MULTABODY Constructs, Compositions and Methods

Abstract

In an embodiment, the fusion protein comprises a nanocage monomer or subunit thereof linked to an Fc monomer, and a plurality of said fusion proteins can self-assemble to form a nanocage comprising one or more Fc dimers. In an embodiment, the fusion protein comprises a nanocage monomer or subunit thereof linked to an Fc monomer or scFc fragment at the C-terminus of the nanocage monomer or subunit thereof, and a plurality of fusion proteins can self-assemble to form a nanocage.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 142,704, filed January 28, 2021, the entire contents of which are incorporated herein by reference in their entirety for all purposes. Sequence Listing

[0002] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on Jan. 28, 2022, is named "Sequence Listing Jan-2022 3206-5048_ST25.txt" and is 8100 bytes in size. Field

[0003] The present invention relates to polypeptides. In particular, the present invention relates to Multabody polypeptides and related constructs, compositions and methods. [Background technology]

[0004] background Nanoparticles have contributed to advances in a variety of fields: their use has the potential to impart targeted delivery, allow for the engineering of ordered microarrays, sustained release and caged microenvironments for catalytic processes.

[0005] Protein self-assembly is an attractive method for the creation of nanoparticles containing sensitive and metastable proteins. Indeed, self-assembled nanoparticles form under physiological conditions through non-covalent interactions, reliably resulting in uniform and often symmetrical nanocapsules or nanocages. Self-assembled protein nanoparticles have three distinct surfaces: exterior, interior and intersubunit surfaces, all of which can be tailored to convey added functionality.

[0006] Fusion proteins involving self-assembling proteins have been described, for example, known to display antigens on the exterior surface of assembled nanocages for use as vaccines.

[0007] There is a need for improved constructs, compositions and methods for designing nanoparticles to treat and / or prevent disease. Summary of the Invention

[0008] Summary of the invention According to one aspect, a fusion protein is provided comprising a first nanocage monomer or a subunit thereof linked to an Fc monomer, wherein multiple fusion proteins can self-assemble to form a nanocage comprising one or more Fc dimers.

[0009] In one embodiment, the Fc monomer is linked to the first nanocage monomer or a subunit thereof at the N-terminus or C-terminus of the first nanocage monomer or a subunit thereof, preferably at the C-terminus.

[0010] In one embodiment, the subunits include an N subunit or a C subunit that substantially corresponds to the N-terminal half of a nanocage monomer and the C-terminal half of a nanocage monomer, respectively, and the N subunit and C subunit are capable of self-assembly to form the nanocage monomer.

[0011] In one embodiment, the Fc monomer is linked to the N or C subunit at the C-terminus of the N or C subunit, preferably the Fc monomer is linked to the C subunit at the C-terminus.

[0012] In one embodiment, the first nanocage monomer or subunit thereof is further linked to a first biologically active moiety.

[0013] In one embodiment, the first biologically active moiety is linked to the first nanocage monomer or subunit thereof at the N-terminus or C-terminus of the first nanocage monomer or subunit thereof, preferably at the N-terminus.

[0014] In one embodiment, the first biologically active moiety is linked to the N subunit or C subunit at the N-terminus of the N subunit or C subunit, preferably the first biologically active moiety is linked to the C subunit at the N-terminus.

[0015] In one embodiment, the first bioactive moiety decorates the interior and / or exterior surface of the assembled nanocage, preferably the exterior surface.

[0016] In one embodiment, the first biologically active moiety comprises a first antigen-binding moiety.

[0017] In one embodiment, the first antigen binding portion comprises an antibody or a fragment thereof.

[0018] In one embodiment, the first antigen binding portion comprises a Fab fragment.

[0019] In one embodiment, the antibody or fragment thereof comprises an scFab fragment, an scFv fragment, an sdAb fragment, a VHH domain, or a combination thereof.

[0020] In one embodiment, the antibody or fragment thereof comprises the heavy and / or light chain of a Fab fragment.

[0021] In one embodiment, the first antigen-binding portion specifically binds to an antigen associated with a condition preventable and / or treatable by an antibody.

[0022] In one aspect, the antigen is associated with an infectious agent, including a virus, bacterium, parasite, fungus, or yeast, cancer, or an immune disease, including an autoimmune disease.

[0023] In one embodiment, the first antigen binding portion comprises an HIV-1 specific antigen binding portion.

[0024] In one embodiment, the HIV-1 specific antigen binding portion binds to BG505 SOSIP_D368R, 93TH057 gp120, and / or MPER peptides.

[0025] In one embodiment, the HIV-1 specific antigen binding portion comprises an HIV-1 specific antigen binding portion derived from PGDM1400, 10E8v4, and / or N49P7.

[0026] In one embodiment, the fusion protein comprises a first nanocage monomer or a subunit thereof linked to an Fc monomer at the C-terminus of the first nanocage monomer or a subunit thereof and linked to a first biologically active moiety at the N-terminus of the first nanocage monomer or a subunit thereof.

[0027] In one embodiment, the fusion protein comprises a C subunit linked at the C-terminus of the C subunit to an Fc monomer and linked at the N-terminus of the C subunit to a first biologically active moiety.

[0028] In one embodiment the fusion protein is provided in combination with the N subunit or a fusion protein comprising the N subunit.

[0029] In one embodiment, the N subunit is linked to the second biologically active moiety at the N-terminus or C-terminus, preferably at the N-terminus.

[0030] In one embodiment, the second biologically active moiety comprises a second antigen-binding moiety, and where the first biologically active moiety comprises a first antigen-binding moiety, the second antigen-binding moiety may be the same as or different from the first antigen-binding moiety.

[0031] In one embodiment, the second antigen binding portion comprises an antibody or a fragment thereof.

[0032] In one embodiment, the second antigen binding portion comprises a Fab fragment.

[0033] In one embodiment, the antibody or fragment thereof comprises an scFab fragment, an scFv fragment, an sdAb fragment, a VHH domain, or a combination thereof.

[0034] In one embodiment, the antibody or fragment thereof comprises the heavy and / or light chain of a Fab fragment.

[0035] In one embodiment, the N subunit is further linked to an Fc monomer at the N-terminus or C-terminus, preferably the C-terminus, of the N subunit.

[0036] In one embodiment, the Fc monomer is derived from IgG, IgA, IgD, IgM, or IgE, and is preferably human.

[0037] In one embodiment, the Fc monomer is derived from an IgG, such as an IgG1, IgG2, IgG3 or IgG4.

[0038] In one embodiment, the Fc monomer is an IgG1 Fc monomer.

[0039] In one embodiment, the Fc monomer comprises one or more mutations or sets of mutations that modulate the half-life of the fusion protein, for example, from minutes or hours to days, weeks or months.

[0040] In one embodiment, the Fc monomer comprises mutations at one or more of L234, L235, G236, G237, M252, I253, S254, T256, P329, A330, M428, N434, or combinations thereof (numbering according to the EU index), e.g., M428L and N434S ("LS"); M252Y, S254T and T256E ("YTE"); L234A and L235A ("LALA"); I253A; L234A, L235A, and P329G ("LALAP"); G236R; G237A; and / or A330L, or combinations thereof.

[0041] In one embodiment, about 3 to about 100 nanocage monomers, e.g., 24, 32, 48, or 60 nanocage monomers, or about 4 to about 200 nanocage monomer subunits, e.g., 4, 6, 8, 10, 12, 14, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or more nanocage monomer subunits, optionally in combination with one or more whole nanocage monomers, can self-assemble to form a nanocage.

[0042] In one embodiment, the first nanocage monomer or subunit thereof is selected from ferritin, apoferritin, encapsulin, SOR, lumazine synthase, pyruvate dehydrogenase, carboxysome, vault protein, GroEL, heat shock proteins, E2P, MS2 coat protein, fragments thereof, and variants thereof.

[0043] In one embodiment, the first nanocage monomer or a subunit thereof is apoferritin, optionally human apoferritin.

[0044] In one embodiment, the first nanocage monomer or a subunit thereof is an apoferritin light chain, optionally a human apoferritin light chain.

[0045] In one embodiment, the fusion protein comprises a first apoferritin subunit, optionally a first human apoferritin subunit, which is capable of self-assembling with a second apoferritin subunit.

[0046] In one embodiment, the first and second apoferritin monomer subunits interchangeably comprise the "N" and "C" regions of apoferritin.

[0047] In one embodiment, the "N" region of apoferritin comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following: [ka]

[0048] In one embodiment, the "C" region of apoferritin comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following: [ka]

[0049] In one embodiment, the Fc monomer is linked to the first nanocage monomer or a subunit thereof via a linker.

[0050] In one embodiment, the first biologically active moiety is linked to the first nanocage monomer or a subunit thereof via a linker.

[0051] In one embodiment, the second biologically active moiety is linked to the N subunit via a linker.

[0052] In one embodiment, the linker is flexible or rigid and comprises about 1 to about 100 amino acid residues, such as about 1 to about 70 amino acid residues, for example, about 1 to about 30 amino acid residues, for example, about 8 to about 16 amino acid residues.

[0053] In one embodiment, the linker comprises a GS domain.

[0054] In one embodiment, the GS domain comprises GS repeats, GGS repeats, GGGS (SEQ ID NO: 11) repeats and / or GGGGS (SEQ ID NO: 12) repeats, such as 1, 2, 3, 4 or more GGGGS (SEQ ID NO: 12) repeats.

[0055] In one embodiment, the linker comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following: GGGGSGGGGSGGGGSGGGGSGGGGSGG (SEQ ID NO: 4)

[0056] In one embodiment, the linker comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following: GGSGGSGGSGGSGGGSGGSGGSGGSG (SEQ ID NO: 5)

[0057] According to one embodiment, there is provided a nanocage comprising at least one fusion protein as described herein and at least one second nanocage monomer or subunit thereof that self-assembles with the fusion protein.

[0058] In one embodiment, the fusion protein comprises a first nanocage monomer subunit, and a second nanocage monomer or a subunit thereof is a second nanocage monomer subunit, and the second nanocage monomer subunit self-assembles with the fusion protein to form the nanocage monomer.

[0059] In one embodiment, from about 1% to about 100%, e.g., from about 1%, 4%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% to about 4%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, e.g., from about 20% to about 80%, of the nanocage monomers or subunits thereof are comprised within a fusion protein described herein.

[0060] In one aspect, each nanocage monomer or subunit thereof is comprised within a fusion protein as described herein.

[0061] In one embodiment, the nanocage comprises one biologically active moiety or at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different biologically active moieties, for example, 2 or 3 different biologically active moieties.

[0062] In one embodiment, the nanocage is multivalent.

[0063] In one embodiment, the nanocages are multispecific.

[0064] In one embodiment, at least one biologically active moiety decorates the exterior surface of the nanocage and at least one Fc dimer decorates the exterior surface of the nanocage.

[0065] In one embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 biologically active moieties decorate the exterior surface of the nanocage and at least two Fc dimers decorate the exterior surface of the nanocage.

[0066] In one aspect, the nanocage comprises: (a) at least one nanocage monomer fused to a first antigen-binding moiety, such as a Fab; (b) at least one N subunit fused to a second antigen-binding moiety, such as a Fab; (c) at least one C subunit, (i) fused at one end to a third antigen-binding portion, such as a Fab; (ii) at least one C subunit fused at its other end to an Fc monomer.

[0067] In one embodiment, each antigen binding moiety is linked to the N-terminus of a nanocage monomer or subunit thereof, and the Fc monomer is linked to the C-terminus of the C subunit.

[0068] In one embodiment, the antigen-binding portion specifically binds to an antigen associated with a condition preventable and / or treatable by an antibody.

[0069] In one aspect, the antigen is associated with an infectious agent, including a virus, bacterium, parasite, fungus, or yeast, cancer, or an immune disease, including an autoimmune disease.

[0070] In one embodiment, each antigen-binding portion is a different HIV-1 specific Fab.

[0071] In one embodiment, the HIV-1 specific Fab binds to BG505 SOSIP_D368R, 93TH057 gp120, and / or MPER peptides.

[0072] In one embodiment, the HIV-1 specific Fab comprises PGDM1400 Fab, 10E8v4 Fab, and / or N49P7 Fab.

[0073] In one embodiment, the HIV-1 specific Fabs include PGDM1400 Fab, 10E8v4 Fab, and N49P7 Fab.

[0074] In one embodiment, the nanocage comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 antigen binding moieties.

[0075] In one embodiment, the nanocage comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 Fc monomers.

[0076] In one embodiment, the nanocages carry a cargo molecule, such as a pharmaceutical agent, a diagnostic agent, and / or an imaging agent.

[0077] In one embodiment, the cargo molecule is contained internally in the nanocage without being fused to a fusion protein, or the cargo molecule is linked to a fusion protein or is attached to the nanocage either internally or externally.

[0078] In one embodiment, the cargo molecule is a protein, and the cargo molecule is fused to a fusion protein such that it is contained within the nanocage.

[0079] In one embodiment, the cargo molecule comprises a flavin-based fluorescent protein, such as a fluorescent protein, such as GFP, EGFP, ametrine, and / or a LOV protein, such as iLOV.

[0080] In one aspect, the nanocages exhibit pan-viral neutralization breadth.

[0081] In one embodiment, the nanocages have an average IC of less than about 0.1 ug / mL, e.g., less than about 0.01 ug / mL, e.g., less than about 0.001 ug / mL, against a multi-clade panel of 118 pseudoviruses (PsVs). 50 The median values ​​are shown.

[0082] In one embodiment, the nanocages have an average IC of less than about 42 pM, e.g., less than about 4.2 pM, e.g., less than about 0.42 pM, against a multi-clade panel of 118 pseudoviruses (PsVs). 50 The median values ​​are shown.

[0083] In one embodiment, the nanocages have an average IC that is at least about 10, at least about 100, at least about 1000, at least about 10,000, or at least about 100,000 more potent than the corresponding cocktail of bNAbs on a mass and / or molar basis against a multi-clade panel of 118 pseudoviruses (PsVs). 50 The median values ​​are shown.

[0084] According to one aspect, a fusion protein is provided comprising a first nanocage monomer or a subunit thereof linked to an scFc fragment at the C-terminus of the first nanocage monomer or a subunit thereof, wherein multiple fusion proteins are capable of self-assembly to form a nanocage.

[0085] In one embodiment, the subunits include an N subunit or a C subunit that substantially corresponds to the N-terminal half of a nanocage monomer and the C-terminal half of a nanocage monomer, respectively, and the N subunit and C subunit are capable of self-assembly to form the nanocage monomer.

[0086] In one embodiment, the scFc fragment is linked to the N or C subunit at the C-terminus of the N or C subunit.

[0087] In one embodiment, the first nanocage monomer or subunit thereof is further linked to a first biologically active moiety.

[0088] In one embodiment, the first biologically active moiety is linked to the first nanocage monomer or subunit thereof at the N-terminus or C-terminus of the first nanocage monomer or subunit thereof, preferably at the N-terminus.

[0089] In one embodiment, the first biologically active moiety is linked to the N subunit or C subunit at the N-terminus of the N subunit or C subunit, preferably the first biologically active moiety is linked to the C subunit at the N-terminus.

[0090] In one embodiment, the first bioactive moiety decorates the interior and / or exterior surface of the assembled nanocage, preferably the exterior surface.

[0091] In one embodiment, the first biologically active moiety comprises a first antigen-binding moiety.

[0092] In one embodiment, the first antigen binding portion comprises an antibody or a fragment thereof.

[0093] In one embodiment, the first antigen binding portion comprises a Fab fragment.

[0094] In one embodiment, the antibody or fragment thereof comprises an scFab fragment, an scFv fragment, an sdAb fragment, a VHH domain, or a combination thereof.

[0095] In one embodiment, the antibody or fragment thereof comprises the heavy and / or light chain of a Fab fragment.

[0096] In one embodiment, the first antigen-binding portion specifically binds to an antigen associated with a condition preventable and / or treatable by an antibody.

[0097] In one aspect, the antigen is associated with an infectious agent, including a virus, bacterium, parasite, fungus, or yeast, cancer, or an immune disease, including an autoimmune disease.

[0098] In one embodiment, the first antigen binding portion comprises an HIV-1 specific antigen binding portion.

[0099] In one embodiment, the HIV-1 specific antigen binding portion binds to BG505 SOSIP_D368R, 93TH057 gp120, and / or MPER peptides.

[0100] In one embodiment, the HIV-1 specific antigen binding portion comprises an HIV-1 specific antigen binding portion derived from PGDM1400, 10E8v4, and / or N49P7.

[0101] In one embodiment, the fusion protein comprises a first nanocage monomer or a subunit thereof linked to an scFc fragment at the C-terminus of the first nanocage monomer or a subunit thereof and linked to a first biologically active moiety at the N-terminus of the nanocage monomer or a subunit thereof.

[0102] In one embodiment, the fusion protein comprises a C subunit linked at the C-terminus of the C subunit to an scFc fragment and linked at the N-terminus of the C subunit to a first biologically active moiety.

[0103] In one embodiment the fusion protein is provided in combination with the N subunit or a fusion protein comprising the N subunit.

[0104] In one embodiment, the N subunit is linked to the second biologically active moiety at the N-terminus or C-terminus, preferably at the N-terminus.

[0105] In one embodiment, when the second biologically active moiety comprises a second antigen-binding moiety and the first biologically active moiety comprises a first antigen-binding moiety, the second antigen-binding moiety may be the same as or different from the antigen-binding moiety linked to the C subunit.

[0106] In one embodiment, the second antigen binding portion comprises an antibody or a fragment thereof.

[0107] In one embodiment, the second antigen binding portion comprises a Fab fragment.

[0108] In one embodiment, the antibody or fragment thereof comprises an scFab fragment, an scFv fragment, an sdAb fragment, a VHH domain, or a combination thereof.

[0109] In one embodiment, the antibody or fragment thereof comprises the heavy and / or light chain of a Fab fragment.

[0110] In one embodiment, the N subunit is further linked to a second scFc fragment at the N-terminus or C-terminus, preferably the C-terminus, of the N subunit.

[0111] In one embodiment, the scFc fragment is derived from IgG, IgA, IgD, IgM or IgE, and is preferably human.

[0112] In one embodiment, the scFc fragment is derived from an IgG, such as an IgG1, IgG2, IgG3 or IgG4.

[0113] In one embodiment the scFc fragment is an IgG1 scFc fragment.

[0114] In one embodiment, the scFc fragment comprises one or more mutations or sets of mutations that modulate the half-life of said fusion protein, for example from minutes or hours to days, weeks or months.

[0115] In one embodiment, the scFc fragment comprises mutations at one or more of L234, L235, G236, G237, M252, I253, S254, T256, P329, A330, M428, N434, or combinations thereof (numbering according to the EU index), e.g., M428L and N434S ("LS"); M252Y, S254T and T256E ("YTE"); L234A and L235A ("LALA"); I253A; L234A, L235A, and P329G ("LALAP"); G236R; G237A; and / or A330L, or combinations thereof.

[0116] In one embodiment, about 3 to about 100 nanocage monomers, e.g., 24, 32, 48, or 60 nanocage monomers, or about 4 to about 200 nanocage monomer subunits, e.g., 4, 6, 8, 10, 12, 14, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or more nanocage monomer subunits, optionally in combination with one or more whole nanocage monomers, can self-assemble to form a nanocage.

[0117] In one embodiment, the first nanocage monomer or subunit thereof is selected from ferritin, apoferritin, encapsulin, SOR, lumazine synthase, pyruvate dehydrogenase, carboxysome, vault protein, GroEL, heat shock proteins, E2P, MS2 coat protein, fragments thereof, and variants thereof.

[0118] In one embodiment, the first nanocage monomer or a subunit thereof is apoferritin, optionally human apoferritin.

[0119] In one embodiment, the first nanocage monomer or a subunit thereof is an apoferritin light chain, optionally a human apoferritin light chain.

[0120] In one embodiment, the fusion protein comprises a first apoferritin subunit, optionally a first human apoferritin subunit, which is capable of self-assembling with a second apoferritin subunit.

[0121] In one embodiment, the first and second apoferritin monomer subunits interchangeably comprise the "N" and "C" regions of apoferritin.

[0122] In one embodiment, the "N" region of apoferritin comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following: [ka]

[0123] In one embodiment, the "C" region of apoferritin comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following: [ka]

[0124] In one embodiment, the scFc fragment is linked to the first nanocage monomer or a subunit thereof via a linker.

[0125] In one embodiment, the first biologically active moiety is linked to the first nanocage monomer or a subunit thereof via a linker.

[0126] In one embodiment, the second biologically active moiety is linked to the N subunit via a linker.

[0127] In one embodiment, the linker is flexible or rigid and comprises about 1 to about 100 amino acid residues, such as about 1 to about 70 amino acid residues, for example, about 1 to about 30 amino acid residues, for example, about 8 to about 16 amino acid residues.

[0128] In one embodiment, the linker comprises a GS domain.

[0129] In one embodiment, the GS domain comprises GS repeats, GGS repeats, GGGS (SEQ ID NO: 11) repeats and / or GGGGS (SEQ ID NO: 12) repeats, such as 1, 2, 3, 4 or more GGGGS (SEQ ID NO: 12) repeats.

[0130] In one embodiment, the linker comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following: GGGGSGGGGSGGGGSGGGGSGGGGSGG (SEQ ID NO: 4)

[0131] In one embodiment, the linker comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following: GGSGGSGGSGGSGGGSGGSGGSGGSG (SEQ ID NO: 5)

[0132] According to one embodiment, there is provided a nanocage comprising at least one fusion protein as described herein and at least one second nanocage monomer or subunit thereof that self-assembles with the fusion protein.

[0133] In one embodiment, the fusion protein comprises a first nanocage monomer subunit, and a second nanocage monomer or a subunit thereof is a second nanocage monomer subunit, and the second nanocage monomer subunit self-assembles with the fusion protein to form the nanocage monomer.

[0134] In one embodiment, from about 1% to about 100%, e.g., from about 1%, 4%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% to about 4%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, e.g., from about 20% to about 80%, of the nanocage monomers or subunits thereof are comprised within a fusion protein described herein.

[0135] In one aspect, each nanocage monomer or subunit thereof is comprised within a fusion protein as described herein.

[0136] In one embodiment, the nanocage comprises one biologically active moiety or at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different biologically active moieties, for example, 2 or 3 different biologically active moieties.

[0137] In one embodiment, the nanocage is multivalent.

[0138] In one embodiment, the nanocages are multispecific.

[0139] In one embodiment, at least one biologically active moiety decorates the exterior surface of the nanocage and at least one scFc fragment decorates the exterior surface of the nanocage.

[0140] In one embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 biologically active moieties decorate the exterior surface of the nanocage and at least two scFc fragments decorate the exterior surface of the nanocage.

[0141] In one aspect, the nanocage comprises: (a) at least one nanocage monomer fused to a first antigen-binding moiety, such as a Fab; (b) at least one N subunit fused to a second antigen-binding moiety, such as a Fab; (c) at least one C subunit, (i) fused at one end to a third antigen-binding portion, such as a Fab; (ii) at least one C subunit fused to an scFc fragment at its other end.

[0142] In one embodiment, each antigen-binding moiety is linked to the N-terminus of a nanocage monomer or subunit thereof, and the scFc fragment is linked to the C-terminus of the C subunit.

[0143] In one embodiment, the first and / or second antigen binding portion specifically binds to an antigen associated with a condition preventable and / or treatable by an antibody.

[0144] In one aspect, the antigen is associated with an infectious agent, including a virus, bacterium, parasite, fungus, or yeast, cancer, or an immune disease, including an autoimmune disease.

[0145] In one embodiment, each antigen-binding portion is a different HIV-1 specific Fab.

[0146] In one embodiment, the HIV-1 specific Fab binds to BG505 SOSIP_D368R, 93TH057 gp120, and / or MPER peptides.

[0147] In one embodiment, the HIV-1 specific Fab comprises PGDM1400 Fab, 10E8v4 Fab, and / or N49P7 Fab.

[0148] In one embodiment, the HIV-1 specific Fabs include PGDM1400 Fab, 10E8v4 Fab, and N49P7 Fab.

[0149] In one embodiment, the nanocage comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 antigen binding moieties.

[0150] In one embodiment, the nanocage comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 scFc fragments.

[0151] In one embodiment, the nanocages carry a cargo molecule, such as a pharmaceutical agent, a diagnostic agent, and / or an imaging agent.

[0152] In one embodiment, the cargo molecule is contained internally in the nanocage without being fused to a fusion protein, or the cargo molecule is linked to a fusion protein or is attached to the nanocage either internally or externally.

[0153] In one embodiment, the cargo molecule is a protein, and the cargo molecule is fused to a fusion protein such that it is contained within the nanocage.

[0154] In one embodiment, the cargo molecule comprises a flavin-based fluorescent protein, such as a fluorescent protein, such as GFP, EGFP, ametrine, and / or a LOV protein, such as iLOV.

[0155] In one aspect, the nanocages exhibit pan-viral neutralization breadth.

[0156] In one embodiment, the nanocages have an average IC of less than about 0.1 ug / mL, e.g., less than about 0.01 ug / mL, e.g., less than about 0.001 ug / mL, against a multi-clade panel of 118 pseudoviruses (PsVs). 50 The median values ​​are shown.

[0157] In one embodiment, the nanocages have an average IC of less than about 42 pM, e.g., less than about 4.2 pM, e.g., less than about 0.42 pM, against a multi-clade panel of 118 pseudoviruses (PsVs). 50 The median values ​​are shown.

[0158] In one embodiment, the nanocages have an average IC that is at least about 10, at least about 100, at least about 1000, at least about 10,000, or at least about 100,000 more potent than the corresponding cocktail of bNAbs on a mass and / or molar basis against a multi-clade panel of 118 pseudoviruses (PsVs). 50 The median values ​​are shown.

[0159] In one aspect, a therapeutic or prophylactic composition is provided that includes the nanocages described herein.

[0160] In one embodiment, the composition is for treating and / or preventing a condition preventable and / or treatable by an antibody.

[0161] In one aspect, the antigen is associated with an infectious agent, including a virus, bacterium, parasite, fungus, or yeast, cancer, or an immune disease, including an autoimmune disease.

[0162] In one embodiment, the composition is for treating and / or preventing an HIV-1 associated condition.

[0163] According to one aspect, there is provided a nucleic acid molecule encoding a fusion protein described herein.

[0164] According to one aspect, there is provided a vector comprising a nucleic acid molecule described herein.

[0165] According to one aspect, there is provided a host cell comprising a vector described herein and producing a fusion protein described herein.

[0166] According to one embodiment, there is provided a method of treating and / or preventing a condition, comprising administering a nanocage or composition described herein to a subject in need thereof.

[0167] In one embodiment, the condition is an HIV-1 associated condition.

[0168] According to one embodiment, there is provided a use of the nanocages or compositions described herein for treating and / or preventing a condition.

[0169] In one embodiment, the condition is an HIV-1 associated condition.

[0170] According to one aspect, there is provided a nanocage or composition described herein for use in the treatment and / or prevention of a condition.

[0171] In one embodiment, the condition is an HIV-1 associated condition.

[0172] In one embodiment, the nanocage does not include a ferritin heavy chain.

[0173] In one embodiment, the nanocage does not contain any moiety capable of ferroxidase activity.

[0174] In one embodiment, the nanocage comprises at least one biologically active moiety comprising an antibody or an antigen-binding fragment thereof, and the ratio of the total number of biologically active moieties to the number of Fc dimers is 6:1.

[0175] In one embodiment, the nanocage comprises at least one biologically active moiety comprising an antibody or an antigen-binding fragment thereof, and the ratio of the total number of biologically active moieties to the number of scFcs is 3:1.

[0176] In one embodiment, the nanocage comprises at least one biologically active moiety comprising an antibody or an antigen-binding fragment thereof, and the ratio of the total number of biologically active moieties to the number of Fc dimers or scFc's is at least 7:1.

[0177] In one embodiment, the nanocage comprises at least one biologically active moiety comprising an antibody or an antigen-binding fragment thereof, and the ratio of the total number of biologically active moieties to the number of Fc dimers or scFc's is at least 4:1.

[0178] According to one aspect, a fusion protein is provided comprising a nanocage monomer or a subunit thereof linked to an Fc monomer, wherein a plurality of the fusion proteins self-assemble to form a nanocage comprising one or more Fc dimers.

[0179] In one embodiment, the subunits include an N subunit or a C subunit that substantially corresponds to the N-terminal half of a nanocage monomer and the C-terminal half of a nanocage monomer, respectively, and the N subunit and C subunit are capable of self-assembly to form the nanocage monomer.

[0180] In one embodiment, the Fc monomer is linked to the nanocage monomer or subunit thereof at the N-terminus or C-terminus of the nanocage monomer or subunit thereof, preferably at the C-terminus.

[0181] In one embodiment, the Fc monomer is linked to the N or C subunit at the C-terminus of the N or C subunit, preferably the Fc monomer is linked to the C subunit at the C-terminus.

[0182] In one embodiment, the nanocage monomer or subunit thereof is further linked to a biologically active moiety.

[0183] In one embodiment, the biologically active moiety is linked to the nanocage monomer or subunit thereof at the N-terminus or C-terminus of the nanocage monomer or subunit thereof, preferably at the N-terminus.

[0184] In one embodiment, the biologically active moiety is linked to the N or C subunit at the N-terminus of the N or C subunit, preferably the biologically active moiety is linked to the C subunit at the N-terminus.

[0185] In one embodiment, the antigen binding moieties decorate the interior and / or exterior surface of the assembled nanocage, preferably the exterior surface.

[0186] In one embodiment, the biologically active portion comprises an antigen-binding portion.

[0187] In one embodiment, the antigen binding portion comprises an antibody or a fragment thereof.

[0188] In one embodiment, the antigen binding portion comprises a Fab fragment.

[0189] In one embodiment, the antibody or fragment thereof comprises an scFab fragment, an scFv fragment, an sdAb fragment, a VHH domain, or a combination thereof.

[0190] In one embodiment, the antibody or fragment thereof comprises the heavy and / or light chain of a Fab fragment.

[0191] In one embodiment, the antigen-binding portion specifically binds to an antigen associated with a condition preventable and / or treatable by an antibody.

[0192] In one aspect, the antigen is associated with an infectious agent, including a virus, bacterium, parasite, fungus, or yeast, cancer, or an immune disease, including an autoimmune disease.

[0193] In one embodiment, the antigen binding portion comprises an HIV-1 antigen binding portion.

[0194] In one embodiment, the HIV-1 specific antigen binding portion binds to the BG505 SOSIP D368R, 93TH057 gp120, and / or MPER peptide.

[0195] In one embodiment, the HIV-1 antigen binding portion comprises PGDM1400, 10E8v4, and / or N49P7.

[0196] In one embodiment, the fusion protein comprises a nanocage monomer linked at the C-terminus of the nanocage monomer to an Fc monomer and linked to a biologically active moiety at the N-terminus of the nanocage monomer.

[0197] In one embodiment, the fusion protein comprises a C subunit linked at the C-terminus of the C subunit to an Fc monomer and linked at the N-terminus of the C subunit to a biologically active moiety.

[0198] In one embodiment, the C subunit is provided in combination with an N subunit.

[0199] In one embodiment, the N subunit is linked to the biologically active moiety at the N-terminus or C-terminus, preferably at the N-terminus.

[0200] In one embodiment, the biologically active portion comprises an antigen binding portion which may be the same as or different from the antigen binding portion linked to the C subunit.

[0201] In one embodiment, the antigen binding portion comprises an antibody or a fragment thereof.

[0202] In one embodiment, the antigen binding portion comprises a Fab fragment.

[0203] In one embodiment, the antibody or fragment thereof comprises an scFab fragment, an scFv fragment, an sdAb fragment, a VHH domain, or a combination thereof.

[0204] In one embodiment, the antibody or fragment thereof comprises the heavy and / or light chain of a Fab fragment.

[0205] In one embodiment, the N subunit is further linked to an Fc monomer at the N-terminus or C-terminus, preferably the C-terminus, of the N subunit.

[0206] In one embodiment, the Fc monomer is derived from IgG, IgA, IgD, IgM, or IgE, and is preferably human.

[0207] In one embodiment, the Fc monomer is derived from an IgG, such as an IgG1, IgG2, IgG3 or IgG4.

[0208] In one embodiment, the Fc monomer is an IgG1 Fc monomer.

[0209] In one embodiment, the Fc monomer comprises one or more mutations or sets of mutations that modulate the half-life of the fusion protein, for example, from minutes or hours to days, weeks or months.

[0210] In one embodiment, the Fc monomer comprises mutations at one or more of L234, L235, G236, G237, M252, I253, S254, T256, P329, A330, M428, N434, or combinations thereof (numbering according to the EU index), e.g., M428L and N434S ("LS"); M252Y, S254T and T256E ("YTE"); L234A and L235A ("LALA"); I253A, and / or L234A, L235A, and P329G ("LALAP"), G236R, G237A, A330L, or combinations thereof.

[0211] In one embodiment, about 3 to about 100 nanocage monomers, e.g., 24, 32, 48, or 60 monomers, or about 4 to about 200 nanocage monomer subunits, e.g., 4, 6, 8, 10, 12, 14, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or more nanocage monomer subunits, optionally in combination with one or more whole nanocage monomers, can self-assemble to form a nanocage.

[0212] In one embodiment, the nanocage monomer or subunit thereof is selected from ferritin, apoferritin, encapsulin, SOR, lumazine synthase, pyruvate dehydrogenase, carboxysome, vault protein, GroEL, heat shock proteins, E2P, MS2 coat protein, fragments thereof, and variants thereof.

[0213] In one embodiment, the nanocage monomer or a subunit thereof is apoferritin, optionally human apoferritin.

[0214] In one aspect, the nanocage monomer or a subunit thereof is an apoferritin light chain, optionally a human apoferritin light chain.

[0215] In one embodiment, the fusion protein comprises a first apoferritin subunit, optionally a first human apoferritin subunit, which is capable of self-assembling with a second apoferritin subunit.

[0216] In one embodiment, the first and second apoferritin monomer subunits interchangeably comprise the "N" and "C" regions of apoferritin.

[0217] In one embodiment, the "N" region of apoferritin comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following: MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEW

[0218] In one embodiment, the "C" region of apoferritin comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following: GKTPDAMKAAMALEKKLNQALLLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD

[0219] In one embodiment, the Fc monomer and / or the biologically active moiety is linked to the nanocage monomer or subunit thereof via a linker.

[0220] In one embodiment, the linker is flexible or rigid and comprises about 1 to about 100 amino acid residues, such as about 1 to about 70 amino acid residues, for example, about 1 to about 30 amino acid residues, for example, about 8 to about 16 amino acid residues.

[0221] In one embodiment, the linker comprises a GS domain.

[0222] In one embodiment the GS domain comprises GS repeats, GGS repeats, GGGS repeats and / or GGGGS repeats, for example 1, 2, 3, 4 or more GGGGS repeats.

[0223] In one embodiment, the linker comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following: GGGGSGGGGSGGGGSGGGGSGGGGSGG

[0224] In one embodiment, the linker comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following: GGSGGSGGSGGSGGSGGSGGSGGSGGSG

[0225] According to one embodiment, there is provided a nanocage comprising at least one fusion protein as described herein and at least one second nanocage monomer or subunit thereof that self-assembles with the fusion protein.

[0226] In one embodiment, the fusion protein comprises a first nanocage monomer subunit, and a second nanocage monomer or a subunit thereof is a second nanocage monomer subunit, and the second nanocage monomer subunit self-assembles with the fusion protein to form the nanocage monomer.

[0227] In one aspect, each nanocage monomer comprises a fusion protein described herein.

[0228] In one embodiment, about 1% to about 100%, e.g., about 1%, 4%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% to about 4%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, e.g., about 20% to about 80%, of the nanocage monomers or subunits thereof comprise a fusion protein described herein.

[0229] In one embodiment, the nanocage comprises one biologically active moiety or at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different biologically active moieties, for example, 2 or 3 different biologically active moieties.

[0230] In one embodiment, the nanocage is multivalent.

[0231] In one embodiment, the nanocages are multispecific.

[0232] In one embodiment, at least one biologically active moiety decorates the exterior surface of the nanocage and at least one Fc dimer decorates the exterior surface of the nanocage.

[0233] In one embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 biologically active moieties decorate the exterior surface of the nanocage and at least two Fc dimers decorate the exterior surface of the nanocage.

[0234] In one embodiment, the nanocage comprises at least one nanocage monomer fused to a first antigen-binding moiety, such as a Fab, at least one N subunit fused to a second antigen-binding moiety, such as a Fab, and at least one C subunit fused at one end to a third antigen-binding moiety, such as a Fab, and at the other end to an Fc monomer.

[0235] In one embodiment, each antigen binding moiety is linked to the N-terminus of a nanocage monomer or subunit thereof, and the Fc monomer is linked to the C-terminus of the C subunit.

[0236] In one embodiment, the antigen-binding portion specifically binds to an antigen associated with a condition preventable and / or treatable by an antibody.

[0237] In one aspect, the antigen is associated with an infectious agent, including a virus, bacterium, parasite, fungus, or yeast, cancer, or an immune disease, including an autoimmune disease.

[0238] In one embodiment, each antigen-binding portion is a different HIV-1 specific Fab.

[0239] In one embodiment, the HIV-1 specific Fab binds to the BG505 SOSIP D368R, 93TH057 gp120, and / or MPER peptides.

[0240] In one embodiment, the HIV-1 specific Fab comprises PGDM1400, 10E8v4, and / or N49P7.

[0241] In one embodiment, the nanocages include PGDM1400, 10E8v4 and N49P7.

[0242] In one embodiment, the nanocage comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 antigen binding moieties.

[0243] In one embodiment, the nanocage comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 Fc monomers.

[0244] In one embodiment, the nanocages carry a cargo molecule, such as a pharmaceutical agent, a diagnostic agent, and / or an imaging agent.

[0245] In one embodiment, the cargo molecule is contained internally in the nanocage without being fused to a fusion protein, or the cargo molecule is linked to a fusion protein or is attached to the nanocage either internally or externally.

[0246] In one embodiment, the cargo molecule is a protein, and the cargo molecule is fused to a fusion protein such that it is contained within the nanocage.

[0247] In one embodiment, the cargo molecule comprises a flavin-based fluorescent protein, such as a fluorescent protein, such as GFP, EGFP, ametrine, and / or a LOV protein, such as iLOV.

[0248] In one aspect, the nanocages exhibit pan-viral neutralization breadth.

[0249] In one embodiment, the nanocages have an average IC of less than about 0.1 ug / mL, e.g., less than about 0.01 ug / mL, e.g., less than about 0.001 ug / mL, against a multi-clade panel of 118 pseudoviruses (PsVs). 50 The median values ​​are shown.

[0250] In one embodiment, the nanocages have an average IC of less than about 42 pM, e.g., less than about 4.2 pM, e.g., less than about 0.42 pM, against a multi-clade panel of 118 pseudoviruses (PsVs). 50 The median values ​​are shown.

[0251] In one embodiment, the nanocages have an average IC that is at least about 10, at least about 100, at least about 1000, at least about 10,000, or at least about 100,000 more potent than the corresponding cocktail of bNAbs on a mass and / or molar basis against a multi-clade panel of 118 pseudoviruses (PsVs). 50 The median values ​​are shown.

[0252] According to one aspect, a fusion protein is provided comprising a nanocage monomer or a subunit thereof linked to an scFc fragment at the C-terminus of the nanocage monomer or a subunit thereof, wherein multiple fusion proteins self-assemble to form a nanocage.

[0253] In one embodiment, the subunits include an N subunit or a C subunit that substantially corresponds to the N-terminal half of a nanocage monomer and the C-terminal half of a nanocage monomer, respectively, and the N subunit and C subunit are capable of self-assembly to form the nanocage monomer.

[0254] In one embodiment, the scFc fragment is linked to the nanocage monomer at the C-terminus of the nanocage monomer.

[0255] In one embodiment, the scFc fragment is linked to the N or C subunit at the C-terminus of the N or C subunit.

[0256] In one embodiment, the nanocage monomer or subunit thereof is further linked to a biologically active moiety.

[0257] In one embodiment, the biologically active moiety is linked to the nanocage monomer or subunit thereof at the N-terminus or C-terminus of the nanocage monomer or subunit thereof, preferably at the N-terminus.

[0258] In one embodiment, the biologically active moiety is linked to the N or C subunit at the N-terminus of the N or C subunit, preferably the biologically active moiety is linked to the C subunit at the N-terminus.

[0259] In one embodiment, the antigen binding moieties decorate the interior and / or exterior surface of the assembled nanocage, preferably the exterior surface.

[0260] In one embodiment, the biologically active portion comprises an antigen-binding portion.

[0261] In one embodiment, the antigen binding portion comprises an antibody or a fragment thereof.

[0262] In one embodiment, the antigen binding portion comprises a Fab fragment.

[0263] In one embodiment, the antibody or fragment thereof comprises an scFab fragment, an scFv fragment, an sdAb fragment, a VHH domain, or a combination thereof.

[0264] In one embodiment, the antibody or fragment thereof comprises the heavy and / or light chain of a Fab fragment.

[0265] In one embodiment, the antigen-binding portion specifically binds to an antigen associated with a condition preventable and / or treatable by an antibody.

[0266] In one aspect, the antigen is associated with an infectious agent, including a virus, bacterium, parasite, fungus, or yeast, cancer, or an immune disease, including an autoimmune disease.

[0267] In one embodiment, the antigen binding portion comprises an HIV-1 antigen binding portion.

[0268] In one embodiment, the HIV-1 specific antigen binding portion binds to the BG505 SOSIP D368R, 93TH057 gp120, and / or MPER peptide.

[0269] In one embodiment, the HIV-1 antigen binding portion comprises PGDM1400, 10E8v4, and / or N49P7.

[0270] In one embodiment, the fusion protein comprises a nanocage monomer linked to an scFc fragment at the C-terminus of the nanocage monomer and linked to a biologically active moiety at the N-terminus of the nanocage monomer.

[0271] In one embodiment, the fusion protein comprises a C subunit linked to an scFc fragment at the C-terminus of the C subunit and linked to a biologically active moiety at the N-terminus of the C subunit.

[0272] In one embodiment, the C subunit is provided in combination with an N subunit.

[0273] In one embodiment, the N subunit is linked to the biologically active moiety at the N-terminus or C-terminus, preferably at the N-terminus.

[0274] In one embodiment, the biologically active portion comprises an antigen binding portion which may be the same as or different from the antigen binding portion linked to the C subunit.

[0275] In one embodiment, the antigen binding portion comprises an antibody or a fragment thereof.

[0276] In one embodiment, the antigen binding portion comprises a Fab fragment.

[0277] In one embodiment, the antibody or fragment thereof comprises an scFab fragment, an scFv fragment, an sdAb fragment, a VHH domain, or a combination thereof.

[0278] In one embodiment, the antibody or fragment thereof comprises the heavy and / or light chain of a Fab fragment.

[0279] In one embodiment, the N subunit is further linked to an scFc fragment at the N-terminus or C-terminus, preferably the C-terminus, of the N subunit.

[0280] In one embodiment, the scFc fragment is derived from IgG, IgA, IgD, IgM or IgE, and is preferably human.

[0281] In one embodiment, the scFc fragment is derived from an IgG, such as an IgG1, IgG2, IgG3 or IgG4.

[0282] In one embodiment the scFc fragment is an IgG1 scFc fragment.

[0283] In one embodiment, the scFc fragment comprises one or more mutations or sets of mutations that modulate the half-life of said fusion protein, for example from minutes or hours to days, weeks or months.

[0284] In one embodiment, the scFc fragment comprises mutations at one or more of L234, L235, G236, G237, M252, I253, S254, T256, P329, A330, M428, N434, or combinations thereof (numbering according to the EU index), e.g., M428L and N434S ("LS"); M252Y, S254T and T256E ("YTE"); L234A and L235A ("LALA"); I253A, and / or L234A, L235A, and P329G ("LALAP"), G236R, G237A, A330L, or combinations thereof.

[0285] In one embodiment, about 3 to about 100 nanocage monomers, e.g., 24, 32, 48, or 60 monomers, or about 4 to about 200 nanocage monomer subunits, e.g., 4, 6, 8, 10, 12, 14, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or more nanocage monomer subunits, optionally in combination with one or more whole nanocage monomers, can self-assemble to form a nanocage.

[0286] In one embodiment, the nanocage monomer or subunit thereof is selected from ferritin, apoferritin, encapsulin, SOR, lumazine synthase, pyruvate dehydrogenase, carboxysome, vault protein, GroEL, heat shock proteins, E2P, MS2 coat protein, fragments thereof, and variants thereof.

[0287] In one embodiment, the nanocage monomer or a subunit thereof is apoferritin, optionally human apoferritin.

[0288] In one aspect, the nanocage monomer or a subunit thereof is an apoferritin light chain, optionally a human apoferritin light chain.

[0289] In one embodiment, the fusion protein comprises a first apoferritin subunit, optionally a first human apoferritin subunit, which is capable of self-assembling with a second apoferritin subunit.

[0290] In one embodiment, the first and second apoferritin monomer subunits interchangeably comprise the "N" and "C" regions of apoferritin.

[0291] In one embodiment, the "N" region of apoferritin comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following: MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEW

[0292] In one embodiment, the "C" region of apoferritin comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following: GKTPDAMKAAMALEKKLNQALLLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD

[0293] In one embodiment, the scFc fragment and / or the biologically active moiety is linked to the nanocage monomer or subunit thereof via a linker.

[0294] In one embodiment, the linker is flexible or rigid and comprises about 1 to about 100 amino acid residues, such as about 1 to about 70 amino acid residues, for example, about 1 to about 30 amino acid residues, for example, about 8 to about 16 amino acid residues.

[0295] In one embodiment, the linker comprises a GS domain.

[0296] In one embodiment the GS domain comprises GS repeats, GGS repeats, GGGS repeats and / or GGGGS repeats, for example 1, 2, 3, 4 or more GGGGS repeats.

[0297] In one embodiment, the linker comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following: GGGGSGGGGSGGGGSGGGGSGGGGSGG

[0298] In one embodiment, the linker comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following: GGSGGSGGSGGSGGSGGSGGSGGSGGSG

[0299] According to one embodiment, there is provided a nanocage comprising at least one fusion protein as described herein and at least one second nanocage monomer or subunit thereof that self-assembles with the fusion protein.

[0300] In one embodiment, the fusion protein comprises a first nanocage monomer subunit, and a second nanocage monomer or a subunit thereof is a second nanocage monomer subunit, and the second nanocage monomer subunit self-assembles with the fusion protein to form the nanocage monomer.

[0301] In one aspect, each nanocage monomer comprises a fusion protein described herein.

[0302] In one embodiment, about 1% to about 100%, e.g., about 1%, 4%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% to about 4%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, e.g., about 20% to about 80%, of the nanocage monomers or subunits thereof comprise a fusion protein described herein.

[0303] In one embodiment, the nanocage comprises one biologically active moiety or at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different biologically active moieties, for example, 2 or 3 different biologically active moieties.

[0304] In one embodiment, the nanocage is multivalent.

[0305] In one embodiment, the nanocages are multispecific.

[0306] In one embodiment, at least one biologically active moiety decorates the exterior surface of the nanocage and at least one scFc fragment decorates the exterior surface of the nanocage.

[0307] In one embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 biologically active moieties decorate the exterior surface of the nanocage and at least two scFc fragments decorate the exterior surface of the nanocage.

[0308] In one embodiment, the nanocage comprises at least one nanocage monomer fused to a first antigen-binding moiety, such as an scFab, at least one N subunit fused to a second antigen-binding moiety, such as an scFab, and at least one C subunit fused at one end to a third antigen-binding moiety, such as an scFab, and at the other end to an scFc fragment.

[0309] In one embodiment, each antigen binding moiety is linked to the N-terminus of a nanocage monomer or subunit thereof, and the scFc fragment is linked to the C-terminus of the C subunit.

[0310] In one embodiment, the antigen-binding portion specifically binds to an antigen associated with a condition preventable and / or treatable by an antibody.

[0311] In one aspect, the antigen is associated with an infectious agent, including a virus, bacterium, parasite, fungus, or yeast, cancer, or an immune disease, including an autoimmune disease.

[0312] In one embodiment, each antigen-binding portion is a different HIV-1 specific Fab.

[0313] In one embodiment, the HIV-1 specific Fab binds to the BG505 SOSIP D368R, 93TH057 gp120, and / or MPER peptides.

[0314] In one embodiment, the HIV-1 specific Fab comprises PGDM1400, 10E8v4, and / or N49P7.

[0315] In one embodiment, the nanocages include PGDM1400, 10E8v4 and N49P7.

[0316] In one embodiment, the nanocage comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 antigen binding moieties.

[0317] In one embodiment, the nanocage comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 Fc monomers.

[0318] In one embodiment, the nanocages carry a cargo molecule, such as a pharmaceutical agent, a diagnostic agent, and / or an imaging agent.

[0319] In one embodiment, the cargo molecule is contained internally in the nanocage without being fused to a fusion protein, or the cargo molecule is linked to a fusion protein or is attached to the nanocage either internally or externally.

[0320] In one embodiment, the cargo molecule is a protein, and the cargo molecule is fused to a fusion protein such that it is contained within the nanocage.

[0321] In one embodiment, the cargo molecule comprises a flavin-based fluorescent protein, such as a fluorescent protein, such as GFP, EGFP, ametrine, and / or a LOV protein, such as iLOV.

[0322] In one aspect, the nanocages exhibit pan-viral neutralization breadth.

[0323] In one embodiment, the nanocages have an average IC of less than about 0.1 ug / mL, e.g., less than about 0.01 ug / mL, e.g., less than about 0.001 ug / mL, against a multi-clade panel of 118 pseudoviruses (PsVs). 50 The median values ​​are shown.

[0324] In one embodiment, the nanocages have an average IC of less than about 42 pM, e.g., less than about 4.2 pM, e.g., less than about 0.42 pM, against a multi-clade panel of 118 pseudoviruses (PsVs). 50 The median values ​​are shown.

[0325] In one embodiment, the nanocages have an average IC that is at least about 10, at least about 100, at least about 1000, at least about 10,000, or at least about 100,000 more potent than the corresponding cocktail of bNAbs on a mass and / or molar basis against a multi-clade panel of 118 pseudoviruses (PsVs). 50 The median values ​​are shown.

[0326] According to one aspect, there is provided a therapeutic or prophylactic composition comprising the nanocages described herein.

[0327] In one embodiment, the composition is for treating and / or preventing a condition preventable and / or treatable by an antibody.

[0328] In one aspect, the antigen is associated with an infectious agent, including a virus, bacterium, parasite, fungus, or yeast, cancer, or an immune disease, including an autoimmune disease.

[0329] In one embodiment, the composition is for treating and / or preventing HIV-1.

[0330] According to one aspect, there is provided a nucleic acid molecule encoding a fusion protein described herein.

[0331] According to one aspect, there is provided a vector comprising a nucleic acid molecule described herein.

[0332] According to one aspect, there is provided a host cell comprising a vector described herein and producing a fusion protein described herein.

[0333] According to one embodiment, there is provided a method of treating and / or preventing a condition comprising administering a nanocage or composition described herein.

[0334] In one embodiment, the condition is HIV-1.

[0335] According to one embodiment, there is provided a use of the nanocages or compositions described herein for treating and / or preventing a condition.

[0336] In one embodiment, the condition is HIV-1.

[0337] In one embodiment, the use is for treating and / or preventing a condition.

[0338] In one embodiment, the condition is HIV-1.

[0339] The novel features of the present invention will become apparent to those skilled in the art upon consideration of the following detailed description of the invention. It should be understood, however, that the detailed description of the invention and the specific examples presented, while indicating particular embodiments of the invention, are provided for illustrative purposes only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the detailed description of the invention and the following claims.

[0340] The invention will be better understood from the following description taken in conjunction with the drawings, in which: [Brief description of the drawings]

[0341] [Figure 1]HIV-1 bNAb multimerization drives increased neutralization potency. Schematic of self-assembly of (a) apoferritin (24 subunits) and (b) single-chain Fab-apoferritin fusion. The Fab light chain (LC) and heavy chain (HC) are shown in light and dark pink, respectively, and are linked to the N-terminus of the light chain of human apoferritin (grey) via a GGS-like flexible linker (dark). (c) Schematic of different Fab densities shown on human apoferritin. Co-transfection of a plasmid encoding scFab-human apoferritin with unconjugated apoferritin in ratios of 1:4 (dark yellow), 1:1 (black), 4:1 (blue) and 1:0 (red) resulted in molecules with different scFab titers, as confirmed by earlier elution volumes and less unconjugated apoferritin in size exclusion chromatography and SDS-PAGE. Negative staining electron micrographs of samples with the lowest and highest valence are shown. (d) Avidity effect on neutralization of five bNAbs against a panel of five PsVs (PVO.04, JRCSF, BG505 T332N, THRO4156.18 and t278-50). For the following cases: N49P7-t278-50, VRC01-T278-50 and 10-1074-THRO4156.18, IC50 fold increase analysis was omitted due to neutralization resistance. Fold increase in potency was calculated as IC50 (μg / mL) of parental IgG divided by IC50 (μg / mL) of Fab-apoferritin fusion. Bars (±SD) represent mean values ​​from n=3 biologically independent samples.

[0342] [Diagram 2]Characterization of scFab-apoferritin fusions. (a) SDS-PAGE bands corresponding to scFab-apoferritin and unconjugated apoferritin were quantified by densitometry using ImageJ software (rsb.info.nih.gov / ij / ). Intensity plots of the bands in each lane (yellow boxes) are shown (b). (c) The approximate number of scFabs displayed on the particles was calculated as follows: intensity of scFab band / total intensity, and compared with the theoretical number inferred from the DNA ratio used for co-transfection.

[0343] [Figure 3-1] Design, assembly and biophysical characterization of the 32-N Multabody. (a) Schematic of the human apoferritin split design that promotes heterodimerization of scFab-human apoferritin subunits. Based on the hetero-oligomerization required to drive self-assembly, purification of the Multabody with four components: Protein A (Fc-bound) and Protein L (PGDM1400-bound) can be achieved by a two-step purification. (b) Negative stain electron micrograph of 32-N. (c) Size-exclusion chromatography along with multi-angle light scattering of the 24-mer PGDM1400 (black) and 32-N Multabody (dark magenta). The molar mass of each elution peak (lines below UV absorbance) indicates that the sample is monodisperse and that the size of the Multabody is larger than the 24-mer format due to the additional antibody fragment in this design. (d) Comparison of Tm and Tagg temperatures of 32-N, 12-mer Multabody and parental IgG. (e) Concentration response curves for binding of 32-N Multabody to multiple epitopes. PGDM1400, N49P7 and 10E8v4 binding sites are colored red, blue and pink, respectively, on the surface representation of HIV Env (grey). Functional binding of Fc to human FcRn was tested by measuring binding at pH 7.5 and pH 5.6. BG505 SOSIP.664_D368R trimer and 93TH057 gp120 monomer were selected as epitope-specific ligands for PGDM1400 and N49P7, respectively. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Diagram 3-4] Same as above. [Figure 3-5] Same as above.

[0344] [Figure 4] Binding profiles of bNAbs PGDM1400, 10E8v4 and N49P7. BLI response curves of IgG binding to 93TH057 gp120, BG505 SOSIP.664_D368R and MPER His-tagged antigens immobilized on Ni-NTA biosensor.

[0345] [Diagram 5] Neutralization properties of the 32-N Multabody against the 14-pseudovirus panel. (a) Breadth and median IC50 (μg / mL) of the 32-N Multabody (red diamond), parental bNAb (black circle) and IgG combination (blue triangle). The IgG cocktail contained each parental antibody (i.e., PGDM1400, N497 and 10E8v4) as well as the Multabody samples. The 14-PsV panel was selected based on sensitivity and resistance to parental IgG. (b) IC50 values ​​(μg / mL) against each PsV variant.

[0346] [Figure 6] Schematic of the second generation Multabody version 2 (MB.v2; see Figure 6, bottom). A single-chain Fc region (green) was fused to the late C-terminus of apoferritin in a split Multabody design. This modification inverts the orientation of the Fc in the Multabody with respect to the orientation adopted in the previous version (MB.v1; see Figure 6, top).

[0347] [Figure 7]Schematic diagram of the second generation Multabody version 3 (MB.v3). (a) The single-chain Fc region (green) fused to the C-terminus of the apoferritin protomer (specifically the C-half of ferritin) is reverted to a monomeric Fc chain. (b) Folding of the functional Fc fragment results from homodimerization of two Fc chains fused to the C-terminus of two independent ferritin subunits at the 4-fold symmetry axis of the apoferritin nanocage.

[0348] [Figure 8-1] Characterization of optimized Multabody versions. (a) Negative stain electron micrographs of 32-N MB.v2 and 32-N MB.v3. Comparison of the three different versions' stability over time under Tagg temperature (b) and temperature stress conditions (c). Functional neutralization assay to evaluate active Multabody before and after accelerated thermostability assay. Concentration-response curves for binding to multiple epitopes (d) and Fc receptors (e-f). 32-N MB.v3 with the Fc half-life extension mutation LS was incorporated into the assay. [Figure 8-2] Same as above. [Figure 8-3] Same as above. [Figure 8-4] Same as above. [Figure 8-5] Same as above.

[0349] [Figure 9-1] Neutralization properties of 32-N Multabody versions 2 and 3. (a) Comparison of bandwidth and median IC50 (μg / mL) of the 32-N Multabody versions (red diamonds of different shades) and the IgG mixture (blue triangles) against a 25-PsV panel containing 44% of PsV mutants resistant to PGDM1400 neutralization. IC50 values ​​(μg / mL) against each PsV are shown. (b) Strength-bandwidth curves comparing the three 32-N Multabody versions as well as the parental IgG and IgG mixture in gram and molar amounts against an extended multiclade panel of 118 PsVs. [Figure 9-2] Same as above.

[0350] [Figure 10] In vivo exposure of 32-N Multabody v3 in immunodeficient mice. Three female NOD / Shi-scid / IL-2Rγnull immunodeficient mouse strain (NCG) mice per group were used to evaluate multabody circulation in the blood following subcutaneous administration of 5 mg / kg 32-N MB.v3 with Fc modification (LS mutation) and parental IgG mix (for comparison). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0351] Detailed Description of Specific Embodiments Described herein are nanocage platforms formed from multiple self-assembled nanocage monomers, referred to as "Multabody" platforms, that allow for tuning of the binding and pharmacokinetic characteristics of the nanocage, for example, by controlling the number or ratio of fusion molecules within the nanocage.

[0352] Each monomer of the nanocage may be expressed independently as is or fused to another moiety, such as an scFc fragment or Fc monomer. To obtain a nanocage that is multivalent and / or multispecific, a biologically active moiety, such as an antibody or fragment thereof, e.g., a Fab fragment, may be fused to one or more or all of the monomers. Additionally, each monomer may be independently split into subunits, such that each subunit contains approximately half of a monomer, providing an additional level or source of control and indication of the scFc fragment, Fc monomer, and / or biologically active moiety.

[0353] Here, it is demonstrated that multimerization of HIV-1 targeting Fabs on the Multabody platform in combination with Fc monomers or scFc fragments results in significantly greater ability to neutralize the HIV-1 virus. As demonstrated herein, the increase in potency and pan-neutralization capacity of Multabody is not simply increased at a 1:1 ratio, but a doubling of valency results in a doubling of potency. Instead, potency is increased synergistically, by at least 10-fold in embodiments, and in some embodiments, much greater. The therapeutic potential of this engineered molecule was demonstrated using Fab specificity resulting in MB potency and a 25-PsV panel containing 44% PsV mutants resistant to an extended multi-clade panel of 118 PsVs. definition

[0354] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Definitions of general terms in molecular biology can be found, for example, in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference (published by: VCH Publishers, Inc., 1995) (ISBN 1-56081-569-8).Although any method and material similar or equivalent to those described herein can be used to carry out the test of the present invention, typical materials and methods are described herein. In describing and claiming the present invention, the following terminology is used.

[0355] It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. Numerous patent applications, patents, and publications are referenced herein to aid in the understanding of the described embodiments. Each of these references is incorporated herein by reference in its entirety.

[0356] In understanding the scope of this application, the articles "a," "an," "the," and "said" are intended to mean that there are one or more elements. Furthermore, as used herein, the term "comprising" and its derivatives are intended to be open-ended terms that specify the presence of stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers, and / or steps. The above also applies to words having similar meanings, such as the terms "including," "having," and their derivatives.

[0357] Any embodiment described as "comprising" a particular component may also be "consist of" or "consist essentially of," with "consisting of" having a closed-ended or restrictive meaning, and "consisting essentially of" being understood to mean including the specified component, but excluding other components, except for materials present as impurities, unavoidable materials present as a result of the process used to provide the component, and components added for purposes other than achieving the technical effect of the invention. For example, a composition defined using the phrase "consisting essentially of" includes any known acceptable additives, excipients, diluents, carriers, and the like. Typically, a composition consisting essentially of a set of components contains less than 5% by weight, typically less than 3% by weight, more typically less than 1% by weight, and even more typically less than 0.1% by weight of an unspecified component.

[0358] It will be understood that any component defined as included herein may be expressly excluded from the claimed invention by condition or negative limitation. For example, in some embodiments, the nanocages and / or fusion proteins described herein may exclude ferritin heavy chains and / or may exclude iron binding components.

[0359] Moreover, all ranges given herein include the range endpoints and any intermediate range points, whether or not expressly stated.

[0360] As used herein, terms of degree such as "substantially," "about," and "approximately" refer to a reasonable amount of deviation from the modified term so that the end result is not materially altered. These terms of degree should be interpreted to include a deviation of at least ±5% of the modified term (unless this deviation negates the meaning of the word it modifies).

[0361] The abbreviation "eg" is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the terms "for example" or "such as." The word "or" is intended to include "and" unless the context clearly dictates otherwise.

[0362] The term "subject" as used herein refers to any member of the animal kingdom, typically a mammal. The term "mammal" refers to any animal classified as a mammal, including humans, other higher primates, domestic and farm animals, and zoo, sport, or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. Typically, the mammal is a human.

[0363] The terms "protein nanoparticle," "nanocage," and "multabody" are used interchangeably herein to refer to protein-based polyhedral shaped structures made from multiple nanocage monomers, each of which is composed of single or multiple features of a protein or polypeptide (e.g., a glycosylated polypeptide), and optionally, nucleic acids, prosthetic groups, organic and inorganic compounds. Non-limiting examples of protein nanoparticles include ferritin nanoparticles (see, e.g., Zhang, Y. Int. J. Mol. Sci., 12:5406-5421, 2011, which is incorporated herein by reference), encapsulin nanoparticles (see, e.g., Sutter et al., Nature Struct, and Mol. Biol., 15:939-947, 2008, which is incorporated herein by reference), sulfur oxygenase reductase (SOR) nanoparticles (see, e.g., Urich et al., Science, 311:996-1000, 2006, which is incorporated herein by reference), lumazine synthase nanoparticles (see, e.g., Zhang et al., J. Mol. Biol., 306:1099-1114, 2001), or pyruvate dehydrogenase nanoparticles (see, e.g., Izard et al., PNAS 2002, which is incorporated herein by reference) that are encapsulated in nanoparticles. 96:1240-1245,1999). Ferritin, apoferritin, encapsulin, SOR, lumazine synthase, and pyruvate dehydrogenase are monomeric proteins that in some cases self-assemble into globular protein complexes consisting of 24, 60, 24, 60, and 60 protein subunits, respectively. Ferritin and apoferritin are generally referred to interchangeably herein, and both are understood to be suitable for use in the fusion proteins, nanocages, and methods described herein. Carboxysomes, vault proteins, GroEL, heat shock proteins, E2P, and MS2 coat proteins also generate nanocages and are contemplated for use herein. Additionally, fully or partially synthetic self-assembling monomers are also contemplated for use herein.

[0364] It will be understood that each nanocage monomer can be split into two or more subunits that self-assemble into a functional nanocage monomer. For example, ferritin or apoferritin can be split into N and C subunits, such as N and C subunits obtained by substantially splitting full-length ferritin into halves, so that each subunit can be separately bound to a different scFc fragment or Fc monomer or bioactive moiety (e.g., Fab fragment) for subsequent self-assembly into nanocage monomers and nanocages. Each subunit can be linked to scFc and / or Fc monomers and / or bioactive moieties at both ends, either the same or different, in embodiments. By "functional nanocage monomer or subunit thereof," it is intended that the nanocage monomer or subunit thereof can self-assemble with a complementary monomer or subunit into a nanocage as described herein.

[0365] The terms "ferritin" and "apoferritin" are used interchangeably herein and generally refer to a polypeptide (e.g., a ferritin chain) that can assemble into a ferritin complex, which typically contains 24 protein subunits. It will be understood that the ferritin can be from any species. Typically, the ferritin is human ferritin. In some embodiments, the ferritin is wild-type ferritin. For example, the ferritin may be wild-type human ferritin. In some embodiments, a ferritin light chain is used as a nanocage monomer, and / or a subunit of the ferritin light chain is used as a nanocage monomer subunit. In some embodiments, the assembled nanocage does not contain a ferritin heavy chain or other ferritin components that can bind iron.

[0366] The term "multispecificity" as used herein refers to the characteristic of having at least two binding sites to which at least two different binding partners, such as antigens or receptors (e.g., Fc receptors), can bind. For example, a nanocage that contains at least two Fab fragments, each of which binds to a different antigen, is "multispecific." As a further example, a nanocage that contains an Fc fragment (capable of binding to an Fc receptor) and a Fab fragment (capable of binding to an antigen) is "multispecific."

[0367] As used herein, the term "multivalent" refers to the characteristic of having at least two binding sites to which a binding partner, e.g., an antigen or a receptor (e.g., an Fc receptor), can bind. The binding partners capable of binding to the at least two binding sites can be the same or different.

[0368] As used herein, the term "antibody," also referred to in the art as "immunoglobulin" (Ig), refers to a protein constructed from paired heavy and light polypeptide chains. There are various Ig isotypes, including IgA, IgD, IgE, IgG, such as IgG1, IgG2, IgG3, and IgG4, and IgM. It will be understood that antibodies can be derived from any species, including human, mouse, rat, monkey, llama, or shark. When an antibody is properly folded, each chain folds into several different globular domains linked by a more linear polypeptide sequence. For example, in the case of IgG, the immunoglobulin light chain is variable (V L ) and constant (CL) domains, and the heavy chains fold into variable (V H ) domain and three constant (C H , C H2 , C H3 ) domain. The heavy and light chain variable domains (V H and V L The interaction of these domains results in the formation of an antigen-binding domain (Fv). Each domain has a well-defined structure that is well known to those skilled in the art.

[0369] The light and heavy chain variable regions are involved in binding the target antigen and can therefore display significant sequence diversity among antibodies. The constant regions display less sequence diversity and are responsible for binding to several natural proteins to trigger important immunological events. The variable regions of an antibody contain the antigen-binding determinants of the molecule and therefore determine the specificity of the antibody for its target antigen. The majority of the sequence variability occurs in six hypervariable regions, three per variable heavy and light chain, respectively. The hypervariable regions combine to form the antigen-binding site and contribute to the binding and recognition of antigenic determinants. The specificity and affinity of an antibody for its antigen is determined by the structure of the hypervariable regions, as well as the size, shape and chemistry of the surface they present to the antigen.

[0370] An "antibody fragment" as referred to herein may include any suitable antigen-binding antibody fragment known in the art. Antibody fragments may be naturally occurring antibody fragments or may be obtained by manipulation of naturally occurring antibodies or by using recombinant methods. For example, antibody fragments include Fv, single chain Fv (scFv; V linked by a peptide linker), and the like. L and V H (e.g., a polypeptide comprising two Fc monomers linked together via a linker, e.g., an amino acid linker), Fc monomer (e.g., a single Fc chain comprising exactly one CH2 domain and exactly one CH3 domain (which can typically dimerize with another Fc monomer)), Fab, single chain Fab, F(ab')2, single domain antibody (sdAb; a molecule consisting of a single V L or V H As used herein, "antigen-binding portion" refers to an antibody or a portion of an antibody that specifically binds to a target antigen.

[0371] The term "synthetic antibody" as used herein refers to an antibody produced using recombinant DNA technology. This term should also be taken to mean an antibody produced by synthesis of a DNA molecule encoding the antibody, which DNA molecule expresses an antibody protein, or an amino acid sequence that specifies the antibody, where the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence techniques that are available and well known in the art.

[0372] The phrases "antibody preventable" and "antibody treatable" as used herein generally refer to symptoms that are known to be associated with the expression or presence of at least one antigen. For example, in the context of infectious disease, the antigen may be an antigen on an infectious disease agent, the antigen may be expressed by infected cells, and / or the antigen may be expressed by cells involved in the immune response to infection. "Antibody preventable" is generally a condition that can be prevented from establishing by antibodies. "Antibody treatable" is generally a condition that can be treated from establishing by antibodies.

[0373] The term "epitope" refers to an antigenic determinant. An epitope is a particular chemical group or peptide sequence on a molecule that is antigenic, i.e., elicits a specific immune response. An antibody specifically binds to a particular antigenic epitope on, for example, a polypeptide. Epitopes can be formed from both contiguous or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically comprises at least 3, more usually at least 5, about 9, about 11, or about 8 to about 12 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, e.g., "Epitope Mapping Protocols" in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0374] The term "antigen" as used herein is defined as a molecule that elicits an immune response. This immune response may include antibody production, or activation of specific immunocompetent cells, or both. Those skilled in the art will understand that any macromolecule, including virtually any protein or peptide, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Thus, those skilled in the art will understand that any DNA that includes a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response will encode an "antigen" as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the embodiments described herein include, but are not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences can be arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be synthesized or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, cells, or biological fluids.

[0375] "Encode" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene codes for a protein if transcription and translation of the mRNA corresponding to the gene produces a protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to code for the protein or other product of that gene or cDNA.

[0376] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0377] The phrase "HIV-1 associated condition," as used herein, refers to a condition of HIV-1 infection (including primary infection, latent infection) and / or a condition resulting from HIV-1 infection (e.g., acquired immune deficiency syndrome).

[0378] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-native environment, such as, for example, a host cell.

[0379] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns, to the extent that a nucleotide sequence encoding a protein may, in some versions, contain an intron(s).

[0380] The term "modulating" as used herein means mediating a detectable increase or decrease in the level of a response in a subject, compared to the level of the response in a subject in the absence of a treatment or compound, and / or compared to the level of the response in an otherwise identical but untreated subject. This term encompasses perturbing and / or affecting a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, typically a human.

[0381] The term "operably linked" refers to a functional link between a regulatory sequence and a heterologous nucleic acid sequence, resulting in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence when it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to link two protein coding regions, in the same reading frame.

[0382] "Parenteral" administration of the compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion techniques. Inhalation and intranasal administration are also included.

[0383] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, as used herein, nucleic acid and polynucleotide are interchangeable. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide that can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes all nucleic acid sequences obtained by any means available in the art, including but not limited to recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR, etc., and by synthetic means.

[0384] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, with no limit on the maximum number of amino acids that may comprise a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides, and oligomers, for example, and longer chains, commonly referred to in the art as proteins, of which there are many varieties. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, mutants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

[0385] The term "specifically binds" as used herein with respect to an antibody means an antibody that recognizes a particular antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind that antigen from one or more species. However, such cross-species reactivity does not in itself change the specific classification of the antibody. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not in itself change the specific classification of the antibody. In some examples, the terms "specific binding" or "specifically binds" can be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species, meaning that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species. For example, antibodies recognize and bind to a particular protein structure rather than a protein in general. If an antibody is specific for epitope "A", then in a reaction containing labeled "A" and an antibody, the presence of a molecule containing epitope A (or free unlabeled A) will reduce the amount of labeled A bound to the antibody.

[0386] As used herein, "treating" a condition (e.g., a condition described herein, such as HIV-1) or "treatment" of a condition is an approach to obtain a beneficial or desired result, such as a clinical result. Beneficial or desired results may include, but are not limited to, alleviation or amelioration of one or more symptoms or symptoms; reduction in the severity of a disease, disorder, or condition; a stabilized (i.e., non-worsening) state of a disease, disorder, or condition; preventing the spread of a disease, disorder, or condition; delaying or slowing the progression of a disease, disorder, or condition; amelioration or reduction of a disease, disorder, or condition; remission (whether partial or total), whether detectable or undetectable. "Relieving" a disease, disorder, or condition means that the severity and / or undesirable clinical symptoms of a disease, disorder, or condition are reduced and / or the time course of progression is slowed or prolonged, compared to the severity or time course without treatment. As used herein, the term "prevention" or "prophylaxis" refers to a reduction in the risk of acquiring or developing a disease or disorder, such as HIV-1, or a reduction or inhibition of recurrence of a disease or disorder, such as HIV-1. Thus, an HIV-1 therapeutic or prophylactic composition refers to a composition comprising an assembled nanocage as described herein, or a fusion protein as described herein that can be assembled into a nanocage and that can treat and / or prevent HIV-1 when administered to a subject.

[0387] The term "therapeutically effective amount", "effective amount" or "sufficient amount" refers to an amount sufficient to achieve a desired result, e.g., an amount effective to cause cell death, when administered to a subject, including a mammal, e.g., a human. The effective amount of the compounds described herein may vary depending on factors such as the molecule, the age, sex, species and weight of the subject. As will be appreciated by those skilled in the art, the dosage or treatment regimen may be adjusted to provide an optimal therapeutic response. For example, administration of a therapeutically effective amount of the fusion proteins described herein is sufficient in some embodiments to treat and / or prevent HIV-1. Furthermore, a treatment regimen for a subject with a therapeutically effective amount may consist of a single administration or, alternatively, may include a series of applications. The frequency and length of the treatment period will depend on various factors such as the molecule, the age of the subject, the concentration of the agent, the patient's responsiveness to the agent, or a combination thereof. It will also be understood that the effective dosage of the agent used for treatment may increase or decrease over the course of a particular treatment regimen. Changes in dosage may occur and be evident by standard diagnostic assays known in the art. The fusion proteins described herein may, in embodiments, be administered before, during or after treatment with conventional therapies for the disease or disorder in question.For example, the fusion proteins described herein may find particular use in combination with conventional treatments for HIV-1.

[0388] The terms "transfected" or "transformed" or "transduced" as used herein refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0389] The phrases "under transcriptional control" or "operably linked" as used herein mean that the promoter is in the correct position and orientation relative to the polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0390] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.

[0391] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0392] The term "pharmaceutical acceptable" means that a compound or combination of compounds is compatible with the other ingredients of a formulation for pharmaceutical use and is generally safe to be administered to humans according to established government standards, including those promulgated by the U.S. Food and Drug Administration.

[0393] The term "pharmaceutically acceptable carrier" includes, but is not limited to, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and / or absorption delaying agents, etc. The use of pharmaceutically acceptable carriers is well known.

[0394] A "variant" is a biologically active fusion protein, antibody, or fragment thereof that has an amino acid sequence that differs from the comparison sequence by insertion, deletion, modification, and / or substitution of one or more amino acid residues in the comparison sequence. A variant generally has less than 100% sequence identity with the comparison sequence. However, a biologically active variant will usually have an amino acid sequence that has at least about 70% amino acid sequence identity with the comparison sequence, such as at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. A variant includes a peptide fragment of at least 10 amino acids that retains some level of biological activity of the comparison sequence. Variants also include polypeptides in which one or more amino acid residues are added to the N-terminus or C-terminus of the comparison sequence or within the comparison sequence. Variants also include polypeptides in which a number of amino acid residues are deleted and / or optionally replaced by one or more amino acid residues. Variants can also be covalently modified, for example, by substitution with a moiety other than naturally occurring amino acids, or by modifying amino acid residues to produce non-naturally occurring amino acids.

[0395] "Percentage amino acid sequence identity" is defined herein as the percentage of amino acid residues in a candidate sequence that are identical to the residues in a sequence of interest, such as a polypeptide of the present invention, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity.Neither N-terminal, C-terminal, or internal extensions, deletions, or insertions into the candidate sequence should be interpreted as affecting sequence identity or homology.Methods and computer programs for alignment are well known in the art, such as "BLAST".

[0396] For purposes herein, "active" or "activity" refers to the biological and / or immunological activity of the fusion proteins described herein, and "biological" activity refers to a biological function (either inhibitory or stimulatory) caused by the fusion protein.

[0397] The fusion proteins described herein may include modifications. Such modifications include, but are not limited to, conjugation to effector molecules. Modifications further include, but are not limited to, conjugation to detectable reporter moieties. Modifications that extend half-life (e.g., pegylation) are also included. Modifications for deimmunization are also included. Proteins and non-protein agents can be conjugated to fusion proteins by methods known in the art. Conjugation methods include direct linkage, linkage via covalently attached linkers, and specific binding pair members (e.g., avidin-biotin). Such methods include, for example, those described by Greenfield et al., Cancer Research 50, 6600-6607 (1990), which is incorporated herein by reference, as well as those described by Amon et al., Adv. Exp. Med. Biol. 303, 79-90 (1991) and Kiseleva et al., MoI. Biol. (USSR) 25, 508-514 (1991), both of which are incorporated herein by reference. Fusion proteins containing Fc monomers

[0398] Fusion proteins are described herein. In an embodiment, the fusion protein comprises a nanocage monomer or subunit thereof linked to an Fc monomer, and multiple fusion proteins self-assemble to form a nanocage comprising one or more Fc dimers. In this way, the assembled one or more Fc dimers can decorate the inner surface of the assembled nanocage, the outer surface of the assembled nanocage, or both. In some embodiments, the assembled one or more Fc dimers decorate the outer surface of the assembled nanocage. Splitting the Fc dimer into monomeric components provides additional control in nanocage design. The Fc monomers help assemble the nanocage and more tightly control the ratio of other linked components, such as bioactive moieties.

[0399] In some embodiments, nanocage monomer subunits are used in the fusion protein instead of the complete nanocage monomer. The nanocage monomer can be divided into two parts, one of which contains the N-terminus of the nanocage monomer, called "N subunit", and the other of which contains the C-terminus of the nanocage monomer, called "C subunit". The N subunit or C subunit can each represent substantially half of the nanopage monomer, or can be divided unevenly. Typically, the N subunit and the C subunit correspond substantially to the N-terminal half of the nanocage monomer and the C-terminal half of the nanocage monomer, respectively. The N subunit and the C subunit can self-assemble to form the nanocage monomer, which itself can self-assemble to form the nanocage as described above.

[0400] It will be understood that each nanocage monomer and each N and C subunit includes an N-terminus and a C-terminus. An Fc monomer can be linked to a nanocage monomer or its subunit at one or both termini. Typically, an Fc monomer is linked to a nanocage monomer or its subunit at the C-terminus. Similarly, an Fc monomer can be linked to either or both termini of an N subunit and / or a C subunit. Typically, an Fc monomer is linked to a C subunit at the C-terminus.

[0401] In a typical embodiment, the nanocage monomer or its subunit is further linked to a biologically active moiety. As with the Fc monomer, the biologically active moiety can be linked to the nanocage monomer or its subunit at the N-terminus or C-terminus of the nanocage monomer or its subunit, typically linked at the N-terminus. When a nanocage monomer subunit is used, the biologically active moiety can also be linked to the N-subunit or C-subunit at either end, typically the end opposite to the Fc monomer. Typically, the biologically active moiety is linked to the C-subunit at the N-terminus.

[0402] The antigen-binding moieties are linked to the nanocage monomers or subunits thereof so as to decorate the interior and / or exterior surface of the assembled nanocage, typically decorating the exterior surface of the assembled nanocage.

[0403] The biologically active portion is typically an antibody or a fragment thereof that specifically binds to an antigen target. It will be understood that the antibody or a fragment thereof can comprise or consist of, for example, the heavy and / or light chains of a Fab fragment. The antibody or a fragment thereof can comprise or consist of, for example, a Fab (e.g., scFab) fragment, a scFv fragment, a sdAb fragment, and / or a VHH region. In some embodiments, the biologically active portion does not comprise a CH2 or CH3 domain. It will be understood that any antibody or a fragment thereof can be used in the fusion proteins described herein.

[0404] Generally, the fusion proteins described herein are associated with a Fab light chain and / or a heavy chain, which may be produced separately or contiguous with the fusion protein.

[0405] In typical embodiments, the antigen-binding portion specifically binds to an antigen associated with a condition preventable and / or treatable by an antibody. For example, the antigen may be associated with an infectious agent, including a virus, bacteria, parasite, fungus, or yeast, cancer, or an immune disease, including an autoimmune disease.

[0406] In exemplary embodiments, the antigen-binding portion comprises an HIV-1 specific antigen-binding portion. The HIV-1 specific antigen-binding portion may bind, for example, to BG505 SOSIP_D368R, 93TH057 gp120, and / or MPER peptide. In specific examples, the HIV-1 specific antigen-binding portion comprises an antigen-binding portion derived from PGDM1400, 10E8v4, and / or N49P7.

[0407] In certain embodiments, the nanocage monomers described herein comprise an N or C subunit linked to an Fc monomer or a bioactive motif (e.g., a Fab fragment). The N or C subunit can self-assemble with a complementary C or N subunit to form a complete nanocage monomer, multiple of which can self-assemble to form a nanocage, thus allowing multiple Fc monomers and / or other moieties to self-assemble into one nanocage. The amount of each different component is controlled by controlling the genes and expression ratios. These nanocage monomer subunits can be used alone or in combination.

[0408] For example, an Fc monomer or a biologically active portion (e.g., a Fab fragment) can be linked to a split apoferritin monomer (either an N subunit or a C subunit, each of which is typically about half of a full-length apoferritin monomer). Each subunit fused to an Fc monomer or a biologically active portion (e.g., a Fab fragment) self-assembles into an apoferritin monomer, which then self-assembles with other apoferritin monomers (either complete apoferritin or assembled apoferritin formed from the N and C subunits) to form a nanocage.

[0409] When a full-length nanocage monomer is used, the nanocage monomer typically comprises an Fc monomer linked to the C-terminus of the nanocage monomer and a biologically active moiety linked to the N-terminus of the nanocage monomer. When a nanocage monomer subunit is used, the subunit typically comprises an Fc monomer linked to the C-terminus of the subunit and a biologically active moiety linked to the N-terminus of the subunit. Typically, a C subunit is used in the fusion proteins described herein, where an Fc monomer is linked to the C-terminus of the C subunit and a biologically active moiety is linked to the N-terminus of the C subunit.

[0410] The N or C subunits described herein are provided in embodiments in combination with a complementary C or N subunit with which the N or C subunit can self-assemble. The complementary C or N subunit may or may not be a fusion protein. In some embodiments, the complementary C or N subunit is linked to a biologically active moiety at its N-terminus or C-terminus, typically at its N-terminus. The biologically active moiety may be, for example, an antigen-binding moiety such as a Fab fragment, which may be the same or different from any biologically active moiety or antigen-binding moiety linked to the N or C subunit. The complementary C or N subunit may be linked to an Fc monomer at its N-terminus or C-terminus, typically at its C-terminus. It will be appreciated that typically, an Fc monomer is fused to the C-terminus of the C subunit of ferritin in order to utilize the four-fold axis of the nanocage and thereby fill the other half.

[0411] For example, in some embodiments, the fusion protein comprises a C subunit linked at its C-terminus to an Fc monomer and linked at its N-terminus to a Fab fragment. In use, the C subunit self-assembles with an N subunit linked at its N-terminus to a Fab fragment (which may be the same or different from the Fab fragment linked to the C subunit) to form a nanocage monomer. As described above, a plurality of nanocage monomers self-assemble to form a nanocage. The nanocage monomer subunits may be provided alone or in combination and may have the same or different biologically active moieties fused thereto.

[0412] The Fc monomer can be any Fc monomer from any antibody type or species. Typically, the Fc monomer is human and is from IgG, IgA, IgD, IgM or IgE. For example, the Fc monomer can be from IgG, such as IgG1, IgG2, IgG3 or IgG4 (e.g., IgG1 Fc monomer).

[0413] The Fc monomer may include one or more mutations or sets of mutations that modulate the half-life of the fusion protein, for example, from minutes or hours to days, weeks or months. For example, the Fc monomer may include mutations at one or more of L234, L235, G236, G237, M252, I253, S254, T256, P329, A330, M428, N434, or combinations thereof (numbering according to the EU index), such as M428L and N434S ("LS"); M252Y, S254T and T256E ("YTE"); L234A and L235A ("LALA"); I253A; L234A, L235A, and P329G ("LALAP"); G236R; G237A; and / or A330L, or combinations thereof.

[0414] Additionally, other substitutions in the fusion proteins and nanocages described herein are contemplated, including Fc sequence modifications and the addition of other agents (e.g., human serum albumin, human serum albumin peptide sequences, and antibodies such as Fabs and / or nanobodies that target human serum albumin), which allow for altered bioavailability and will be understood by one of skill in the art. Additionally, the fusion proteins and nanocages described herein can be modulated in sequence or by the addition of other agents to mute immunogenicity and anti-drug responses (therapeutic, e.g., adapting the sequence to the host, or adding immunosuppressive therapy [e.g., methotrexate when administering infliximab for the treatment of rheumatoid arthritis, or induction of neonatal tolerance, which is a major strategy to reduce the incidence of inhibitors to FVIII] (review: DiMichele DM, Hoots WK, Pipe SW, Rivard GE, Santagostino E. International workshop on immune tolerance induction: consensus recommendations. Haemophilia. 2007;13:1-22, incorporated herein by reference in its entirety]).

[0415] As described herein, nanocage monomers can be formed from subunits. Thus, the nanocage monomer subunits can include a first apoferritin subunit, optionally a first human apoferritin subunit, for example, apoferritin N or C subunit, which can self-assemble with a second apoferritin subunit, for example, a complementary C or N subunit. It will be understood that other nanocage monomers can be split into two-component subunits similar to the apoferritin described herein, such that the subunits are self-assembled and each is amenable to fusion with a bioactive moiety.

[0416] The "N" region of apoferritin typically comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following: MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEW (SEQ ID NO: 1)

[0417] It will be understood that anywhere herein where the "N" region of apoferritin is described in whole or in part, alone or as a component of another fusion protein, the sequence MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEW (SEQ ID NO: 1) can be replaced with SSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEW (SEQ ID NO: 15). Thus, in embodiments, the "N" region of apoferritin typically comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following: SSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEW (SEQ ID NO: 15)

[0418] The "C" region of apoferritin typically comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following: GKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD (SEQ ID NO: 2)

[0419] Anywhere herein where the "C" region of apoferritin is described in whole or in part, either alone or as a component of another fusion protein, the sequence GKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD (SEQ ID NO: 2) is [ka] It will be understood that the "C" region of apoferritin may be substituted with. Thus, in embodiments, the "C" region of apoferritin typically comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following: GKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLKHD (SEQ ID NO: 16)

[0420] When the antibody or fragment thereof comprises two chains, such as a first and a second chain, or a heavy and a light chain, the two chains are optionally separated by a linker. The linker can be flexible or rigid, but is typically flexible to allow the chains to fold appropriately. Similarly, the Fc monomer and / or biologically active moiety is typically linked to the nanocage monomer or subunit thereof via a linker.

[0421] The linker will generally be of sufficient length to provide some flexibility to the fusion protein, although it will be understood that the length of the linker will vary depending on the sequence of the nanocage monomer or subunits thereof and the Fc monomer and biologically active portion, as well as the three-dimensional conformation of the fusion protein. Thus, the linker will typically be of length from about 1 to about 130 amino acid residues, e.g., from about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, or 125 to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, or 125. , 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acid residues, or about 50 to about 90 amino acid residues, for example 70 amino acid residues, or about 1 to about 100 amino acid residues, for example about 1 to about 70 amino acid residues, for example about 1 to about 30 amino acid residues, for example about 8 to about 16 amino acid residues.

[0422] The linker may be of any amino acid sequence, and in one typical example, the linker comprises a GS domain, or a series of G and S amino acids, such as a series of GS, GGS, GGGS and / or GGGGS repeats. Typically, the linker comprises GGGGS and / or GGGS repeats, and more typically, the linker comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 GGGS and / or GGGGS repeats, such as about 5 GGGS repeats and / or about 14 GGGGS repeats. In certain embodiments, the linker comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following: [ka]

[0423] In an exemplary embodiment, the antibody or fragment thereof specifically binds to an antigen associated with HIV-1.

[0424] In embodiments, the fusion proteins described herein comprise or consist of a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to one or more of the following sequences, where the ferritin subunits are in bold and the linker is underlined. In each of these cases, the complete ferritin monomer is shown ( [ka] ) , or N- or C-ferritin monomers are shown (for N-ferritin ( ( [ka] ) and for C-ferritin ( [ka] )), it being understood that these can be used interchangeably or that another monomer or portion thereof can be used in its place.

[0425] 32-N MB.v3: N49P7_N-ferritin (SEQ ID NO: 7) [ka] PGDM1400_h Ferritin (SEQ ID NO: 8) [ka] 10E8v4_C-Ferritin_Fc1 (SEQ ID NO: 9) [ka] 32-N MB.v3 LS: N49P7_N-ferritin (SEQ ID NO: 7) [ka] PGDM1400_h Ferritin (SEQ ID NO: 8) [ka] 10E8v4_C-Ferritin_Fc1-LS (SEQ ID NO: 10) [ka] Or a combination thereof.

[0426] In further embodiments, the fusion protein may be conjugated or associated with an additional moiety, such as a detectable moiety (e.g., a small molecule, a fluorescent molecule, a radioisotope, or a magnetic particle), a pharmaceutical agent, a diagnostic agent, or combinations thereof, including, for example, an antibody-drug conjugate.

[0427] In embodiments where the additional moiety is a detectable moiety, the detectable moiety may comprise a fluorescent protein such as GFP, EGFP, ametrine, and / or a flavin-based fluorescent protein such as a LOV protein, such as iLOV.

[0428] In aspects in which the additional moiety is a pharmaceutical agent, the pharmaceutical agent can include, for example, a small molecule, a peptide, a lipid, a carbohydrate, or a toxin.

[0429] In typical embodiments, nanocages assembled from the fusion proteins described herein are comprised of about 3 to about 100 nanocage monomers, any of which may be a bicomponent nanocage monomer subunit, e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 55, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, or the like. or 98 to about 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 55, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or 100 nanocage monomers, e.g., 24, 32, or 60 nanocage monomers, some or all of which may be provided as such nanocage monomers. The nanocage monomer or subunit thereof may be any known nanocage monomer, natural, synthetic, or partially synthetic, and in embodiments is selected from ferritin, apoferritin, encapsulin, SOR, lumazine synthase, pyruvate dehydrogenase, carboxysome, vault protein, GroEL, heat shock protein, E2P, MS2 coat protein, fragments thereof, and mutants thereof. Typically, the nanocage monomer or subunit thereof is ferritin or apoferritin or subunit thereof.

[0430] Also described herein are pairs of the above fusion proteins, where each pair self-assembles to form a nanocage monomer, and the first and second nanocage monomer subunits are fused to different biologically active moieties as described herein, providing multivalency and / or multispecificity to a single nanocage monomer assembled from the pair of subunits. Fusion protein containing a C-terminal scFc fragment

[0431] Fusion proteins are described herein. In an embodiment, the fusion protein comprises a nanocage monomer or a subunit thereof linked to an scFc fragment at the C-terminus of the nanocage monomer or a subunit thereof, and a plurality of fusion proteins self-assemble to form a nanocage. In this manner, the scFc fragment may decorate the inner surface of the assembled nanocage, the outer surface of the assembled nanocage, or both. In some embodiments, the scFc fragment decorates the outer surface of the assembled nanocage. By placing the scFc fragment at the C-terminus of the nanocage monomer or a subunit thereof, surprisingly, improved potency and neutralization efficiency have been observed.

[0432] In some embodiments, nanocage monomer subunits are used in the fusion protein instead of the complete nanocage monomer. The nanocage monomer can be divided into two parts, one of which contains the N-terminus of the nanocage monomer, called "N subunit", and the other of which contains the C-terminus of the nanocage monomer, called "C subunit". The N subunit or C subunit can each represent substantially half of the nanopage monomer, or can be divided unevenly. Typically, the N subunit and the C subunit correspond substantially to the N-terminal half of the nanocage monomer and the C-terminal half of the nanocage monomer, respectively. The N subunit and the C subunit can self-assemble to form the nanocage monomer, which itself can self-assemble to form the nanocage as described above.

[0433] It will be understood that each nanocage monomer and each N and C subunit includes an N-terminus and a C-terminus. The scFc fragment can be linked to the nanocage monomer or its subunit at one or both termini. Typically, the scFc fragment is linked to the nanocage monomer or its subunit at the C-terminus. Similarly, the scFc fragment can be linked to either or both termini of the N subunit and / or the C subunit. Typically, the scFc fragment is linked to the C subunit at the C-terminus.

[0434] In a typical embodiment, the nanocage monomer or its subunit is further linked to a biologically active moiety. As with the scFc fragment, the biologically active moiety can be linked to the nanocage monomer or its subunit at the N-terminus or C-terminus of the nanocage monomer or its subunit, typically linked at the N-terminus. When a nanocage monomer subunit is used, the biologically active moiety can also be linked to the N-subunit or C-subunit at either end, typically the end opposite to the scFc fragment. Typically, the biologically active moiety is linked to the C-subunit at the N-terminus.

[0435] The antigen-binding moieties are linked to the nanocage monomers or subunits thereof so as to decorate the interior and / or exterior surface of the assembled nanocage, typically decorating the exterior surface of the assembled nanocage.

[0436] The biologically active portion is typically an antibody or a fragment thereof that specifically binds to an antigen target. It will be understood that the antibody or a fragment thereof can comprise or consist of, for example, the heavy and / or light chains of a Fab fragment. The antibody or a fragment thereof can comprise or consist of, for example, a Fab (e.g., scFab) fragment, a scFv fragment, a sdAb fragment, and / or a VHH region. In some embodiments, the biologically active portion does not comprise a CH2 or CH3 domain. It will be understood that any antibody or a fragment thereof can be used in the fusion proteins described herein.

[0437] Generally, the fusion proteins described herein are associated with a Fab light chain and / or a heavy chain, which may be produced separately or contiguous with the fusion protein.

[0438] In typical embodiments, the antigen-binding portion specifically binds to an antigen associated with a condition preventable and / or treatable by an antibody. For example, the antigen may be associated with an infectious agent, including a virus, bacteria, parasite, fungus, or yeast, cancer, or an immune disease, including an autoimmune disease.

[0439] In exemplary embodiments, the antigen-binding portion comprises an HIV-1 specific antigen-binding portion. The HIV-1 specific antigen-binding portion may bind, for example, to BG505 SOSIP_D368R, 93TH057 gp120, and / or MPER peptide. In specific examples, the HIV-1 specific antigen-binding portion comprises an antigen-binding portion derived from PGDM1400, 10E8v4, and / or N49P7.

[0440] In certain embodiments, the nanocage monomers described herein comprise an N or C subunit linked to an scFc fragment or a bioactive motif (e.g., a Fab fragment). The N or C subunit can self-assemble with a complementary C or N subunit to form a complete nanocage monomer, multiple of which can self-assemble to form a nanocage, thus allowing multiple scFc fragments and / or other moieties to self-assemble into one nanocage. The amount of each different component is controlled by controlling the genes and expression ratios. These nanocage monomer subunits can be used alone or in combination.

[0441] For example, an scFc fragment or biologically active portion (e.g., a Fab fragment) can be linked to a split apoferritin monomer (either the N subunit or the C subunit, each of which is typically about half of a full-length apoferritin monomer). Each subunit fused to an scFc fragment or biologically active portion (e.g., a Fab fragment) self-assembles into an apoferritin monomer, which then self-assembles with other apoferritin monomers (either complete apoferritin or assembled apoferritin formed from the N and C subunits) to form a nanocage.

[0442] When a full-length nanocage monomer is used, the nanocage monomer typically comprises an scFc fragment linked to the C-terminus of the nanocage monomer and a biologically active moiety linked to the N-terminus of the nanocage monomer. When a nanocage monomer subunit is used, the subunit typically comprises an scFc fragment linked to the C-terminus of the subunit and a biologically active moiety linked to the N-terminus of the subunit. Typically, a C subunit is used in the fusion proteins described herein, where an scFc fragment is linked to the C-terminus of the C subunit and a biologically active moiety is linked to the N-terminus of the C subunit.

[0443] The N or C subunits described herein are provided in embodiments in combination with complementary C or N subunits that allow the N or C subunits to self-assemble. Complementary C or N subunits may or may not be fusion proteins. In some embodiments, complementary C or N subunits are linked to biologically active moieties at their N-terminus or C-terminus, typically at their N-terminus. Biologically active moieties may be, for example, antigen-binding moieties, such as Fab fragments, which may be the same or different from any biologically active moieties or antigen-binding moieties linked to the N or C subunits. Complementary C or N subunits may be linked to scFc fragments at their N-terminus or C-terminus, typically at their C-terminus.

[0444] For example, in some embodiments, the fusion protein comprises a C subunit linked at its C-terminus to an scFc fragment and linked at its N-terminus to a Fab fragment. In use, the C subunit self-assembles with an N subunit linked at its N-terminus to a Fab fragment (which may be the same or different from the Fab fragment linked to the C subunit) to form a nanocage monomer. As described above, a plurality of nanocage monomers self-assemble to form a nanocage. The nanocage monomer subunits may be provided alone or in combination and may have the same or different biologically active moieties fused thereto.

[0445] The scFc fragment can be any scFc fragment derived from any antibody type or species. Typically, the scFc fragment is human and derived from IgG, IgA, IgD, IgM or IgE. For example, the scFc fragment can be derived from IgG, such as IgG1, IgG2, IgG3 or IgG4 (e.g., an IgG1 scFc fragment).

[0446] The scFc fragment may comprise one or more mutations or a set of mutations that modulate the half-life of the fusion protein, for example, from minutes or hours to days, weeks or months. For example, the scFc fragment may comprise mutations at one or more of L234, L235, G236, G237, M252, I253, S254, T256, P329, A330, M428, N434, or combinations thereof (numbering according to the EU index), such as M428L and N434S ("LS"); M252Y, S254T and T256E ("YTE"); L234A and L235A ("LALA"); I253A; L234A, L235A, and P329G ("LALAP"); G236R; G237A; and / or A330L, or combinations thereof.

[0447] Additionally, other substitutions in the fusion proteins and nanocages described herein are contemplated, including Fc sequence modifications and the addition of other agents (e.g., human serum albumin, human serum albumin peptide sequences, and antibodies such as Fabs and / or nanobodies that target human serum albumin), which allow for altered bioavailability and will be understood by one of skill in the art. Additionally, the fusion proteins and nanocages described herein can be modulated in sequence or by the addition of other agents to mute immunogenicity and anti-drug responses (therapeutic, e.g., adapting the sequence to the host, or adding immunosuppressive therapy [e.g., methotrexate when administering infliximab for the treatment of rheumatoid arthritis, or induction of neonatal tolerance, which is a major strategy to reduce the incidence of inhibitors to FVIII] (review: DiMichele DM, Hoots WK, Pipe SW, Rivard GE, Santagostino E. International workshop on immune tolerance induction: consensus recommendations. Haemophilia. 2007;13:1-22, incorporated herein by reference in its entirety]).

[0448] As described herein, nanocage monomers can be formed from subunits. Thus, the nanocage monomer subunits can include a first apoferritin subunit, optionally a first human apoferritin subunit, for example, apoferritin N or C subunit, which can self-assemble with a second apoferritin subunit, for example, a complementary C or N subunit. It will be understood that other nanocage monomers can be split into two-component subunits similar to the apoferritin described herein, such that the subunits are self-assembled and each is amenable to fusion with a bioactive moiety.

[0449] The "N" region of apoferritin typically comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following: MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEW (SEQ ID NO: 1)

[0450] The "C" region of apoferritin typically comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following: GKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD (SEQ ID NO: 2)

[0451] When the antibody or fragment thereof comprises two chains, such as a first and a second chain, or a heavy and a light chain, the two chains are optionally separated by a linker. The linker can be flexible or rigid, but is typically flexible to allow the chains to fold appropriately. Similarly, the scFc fragment and / or the biologically active moiety is typically linked to the nanocage monomer or subunit thereof via a linker.

[0452] The linker is generally long enough to provide some flexibility to the fusion protein, although it will be understood that the length of the linker will vary depending on the sequence of the nanocage monomer or subunits thereof and the scFc fragment and biologically active portion, as well as the three-dimensional conformation of the fusion protein. Thus, the linker will typically be from about 1 to about 130 amino acid residues, e.g., from about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, or 125 to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, or 125. , 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acid residues, or about 50 to about 90 amino acid residues, for example 70 amino acid residues, or about 1 to about 100 amino acid residues, for example about 1 to about 70 amino acid residues, for example about 1 to about 30 amino acid residues, for example about 8 to about 16 amino acid residues.

[0453] The linker may be of any amino acid sequence, and in one typical example, the linker comprises a GS domain, or a series of G and S amino acids, such as a series of GS, GGS, GGGS and / or GGGGS repeats. Typically, the linker comprises GGGGS and / or GGGS repeats, and more typically, the linker comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 GGGS (SEQ ID NO:11) and / or GGGGS (SEQ ID NO:12) repeats, such as about 5 GGGS (SEQ ID NO:11) repeats and / or about 14 GGGGS (SEQ ID NO:12) repeats. In certain embodiments, the linker comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following: [ka]

[0454] In an exemplary embodiment, the antibody or fragment thereof specifically binds to an antigen associated with HIV-1.

[0455] In embodiments, the fusion proteins described herein comprise or consist of a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to one or more of the following sequences, where the ferritin subunits are in bold and the linker is underlined. In each of these cases, the complete ferritin monomer is shown ( [ka] ), or N- or C-ferritin monomers are shown (for N-ferritin (( [ka] [ka] ) and for C-ferritin ( [ka] )), it being understood that these can be used interchangeably or that another monomer or portion thereof can be used in its place.

[0456] 32-N MB.v2: N49P7_N-ferritin (SEQ ID NO: 7) [ka] PGDM1400_h Ferritin (SEQ ID NO: 8) [ka] 10E8v4_C-Ferritin_Fc (SEQ ID NO: 13) [ka] [ka] Or a combination thereof.

[0457] In further embodiments, the fusion protein may be conjugated or associated with an additional moiety, such as a detectable moiety (e.g., a small molecule, a fluorescent molecule, a radioisotope, or a magnetic particle), a pharmaceutical agent, a diagnostic agent, or combinations thereof, including, for example, an antibody-drug conjugate.

[0458] In embodiments where the additional moiety is a detectable moiety, the detectable moiety may comprise a fluorescent protein such as GFP, EGFP, ametrine, and / or a flavin-based fluorescent protein such as a LOV protein, such as iLOV.

[0459] In aspects in which the additional moiety is a pharmaceutical agent, the pharmaceutical agent can include, for example, a small molecule, a peptide, a lipid, a carbohydrate, or a toxin.

[0460] In typical embodiments, nanocages assembled from the fusion proteins described herein are comprised of about 3 to about 100 nanocage monomers, any of which may be a bicomponent nanocage monomer subunit, e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 55, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, or the like. or 98 to about 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 55, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or 100 nanocage monomers, e.g., 24, 32, or 60 nanocage monomers, some or all of which may be provided as such nanocage monomers. The nanocage monomer or subunit thereof may be any known nanocage monomer, natural, synthetic, or partially synthetic, and in embodiments is selected from ferritin, apoferritin, encapsulin, SOR, lumazine synthase, pyruvate dehydrogenase, carboxysome, vault protein, GroEL, heat shock protein, E2P, MS2 coat protein, fragments thereof, and mutants thereof. Typically, the nanocage monomer or subunit thereof is ferritin or apoferritin or subunit thereof.

[0461] Also described herein are pairs of the above fusion proteins, where each pair self-assembles to form a nanocage monomer, and the first and second nanocage monomer subunits are fused to different biologically active moieties as described herein, providing multivalency and / or multispecificity to a single nanocage monomer assembled from the pair of subunits. Nanocage

[0462] Also disclosed herein are nanocages comprising at least one fusion protein disclosed herein, where the nanocage self-assembles from at least one fusion protein and additional fusion protein(s) and / or nanocage monomer(s) or subunits thereof, such as ferritin chain(s) (e.g., human ferritin light chain).

[0463] Also described herein is a nanocage comprising at least one fusion protein described herein and at least one nanocage monomer or subunit thereof that self-assembles with the fusion protein to form a nanocage. Additionally, described herein are pairs of fusion proteins that self-assemble to form a nanocage monomer, where the first and second nanocage monomer subunits are fused to different biologically active moieties.

[0464] It will be understood that nanocages can self-assemble from multiple identical fusion proteins, multiple different fusion proteins (thus being multivalent and / or multispecific), combinations of fusion proteins and wild-type proteins, and any combination thereof. For example, nanocages can be decorated internally and / or externally with at least one of the fusion proteins described herein in combination with at least one Fc monomer and / or scFc fragment. In some embodiments, at least one Fc monomer and / or scFc fragment and at least one Fab fragment decorate the exterior surface of the nanocage. In some embodiments, at least two Fc monomers and / or scFc fragments and at least two Fab fragments decorate the exterior surface of the nanocage.

[0465] In typical embodiments, about 20% to about 80% of the nanocage monomers or subunits thereof comprise a fusion protein as described herein. Considering the modular solutions described herein, the nanocage can theoretically contain up to 3 or 4 times as many bioactive moieties, e.g., antibody fragments and / or Fc monomers / scFc fragments, as there are monomers in the nanocage, since each nanocage monomer can be split into two subunits, each of which can be independently bound to a different bioactive moiety at each end. Notwithstanding this, a lower number is typically used to avoid steric hindrance. It will be appreciated that this modularity can be exploited to achieve any desired ratio of bioactive moieties.

[0466] In some examples, the nanocages described herein may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 or more copies of identical or substantially identical or functionally equivalent Fc monomers or scFc fragments. In additional or alternative examples, the nanocages described herein may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 or more copies of the same or substantially identical or functionally equivalent biologically active moiety, such as a Fab fragment. In additional or alternative examples, the nanocages described herein may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 different Fc monomers and / or scFc fragments and / or other biologically active moieties. In this manner, the nanocages may be multivalent and / or multispecific, the extent of which can be relatively easily controlled with the systems described herein. In some embodiments, the nanocages are both multivalent and multispecific.

[0467] In some embodiments, the nanocages described herein may further comprise at least one entire nanocage monomer optionally fused to a biologically active moiety, which may be the same as or different from the biologically active moiety described herein as being linked to the nanocage monomer subunit.

[0468] In some embodiments, the nanocages described herein comprise first and second fusion proteins, each comprising a different antigen-binding moiety fused to a nanocage monomer or subunit thereof, and optionally a third fusion protein comprising an Fc monomer or scFc fragment fused to the nanocage monomer or subunit thereof.

[0469] In some embodiments, the first, second, and third fusion proteins each comprise a biologically active portion or portion thereof fused to an N or C half ferritin, wherein at least one of the first, second, and third fusion proteins is fused to an N half ferritin, at least one of the first, second, and third fusion proteins is fused to a C half ferritin, and an Fc monomer or scFc fragment is fused to the C-terminus of the C half ferritin.

[0470] In some embodiments, the first and optionally the second fusion proteins each comprise an antigen-binding portion fused to a complete apoferritin.Similarly, in some embodiments, the third protein comprises a biologically active portion fused to a complete apoferritin.It will be understood that combinations of complete nanocage monomers and subunits of nanocage monomers are contemplated for use in the modular nanocages described herein.

[0471] It will be understood that typically, at least one complete nanocage monomer linked at its N-terminus to a biologically active moiety such as a Fab is used in the nanocage described herein. Typically, there is one such complete nanocage monomer per pair of N- and C-ferritin subunits. This allows the nanocage to multimerize properly without excessive steric hindrance, and also reduces the number of Fc monomers and / or scFc fragments on the nanocage, which may result in undesirable excess binding activity.

[0472] While the protein can contain any number or ratio of fusion proteins, in some embodiments, the nanocages described herein contain three Fabs, each specific for a different antigen associated with the same disease, and optionally an Fc monomer or scFc fragment in a 2:2:2:1 Fab1:Fab2:Fab:Fc monomer / scFc ratio.

[0473] Also described herein are compositions comprising the nanocages, such as therapeutic or prophylactic compositions. Related methods and uses for treating and / or preventing HIV-1 are also described, which methods or uses comprise administering the nanocages or compositions described herein to a subject in need thereof.

[0474] In certain embodiments relating to HIV-1, the nanocages, in aspects, exhibit a pan-viral neutralization breadth. In further or alternative aspects, the nanocages exhibit an average IC of less than about 0.1 ug / mL, e.g., less than about 0.01 ug / mL, e.g., less than about 0.001 ug / mL, against a multi-clade panel of 118 pseudoviruses (PsVs). 50 In further or alternative embodiments, the nanocages have an average IC of less than about 42 pM, e.g., less than about 4.2 pM, e.g., less than about 0.42 pM, against a multi-clade panel of 118 pseudoviruses (PsVs). 50 In further or alternative embodiments, the nanocages have an average IC that is at least about 10, at least about 100, at least about 1000, at least about 10,000, or at least about 100,000 more potent than the corresponding cocktail of bNAbs on a mass and / or molar basis against a multi-clade panel of 118 PsVs. 50 The median values ​​are shown.

[0475] It is understood that substantially identical polypeptides to the polypeptides described herein are also contemplated. Substantially identical sequences may include one or more conservative amino acid mutations. It is known in the art that one or more conservative amino acid mutations to a reference sequence may result in a mutant peptide that does not have substantial changes in physiological, chemical or functional properties compared to the reference sequence. In such cases, the reference sequence and the mutant sequence will be considered as "substantially identical" polypeptides. Conservative amino acid mutations may include addition, deletion or substitution of amino acids. Conservative amino acid substitution is defined herein as the replacement of an amino acid residue with another amino acid residue that has similar chemical properties (e.g., size, charge, or polarity).

[0476] In a non-limiting example, a conservative mutation can be an amino acid substitution. Such a conservative amino acid substitution can replace a basic, neutral, hydrophobic or acidic amino acid with another of the same group. The term "basic amino acid" refers to a hydrophilic amino acid with a side chain pK value greater than 7, which is typically positively charged at physiological pH. Basic amino acids include histidine (His or H), arginine (Arg or R) and lysine (Lys or K). The term "neutral amino acid" (also "polar amino acid") refers to a hydrophilic amino acid that is uncharged at physiological pH but has a side chain with at least one bond in which the electron pair commonly shared by two atoms is held closer by one of the atoms. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine ​​(Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N) and glutamine (Gln or Q). The term "hydrophobic amino acid" (also "nonpolar amino acid") is meant to include amino acids that exhibit a hydrophobicity greater than 0 according to the normalized consensus hydrophobicity scale of Eisenberg (1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (Ile or I), phenylalanine (Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G).

[0477] "Acidic amino acid" refers to a hydrophilic amino acid having a side chain pK value of less than 7, which is typically negatively charged at physiological pH. Acidic amino acids include glutamic acid (Glu or E) and aspartic acid (Asp or D).

[0478] Sequence identity is used to evaluate the similarity of two sequences. It is determined by calculating the percentage of residues that are the same when two sequences are aligned for maximum correspondence between residue positions. Any known method can be used to calculate sequence identity. For example, computer software is available to calculate sequence identity. Without wishing to be limiting, sequence identity can be calculated by software such as NCBI BLAST2 service, BLAST-P, Blast-N or FASTA-N, which are maintained by the Swiss Institute of Bioinformatics (and can be found at ca.expasy.org / tools / blast / ), or any other suitable software known in the art.

[0479] The substantially identical sequences of the present invention may be at least 85% identical. In another example, the substantially identical sequences may be at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% (or any percentage therebetween) identical at the amino acid level to the sequences described herein. In certain aspects, the substantially identical sequences retain the activity and specificity of the reference sequence. In a non-limiting embodiment, the difference in sequence identity may be due to conservative amino acid mutation(s).

[0480] The polypeptides or fusion proteins of the invention may also contain additional sequences to aid in their expression, detection, purification, or any other desired properties, such as peptides that inhibit transcytosis, etc. Any such sequences or tags known to those of skill in the art can be used. For example, and without wishing to be limiting, the fusion protein may comprise an exemplary tag cassette including a targeting or signal sequence (such as, but not limited to, ompA), a detection tag, a Strep tag, or any variant thereof (see, for example, U.S. Patent No. 7,981,632), a His tag, a Flag tag having the sequence motif DYKDDDDK (SEQ ID NO: 14), an Xpress tag, an Avi tag, a calmodulin tag, a polyglutamate tag, an HA tag, a Myc tag, a Nus tag, an S tag, an SBP tag, a Softag 1, a Softag 3, a V5 tag, a CREB binding protein (CBP), a glutathione S-transferase (GST), a maltose binding protein (MBP), a green fluorescent protein (GFP), a thioredoxin tag, or any combination thereof; a purification tag (such as, but not limited to, His5 or His6), or any combination thereof.

[0481] In another example, the additional sequence may be a biotin recognition site as described by Cronan et al. in WO 1995 / 004069 or Voges et al. in WO 2004 / 076670. As is known to those skilled in the art, linker sequences may be used with additional sequences or tags.

[0482] More specifically, the tag cassette may comprise an extracellular moiety that can specifically bind to an antibody with high affinity or avidity. In the single-chain fusion protein structure, the tag cassette may be located (a) immediately amino-terminal to the connector region, (b) intervening between the linker module, (c) immediately carboxy-terminal to the binding domain, (d) intervening between the binding domain (e.g., scFv or scFab), (e) intervening between the subunits of the binding domain, or (f) at the amino-terminus of the single-chain fusion protein. In certain embodiments, one or more junction amino acids may be located between and connecting the tag cassette and the hydrophobic moiety, between and connecting the tag cassette and the connector region, between and connecting the tag cassette and the linker module, or between and connecting the tag cassette and the binding domain.

[0483] Also encompassed herein are isolated or purified fusion proteins, polypeptides, or fragments thereof immobilized on surfaces using various methodologies.For example, and without wishing to be limited thereto, polypeptides can be linked or coupled to surfaces via His-tag coupling, biotin binding, covalent binding, adsorption, etc.The solid surface can be any suitable surface, for example, but not limited to, the well surface of a microtiter plate, the channel of a surface plasmon resonance (SPR) sensor chip, a membrane, a bead (such as magnetic-based or sepharose-based beads or other chromatographic resins), glass, film, or any other useful surface.

[0484] In other aspects, the fusion protein can be linked to a cargo molecule. The fusion protein can deliver the cargo molecule to a desired site and can be linked to the cargo molecule using any method known in the art (recombinant technology, chemical conjugation, chelation, etc.). The cargo molecule can be any type of molecule, such as a therapeutic or diagnostic agent.

[0485] In some embodiments, the cargo molecule is a protein and is fused to a fusion protein such that the cargo molecule is contained internally or externally in the nanocage. In other embodiments, the cargo molecule is not fused to a fusion protein and is contained internally in the nanocage. The cargo molecule is typically a protein, a small molecule, a radioisotope, or a magnetic particle.

[0486] The fusion proteins described herein specifically bind to their targets. Antibody specificity, which refers to the selective recognition of an antibody to a specific epitope of an antigen, of the antibodies or fragments described herein can be determined based on affinity and / or avidity. Affinity is determined by the equilibrium constant for the dissociation of an antigen with an antibody (K D ) and measures the binding strength between an antigenic determinant (epitope) and an antibody binding site. Avidity is a measure of the strength of binding between an antibody and its antigen. Antibodies are typically -5 ~10 -11 K of M D Combine with 10 -4 K exceeds M D is generally considered to represent non-specific binding. D The smaller the value of , the stronger the binding strength between the antigenic determinant and the antibody binding site. In an embodiment, the antibodies described herein have a 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M, 10 -14 M, or 10 -15 K less than M D has.

[0487] Also described herein are nucleic acid molecules encoding the fusion proteins and polypeptides described herein, as well as vectors containing the nucleic acid molecules and host cells containing the vectors.

[0488] Polynucleotides encoding fusion proteins described herein include polynucleotides having substantially the same nucleic acid sequence as the nucleic acid sequence of the polynucleotides of the present invention. A "substantially the same" nucleic acid sequence is defined herein as a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% identity to another nucleic acid sequence when the two sequences are optimally aligned (with appropriate nucleotide insertions or deletions) and compared to determine the exact nucleotide correspondence between the two sequences.

[0489] Suitable sources of polynucleotides encoding antibody fragments include any cell that expresses full-length antibodies, such as hybridomas and spleen cells. Fragments may be used alone as antibody equivalents, or may be recombined into equivalents as described above. The deletions and recombinations of DNA described in this section can be performed by known methods, such as those described in the published patent applications listed in the section entitled "Functional Equivalents of Antibodies" above, and / or other standard recombinant DNA techniques, such as those described below. Another source of DNA is single-chain antibodies produced from phage display libraries, as known in the art.

[0490] Furthermore, expression vectors are provided that contain the aforementioned polynucleotide sequences operably linked to expression sequences, promoters and enhancer sequences.Various expression vectors have been developed for efficient synthesis of antibody polypeptides in prokaryotes, such as bacteria and eukaryotic systems, including but not limited to yeast and mammalian cell culture systems.Vector of the present invention can include segments of chromosomal, non-chromosomal and synthetic DNA sequences.

[0491] Any suitable expression vector can be used. For example, prokaryotic cloning vectors include plasmids derived from E. coli, such as colEl, pCRl, pBR322, pMB9, pUC, pKSM and RP4. Prokaryotic vectors also include derivatives of phage DNA, such as Ml3 and other filamentous single-stranded DNA phages. An example of a vector useful in yeast is the 2μ plasmid. Suitable vectors for expression in mammalian cells include well-known derivatives of DNA sequences derived from SV-40, adenovirus, retrovirus, and shuttle vectors derived from the combination of functional mammalian vectors (such as those described above) with functional plasmids and phage DNA.

[0492] Additional eukaryotic expression vectors are known in the art (see, e.g., P J. Southern & P. ​​Berg, J. Mol. Appl. Genet, 1:327-341 (1982); Subramani et al, Mol. Cell. Biol, 1:854-864 (1981); Kaufman & Sharp, "Amplification And Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene", J. Mol. Biol, 159:601-621 (1982); Kaufman & Sharp, Mol. Cell. Biol, 159:601-664 (1982); Scahill et al., "Expression And Characterization Of The Product Of A Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells", Proc. Nat'l Acad. Sci. USA, 80:4654-4659 (1983); Urlaub & Chasin, Proc. Nat'l Acad. Sci USA, 77:4216-4220, (1980) (all of which are incorporated herein by reference).

[0493] An expression vector typically contains at least one expression control sequence operably linked to the DNA sequence or fragment to be expressed. The control sequence is inserted into the vector to control and regulate the expression of the cloned DNA sequence. Examples of useful expression control sequences are the lac system, the trp system, the tac system, the trc system, the major operator and promoter region of phage lambda, the control region of fd coat protein, yeast glycolytic promoters such as the promoter of 3-phosphoglycerate kinase, the promoter of yeast acid phosphatase such as Pho5, the promoter of yeast alpha-mating factor, and promoters from polyoma, adenovirus, retrovirus and simian virus such as the early and late promoters or SV40, and other sequences known to control the expression of genes in prokaryotic or eukaryotic cells and their viruses or combinations thereof.

[0494] Also described herein are recombinant host cells comprising the expression vectors described above. The fusion proteins described herein can be expressed in cell lines other than hybridomas. Nucleic acids comprising sequences encoding the polypeptides according to the invention can be used to transform suitable mammalian host cells.

[0495] Particularly preferred cell lines are selected based on high levels of expression, constitutive expression of the protein of interest, and minimal contamination from host proteins. Mammalian cell lines available as hosts for expression are well known in the art, including many immortalized cell lines, such as, but not limited to, HEK293 cells, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, and the like. Additional suitable eukaryotic cells include yeast and other fungi. Useful prokaryotic hosts include, for example, E. coli, such as E. coli SG-936, E. coli HB 101, E. coli W3110, E. coli X1776, E. coli X2282, E. coli DHI, E. coli T7 Shuffle, and E. coli MRC1, Pseudomonas, Bacillus, such as Bacillus subtilis, and Streptomyces.

[0496] These subject recombinant host cells can be used to produce fusion proteins by culturing the cells under conditions that allow expression of the polypeptide and purifying the polypeptide from the host cells or the medium surrounding the host cells. Targeting of an expressed polypeptide for secretion in a recombinant host cell can be facilitated by inserting a signal or secretory leader peptide coding sequence (see Shokri et al, (2003) Appl Microbiol Biotechnol. 60(6):654-664, Nielsen et al, Prot. Eng., 10:1-6 (1997); von Heinje et al., Nucl. Acids Res., 14:4683-4690 (1986), all of which are incorporated herein by reference) at the 5' end of the antibody-encoding gene of interest. These secretory leader peptide elements can be derived from either prokaryotic or eukaryotic sequences. Thus, suitably, a secretory leader peptide is used that is an amino acid attached to the N-terminus of a polypeptide to direct the translocation of the polypeptide from the host cell cytosol and secretion into the medium.

[0497] The fusion proteins described herein can be fused to additional amino acid residues. Such amino acid residues can be, for example, peptide tags to facilitate isolation. Other amino acid residues for homing the antibody to a specific organ or tissue are also contemplated.

[0498] It will be appreciated that Fab nanocages can be generated, for example, by co-transfection of a plasmid with one encoding a fusion protein comprising a Fab heavy chain fused to a ferritin chain (e.g., ferritin light chain) and another encoding a Fab light chain. Typically, the construct is arranged as a LC-linker-HC-linker-nanocage monomer / subunit, but can also be arranged as a HC-linker-LC-linker-nanocage monomer / subunit. As described herein, scFv or any other antigen-binding moiety can be used. Alternatively, a single-chain Fab-ferritin nanocage can be used, which requires only one plasmid transfection (e.g., using a plasmid encoding a fusion protein comprising a Fab light chain, a Fab heavy chain and a ferritin chain (e.g., ferritin light chain)). This can be done with different lengths of linkers between the Fab light chain and the Fab heavy chain, e.g., 60 or 70 amino acids. When using a single-chain Fab, it can be ensured that the heavy and light chains are paired. Tags (e.g., Flag, HA, myc, His6x, Strep, etc.) can also be added to the N-terminus of the construct or within the linker to facilitate purification as described above. Additionally, if the tag system is used to co-transfect different Fab nanoparticle plasmids, successive / additional affinity chromatography steps can be used to ensure that many different Fabs are present on the same nanoparticle. This provides multispecificity to the nanoparticles. Protease sites (e.g., TEV, 3C, etc.) can be inserted to cleave the linker and tag after expression and / or purification, if desired.

[0499] Any suitable method or route can be used to administer the fusion proteins described herein, including, for example, oral, intranasal, intravenous, intraperitoneal, subcutaneous, or intramuscular administration.

[0500] It is understood that when the fusion protein described herein is used in mammals for prevention or treatment, it is administered in the form of a composition further comprising a pharmaceutically acceptable carrier.Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof.Pharmaceutically acceptable carriers may further comprise small amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the binding protein.The composition of injection can be formulated to provide quick, sustained or delayed release of active ingredient after administration to mammals, as is well known in the art.

[0501] The fusion peptides and multibodies described herein are particularly useful for administration to humans, but can also be administered to other mammals. As used herein, the term "mammal" is intended to include, but is not limited to, humans, laboratory animals, domestic pets and farm animals.

[0502] The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited to the following examples in any way, but rather as embracing any and all variations that become evident as a result of the teachings provided herein.

[0503] The following examples do not include detailed descriptions of conventional methods such as those used for constructing vectors and plasmids, inserting genes encoding polypeptides into such vectors and plasmids, or introducing plasmids into host cells. Such methods are well known to those of skill in the art and are described in numerous publications, including Sambrook, J., Fritsch, EF and Maniatis, T. (1989), Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, which is incorporated herein by reference.

[0504] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. Thus, the following examples specifically point out exemplary aspects of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure. EXAMPLES

[0505] Example 1 summary The rapid evolution of HIV continues to be a major barrier to the development of therapeutics for prevention and treatment. Here, we describe the design of second-generation antibody platforms, termed Multabody.v2 (MB.v2) and Multabody.v3 (MB.v3), that allow the formation of a single molecule capable of establishing multiple simultaneous interactions with three independent epitopes of a target, in this case the HIV envelope. The multivalent and multispecific properties of this technology lead to extraordinary potency and complete pan-neutralization of a variety of diverse HIV-1 strains, surpassing those of the most potent anti-HIV bnAbs. Thus, these new MB.v2 and MB.v3 platforms have prophylactic and therapeutic potential against HIV-1. Introduction

[0506] Despite decades of research, there are no effective vaccines or treatments against human immunodeficiency virus type I (HIV-1). However, the fact that a small proportion of HIV-1-infected individuals develop antibodies with extraordinary neutralizing potency across circulating HIV-1 isolates highlights the possibility of antibody-mediated control of HIV-1. Since the discovery of the first generation broadly neutralizing antibodies (bNAbs) 2F5 (1), 4E10 (2, 3), 2G12 (4) and b12 (5, 6), the catalogue of bNAbs has increased dramatically due to the implementation of new techniques of Env-specific single B cell sorting (7–9), antibody cloning and high-throughput neutralization assays (10–13), and more recently proteome deconvolution (14). Several dozen HIV bNAbs have now been described that target six conserved sites on the trimeric HIV envelope (Env), including the V1 / V2 loop at the trimer apex, the V3 loop glycan, the CD4 binding site (CD4bs), the gp120-g41 interface, the fusion peptide, and the membrane-proximal external region (MPER) ( 7 , 9 , 10 , 12 – 19 ).

[0507] Interest in bNAbs as therapeutic molecules in the fight against HIV-1 stems from the potent antiviral activity observed in challenge studies in macaques (20–23, 23) and humanized mice (24–27), as well as the reduction in viremia achieved in infected humans after infusion of bNAbs (28–32). Furthermore, antibodies have important advantages compared to oral antiretroviral therapy (ART): they have a longer circulating half-life and can form immune complexes that enhance host immunity against the virus. These observations have led to the clinical evaluation of antibody-based therapies to confer protection against HIV-1 via passive administration of bNAbs, as well as efforts to control and / or eliminate HIV-1 in infected individuals.

[0508] One of the main limitations to the clinical use of bNAbs is the rapid evolution of neutralization-resistant virus populations (29–31, 33, 34). RNA viruses such as HIV-1 exhibit extraordinary genetic diversity that allows the virus to generate resistance mutations to escape mAb recognition (35). However, mutations that abrogate binding to a specific bNAb may entail a significant penalty in viral fitness (36–38). Similar to the combination of different drugs in HIV-1 treatment regimens, this observation suggests that successful antibody-based therapy against HIV-1 should include a combination of bNAb specificities. As a result, the development of various formats of antibody-like molecules with dual specificities (39–41) or trispecificities (42–44) against Env has been explored recently. An additional consideration is the amount of antibody required for in vivo efficacy. Indeed, extensive efforts have been made to engineer bNAbs to improve their potency using structure-guided design or bioinformatics approaches (e.g., VRC01 (45), 10E8 (46, 47) and NIH45-46 (48)), but have so far met with limited success. For bispecific and trispecific antibodies targeting multiple epitopes in Env, potency is generally limited by the potency of their parent mAb. As a result, significant improvements in neutralization breadth have been achieved, but relatively little in antiviral potency has been achieved so far. 40,43, (43) ,45 .

[0509] We recently described a MULTI-specific multi-affinity anti-BODY (Multabody) platform to drive oligomerization of antibody fragments and convert SARS-CoV-2-targeting antibodies into highly potent neutralizers (49). Utilizing this platform, we now combine multiple copies of the three best bNAbs against HIV-1 with a crystallizable fragment (Fc) derived from IgG1 in a single molecule. By further engineering the Multabody to maximize the number of Fabs in the particle, we significantly improved the potency, breadth and uniformity achieved by Multabody technology. The resulting anti-HIV Multabody.v3 achieves complete pan-viral neutralization breadth and mean IC against a multi-clade panel of 118 pseudoviruses (PsVs) at 0.0009 μg / mL (0.4 pM), 32- and 460-fold lower in mass and molar concentration, respectively, compared to a cocktail made with the current best known bNAbs. 50 Median values ​​are shown. The Multabody.v3 design described herein represents a robust and powerful platform for the development of next generation biologics against HIV-1. material and method

[0510] Expression and purification of Fab-only apoferritin-based multimers. Genes encoding the light chain of human apoferritin and scFab-human apoferritin fusions were synthesized and cloned into the pHLsec expression vector by GeneArt (Life Technologies). 200 ml of HEK293F cells (Thermo Fisher Scientifics) were cultured in Freestyle expression medium for 0.8–10 min. 6Cells were seeded at a density of 10000 cells / mL and incubated in a Multitron Pro shaker (Infors HT) at 37 °C, 8% CO2 and 70% humidity with 125 rpm shaking. Within 24 h after seeding, cells were transiently transfected with 50 μg of filtered DNA in a 1:1 ratio, preincubated for 10 min at room temperature (RT) with the transfection reagent FectoPRO (Polyplus Transfections). scFab-human apoferritin and human apoferritin-encoding plasmids were mixed in ratios of 1:4, 1:1, 4:1 and 1:0 to obtain nanoparticles with 20%, 50%, 80% and 100% scFab conjugation valency, respectively. After 6–7 days, cell suspensions were harvested by centrifugation at 5000 × g for 15 min and the supernatant was filtered through a 0.22 μm Steritop filter (EMD Millipore). The nanoparticles were purified by affinity chromatography against the Fab and eluted after washing. Fractions containing the protein were pooled, concentrated and loaded onto a Superose 6 10 / 300 GL size exclusion column (GE Heathcare) in 20 mM sodium phosphate pH 8.0, 150 mM NaCl.

[0511] Design, Expression, and Purification of 32-N MB.v1, 32-N MB.v2, and 32-N MB.v3 Multabodies. Genes encoding scFab, scFc, and Fc fragments linked to hemi-ferritin were generated by deleting residues 1–95 (leaving the C-terminal part of ferritin, "C-ferritin") or residues 95–175 (leaving the N-terminal part of ferritin, "N-ferritin") of the light chain of human apoferritin using the KOD-Plus Mutagenesis Kit (Toyobo, Osaka, Japan). Transient transfection of 32-N MB.v1 in HEK293F cells was obtained by mixing 67 μg of plasmids PGDM1400 scFab-human apoferritin:scFcN-ferritin:N49P7 scFab-C-ferritin:10E8v4 scFab-C-ferritin in a ratio of 4:2:1:1. For 32-N MB.v2, 59 μg of the plasmid PGDM1400 scFab-human apoferritin:N49P7 scFab-N-ferritin:10E8v4 scFab-C-ferritin-scFc was used in a ratio of 5:1:1, and for 32-N MB.v3, 63 μg of the plasmid PGDM1400 scFab-human apoferritin:N49P7 scFab-N-ferritin:10E8v4 scFab-C-ferritin-Fc was used in a ratio of 3:1:1. The characteristics of the different constructs used in this example are summarized in Table 1 below.

[0512] Table 1: Characteristics of multibody constructs. [Table 1]

[0513] The DNA mixture was filtered and incubated with FectoPRO in a 1:1 ratio at RT before being added to the cell culture. Multabodies were first purified by affinity chromatography using a HiTrap Protein A HP column (GE Healthcare) with 20 mM Tris pH 8.0, 3 M MgCl2 and 10% glycerol elution buffer. After buffer exchange using a PD-10 desalting column (GE Healthcare), Multabodies were further purified by a second affinity chromatography using a HiTrap Protein L column (GE Healthcare). Protein-containing fractions were concentrated and further purified by gel filtration on a Superose 6 10 / 300 GL column (GE Healthcare) in 20 mM sodium phosphate pH 8.0, 150 mM NaCl.

[0514] Negative staining electron microscopy. Three microliters of Multabody at a concentration of approximately 0.02 mg / mL was added to carbon-coated copper grids for 30 seconds and stained with 3 μL of 2% uranyl formate. The stained excess was immediately removed from the grids using Whatman No. 1 filter paper, and an additional 3 μL of 2% uranyl formate was added for 20 seconds. Grids were imaged using a field emission FEI Tecnai F20 electron microscope operated at 200 kV and equipped with an Orius charge-coupled device (CCD) camera (Gatan Inc.).

[0515] Biolayer Interferometry. Binding kinetics measurements were performed using an Octet RED96 BLI system (Pall ForteBio) in PBS pH 7.4, 0.01% BSA and 0.002% Tween®. Unique His-tagged ligands for each Multabody component were selected and loaded onto Ni-NTA biosensors to reach a signal response of 0.8 nm. Association rates were measured by transferring the loaded biosensors into wells containing serial dilutions of Multabody (50-25-12.5-6.25-3.1-1.5 nM) and into buffer-containing wells, respectively. Dissociation rates were measured by immersing the biosensors into buffer-containing wells. The duration of these two steps was 180 s each. To achieve selective binding to PGDM1400, the D368R mutation in the CD4bs of the BG505 SOSIP.664 trimer was introduced, thus disrupting the binding of N49P7 to this antigen. Similarly, gp120 subunit 93TH057, MPER peptide and Fc receptors (FcγRI, FcγRIIa, FcγRIIb and hFcRn complexed with β2-microglobulin) were produced as ligands for N49P7, 10E8 and Fc binding, respectively. The ability of the multibodies to undergo endosomal recycling was tested by measuring their binding to the hFcRnβ2-microglobulin complex at physiological pH (7.5) and acidic pH (5.6).

[0516] Size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS). A MiniDAWN TREOS and Optilab T-rEX refractometer (Wyatt) were used coupled to an Agilent Technologies 1260 infinity II HPLC. 50 μg of 24-mer PGDM1400 scFab multimer and Multabody 32-N were loaded onto a Superose 6 10 / 300 (GE Healthcare) column in 20 mM sodium phosphate pH 8.0, 150 mM NaCl. Data collection and analysis were performed using ASTRA software (Wyatt).

[0517] Measurement of melting and aggregation temperatures. The melting temperatures (T m ) and aggregation temperature (T agg ) was determined using the UNit system (Unchained Labs). m is obtained by measuring the centroid mean fluorescence, while T agg was determined as the temperature at which a 50% increase in static light scattering at a wavelength of 266 nm was observed relative to the baseline. Samples were concentrated to 1.0 mg / mL and subjected to a temperature gradient from 25 to 95 °C in 1 °C increments. The mean and standard error of three independent measurements were calculated using UNit analysis software.

[0518] Accelerated thermostability assay. The different Multabody versions were subjected to extreme conditions of temperature and concentration for 4 consecutive weeks. 10 mg / ml of each sample was incubated at 40 °C and the percentage of properly folded protein was calculated every week and analyzed by the area of ​​each peak obtained by size exclusion chromatography when 10 μL of sample was loaded onto a Superose 6 10 / 300 GL column (GE Healthcare). A functional assay was added to this analysis, in this case a neutralization assay, to determine the amount of active protein at the beginning (week 0) and end (week 4) of the experiment.

[0519] Virus production and TZM-bl neutralization assays. As previously described ( 50 ), HIV-1 subtype B backbone NL4-3.Luc.R -A panel of 25 HIV-1 pseudotyped viruses was generated by cotransfecting 293T cells with E plasmid (AIDS Research and Reference Reagent Program (ARRRP)) and a plasmid encoding a full-length Env clone. HIV isolates X2088, ZM106.9, NL4.3 and 3817 were kindly provided by the Collaboration for AIDS Vaccine Discovery (CAVD), SF162 was kindly provided by JL Nieva. (Biofisika Institute), and pCNE8, 1632, THRO, 278, ZM197, JRCSF, t257, Du422, BG505, p1054.TC4.1499, 6535, ZM214M.PL15, AC10.29, p16845, P6244_13.B5.4576, pM246F_C1G, TRJO4551, QH0692 and pCAAN5342 were kindly provided by the NIH ARRRP. Neutralization was determined in a one-cycle neutralization assay using a standard TZM-bl neutralization assay. Briefly, antibodies and antibody-based particles were incubated with 10–15% tissue culture infectious dose of pseudovirus for 1 h at 37°C prior to 44–72 h incubation with TZM-bl cells. Virus neutralization was monitored by adding Britelite plus reagent (PerkinElmer) to the cells and measuring luminescence in relative light units (RLU) using a Synergy Neo2 Multi-Mode Assay Microplate Reader (Biotek Instruments). An extended multiclade panel of 118 PsVs was performed at the Center for Virology and Vaccine Research at Harvard Medical School by Dr. Michael Seaman following standard protocols. Antibody breadth was determined using a cutoff limit of 10 μg / mL.

[0520] Pharmacokinetics. In vivo studies were performed using female NOD / Shi-scid / IL-2Rγnull immunodeficient mouse strain (NCG). 32-N MB.v3, composed of scFabs of antibodies PGDM1400, N49P7 and 10E8v4 and scFc fragment of IgG1 Fc containing half-life extension mutations (M428L / N434S), was used for the study. A single injection of 5 mg / kg Multabody or control sample (IgG mixture matching Fab specificity of Multabody) in 200 μL PBS (pH 7.5) was subcutaneously injected. Blood samples were collected at multiple time points and serum samples were evaluated for levels of circulating antibodies by ELISA. Briefly, 96-well Pierce nickel-coated plates (Thermo Fisher) were plated with BG505 SOSIP.664_D368R trimer, gp120 subunit 93TH057 and His recognized by MPER peptide. 6x Circulating sample concentrations were determined using reagent-specific standard curves for IgG and Multabody, with 50 μL coating at 0.5 μg / ml of each tagged antigen. HRP-Protein A (Invitrogen) was used as the secondary molecule, and chemiluminescent signals were quantified using a Synergy Neo2 Multi-Mode Assay Microplate Reader (Biotek Instruments).

[0521] result The potency of HIV-1 bNAbs can be enhanced with avidity Apoferritin is a spherical nanocage with a hydrodynamic radius of approximately 6 nm formed by the self-oligomerization of 24 identical subunits (Figure 1a). To investigate the effect of multivalency on neutralization potency, we used the self-assembly properties of the light chain of human apoferritin to multimerize antigen-binding (Fab) fragments derived from the most potent and broad-spectrum HIV-1 bNAbs targeting different HIV-1 Env epitopes. Apoferritin subunits were genetically fused to single-chain Fabs (scFabs). To ensure correct Fab heterodimerization, a flexible linker between the light and heavy chains was used to generate the scFabs. Apoferritin self-assembly promoted the multimerization of the scFabs, displaying the antibody fragments around the nanocage (Figure 1b). Different densities of multimerized Fabs were achieved by co-transfection of plasmids encoding scFab-human apoferritin together with different ratios of non-genetically modified human apoferritin (Figures 1c and 3). The ability of scFab-apoferritin fusions to block HIV-1 infection was compared to the corresponding IgG using a panel of small HIV-1 pseudoviruses (PsVs) (Fig. 1d). Strikingly, PGDM1400, one of the most potent anti-HIV bNAbs described to date, showed 10-40-fold higher neutralization potency when multimerized via the light chain of apoferritin compared to its conventional IgG format. bNAb 10-1074 also showed a considerable improvement in neutralization potency (4-40-fold), whereas bNAbs 10E8, N49P7 and VRC01 showed no effect or more modest enhancement.

[0522] Characterization of scFab-apoferritin fusions. SDS-PAGE bands corresponding to scFab-apoferritin and unconjugated apoferritin were quantified by densitometry using ImageJ software (rsb.info.nih.gov / ij / ) (Fig. 2a). Intensity plots of the bands in each lane (yellow boxes) are shown in Fig. 2b. The approximate number of scFabs displayed on the particles was calculated as follows: intensity of scFab band / total intensity, and compared with the theoretical number inferred from the DNA ratio used for co-transfection (Fig. 2c).

[0523] Multibodies potently and broadly neutralize HIV-1 Considering these results, we sought to increase the coverage of PGDM1400 using our previously described Multabody platform based on an apoferritin split design (49). This strategy consists of splitting the four-helical apoferritin subunit into two halves (N-ferritin and C-ferritin) and fusing their N-termini to scFabs of different specificities (Figure 3a). This approach allows the inclusion of a higher number of Fabs on the surface of the nanocage, resulting in a final molecule with higher binding activity. Furthermore, this design allows the efficient combination of three different antibody specificities as well as fragment crystallizable (Fc) to endow the molecule with IgG-like properties such as ease of purification leveraging Protein A affinity. Specifically, we combined the scFab PGDM1400 with the scFabs of the near-pan neutralizing antibodies 10E8v4 (solubility-improved modified 10E8(51)) and N49P7 and the Fc fragment of human IgG1 isotype (Figure 3a). As expected, the resulting Multabody, called 32-N, formed highly decorated homogenous particles (Figures 3b and c) with similar biophysical and functional properties as the corresponding IgG (Figure 3d). Epitope binding by the trispecific Multabody was evaluated in binding kinetics experiments using epitope-specific molecules: BG505 SOSIP D368R (PGDM1400), 93TH057 gp120 (N49P7), MPER peptide (10E8v4) and human Fc receptors (FcRn and FcγR) (Fc) (Figure 3e). Binding to the three epitope-specific antigens with high apparent binding affinity and no detectable dissociation confirms the presence of all specificities in the multibody. Individual IgG molecules did not have the capacity to bind all antigens (Figure 4).

[0524] The neutralization potency and breadth of Multabody 32-N was evaluated against a panel of 14 PsVs in a standardized in vitro TZM-bl neutralization assay (50). The 14-PsV panel was designed to include low-sensitivity PsVs with a minimum of one resistant PsV for each bNAb evaluated (cutoff IC50 (Set the concentration to 10 μg / mL). IC of multibody 50 Values ​​and ranges were compared to individual IgGs and IgG cocktails containing the same relative amounts of IgG present in the multibody. IC of 32-N MB 50 The median was 0.0071 μg / mL (3 pM) (Figure 5). However, despite the 12-fold improved potency achieved with respect to the parental IgG mixture, the multibody was unable to reach 100% neutralization against this panel (a width of 93% at the cut-off limit of 10 μg / mL). Furthermore, the individual IC 50 Examination of the values ​​revealed that Multabody exhibited low potency against PsV resistant to PGDM1400 neutralization (Figure 5b). This data suggests that the neutralizing properties of Multabody are highly dependent on one of the three antibody specificities within the particle, in this case PGDM1400.

[0525] Engineering the apoferritin scaffold To further improve the neutralization properties of Multabody, several modifications were introduced into its design to generate second generation versions (MB.v2 and MB.v3). In the original Multabody (hereafter referred to as MB.v1), the scFc is located at the N-terminus of the N-ferritin half, and only one Fab (Fab2 or Fab3) per functional Fc homodimer is incorporated into the Multabody (top row of Figure 6).

[0526] In comparison, in the optimized MB.v2, the Fc fragment is located at the C-terminus of the C-ferritin half. Driven by split apoferritin complementation, two different Fabs (Fab2 and Fab3) and one Fc domain self-associate in a 1:1:1 ratio at defined positions relative to each other (Figure 6; bottom row; see "MB.v2 format").

[0527] In the case of MB.v3, this optimized version contains more Fabs per Fc homodimer. In this design, two Fab2s and two Fab3s per dimeric Fc are incorporated into the Multabody. To achieve this, a monomeric Fc fragment (i.e., one Fc chain) and a scFab are placed at the C-terminus and N-terminus of the C-ferritin half, respectively (Figure 7a). As a result, dimerization of functional Fc homodimers drives the assembly of MB.v3 particles along with split-ferritin complementation and ferritin subunit oligomerization (Figure 7b).

[0528] Importantly, these designs ensure the assembly of a greater number of Fabs distinct from PGDM1400 (i.e., Fab2 and Fab3), thus supporting more balanced binding activity for each of the three Fabs in the fully assembled Multabody.

[0529] The resulting Multabody particles (MB.v2 and MB.v3) assembled into highly stable, well-formed spherical particles with no significant differences in morphology and biophysical properties between the different Multabody versions (Figure 8a-c). Antigen (Figure 8d) and Fc receptor binding (Figure 8e) confirmed proper folding of the Multabody, including proper dimerization of the Fc fragment in the case of MB.v3. In the MB.v2 and MB.v3 formats, placing the Fc at the C-terminus of ferritin leads to the formation of particles with an inverted, more distantly positioned Fc domain, resulting in reduced Fc binding activity. Accordingly, binding of these Multabodies to FcγRI and FcRn at acidic pH was reduced compared to 32-N MB.v1 (Fig. 3e and Fig. ​Fig.8e8),8e, and binding to FcRn at acidic pH did not show any measurable K, even in the presence of the half-life extended mutant LS (M428L / N434S), as shown for the 32-N MB.v3 LS mutant (Fig.8f). off This resulted in...

[0530] Exceptional Potency and Pan-Neutralization Breadth Achieved by Second-Generation Multabody Particles Next, we evaluated the neutralization potency of the optimized Multabody versions (MB.v2 and MB.v3) against a PsV panel generated by spiking our previous panel with 11 HIV-1 strains highly resistant to PGDM1400. The resulting 25-PsV panel showed that 56% of PsV mutants resistant to PGDM1400 neutralization (cutoff IC 50 The neutralization profiles of antibodies N49P7 and 10E8v4, as engineered, were more prevalent in 32-N MB.v2 and 32-N MB.v3, allowing this optimized multabody to achieve pan-neutralization while maintaining the enhanced neutralization potency previously observed for this type of molecule (Fig. 9a). When tested against an expanded multi-clade panel of 118 PsVs, 32-N MB.v3 outperformed the corresponding IgG cocktail (100% viral coverage, cut-off IC 50 The IC50-100 of IgG cocktail and 32-N MB.v1 were consistent with the pan-neutralization breadth of the antibody (set at 10 μg / mL), but showed remarkable neutralization potency (Figure 9b). Specifically, the IgG cocktail and 32-N MB.v1 each had an IC50-100 of 0.001 μg / mL. 50 Although 32-N MB.v3 was only able to neutralize 9% and 8% of PsV with IC values ​​of only 0.001 μg / mL, 50 Remarkably, 32-N MB.v3 had an IC value of only 0.0009 μg / mL (0.4 pM). 50 The median neutralization was achieved and thus 32-fold and 490-fold more potent by mass and molar concentration, respectively, compared to the IgG cocktail (FIG. 9b).

[0531] In vivo pharmacokinetics of multibodies are similar to that of the corresponding IgG Next, we investigated the in vivo bioavailability of 32-N MB.v3, which contains an IgG1 Fc fragment with LS mutations. A single dose of 5 mg / kg was administered subcutaneously to NOD / Shi-scid / IL-2Rγnull immunodeficient mouse strain (NCG), and the levels of Multabody in serum were detected every 2 days for 15 consecutive days. Multabody administration was well tolerated, with no weight loss or visible signs of toxicity. Furthermore, the Multabody showed several days of in vivo exposure with a decay rate similar to the parent IgG cocktail (Figure 10). This data demonstrates the feasibility of the Multabody platform to generate molecules with bioavailability properties and provide a promising set of early in vivo validation for their development potential.

[0532] References [ka] [ka] [ka] [ka]

Claims

1. A fusion protein comprising a first nanocage monomer or a subunit thereof linked to an Fc monomer, wherein a plurality of said fusion proteins can self-assemble to form a nanocage comprising one or more Fc dimers.

2. The fusion protein of claim 1 , wherein the Fc monomer is linked to the first nanocage monomer or its subunit at the N-terminus or C-terminus, preferably the C-terminus, of the first nanocage monomer or its subunit.

3. The fusion protein of claim 1, wherein the subunits include an N subunit or a C subunit that substantially corresponds to the N-terminal half of a nanocage monomer and the C-terminal half of a nanocage monomer, respectively, and the N subunit and the C subunit are capable of self-assembly to form a nanocage monomer.

4. The fusion protein of claim 3, wherein the Fc monomer is linked to the N subunit or C subunit at the C-terminus of the N subunit or C subunit, preferably the Fc monomer is linked to the C subunit at the C-terminus.

5. The fusion protein of claim 1 , wherein the first nanocage monomer or subunit thereof is further linked to a first biologically active moiety.

6. The fusion protein of claim 5, wherein the first biologically active moiety is linked to the first nanocage monomer or its subunit at the N-terminus or C-terminus, preferably the N-terminus, of the first nanocage monomer or its subunit.

7. The fusion protein of claim 6, wherein the first biologically active moiety is linked to the N subunit or C subunit at the N-terminus of the N subunit or C subunit, preferably the first biologically active moiety is linked to the C subunit at the N-terminus.

8. The fusion protein of claim 5 , wherein the first biologically active portion comprises a first antigen-binding portion.

9. The fusion protein of claim 8 , wherein the first antigen-binding portion comprises an antibody or a fragment thereof.

10. The fusion protein of claim 5 , wherein the first antigen-binding portion specifically binds to an antigen associated with a condition preventable and / or treatable by an antibody.

11. The fusion protein of claim 10, wherein the antigen is associated with an infectious agent, including a virus, a bacterium, a parasite, a fungus, or a yeast, a cancer, or an immune disease, including an autoimmune disease.

12. The fusion protein of claim 11, wherein the first antigen-binding portion comprises an HIV-1 specific antigen-binding portion.

13. 13. The fusion protein of claim 12, wherein the HIV-1 specific antigen binding portion binds to BG505 SOSIP_D368R, 93TH057 gp120, and / or MPER peptide.

14. 14. The fusion protein of claim 13, wherein the HIV-1 specific antigen-binding portion comprises an HIV-1 specific antigen-binding portion derived from PGDM1400, 10E8v4, and / or N49P7.

15. The fusion protein of claim 1, comprising a first nanocage monomer or a subunit thereof linked to the Fc monomer at the C-terminus of the first nanocage monomer or a subunit thereof and linked to the first biologically active moiety at the N-terminus of the first nanocage monomer or a subunit thereof.

16. The fusion protein of claim 5, wherein the fusion protein comprises a C subunit linked to the Fc monomer at the C-terminus of the C subunit and linked to the first biologically active moiety at the N-terminus of the C subunit.

17. The fusion protein of claim 1, wherein the first nanocage monomer or a subunit thereof is selected from ferritin, apoferritin, encapsulin, SOR, lumazine synthase, pyruvate dehydrogenase, carboxysome, vault protein, GroEL, heat shock proteins, E2P, MS2 coat protein, fragments thereof, and mutants thereof.

18. The fusion protein of claim 17 , wherein the first nanocage monomer or subunit thereof is apoferritin, optionally human apoferritin.

19. 19. The fusion protein of claim 18, wherein the first nanocage monomer or a subunit thereof is an apoferritin light chain, optionally a human apoferritin light chain.

20. The fusion protein of claim 18, wherein the fusion protein comprises a first apoferritin subunit, optionally a first human apoferritin subunit, and the first apoferritin subunit is capable of self-assembling with a second apoferritin subunit.

21. The fusion protein of claim 20, wherein the first and second apoferritin monomer subunits interchangeably comprise the "N" and "C" regions of apoferritin.

22. A nanocage comprising at least one fusion protein according to claim 1 and at least one second nanocage monomer or subunit thereof that self-assembles with said fusion protein.

23. A fusion protein comprising a first nanocage monomer or a subunit thereof linked to an scFc fragment at the C-terminus of the first nanocage monomer or a subunit thereof, wherein a plurality of the fusion proteins can self-assemble to form a nanocage.

24. 24. The fusion protein of claim 23, wherein the first nanocage monomer or a subunit thereof is selected from ferritin, apoferritin, encapsulin, SOR, lumazine synthase, pyruvate dehydrogenase, carboxysome, vault protein, GroEL, heat shock proteins, E2P, MS2 coat protein, fragments thereof, and mutants thereof.

25. 25. The fusion protein of claim 24, wherein the first nanocage monomer or subunit thereof is apoferritin, optionally human apoferritin.

26. 26. The fusion protein of claim 25, wherein the first nanocage monomer or a subunit thereof is an apoferritin light chain, optionally a human apoferritin light chain.

27. 24. A nanocage comprising at least one fusion protein of claim 23 and at least one second nanocage monomer or subunit thereof that self-assembles with the fusion protein.

28. A therapeutic or prophylactic composition comprising the nanocage of claim 22.

29. A nucleic acid molecule encoding the fusion protein of claim 1.

30. 30. A vector comprising the nucleic acid molecule of claim 29.

31. A host cell comprising the vector of claim 30 and producing the fusion protein of claim 1.

32. A pharmaceutical composition comprising the nanocages of claim 22 or the composition of claim 28 for use in the treatment and / or prevention of a condition.

33. 33. The pharmaceutical composition or composition of claim 32, wherein the condition is an HIV-1 associated condition.

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