDQKEEVQLVVVG
SEQ ID NO: 154 Bispecific 35-3
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSKKWYGKKGDTVELTCTASQKK NIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVE DQKEEVQLWVG
SEQ ID NO: 155 Bispecific 35-2
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWG QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSKKVVYGKKGDTVELTCTASQKKNIQFH WKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKE
EVQLVVVG
SEQ ID NO: 156 Bispecific 35-1
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWG QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGKGGGGSKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQ IKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWV
G
SEQ ID NO: 157 Bispecific 35-0
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWG QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGKKKWYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGN
QGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLWVG
SEQ ID NO: 158 Bispecific 35-EP
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGKESPEPETPEDESPEPETPEDEKKVVYGKKGDTVELTCTA
SQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYI CEVEDQKEEVQ LVVVG
SEQ ID NO: 159 Bispecific 35-6-D2
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSKKWL
GKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFP
LIIKNLKIEDSDTYICEVEDQKEEVQLWFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSP
RGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIWLAF SEQ ID NO: 160 Bispecific 35-5-D2
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSKKVVLGKKGD
TVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNL
KIEDSDTYICEVEDQKEEVQLWFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNI
QGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIWLAF
SEQ ID NO: 161 Bispecific 35-4-D2
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSKKVVLGKKGDTVELTC
TASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKIEDSDT
YICEVEDQKEEVQLWFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLS
VSQLELQDSGTWTCTVLQNQKKVEFKIDIWLAF
SEQ ID NO: 162 Bispecific 35-3-D2
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSKKWLGKKGDTVELTCTASQKK
NIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKIEDSDTYICEVE
DQKEEVQLVVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLE
LQDSGTWTCTVLQNQKKVEFKIDIWLAF
SEQ ID NO: 163 Bispecific 35-2-D2
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSKKVVLGKKGDTVELTCTASQKKNIQFH
WKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEE
VQLVVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSG
TWTCTVLQNQKKVEFKIDIWLAF
SEQ ID NO: 164 Bispecific 35-1 -D2
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGKGGGGSKKVVLGKKGDTVELTCTASQKKNIQFHWKNSNQ
IKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLWF
GLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTV
LQNQKKVEFKIDIWLAF SEQ ID NO: 165 Bispecific 35-0-D2
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGKKKWLGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGN
QGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLWFGLTAN
SDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQ KKVEFKIDIWLAF
SEQ ID NO: 166 Bispecific 35-EP-D2
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGKESPEPETPEDESPEPETPEDEKKWLGKKGDTVELTCTA
SQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKIEDSDTYI
CEVEDQKEEVQLVVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSV SQLELQDSGTWTCTVLQNQKKVEFKIDIWLAF
SEQ ID NO: 167 Bispecific 35-4-LS
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVLHEALHSHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSKKVVYGKKGDTVELTCT
ASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDT YICEVEDQKEEVQLVVVG
SEQ ID NO: 168 bNAb33 CDRH1
NAWMT
SEQ ID NO: 169 bNAb33 CDRH2
RITGPGEGWSVDYAESVKG
SEQ ID NO: 170 bNAb33 CDRH3
TGKYYDFWSGYPPGEEYFQD
SEQ ID NO: 171 bNAb33 CDRL1
RGDSLRSHYAS
SEQ ID NO: 172 bNAb33 CDRL2
GKNNRPS
SEQ ID NO: 173 bNAb33 CDRL3
SSRDKSGSRLSV
SEQ ID NO: 174 bNAb33 VH
EVRLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWG QGTLVIVSS SEQ ID NO: 175 bNAb33 VL
ASELTQDPAVSVALKQTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSG
SASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVL
SEQ ID NO: 176 bNAb33 HO (no LS)
EVRLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SOS VM H EALH N H YTQ KS LS LS PG K
SEQ ID NO: 177 bNAb33 HO (LS)
EVRLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVLHEALHSHYTQKSLSLSPGK
SEQ ID NO: 178 bNAb33 LC
ASELTQDPAVSVALKQTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSG
SASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVLSQPKAAPSVTLFPPSSE
ELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWK SHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO: 179 bNAb34 CDRH1
NAWMT
SEQ ID NO: 180 bNAb34 CDRH2
RITGPGEGWSVDYAESVKG
SEQ ID NO: 181 bNAb34 CDRH3
TGKYYDFWFGYPPGEEYFQD
SEQ ID NO: 182 bNAb34 CDRL1
RGDSLRSHYAS
SEQ ID NO: 183 bNAb34 CDRL2
GKNNRPS
SEQ ID NO: 184 bNAb34 CDRL3
SSRDKSGSRLSV
SEQ ID NO: 185 bNAb34 VH
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWFGYPPGEEYFQDWG QGTLVIVSS
SEQ ID NO: 186 bNAb34 VL
ASELTQDPAVSVALKQTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSG
SASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVL SEQ ID NO: 187 bNAb34 HC (no LS)
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWFGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCS VM H EALH N H YTQ KS LS LS PG K
SEQ ID NO: 188 bNAb34 HC (LS)
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWFGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVLHEALHSHYTQKSLSLSPGK
SEQ ID NO: 189 bNAb34 LC
ASELTQDPAVSVALKQTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSG
SASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVLSQPKAAPSVTLFPPSSE
ELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWK SHRSYSCQVTHEGSTVEKTVAPTECS
SEQ ID NO: 190 bNAb35 CDRH1
NAWMT
SEQ ID NO: 191 bNAb35 CDRH2 RITGPGEGWSVDYAESVKG
SEQ ID NO: 192 bNAb35 CDRH3
TGKYYDFWSGYPPGEEYFQD
SEQ ID NO: 193 bNAb35 CDRL1
RGDSLRSHYAS
SEQ ID NO: 194 b N Ab35
GKNNRPS
SEQ ID NO: 195 bNAb35 CDRL3
SSRDKSGSRLSV
SEQ ID NO: 196 bNAb35 VH
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWG QGTLVIVSS
SEQ ID NO: 197 bNAb35 VL
ASELTQDPAVSVALKQTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSG
SASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVL
SEQ ID NO: 198 bNAb35 HO (no LS)
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCS VM H EALH N H YTQ KS LS LS PG K
SEQ ID NO: 199 bNAb35 HC (LS)
EVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSV
DYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGEEYFQDWG
QGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVLHEALHSHYTQKSLSLSPGK
SEQ ID NO: 200 bNAb35 LC
ASELTQDPAVSVALKQTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSG
SASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVLSQPKAAPSVTLFPPSSE
ELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWK SHRSYSCQVTHEGSTVEKTVAPTECS
SEQ ID NO: 201 EP linker
ESPEPETPEDESPEPETPEDE

Claims

1. A multimeric anti-HIV envelope spike complex-binding protein comprising: i. a broadly neutralizing anti-MPER antibody, comprising at least one heavy chain or light chain, wherein the antibody binds to the MPER domain of a gp41 protein; and ii. at least one CD4 domain which binds to gpl20, wherein the CD4 domain is attached directly or by a linker to the N-terminus or C-terminus of one of the heavy chains or light chains.
2. The binding protein of any preceding claim, wherein the binding protein is a bispecific binding protein.
3. The binding protein of any preceding claim, wherein the CD4 domain is attached via a linker to the N-terminus or C-terminus of at least one heavy or light chain of the broadly neutralizing antibody.
4. The binding protein of any preceding claim, wherein the CD4 domain is attached via a linker to the C-terminus of at least one heavy chain of the broadly neutralizing antibody.
5. The binding protein of any preceding claim, wherein the binding protein comprises at least four CD4 domains.
6. The binding protein of claim 5, wherein the broadly neutralizing antibody has two heavy chains and two light chains; and the C-terminus of a first CD4 domain is attached by a linker to the N-terminus of a first heavy chain, a second CD4 domain is attached by a linker to the N-terminus of a second heavy chain, a third CD4 domain is attached by a linker to the N- terminus of a first light chain, an a fourth CD4 domain is attached by a linker to the N- terminus of a second light chain.
7. The binding protein of any preceding claim, wherein the linker is selected from: SEQ ID NOs: 30 to 35.
8. The binding protein of any preceding claim, wherein the CD4 domain comprises at least one of: L5Y, S23N, A55V, I79P, L96V and F98V mutations.
9. The binding protein of any preceding claim, wherein the CD4 domain comprises any one of SEQ ID NO: 4 - 21.
10. The binding protein of any preceding claim, wherein the broadly neutralizing antibody comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3 as set out in any one row of Table 3.
11. The binding protein of any preceding claim, wherein the broadly neutralizing antibody comprises a heavy chain variable domain and a light chain variable domain pair that is at least 95% identical to any of the variable domain pairs set out in any one row of Table 4.
12. The binding protein of any preceding claim, wherein the broadly neutralizing antibody comprises a heavy chain and a light chain pair that is at least 95% identical to any of the chain pairs set out in any one row of Table 4.
13. The binding protein of any preceding claim, wherein the binding protein comprises or consists of a sequence at least 95% identical to SEQ ID NO: 147-167 or 59-65.
14. A pharmaceutical composition comprising the binding protein as defined in any one of the preceding claims and a pharmaceutically acceptable excipient.
15. A method of treating or preventing an HIV infection in a human comprising administering to the human an anti-HIV binding protein according to any one of claims 1 to 14, or a pharmaceutical composition according to claim 15
16. An anti-HIV binding protein according to any one of claims 1 to 14, or a pharmaceutical composition according to claim 15, for use in treating or preventing an HIV infection in a human.
17. Use of an anti-HIV binding protein according to any one of claims 1 to 14, or a pharmaceutical composition according to claim 15, in the manufacture of a medicament for treating or preventing an HIV infection in a human.
18. The method of claim 15, protein or composition for use according to claim 16, or use according to claim 17, wherein viral load in the human is decreased.
19. A kit comprising in separate containers: an anti-HIV binding protein according to any one of claims 1 to 14 and an anti-viral drug that inhibits cellular entry, replication, or transcription of HIV in a human.
20. The kit as claimed in claim 19, wherein the antiviral drug is selected from: Nucleoside Reverse Transcriptase Inhibitors (NRTIs), Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs), Protease Inhibitors (Pls), Entry Inhibitors, Integrase Strand Transfer Inhibitors (INSTI), Maturation Inhibitors (Mis), Capsid Inhibitors (Cis) and Nucleoside Reverse Transcriptase Translocation Inhibitors (NRTTIs).
21. A nucleic acid sequence that encodes an anti-HIV binding protein according to any one of claims 1 to 14.
22. An expression vector that comprises the nucleic acid sequence of claim 21.
23. A recombinant host cell that comprises the nucleic acid sequence of claim 21 or the expression vector of claim 22.
24. A method of producing an anti-HIV binding protein, comprising culturing the host cell as defined in claim 23 under conditions suitable for expression of said nucleic acid sequence or vector, whereby an anti-HIV binding protein is produced.
25. An anti-HIV binding protein produced by the method of claim 24.
PCT/EP2025/061759 2024-04-30 2025-04-29 Neutralizing antibody constructs against hiv Pending WO2025229018A1 (en)

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