Multivalent and multispecific nanoparticle platforms and methods

Abstract

The present invention relates to multivalent and multispecific nanoparticle platforms and methods. A fusion protein comprising: a first nanocage monomer subunit of a nanocage monomer; and a bioactive moiety attached to the first nanocage monomeric subunit; wherein the fusion protein is self-assembled with a protein comprising a second nanocage monomer subunit to form a nanocage monomer.

Description

TECHNICAL FIELD

[0001] The present invention relates to nanoparticles. In particular, the present invention relates to nanoparticle subunit fusion proteins, vaccines, prophylactic and therapeutic agents comprising the nanoparticles, and related compositions and methods. BACKGROUND

[0002] Nanoparticles have contributed to advances in various disciplines. Their use has the potential to confer targeted delivery, and can enable ordered microchip engineering, cage-like microenvironments for slow release and catalytic processes.

[0003] For the manufacture of nanoparticles containing sensitive and metastable proteins, protein self-assembly is an attractive approach. Indeed, self-assembled nanoparticles form under physiological conditions through non-covalent interactions and reliably produce uniform and often symmetrical nanocapsules or nanocages. Self-assembled protein nanoparticles have three distinct surfaces: the outer surface, the inner surface, and the inter-subunit surface, all of which can be tailored to increase their functionality.

[0004] Fusion proteins comprising self-assembling proteins have been reported. For example, it is known to display antigens on the outer surface of assembled nanocages for use as vaccines.

[0005] However, there remains a need for improved compositions and methods involving nanocages. SUMMARY

[0006] Aspects herein describe fusion proteins and self-assembling nanocages that allow for the presentation and tailoring of multiple cargo molecules (e.g., multiple copies of the same cargo molecule and / or different cargo molecules) on a single nanoparticle, as well as related compositions and methods. In some embodiments, the disclosed fusion proteins, nanocages, compositions, and methods allow for control over the ratio of different cargo molecules, e.g., to optimize the self-assembling nanocage for a particular therapeutic and / or prophylactic purpose.

[0007] According to one aspect, the present invention provides a fusion protein comprising:

[0008] a first nanocage monomer subunit of a nanocage monomer; and

[0009] a biologically active moiety linked to the first nanocage monomer subunit;

[0010] wherein the fusion protein self-assembles with a protein comprising a second nanocage monomer subunit to form a nanocage monomer.

[0011] In one aspect, the biologically active moiety modifies the inner surface and / or the outer surface of the assembled nanocage.

[0012] In an aspect, the biologically active moiety comprises: an antibody or fragment thereof, an antigen, a detectable moiety, a pharmaceutical agent, a diagnostic agent, or a combination thereof.

[0013] In an aspect, the antibody or fragment thereof comprises an Fc fragment.

[0014] In an aspect, the Fc fragment is an IgGl Fc fragment.

[0015] In an aspect, the Fc fragment comprises one or more mutations, e.g., LS, YTE, LALA, and / or LALAP, that modulate the half-life of the fusion protein, e.g., from minutes or hours to days, weeks, or months.

[0016] In an aspect, the antibody or fragment thereof comprises a Fab fragment.

[0017] In an aspect, the antibody or fragment thereof comprises a scFab fragment, a scFv fragment, or a sdAb fragment.

[0018] In an aspect, the antibody or fragment thereof comprises a heavy chain and / or a light chain of a Fab fragment.

[0019] In an aspect, the antibody or fragment thereof comprises both a light chain and a heavy chain, or in the case of an Fc fragment, a first chain and a second chain, optionally separated by a linker.

[0020] In an aspect, the linker comprises or consists of a sequence having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to:

[0021] GGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGTATAGASSGSGSSGSSSSGGTG.

[0022] In an aspect, the fusion protein is associated with a separately produced Fab light chain and / or heavy chain.

[0023] In an aspect, the antibody or fragment thereof specifically binds to an antigen associated with an antibody preventable condition and / or an antibody treatable condition.

[0024] In one aspect, the antigen is associated with an infectious agent, including a virus (e.g., HIV (including HIV-1), influenza, RSV, rotavirus), a bacterium (e.g., TB, C. difficile), a parasite (e.g., malaria), a fungus, or a yeast; a cancer (e.g., CD19, CD22, CD79, BCMA, or CD20), including a solid cancer and a liquid cancer; or an immune disease, including an autoimmune disease.

[0025] In one aspect, the antigen is associated with HIV-1, and the antibody or fragment thereof includes, for example, ibalizumab-A12P, 10E8, 10E8.v4, N49P7, PGDM1400, 10-1074, VRC01, or a combination thereof.

[0026] In one aspect, the antibody or fragment thereof includes or consists of a sequence having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the following sequence:

[0027] Fc chain 1:

[0028] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

[0029] GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK

[0030] AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK.

[0031] Fc chain 2:

[0032] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

[0033] GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK

[0034] AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK; Ibalizumab-A12P light chain:

[0035] DIVMTQSPDSLPVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWAST

[0036] RESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIKRTVAAPS

[0037] VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0038] Ibalizumab-A12P heavy chain:

[0039] QVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGT

[0040] DYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQG

[0041] TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGSR; 10E8.v4 light chain:

[0042] SELTQDPAVSVALKQTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDR

[0043] FSGSASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVLSQPKAAPSVTL

[0044] FPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC;

[0045] 10E8.v4 heavy chain:

[0046] EVRLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEG

[0047] WSVDYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGE

[0048] EYFQDWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS

[0049] GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCSR;

[0050] N49P7 light chain:

[0051] QSALTQPRSVSASPGQSVTISCTGTHNLVSWCQHQPGRAPKLLIYDFNKRPSGVPDRFSG

[0052] SGSGGTASLTITGLQDDDDAEYFCWAYEAFGGGTKLTVLGQPKAAPSVTLFPPSSEELQ

[0053] ANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC;

[0054] N49P7 heavy chain:

[0055] ADLVQSGAVVKKPGDSVRISCEAQGYRFPDYIIHWIRRAPGQGPEWMGWMNPMGGQV

[0056] NIPWKFQGRVSMTRDTSIETAFLDLRGLKSDDTAVYYDRSNGSGKRFESSNWFLDLWG

[0057] RGTAVTIQSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDSR;

[0058] PGDM1400 light chain:

[0059] DFVLTQSPHSLSVTPGESASISCKSSHSLIHGDRNNYLAWYVQKPGRSPQLLIYLASSRAS

[0060] GVPDRFSGSGSDKDFTLKISRVETEDVGTYYCMQGRESPWTFGQGTKVDIKRTVAAPSV

[0061] FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0062] PGDM1400 heavy chain:

[0063] QAQLVQSGPEVRKPGTSVKVSCKAPGNTLKTYDLHWVRSVPGQGLQWMGWISHEGDK

[0064] KVIVERFKAKVTIDWDRSTNTAYLQLSGLTSGDTAVYYCAKGSKHRLRDYALYDDDGA

[0065] LNWAVDVDYLSNLEFWGQGTAVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY

[0066] FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDSR; or

[0067] combinations thereof.

[0068] In one aspect, the antibody or fragment thereof is coupled to or associated with another moiety, such as an antigen, a detectable moiety (e.g., a small molecule, a fluorescent molecule, a radioisotope, or a magnetic particle), an agent, a diagnostic agent, or a combination thereof.

[0069] In an aspect, the antibody or fragment thereof comprises an antibody drug conjugate.

[0070] In an aspect, the antigen is associated with a vaccine-preventable condition and / or a vaccine-treatable condition.

[0071] In an aspect, the antigen is associated with an infectious agent, including a virus, a bacterium, a parasite, a fungus, or a yeast; a cancer, including a solid cancer and a liquid cancer; or an immune disease, including an autoimmune disease.

[0072] In an aspect, the detectable moiety comprises a fluorescent protein, such as GFP, EGFP, sapphyrin, and / or a flavin-based fluorescent protein such as a LOV protein (such as iLOV).

[0073] In an aspect, the agent comprises a small molecule, a peptide, a lipid, a carbohydrate, or a toxin.

[0074] In an aspect, about 3 to about 100 nanocage monomers, such as 24, 32, or 60 monomers, or about 4 to about 200 nanocage monomer subunits, such as 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 subunits, optionally in combination with one or more complete nanocage monomers, self-assemble to form a nanocage.

[0075] In an aspect, the nanocage monomer is selected from the group consisting of an ferritin, an apoferritin, an encapsulin, an SOR, a lumazine synthase, a pyruvate dehydrogenase, a carboxysome, an vault protein, a GroEL, a heat shock protein, an E2P, an MS2 coat protein, a fragment thereof, and a variant thereof.

[0076] In an aspect, the nanocage monomer is an apoferritin.

[0077] In an aspect, the first nanocage monomer subunit and the second nanocage monomer subunit interchangeably comprise the “N” region and the “C” region of an apoferritin.

[0078] In an aspect, the “N” region of an apoferritin comprises, or consists of, a sequence having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence:

[0079] MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEW.

[0080] In an aspect, the "C" region of an apo-transferrin includes or consists of a sequence having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence:

[0081] GKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD.

[0082] In an aspect, the fusion protein further includes a linker between the nanocage monomer subunit and the biologically active moiety.

[0083] In an aspect, the linker is flexible or rigid and includes from about 1 to about 30 amino acid residues, such as from about 8 to about 16 amino acid residues.

[0084] In an aspect, the linker includes GGS repeats, such as 1, 2, 3, 4, or more GGS repeats.

[0085] In an aspect, the linker includes or consists of a sequence having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence:

[0086] ASTASSASSGGGGGGSGGSGGSGGS.

[0087] In an aspect, the fusion protein further includes a C-terminal linker.

[0088] In an aspect, the C-terminal linker includes or consists of a sequence having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence:

[0089] GGSGGSGGSGGSGGGASGGS.

[0090] According to an aspect, the present application provides a pair of fusion proteins of a fusion protein described herein, wherein the pair of fusion proteins self-assemble to form a nanocage monomer, wherein the first and second nanocage monomer subunits are fused to different biologically active moieties.

[0091] According to an aspect, the present application provides a nanocage including at least one fusion protein described herein and at least one second nanocage monomer subunit that self-assembles with the fusion protein to form a nanocage monomer.

[0092] According to one aspect, the application provides a nanocage comprising at least one fusion protein pair described herein.

[0093] In one aspect, each nanocage monomer comprises a fusion protein or fusion protein pair described herein.

[0094] In one aspect, about 20% to about 80% of the nanocage monomers comprise a fusion protein or fusion protein pair described herein.

[0095] In one aspect, the fusion protein comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different biologically active moieties.

[0096] In one aspect, the nanocage comprises at least one complete nanocage monomer optionally fused to a biologically active moiety that can be the same or different from the biologically active moieties described herein.

[0097] In one aspect, the nanocage is multivalent and / or multispecific.

[0098] In one aspect, the nanocage comprises a first fusion protein, a second fusion protein, and a third fusion protein described herein, and at least one complete nanocage monomer optionally fused to a biologically active moiety, wherein the biologically active moieties of the first, second, and third fusion proteins and the biologically active moiety of the complete nanocage monomer are each different from one another.

[0099] In one aspect, the first, second, and third fusion proteins each comprise an antibody or fragment thereof fused to an N or C ferritin, wherein at least one of the first, second, and third fusion proteins is fused to an N ferritin, and at least one of the first, second, and third fusion proteins is fused to a C ferritin.

[0100] In one aspect, the antibody or fragment thereof of the first fusion protein is an Fc fragment; wherein the second and third fusion proteins each comprise an antibody or fragment thereof specific for a different antigen of a virus, such as HIV, or wherein one of the second and third fusion proteins comprises an antibody or fragment thereof specific for an antigen of a virus, such as HIV, and the third fusion protein comprises an antibody or fragment thereof specific for a different antigen, such as a CD4 receptor; and wherein the complete nanocage monomer is fused to a biologically active moiety specific for another different antigen, optionally a different antigen of the same virus, such as HIV.

[0101] In an aspect, the Fc fragment comprises one or more mutations, e.g., LS, YTE, LALA, and / or LALAP, that modulate the half-life of the fusion protein, e.g., from minutes or hours to days, weeks, or months.

[0102] In an aspect, the antibody or fragment thereof of the second fusion protein is N49P7 or iMab A12P; wherein the antibody or fragment thereof of the third fusion protein is 10E8v4.

[0103] In an aspect, the nanocage comprises or consists of four fusion proteins:

[0104] a. PGDM1400 (optionally scPGDM1400) fused to a full length ferritin;

[0105] b. Fc (optionally scFc) fused to N ferritin;

[0106] c. N49P7 or iMab A12P (optionally scN49P7 or sciMab A12P) fused to C ferritin; and

[0107] d. 10E8v4 (optionally sc10E8v4) fused to C ferritin.

[0108] In an aspect, the nanocage comprises a 4:2:1:1 ratio of a:b:c:d.

[0109] In an aspect, the nanocage comprises or consists of a sequence having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to one or more of the following sequences, wherein ferritin subunits are shown in bold, linkers are underlined, light chains are shown in italics, and heavy chains are shown in lower case:

[0110]

[0111]

[0112] In an aspect, the nanocage carries a cargo molecule, e.g., a pharmaceutical agent, a diagnostic agent, and / or an imaging agent.

[0113] In an aspect, the cargo molecule is not fused to the fusion protein and is contained inside the nanocage.

[0114] In an aspect, the cargo molecule is a protein and is fused to the fusion protein such that the cargo molecule is contained inside the nanocage.

[0115] In one aspect, the cargo molecule is a fluorescent protein, such as GFP, EGFP, violacein, and / or a flavin-based fluorescent protein such as a LOV protein (such as iLOV).

[0116] In one aspect, the cargo molecule is contained internally to provide a T cell epitope, but optionally not a B cell epitope.

[0117] In one aspect, the cargo molecule is fused to the fusion protein and contained internally to provide a T cell epitope, but optionally not a B cell epitope.

[0118] In one aspect, the cargo molecule is a small molecule, a radioisotope, or a magnetic particle.

[0119] In one aspect, the nanocage further comprises an antigen on the surface.

[0120] In one aspect, the antigen is expressed as a fusion protein with the nanocage monomer.

[0121] According to one aspect, the present application provides a vaccine comprising a nanocage as described herein.

[0122] According to one aspect, the present application provides a therapeutic or prophylactic composition comprising a nanocage as described herein.

[0123] According to one aspect, the present application provides a nucleic acid molecule encoding a fusion protein or pair of fusion proteins as described herein.

[0124] According to one aspect, the present application provides a vector comprising a nucleic acid molecule as described herein.

[0125] According to one aspect, the present application provides a host cell comprising a vector as described herein and producing a fusion protein or pair of fusion proteins as described herein.

[0126] According to one aspect, the present application provides a method of immunizing a subject comprising administering a nanocage or vaccine as described herein.

[0127] According to one aspect, the present application provides a method for treating and / or preventing a disease or disorder comprising administering a nanocage or vaccine as described herein.

[0128] In one aspect, the disease or disorder is a cancer, an infectious disease such as HIV, malaria, influenza, RSV, rotavirus, or an autoimmune disease.

[0129] According to one aspect, the present application provides a method for diagnostic imaging, the method comprising: administering to a subject, tissue or sample a nanocage as described herein, wherein the nanocage comprises a diagnostic label, such as a fluorescent protein or a magnetic imaging moiety; and imaging the subject, tissue or sample.

[0130] According to one aspect, the present application provides the use of a nanocage or vaccine as described herein for immunizing a subject.

[0131] According to one aspect, the present application provides the use of a nanocage or vaccine as described herein for treating and / or preventing a disease or disorder.

[0132] In one aspect, the disease or disorder is cancer, an infectious disease such as HIV, malaria, influenza, RSV, rotavirus or an autoimmune disease.

[0133] According to one aspect, the present application provides the use of a nanocage as described herein for diagnostic imaging of a subject, tissue or sample, wherein the nanocage comprises a diagnostic label, such as a fluorescent protein or a magnetic imaging moiety, and imaging the subject, tissue or sample.

[0134] According to one aspect, the present application provides the use of a fusion protein, fusion protein pair or nanocage as described herein as a research tool, for example in FACS or ELISA.

[0135] According to one aspect, the present application provides a nanocage or vaccine as described herein for immunizing a subject.

[0136] According to one aspect, the present application provides a nanocage or vaccine as described herein for treating and / or preventing a disease or disorder.

[0137] In one aspect, the disease or disorder is cancer, an infectious disease such as HIV, malaria, influenza, RSV, rotavirus or an autoimmune disease.

[0138] According to one aspect, the present application provides a nanocage as described herein for diagnostic imaging of a subject, tissue or sample, wherein the nanocage comprises a diagnostic label, such as a fluorescent protein or a magnetic imaging moiety, and imaging the subject, tissue or sample.

[0139] According to one aspect, the present application provides a fusion protein, fusion protein pair or nanocage as described herein for use as a research tool, for example in FACS or ELISA.

[0140] According to one aspect, the present application provides a nanocage, the nanocage comprising a plurality of fusion proteins,

[0141] wherein each fusion protein comprises a ferritin light chain and a Fab fragment,

[0142] wherein each Fab fragment is capable of specifically binding an antigen,

[0143] wherein each Fab fragment modifies the outer surface of the nanocage, and

[0144] wherein the plurality of fusion proteins comprises at least 12 fusion proteins.

[0145] In one aspect, the plurality of fusion proteins comprises at least 19 fusion proteins.

[0146] In one aspect, the plurality of fusion proteins comprises at least 24 fusion proteins.

[0147] In one aspect, the plurality of fusion proteins is 24 fusion proteins.

[0148] In one aspect, the Fab fragments of the plurality of fusion proteins are capable of specifically binding the same antigen.

[0149] In one aspect, the nanocage does not comprise any ferritin heavy chain.

[0150] In one aspect, the Fab fragment is a Fab fragment of a neutralizing antibody.

[0151] In one aspect, the antigen is associated with an infectious agent.

[0152] In one aspect, the infectious agent is a virus.

[0153] In one aspect, the virus is a human immunodeficiency virus (HIV).

[0154] In one aspect, the nanocage is capable of neutralizing the infectious agent with a neutralizing activity that is at least 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, or 500-fold relative to a control.

[0155] In one aspect, the control comprises a full-length form of the neutralizing antibody.

[0156] In one aspect, the neutralizing antibody is an IgG antibody.

[0157] According to one aspect, the present application provides a nanocage comprising a plurality of first fusion proteins and a plurality of second fusion proteins,

[0158] wherein each first fusion protein comprises a nanocage monomer or a subunit thereof and a Fab fragment capable of specifically binding an antigen, and

[0159] wherein each second fusion protein comprises a nanocage monomer or a subunit thereof and a Fc fragment.

[0160] In one aspect, the nanocage monomer is selected from the group consisting of ferritin, apo-ferritin, encapsulin, sulfur oxygenase reductase (SOR), lumazine synthase, pyruvate dehydrogenase, carboxysome, vault protein, GroEL, heat shock protein, E2P, MS2 coat protein, fragments thereof, and variants thereof.

[0161] In one aspect, the nanocage monomer is apo-ferritin or ferritin.

[0162] In one aspect, the nanocage monomer is a ferritin light chain.

[0163] In one aspect, the nanocage monomer does not include any ferritin heavy chain.

[0164] According to one aspect, the present application provides a nanocage comprising a plurality of first fusion proteins and a plurality of second fusion proteins, wherein

[0165] (a) (i) the first fusion protein comprises a ferritin light chain and a Fab fragment capable of specifically binding a first antigen, and

[0166] (ii) the second fusion protein comprises a ferritin light chain and a Fab fragment capable of specifically binding a second antigen, or

[0167] (b) (i) the first fusion protein comprises an N-ferritin and a Fab fragment capable of specifically binding a first antigen, and

[0168] (ii) the second fusion protein comprises a C-ferritin and a Fab fragment capable of specifically binding a second antigen,

[0169] wherein, within each fusion protein, the Fab fragment is fused to the N-terminus of the ferritin light chain, the N-terminus of the N-ferritin, or the N-terminus of the C-ferritin, and

[0170] wherein the first antigen is different from the second antigen.

[0171] According to one aspect, the present application provides a nanocage comprising a plurality of first fusion proteins, a plurality of second fusion proteins, and a plurality of third fusion proteins, wherein

[0172] (a) the first fusion protein comprises a ferritin light chain and a Fab fragment capable of specifically binding a first antigen,

[0173] (b) the second fusion protein comprises a C-ferritin and a Fab fragment capable of specifically binding a second antigen, and

[0174] (c) the third fusion protein comprises an N-ferritin and a Fc fragment,

[0175] wherein, within each fusion protein, the Fab fragment or the Fc fragment is fused to the N-terminus of the ferritin light chain, the N-terminus of the C-ferritin, or the N-terminus of the N-ferritin, and

[0176] wherein the first antigen is different from the second antigen.

[0177] In one aspect, the nanocage further comprises a plurality of fourth fusion proteins, wherein the fourth fusion protein comprises a C-ferritin and a Fab fragment capable of specifically binding a third antigen, wherein the third antigen is different from the first antigen and the second antigen.

[0178] In one aspect, the Fab fragment is a Fab fragment of a neutralizing antibody.

[0179] In one aspect, the first antigen and the second antigen are each associated with an infectious agent.

[0180] In one aspect, the first antigen and the second antigen are associated with the same infectious agent.

[0181] In one aspect, the infectious agent is a virus.

[0182] In one aspect, the virus is a human immunodeficiency virus (HIV).

[0183] In one aspect, the first antigen and the second antigen are each associated with a virus,

[0184] wherein the nanocage is capable of neutralizing 100% of the pseudovirus panel, and

[0185] wherein for each Fab fragment within the nanocage capable of specifically binding an antigen associated with the virus, the pseudovirus panel comprises at least one pseudovirus resistant to a neutralizing antibody corresponding to the Fab fragment.

[0186] In one aspect, the pseudovirus panel comprises at least 10, at least 11, at least 12, at least 13, or at least 14 pseudoviruses.

[0187] In one aspect, the first antigen and the second antigen are each associated with a virus,

[0188] wherein the nanocage is capable of neutralizing a panel of pseudoviruses with an IC 50 of less than 1 nM, less than 500 pM, less than 250 pM, less than 100 pM, less than 50 pM, less than 10 pM, or less than 5 pM,

[0189] wherein for each Fab fragment within the nanocage capable of specifically binding an antigen associated with the virus, the panel of pseudoviruses comprises at least one pseudovirus resistant to a neutralizing antibody corresponding to the Fab fragment.

[0190] In one aspect, the first antigen and the second antigen are each associated with a virus,

[0191] wherein the nanocage is capable of neutralizing one or more controls at an IC 50 at most 1 / 10, at most 1 / 20, at most 1 / 30, at most 1 / 40, at most 1 / 50, at most 1 / 60, at most 1 / 70, at most 1 / 80, at most 1 / 90, or at most 1 / 100 of the IC 50 neutralizes a panel of pseudoviruses, and

[0192] wherein for each Fab fragment within the nanocage that is capable of specifically binding to an antigen associated with the virus, the panel of pseudoviruses includes at least one pseudovirus that is resistant to neutralization by a neutralizing antibody corresponding to the Fab fragment.

[0193] In one aspect, the one or more controls include a neutralizing antibody corresponding to a Fab fragment within the nanocage that is capable of specifically binding to an antigen associated with the virus.

[0194] In one aspect, the neutralizing antibody is an IgG antibody.

[0195] In one aspect, the one or more controls include a mixture of neutralizing antibodies, wherein for each Fab fragment within the nanocage that is capable of specifically binding to an antigen associated with the virus, the mixture includes a neutralizing antibody corresponding to the Fab fragment.

[0196] In one aspect, the neutralizing antibodies are IgG antibodies.

[0197] In one aspect, the one or more controls include one or more multispecific antibodies, wherein the one or more multispecific antibodies collectively are capable of binding the first antigen and the second antigen, and optionally the third antigen.

[0198] In one aspect, the one or more controls include a trispecific antibody that is capable of specifically binding the first antigen, the second antigen, and the third antigen.

[0199] In one aspect, the first antigen, the second antigen, and the third antigen are associated with HIV-1; and wherein:

[0200] the Fab fragment of the first fusion protein is PDGM1400 Fab,

[0201] the Fab fragment of the second fusion protein is 10E8v4 Fab,

[0202] the Fc fragment of the third fusion protein is a human IgG1 Fc fragment, and

[0203] The Fab fragment of the fourth fusion protein is N49P7 Fab.

[0204] In one aspect, the first antigen and the second antigen are associated with HIV-1; wherein the third antigen is associated with CD4; and wherein:

[0205] The Fab fragment of the first fusion protein is PDGM1400 Fab,

[0206] The Fab fragment of the second fusion protein is 10E8v4 Fab,

[0207] The Fc fragment of the third fusion protein is a human IgGl Fc fragment, and

[0208] The Fab fragment of the fourth fusion protein is iMab Fab.

[0209] According to one aspect, the present application provides a therapeutic or prophylactic composition comprising a nanocage as described herein.

[0210] According to one aspect, the present application provides a method for treating or preventing a disease or disorder, the method comprising administering to a subject in need thereof a nanocage or a composition as described herein.

[0211] According to one aspect, the present application provides a method of producing a multispecific self-assembling nanocage, the nanocage being characterized by a preselected ratio of different specificities, the method comprising the steps of:

[0212] co-transfecting a host cell with one or more expression plasmids comprising a plurality of polynucleotides, each polynucleotide encoding a fusion protein,

[0213] wherein each fusion protein comprises: (i) a nanocage monomer or a subunit thereof and (ii) an antibody or antibody fragment having a given specificity,

[0214] wherein the step of co-transfecting comprises: co-transfecting the polynucleotides at a ratio based on the preselected ratio;

[0215] obtaining polypeptides produced by the host cell; and

[0216] purifying by affinity selection all polypeptides of different specificities present in the assembled nanocage.

[0217] In one aspect, the plurality of polynucleotides comprises at least one polynucleotide encoding a first fusion protein and at least one polynucleotide encoding a second fusion protein,

[0218] wherein the first fusion protein comprises a first nanocage monomer subunit, and

[0219] wherein the second fusion protein comprises a second nanocage monomer subunit capable of self-assembly with the first nanocage monomer subunit.

[0220] The novel features of the application will become apparent to those skilled in the art upon examination of the following detailed description of the application. It should be understood, however, that the detailed description and specific examples, while indicating certain aspects of the application, are given by way of illustration only, since various changes and modifications within the spirit and scope of the application will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF DRAWINGS

[0221] The application will be further understood by the following description, with reference to the drawings, in which:

[0222] Figure 1 Schematic of the self-assembly of the MULTi-specific, multi-Affinity antiBODY (multabody) platform. Single chain Fabs (light chain (LC) and heavy chain (HC) are light pink and dark pink, respectively) and single chain Fc region (green) are linked via GGS-like flexible linkers (dark) to the N-terminus of the light chain of human apoferritin (gray). The 24 subunits of apoferritin self-assemble into a 12 nm spherical core that is surrounded by the spatially dispersed antibody fragments.

[0223] Figure 2 Characterization of HIV-1 multispecific, multi-affinity antibodies of different valency. (a) Schematic of different Fab densities displayed on human apoferritin. Co-transfection of plasmids encoding scFab-apoferritin with different ratios of unconjugated apoferritin yielded scFabs of 5-valency (dark yellow), 12-valency (black), 19-valency (blue), and 24-valency (red), as evidenced by early elution volumes and less unconjugated apoferritin in size exclusion chromatography and SDS-PAGE. Negative-stain electron micrographs of samples with the lowest (20%) and highest (100%) valency are shown. (b) Allosteric effect of the five bNAb neutralization of the 5-PsV panel (PVO.04, JRCSF, BG505 T332N, THRO4156.18, and t278-50). IC 50 Fold increase analysis: N49P7-t278-50, VRC01-t278-50, and 10-1074-THRO4156.18. Fold increase in potency was calculated as the parental IgG IC 50 (nM) divided by the multispecific, multi-affinity antibody IC 50 (nM).

[0224] Figure 3. Design, assembly, and biophysical characterization of 32-N multispecific multi-affinity antibody and 32-I multispecific multi-affinity antibody. (a) Schematic of the split design of human apoferritin that favors heterodimerization of scFab-human apoferritin subunits. The resulting halves, called N ferritin and C ferritin, span residues 1 to 95 and residues 95 to 175, respectively. scFc is attached at the N-terminus of the N ferritin half, while N49P7, iMab (also referred to herein as ibalizumab), and 10E8v4 are attached at the N-terminus of the C ferritin half. Heterodimerization of the two halves of the split drives self-assembly of the different antibody fragments, resulting in a single human apoferritin subunit with two cargo molecules. These constructs are further combined with PGDM1400 scFab attached to full-length human apoferritin subunits, leading to the assembly of nanoparticles displaying 32 scFab / scFc mixtures on the surface of the multispecific multi-affinity antibody. Negative-stain electron micrographs, model schematics of the scFab / scFc 32-N / 32-I design, and the specific composition of the 32-N and 32-I multispecific multi-affinity antibodies are shown. Based on the hetero-oligomerization necessary to drive self-assembly, purification of the multispecific multi-affinity antibody with four components can be achieved in two steps: protein A (Fc binding) and protein L (PGDM1400 binding). (b) Size exclusion chromatography with multi-angle light scattering of 24-mer PGDM1400 multispecific multi-affinity antibody (black), 32-N multispecific multi-affinity antibody (dark magenta), and 32-I multispecific multi-affinity antibody (blue). The molar mass of each elution peak (below the UV absorbance line) shows that the samples are monodisperse, and the 32-N / 32-I multispecific multi-affinity antibodies are significantly larger than the 24-mer version of the multispecific multi-affinity antibody due to the additional antibody fragments in the design. (c) T m emperature and T agg emperature comparison. (d) Concentration-response curves of 32-N and 32-I multispecific multi-affinity antibodies binding to multiple epitopes. PGDM1400 binding sites, N49P7 binding sites, and 10E8 binding sites are colored red, blue, and pink, respectively, in a surface rendering of HIV Env (gray). iMab binding was assessed using soluble CD4, while 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 chosen as epitope-specific ligands for PGDM1400 and N49P7, respectively.

[0225] Figure 4 . Split design impact on biophysical and functional properties of multispecific, multi-affinity antibodies. Comparison of 12-mer multispecific, multi-affinity antibody composed of 6 copies of PGDM1400 and 6 copies of Fc multimerized with full-length apo-ferritin subunit (left panel) or ferritin half (Fc attached to N ferritin half and PGDM1400 attached to C ferritin half, right panel). a) Bio-layer interferometry (BLI) concentration-response curves for binding of BG505.664 to 12-mer multispecific, multi-affinity antibodies loaded onto anti-hIgG Fc capture (AHC) biosensors. b) Plot of barycenter mean fluorescence (BCM) and static light scattering (SLS) at 266 nm versus temperature. T m and T agg Indicated with yellow line. c) Neutralization assay for BG505 T332N PsVs.

[0226] Figure 5 . Multispecific, multi-affinity antibody affinity purification scheme. Sequential affinity purification with protein A and protein L. Binding to protein A enriches the multispecific, multi-affinity antibody with Fc (green), while binding to protein L enriches the multispecific, multi-affinity antibody with kappa chain Fab PGDM1400 (blue). Introduction of alanine to proline point mutation at position 12 of the kappa chain of iMab to disrupt binding to protein L 75 . Complementation of the two halves of human apo-ferritin ensures the presence of (fused to C ferritin) N49P7 / iMab scFab (orange) and 10E8 scFab (pink) during the protein A purification step. Gel filtration was performed to isolate any aggregated material.

[0227] Figure 6 . Minimal batch-to-batch variation for 32-N multispecific, multi-affinity antibody. a) SEC chromatogram. b) Plot of BCM and SLS at 266 nm versus temperature. Thermal transition temperature (T m and T agg ) indicated with yellow line. c) Concentration-response curves for 93TH057 gp120 binding to N49P7 within 32-N. d) Neutralization profile of 32-N against the 4-PsV panel selected to include one PsV resistant to each Fab in the multispecific, multi-affinity antibody.

[0228] Figure 7Thermal stability analysis. Plot of BCM (top panel) and SLS at 266 nm (bottom panel) vs. temperature for 32-N and 32-I multispecific multimeric antibodies, their respective 12-mer multispecific multimeric antibodies, parental IgGs, and N6 / PGDM1400 x 10E8 trispecific antibodies. Thermal transition temperature (T m and T agg are indicated with yellow lines.

[0229] Figure 8 Binding characteristics of bNAbs PGDM1400, 10E8v4, N49P7, and iMab. BLI response curves of IgG binding to 93TH057 gp120, BG505 SOSIP.664_D368R, MPER-mVenus, and CD4 immobilized on a Ni-NTA biosensor.

[0230] Figure 9 Neutralization properties of 32-N and 32-I multispecific multimeric antibodies against a panel of 14 pseudoviruses. Width and IC 50 median (pg / mL) of multispecific multimeric antibodies (red diamonds), parental bNAbs (black circles), IgG combinations (black triangles), and N6 / PGDM1400 x 10E8v4 trispecific antibodies (black squares). The relative content of each parental antibody in the IgG mixture was identical to the relative content in the multispecific multimeric antibody sample (i.e., 66% PGDM1400, 17% N49P7 / iMab, and 17% 10E8v4). The panel of 14-PsVs was selected based on susceptibility and resistance to parental IgGs.

[0231] Figure 10 Neutralization properties of 32-N and 32-I multispecific multimeric antibodies against a panel of 14 pseudoviruses. Width and IC 50 median (nM) of multispecific multimeric antibodies (red diamonds), parental bNAbs (black circles), IgG combinations (66% PGDM1400, 17% N49P7 / iMab, and 17% 10E8v4, black triangles), and N6 / PGDM1400 x 10E8v4 trispecific antibodies (black squares).

[0232] Figure 11Exposure of immunogenicity and multispecific multivalent antibodies in mice. Five male C57BL / 6 mice per group were used to assess the circulation of anti-drug antibodies and multispecific multivalent antibodies in the blood after subcutaneous administration of 5 mg / kg mouse surrogate multispecific multivalent antibodies and Fc-modified multispecific multivalent antibodies (LALAP mutation to disrupt Fc receptor binding). Reference samples of HpFerritin-PfCSP malaria peptide as well as parental mouse IgGl and parental mouse IgG2a isotypes were used for immunogenicity and exposure comparisons, respectively. DETAILED DESCRIPTION

[0233] DEFINITIONS

[0234] Unless otherwise indicated, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). Although exemplary materials and methods are described herein, any method and materials similar or equivalent to those described herein can be used in the practice of the present application. In describing and claiming the present application, the following terminology will be used.

[0235] It should also be understood that the terms used herein are not intended to limit the present application to the particular field of use, but rather are used for descriptive purposes only. Numerous patent applications, patents, and publications are cited and discussed in the description of the aspects. Each of these references is incorporated by reference herein in its entirety.

[0236] In understanding the scope of the present application, the articles "a," "an," "the" and "said" are intended to mean that there are one or more of the elements. Further, as used herein, the term "include" and its derivatives are intended to be open-ended terms that specifically mean the inclusion of the stated features, elements, components, compositions, integers and / or steps but do not exclude the presence of other unstated features, elements, components, compositions, integers and / or steps. The foregoing also applies to words having similar meanings, such as the terms "comprise," "have," "contain," and "comprising," "having," and "containing."

[0237] It is understood that any aspects described as comprising certain components can also "consist of or "consist essentially of the components, where "consisting of has a closed or limiting meaning and "consisting essentially of means including the specified components but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for purposes other than to achieve the technical effect of the invention. For example, compositions defined using the phrase "consisting essentially of encompass any known acceptable additives, excipients, diluents, and carriers, etc. Generally, a composition consisting essentially of a group of components will contain less than 5 wt%, often less than 3 wt%, more often less than 1 wt%, even more often less than 0.1 wt% of unspecified components.

[0238] It is understood that any components defined herein as being included can be expressly excluded from the claimed invention by recital or negative limitation. For example, in some aspects, the nanocages and / or fusion proteins described herein can not include ferritin heavy chains and / or can not include iron-binding components.

[0239] Further, all ranges given herein include the range end points and any intermediate range point, whether or not explicitly stated.

[0240] As used herein, the degree terms such as "substantially," "about," and "approximately" mean an acceptable quantity of deviation of the modified term as understood by one of ordinary skill in the art, and are not to be construed as indicating any deviance beyond that meaning. These degree terms should be interpreted in the context based on the desired technical effects. These degree terms are to be understood to include at least ±5% of the modified term.

[0241] It is also to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and provided for descriptive purposes only. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus the abbreviation "e.g." is synonymous with the term "for example." The word "or" is intended to mean an inclusive "and" unless the context clearly indicates to the contrary.

[0242] The terms "protein nanoparticle," "nanocage," and "multispecific, multivalent antibody" are used interchangeably herein and refer to a multimeric protein polyhedral structure. The subunits or nanocage monomers are each composed of a protein or polypeptide (e.g., glycosylated polypeptide) and optionally composed of one or more of the following individual or multiple features: 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, incorporated herein by reference), encapsulin nanoparticles (see, e.g., Sutter et al., Nature Struct. and Mol. Biol., 15:939-947, 2008, incorporated herein by reference), sulfur oxygenase reductase (SOR) nanoparticles (see, e.g., Urich et al., Science, 311:996-1000, 2006, incorporated herein by reference), dihydropteridine synthase (see, e.g., Zhang et al., J. Mol. Biol., 306:1099-1114, 2001), or pyruvate dehydrogenase nanoparticles (see, e.g., Izard et al., PNAS, 96:1240-1245, 1999, incorporated herein by reference). Ferritin, apoferritin, encapsulin, SOR, dihydropteridine synthase, and pyruvate dehydrogenase are monomeric proteins that self-assemble into globular protein complexes, in some cases consisting of 24, 60, 24, 60, and 60 protein subunits, respectively. Ferritin and apoferritin are generally used interchangeably herein and are understood to apply equally to the fusion proteins, nanocages, and methods described herein. The use of carboxysome, vault protein, GroEL, heat shock proteins, E2P, and MS2 coat proteins, which also produce nanocages, are contemplated herein. In addition, wholly or partially synthetic self-assembling monomers are also contemplated for use herein.

[0243] It is understood that each nanocage monomer can be divided into two or more subunits that will self-assemble into a functional nanocage monomer. For example, a ferritin or apo-ferritin can be divided into an N subunit and a C subunit, e.g., an N subunit and a C subunit obtained by dividing a full-length ferritin substantially in half, such that each subunit can individually bind a different biologically active moiety for subsequent self-assembly into a nanocage monomer, and further self-assembly into a nanocage. By "functional nanocage monomer" is meant a nanocage monomer that is capable of self-assembly with other such monomers into a nanocage as described herein.

[0244] The terms "ferritin" and "apo-ferritin" are used interchangeably herein, and generally refer to a polypeptide (e.g., a ferritin chain) that is capable of assembling into a ferritin complex that typically includes 24 protein subunits. It is understood that the ferritin can be from any species. Typically, the ferritin is a human ferritin. In some embodiments, the ferritin is a wild-type ferritin. For example, the ferritin can be a wild-type human ferritin. In some embodiments, the 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 include any ferritin heavy chain or other ferritin component capable of binding iron.

[0245] As used herein, the term "multispecific" refers to the feature of having at least two binding sites, and at least two different binding partners, e.g., antigens or receptors (e.g., Fc receptors), can bind at the at least two binding sites. For example, a nanocage that includes at least two Fab fragments and wherein each of the two Fab fragments binds a different antigen is "multispecific." As a further example, a nanocage that includes an Fc fragment (capable of binding an Fc receptor) and an Fab fragment (capable of binding an antigen) is "multispecific."

[0246] As used herein, the term "multivalent" refers to the feature of having at least two binding sites, and a binding partner, e.g., an antigen or a receptor (e.g., an Fc receptor), can bind at the at least two binding sites. The binding partners that can bind to the at least two binding sites can be the same or different.

[0247] A "vaccine" is a pharmaceutical composition that induces a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine induces an antigen-specific immune response against an antigen of a pathogen, such as a viral pathogen, or against a cellular component associated with a pathological condition. A vaccine can include a polynucleotide (e.g., a nucleic acid encoding a disclosed antigen), a peptide or polypeptide (e.g., a disclosed antigen), a virus, a cell, or one or more cellular components. In one particular, non-limiting example, a vaccine induces an immune response that reduces the severity of symptoms associated with a malaria infection and / or reduces the parasite load compared to a control. In another non-limiting example, a vaccine induces an immune response that reduces and / or prevents a malaria or HIV infection.

[0248] The term "antibody", also referred to in the art as "immunoglobulin" (Ig), as used herein refers to a protein constructed from pairs of heavy and light polypeptide chains; there are multiple Ig isotypes, including IgA, IgD, IgE, IgG (such as IgGl, IgG2, IgG3, and IgG4), and IgM. It is understood that the antibody can be from any species, including human, mouse, rat, monkey, llama, or shark. When an antibody is properly folded, each chain is folded into multiple distinct globular domains connected by more linear polypeptide sequences. For example, an immunoglobulin light chain is folded into one variable domain (V L ) and one constant domain (C L ), while a heavy chain is folded into one variable domain (V H ) and three constant domains (C H , C H2 , C H3 ). The interaction of the heavy and light chain variable domains (V H and V L ) results in the formation of the antigen binding region (Fv). Each domain has an established structure familiar to those skilled in the art.

[0249] The light and heavy chain variable regions are responsible for binding the target antigen, and thus can show significant sequence diversity between antibodies. The constant regions show lower sequence diversity, and are responsible for binding to a number of natural proteins to initiate important immune events. The variable regions of an antibody contain the antigen-binding determinants of the molecule, and thus determine the specificity of the antibody for its target antigen. Most of the sequence variability occurs in six hypervariable regions, three in the variable heavy chain and three in the variable light chain; the hypervariable regions combine to form the antigen binding site and help the binding and recognition of the antigenic determinant. The specificity and affinity of an antibody for its antigen is determined by the structure of the hypervariable regions and the size, shape, and chemical properties of the surface they present to the antigen.

[0250] “Antibody fragments” as referred to herein can include any suitable antigen-binding antibody fragment known in the art. Antibody fragments can be naturally occurring antibody fragments, or can be obtained by manipulation of naturally occurring antibodies or by using recombinant methods. For example, antibody fragments can include, but are not limited to, Fv, single-chain Fv (scFv; composed of V L and V H connected to a connecting peptide), Fc, single-chain Fc, Fab, single-chain Fab, F(ab')2, single-domain antibody (sdAb; composed of a single V L or V H fragment), and multivalent forms of any of these.

[0251] The term “synthetic antibody” as used herein refers to an antibody produced using recombinant DNA technology. The term is also to be construed to mean an antibody produced by synthesizing a DNA molecule encoding the antibody, and the DNA molecule expresses the antibody protein or indicates the amino acid sequence of the antibody, wherein the DNA or amino acid sequence is obtained using DNA or amino acid sequence synthesis techniques available and well known in the art.

[0252] The term “epitope” refers to an antigenic determinant. An epitope is a particular antigenic (i.e., elicits a specific immune response) chemical group or peptide sequence on a molecule. An antibody specifically binds to a particular antigenic epitope (e.g., on a polypeptide). Epitopes can be formed either from contiguous amino acids or noncontiguous amino acids juxtaposed by protein folding. Epitopes formed from contiguous amino acids are typically preserved when exposed to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost under such conditions. Epitopes usually include 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 of determining the spatial conformation of an epitope include, for example, X-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Methods in Molecular Biology, Vol. 66, “Epitope Mapping Protocols,” ed. Glenn E. Morris, 1996.

[0253] As used herein, the term "antigen" is defined as a molecule that elicits an immune response. This immune response can involve antibody production or activation of specific immunocompetent cells or both. The skilled artisan will appreciate that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, the antigen can be derived from recombinant or genomic DNA. The skilled artisan will appreciate that any DNA that includes a nucleotide sequence or a portion of a nucleotide sequence that encodes a protein that elicits an immune response thus encodes an "antigen" as used herein. Furthermore, the skilled artisan will appreciate that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that aspects described herein include, but are not limited to, the use of more than one partial nucleotide sequence of a gene, and these nucleotide sequences can be arranged in various combinations to elicit the desired immune response. Furthermore, the skilled artisan will appreciate that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be synthetic or can be derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, cells, or biological fluids.

[0254] Accordingly, the compositions described herein can be suitable for protecting or treating a vertebrate subject against a variety of disease states, such as viral, bacterial, fungal, or parasitic infections, cancer, and autoimmune diseases. It should be recognized that these particular disease states are mentioned by way of example only and are not intended to be limiting.

[0255] Suitable antigens that can be used in combination with the compositions described herein include any antigen as defined herein. Antigens are commercially available or can be produced by one skilled in the art. Antigens can be modified live microorganisms or inactivated microorganisms, or natural products purified from microorganisms or other cells, including but not limited to tumor cells, synthetic products, genetically engineered proteins, peptides, polysaccharides, or the like, or allergens. Antigenic portions can also be subunits of proteins, peptides, polysaccharides, or the like. Antigens can also be genetic antigens, i.e., DNA or RNA that elicit an immune response.

[0256] Representative of antigens that can be used include, but are not limited to, natural, recombinant or synthetic products derived from viruses, bacteria, fungi, parasites and other infectious agents other than autoimmune diseases, hormones, or tumor antigens that can be used in prophylactic or therapeutic vaccines, and allergens. In one embodiment, the antigen includes a virus-like particle (VLP) from various viruses such as influenza virus, HIV, RSV, Newcastle Disease Virus (NDV), and the like. See PCT / US2006 / 40862, PCT / US2004 / 022001, U.S. Patent Serial No. 11 / 582,540, U.S. Patent Serial No. 60 / 799,343, U.S. Patent Serial No. 60 / 817,402, and U.S. Patent Serial No. 60 / 859,240, which are incorporated herein by reference in their entirety. In another embodiment, the antigen includes a chimeric VLP. By "chimeric VLP" is meant a VLP that contains proteins or portions thereof from at least two different sources (organisms). Typically, one of the proteins is derived from a virus that can drive a host cell to form a VLP. Thus, in one embodiment, the chimeric VLP includes an RSV M protein. In another embodiment, the chimeric VLP includes an NDV M protein. In another embodiment, the chimeric VLP includes an influenza virus M protein.

[0257] The viral or bacterial product can be a component of the organism produced by enzymatic cleavage, or can be a component of the organism produced by recombinant DNA techniques well known to those of ordinary skill in the art.

[0258] Some specific examples of antigens are antigens derived from hepatitis B, C, D, and E types 3, human immunodeficiency virus (HIV), herpes viruses types 1, 2, 6, and 7, cytomegalovirus, varicella zoster virus, papilloma virus, Epstein-Barr virus, parainfluenza virus, adenovirus, bunyaviruses (e.g., hantavirus), coxsackie virus, picornaviruses, rotaviruses, respiratory syncytial virus, rhinovirus, rubella virus, papovaviruses, mumps virus, measles virus, poliovirus (multiple types), adenovirus (multiple types), parainfluenza virus (multiple types), avian flu or pandemic influenza virus (multiple types), seasonal influenza virus, marine heat virus, western and eastern equine encephalitis virus, Japanese B encephalitis virus, Russian spring-summer encephalitis virus, swine fever virus, newcastle disease virus, fowlpox virus, rabies virus, feline and canine distemper virus, slow virus, Rous sarcoma virus (RSV), papovaviridae, parvoviridae, picornaviridae, poxviridae (e.g., smallpox or vaccinia virus), reoviridae (e.g., rotavirus), retroviridae (HTLV-I, HTLV-II, lentivirus), and togaviridae (rubella virus genus) that cause viral infections. Viruses belonging to these families can cause a variety of diseases or symptoms, including but not limited to: arthritis, bronchiolitis, encephalitis, ocular infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, Japanese B encephalitis, Argentine hemorrhagic fever, Kyasanur fever, Rift Valley fever, yellow fever, meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's lymphoma, chickenpox, hemorrhagic fever, measles, mumps, parainfluenza, rabies, common cold, polio, leukemia, rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's disease, warts), and viremia.

[0259] Antigens can also be derived from bacterial and fungal infections, for example: antigens derived from Mycobacteria causing TB and leprosy, Pneumococcus, aerobic gram negative bacilli, Mycoplasma, Staphylococcal infections, Streptococcal infections, Salmonella and Chlamydia, B. pertussis, Leptospira pomona and Haemorrhagicae causing infection. Specific embodiments include S. Paratyphi, C. diphtheriae, C. tetani, C. botulinum, C. perfringens, C. feseri and other gas gangrene bacteria, B. anthracis, P. pestis, P. multocida, Neisseria meningitidis, N. gonorrheae, Hemophilus influenzae, Actinomyces (e.g. Norcardia), Acinetobacter, Bacillaceae (e.g. B. anthrasis), Bacteroides (e.g. Bacteroides fragilis), Blastomyces dermatitidis, Bordetella, Borrelia (e.g. Borrelia burgdorferi), Brucella, Candidia, Campylobacter, Chlamydia, Coccidioides, Corynebacterium (e.g. Corynebacterium diptheriae), Cryptococcus, Dermatocycoses, E. coli, Enterobacter, Escherichia, Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Nocardia, Pasteurella, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Vibrio, Yersinia, and the like.Enterobacter; e.g. Enterobacter aerogenes), Enterobacteriaceae (Klebsiella, Salmonella; e.g. Salmonella typhi, Salmonella enteritidis), Serratia, Yersinia, Shigella), Erysipelothrix, Haemophilus; e.g. Haemophilus influenzae type B, Helicobacter, Legionella; e.g. Legionella pneumophila, Leptospira, Listeria; e.g. Listeria monocytogenes, Mycoplasma, Mycobacterium; e.g. Mycobacterium leprae and Mycobacterium tuberculosis, Vibrio; e.g. Vibrio cholerae, Pasteurella, Proteus, Pseudomonas; e.g. Pseudomonas aeruginosa, Rickettsiaceae, Spirochaetes (e.g. Treponema species, Leptospira species, Borrelia species), Shigella species, Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus; e.g. S. pneumoniae and types 1, 2 and 3 Streptococcus, Ureaplasma urealyticum, Treponema pollidum, etc.; Staphylococcus aureus, Plasmodium species (P. falciparum, P. vivax), etc.Aspergillus species, Candida albicans, Pasteurella haemolytica, diphtheria toxoid, meningococcal polysaccharide, Bordetella pertusis, Streptococcus pneumoniae (pneumococcal) polysaccharide, Clostridium tetani toxoid, Mycobacterium bovis, and inactivated cells of Salmonella typhi, Cryptococcus neoformans, and Aspergillus.

[0260] Antigens can also be derived from parasitic malaria, leishmaniasis, trypanosomiasis, toxoplasmosis, schistosomiasis, filariasis malaria, amebiasis, babesiosis, coccidiosis, cryptosporidiosis, binucleate amebiasis, equine trypanosomiasis, ectoparasites, giardiasis, helminthiasis, theileriasis, trichomoniasis, and sporozoa (e.g., Plasmodium vivax, Plasmodium falciparum, Plasmodium malariae, Plasmodium knowlesi, and Plasmodium ovale). These parasites can cause a variety of diseases or symptoms, including but not limited to: scabies, acariasis, ocular infections, intestinal diseases (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), malaria, complications of pregnancy, and toxoplasmosis.

[0261] Tumor-associated antigens suitable for use in the compositions described herein include mutated and non-mutated molecules that can be indicative of a single tumor type, shared by several classes of tumors, and / or specifically expressed or overexpressed in tumor cells compared to normal cells. In addition to proteins and glycoproteins, tumor-specific expression patterns have been documented for carbohydrates, gangliosides, glycolipids, and mucins. Exemplary tumor-associated antigens for use in the cancer vaccines of the present application include protein products of oncogenes, tumor suppressor genes, and other genes with mutations or rearrangements specific to tumor cells, reactivated embryonic gene products, oncofetal antigens, tissue-specific (but not tumor-specific) differentiation antigens, growth factor receptors, cell surface sugar residues, foreign viral proteins, and many other self-proteins. Particular embodiments of tumor-associated antigens include, for example: mutated antigens such as protein products of the Ras p21 proto-oncogene, the tumor suppressor gene p53, and the HER-2 / neu and BCR-abl oncogenes, as well as CDK4, MUM1, Caspase 8, and beta catenin; overexpressed antigens such as galectin 4, galectin 9, carbonic anhydrase, aldolase A, PRAME, Her2 / neu, ErbB-2, and KSA, oncofetal antigens such as alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG); self antigens such as carcinoembryonic antigen (CEA), and melanocyte differentiation antigens such as Mart 1 / Melan A, gplOO, gp75, tyrosinase, TRP1, and TRP2; prostate-associated antigens such as PSA, PAP, PSMA, PSM-P1, and PSM-P2; reactivated embryonic gene products such as MAGE 1, MAGE 3, MAGE 4, GAGE 1, GAGE 2, BAGE, RAGE, and other cancer testis antigens such as NY-ESO1, SSX2, and SCP1; mucins such as Muc-1 and Muc-2; gangliosides such as GM2, GD2, and GD3, neutral glycolipids and glycoproteins such as Lewis (y) and globo-H; and glycoproteins such as Tn, Thompson-Freidenreich antigen (TF), and sTn. Also included as tumor-associated antigens herein are whole cells and tumor cell lysates and immunogenic portions thereof, as well as immunoglobulin idiotypes expressed on monoclonal proliferation of B lymphocytes for anti-B cell lymphomas. Tumor-associated antigens and their respective tumor cell targets include, for example, cytokeratins, particularly cytokeratin 8, 18, and 19, as cancer antigens. Epithelial membrane antigen (EMA), EphAl, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphAlO, EphBl, EphB2, EphB3, EphB4, EphB6, human embryonic antigen (HEA-125), human milk fat globules, MBr1, MBr8, Ber-EP4, 17-1A, C26, and T16 are also known cancer antigens.Desmin and muscle-specific actin are antigens for myogenic sarcomas. Placental alkaline phosphatase, beta-human chorionic gonadotropin, and alpha-fetoprotein are antigens for trophoblastic and germ cell tumors. Prostate-specific antigen is an antigen for prostate cancer, and carcinoembryonic antigen for colon adenocarcinoma. HMB-45 is an antigen for melanoma. In cervical cancer, useful antigens can be encoded by human papilloma virus. Chromogranin-A and synaptophysin are antigens for neuroendocrine and neuroectodermal tumors. Of particular interest are invasive tumors that form solid tumor masses with necrotic areas. The lysis of this necrotic cell is a rich source of antigen for antigen presenting cells, so the present treatment can have utility in conjunction with conventional chemotherapy and / or radiation therapy. Antigens can be derived from any tumor or malignant cell line.

[0262] Antigens can also be derived from common allergens that cause allergies. Allergens include organic or inorganic materials derived from a variety of artificial or natural sources, such as plant material, metals, ingredients in cosmetics or detergents, latex, etc. Classes of suitable allergens for use in the compositions and methods described herein can include, but are not limited to, pollen, animal dander, grass, mold, dust, antibiotics, stinging insect venom, and various environmental (including chemicals and metals) drug and food allergens. Common tree allergens include pollen from cottonwood, black cottonwood, ash tree, birch, maple, oak, elm, pecan, and hickory; common plant allergens include allergens from rye grass, ragweed, ribgrass, sow thistle, and goosefoot; plant contact allergens include allergens from poison oak, poison ivy, and stinging nettle; common grass allergens include cat's tail, quack grass, Bermuda grass, fescue, and bluegrass allergens; common allergens can also be obtained from molds or fungi, such as Alternaria, Fusarium, Hormodendrum, Aspergillus, Micropolyspora, Mucor, and thermophilic actinomycetes; penicillin and tetracycline are common antibiotic allergens; dermal allergens can be obtained from household or organic dust (usually derived from fungi), from insects such as house dust mites (dermalphagoides pterosinyssis), or from animal sources such as feathers and cat and dog dander; common food allergens include milk and cheese (dairy), egg, wheat, nuts (e.g., peanuts), seafood (e.g., shellfish), pea, bean, and gluten allergens; common environmental allergens include metals (nickel and gold), chemicals (formaldehyde, trinitrophenol, and turpentine), latex, rubber, fibers (cotton or wool), burlap, hair dyes, cosmetics, detergents, and perfume allergens; common drug allergens include local anesthetics and salicylate allergens; antibiotic allergens include penicillin and sulfonamide allergens; common insect allergens include honeybee, yellow jacket, and ant venom and cockroach cupula allergens. Particularly well-characterized allergens include, but are not limited to, the Der pl allergen (Hoyne et al., 1994, Immunology, 83:190-195), the major and cryptic epitopes of bee venom phospholipase A2 (PLA; Akdis et al., 1996, J. Clin. Invest., 98:1676-1683), the birch pollen allergen Bet v 1 (Bauer et al., 1997, Clin. Exp. Immunol., 107:536-541), and the polyepitopic recombinant grass allergen rKBG 8.3 (Cao et al., 1997, Immunology, 90:46-51).These and other suitable allergens are commercially available and / or can be readily prepared as extracts according to known techniques.

[0263] Antigens can be in the form of purified or partially purified antigens, and can be derived from any of the above antigens, antigenic peptides, proteins known and available in the art, and other antigens identifiable using routine techniques. Antigens are typically in a form in which the toxin or virulence properties have been reduced or destroyed, and when introduced in the appropriate form, will induce an immune response against the particular microorganism, extract or microbial product used for the antigen preparation, or in the case of allergens, will help alleviate the symptoms of an allergy due to the particular allergen. Antigens can be used alone or in combination; for example, multiple bacterial antigens, multiple viral antigens, multiple bacterial antigens, multiple parasitic antigens, multiple bacterial, viral toxoids, multiple tumor antigens, multiple allergens, or combinations of any of the foregoing products can be combined with an adjuvant composition to produce a multivalent antigen composition and / or vaccine. In the compositions described herein, the antigens can be antigens encapsulated in, adsorbed to, or mixed with the vesicle component of the composition.

[0264] In one embodiment, suitable antigens for use in the compositions described herein include poorly immunogenic antigens, such as malaria antigens, dengue antigens, and HIV antigens, or antigens intended to confer immunity against a pandemic disease, such as influenza antigens. Combinations of any such antigens described or known herein are contemplated for use in the fusion proteins, fusion protein pairs, and nanocages described herein.

[0265] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of the sequences of nucleotides that, when translated, result in the synthesis of a protein, such as an rRNA, tRNA, and mRNA, or other

[0266] The term “expression” as used herein is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0267] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely removed from the natural environment of the animal is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as a host cell.

[0268] Unless otherwise indicated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences which are degenerate variations of each other, and which encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include an intron, to the extent that the nucleotide sequence encoding the protein can contain the intron in certain variations.

[0269] As used herein, the term "modulate" refers to mediating a detectable increase or decrease in a level of a response in a subject as compared to the level of the response in a subject in the absence of the treatment or the compound, and / or as compared to the level of the response in an otherwise identical subject that is not treated. The term encompasses perturbing and / or affecting a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, typically a human.

[0270] The term "operably linked" refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Typically, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0271] "Parenteral" administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection or infusion techniques.

[0272] The term "polynucleotide" as used herein is defined as a chain of nucleotides. In addition, a nucleic acid is a polymer of nucleotides. Thus, as used herein, nucleic acid and polynucleotide are interchangeable. One of skill in the art has general knowledge that nucleic acids are polynucleotides and are hydrolyzable to monomeric "nucleotides." The monomeric nucleotides are hydrolyzable to nucleosides. As used herein, polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means (i.e., cloning of nucleic acid sequences from a recombinant library or the genome of a cell using ordinary cloning techniques and PCR and the like) as well as by synthetic means.

[0273] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound composed of amino acid residues connected to one another by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can comprise the sequence of a protein or peptide. A polypeptide includes any peptide or protein that includes two or more amino acids connected to one another by peptide bonds. As used herein, the term refers not only to short chains (which are also commonly referred to in the art as, e.g., peptides, oligopeptides, and oligomers), but also to long chains (which are commonly referred to in the art as proteins, of which there are numerous types). "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and the like. The polypeptides include natural, recombinant, synthetic, or combinations thereof.

[0274] As used herein in reference to antibodies, the term "specifically binds" refers to 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 an antigen from one species can also bind the antigen from one or more species. However, such cross-species reactivity does not by itself alter the antibody's classification as specific. In another example, an antibody that specifically binds an antigen can also bind different allelic forms of the antigen. However, such cross-reactivity does not by itself alter the antibody's classification as specific. In some contexts, the term "specifically binds" can be used to refer to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction depends on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds a particular protein structure and not a general protein. If an antibody is specific for an epitope "A," then the presence of a molecule containing epitope A (or free, unlabelled A) will decrease the amount of labelled A bound to the antibody in a reaction containing labelled "A" and the antibody.

[0275] The terms "therapeutically effective amount," "effective amount," or "sufficient amount" refer to an amount that, when administered to a subject (including a mammal, such as a human), is sufficient to achieve the desired result, e.g., an amount effective to elicit a protective immune response. The effective amount of a compound described herein can vary according to factors such as the immunogen, the age, sex, and weight of the subject. Dosage or treatment regimens can be adjusted to provide the optimum therapeutic response, as is understood by the skilled artisan. For example, administration of a therapeutically effective amount of a fusion protein described herein is, in certain aspects, sufficient to enhance immunity against a pathogen, such as a malaria parasite or HIV. In other aspects, administration of a therapeutically effective amount of a fusion protein described herein is sufficient to treat a disease or disorder, such as cancer, HIV, malaria, or an autoimmune disease. In still other aspects, administration of a therapeutically effective amount of a fusion protein described herein is sufficient to act as an adjuvant to increase the effectiveness of a vaccine. In yet other aspects, administration of a therapeutically effective amount of a fusion protein described herein is sufficient to prevent the development of a disease or infection.

[0276] Further, a treatment regimen that administers a therapeutically effective amount to a subject can consist of a single administration, or, alternatively, include a series of administrations. The length of a treatment period depends on a variety of factors, such as the immunogen, the age of the subject, the concentration of the drug, the patient's responsiveness to the drug, or a combination thereof. It is also understood that the effective dose of a drug for treatment can increase or decrease during the course of a particular treatment regimen. Variations in dosage can thus occur and be evident by standard diagnostic assays known in the art. In some aspects, a fusion protein described herein can be administered prior to, during, or after treatment of a target disease or disorder, such as malaria, HIV, or cancer, with conventional therapies. For example, a fusion protein described herein can be used in particular in combination with immunotherapy to treat cancer.

[0277] As used herein, the term "transfected" or "transformed" or "transduced" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is one which has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the original subject cell and its progeny.

[0278] As used herein, the phrase "under the control of" or "operably linked" means that a promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription performed by RNA polymerase and expression of the polynucleotide.

[0279] A "vector" is a composition of matter that comprises an isolated nucleic acid and is useful for delivering the isolated nucleic acid into the interior of a cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions 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 acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

[0280] 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 primates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, and the like. Typically, the mammal is a human.

[0281] Administration "in combination" with one or more other therapeutic agents includes concurrent (parallel) and consecutive administration in any order.

[0282] The term "pharmaceutically acceptable" means that the compound or combination of compounds is compatible with the other ingredients of a pharmaceutical formulation and is generally safe for administration to humans according to established government standards, including those published by the U.S. Food and Drug Administration.

[0283] The term "pharmaceutically acceptable carrier" includes, but is not limited to, solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and / or absorption delaying agents, and the like. The use of pharmaceutically acceptable carriers is well known.

[0284] The term "adjuvant" refers to a compound or mixture that is present in a vaccine and enhances the immune response to an antigen present in the vaccine. For example, an adjuvant can enhance the immune response to a polypeptide present in a vaccine as contemplated herein or to an immunogenic fragment or variant thereof as contemplated herein. An adjuvant can act as a tissue reservoir for slow release of antigen or as an activator of the lymphoid system that nonspecifically enhances the immune response. Examples of adjuvants that can be used include the MPL-TDM adjuvant (monophosphoryl lipid A / synthetic trehalose dimycolate ester, available for example from GSK Biologies). Another suitable adjuvant is the immunostimulatory adjuvant AS021 / AS02 (GSK). These immunostimulatory adjuvants are formulated to generate strong T cell responses and include QS-21, a saponin from Quillay saponaria, a TL4 ligand, monophosphoryl lipid A, and a lipid or liposome carrier. Other adjuvants include, but are not limited to, non-ionic block copolymer adjuvants (e.g., CRL 1005), aluminum phosphate (e.g., AIPO4), R-848 (a Thl-like adjuvant), imiquimod, PAM3CYS, poly (I:C), rosoxstatin, BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum, CpG oligodeoxynucleotides (ODNs), cholera toxin derivative antigens (e.g., CTA 1-DD), lipopolysaccharide adjuvants, complete Freund's adjuvant, incomplete Freund's adjuvant, saponins, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions (e.g., MF59 or Montanide ISA 720 available from Novartis Vaccines), keyhole limpet hemocyanin, and dinitrophenol.

[0285] A "variant" is a biologically active fusion protein, antibody or fragment thereof having a sequence that differs from a comparison sequence due to the insertion, deletion, modification and / or substitution of one or more amino acid residues in the comparison sequence. A variant typically has less than 100% sequence identity compared to the comparison sequence. However, typically, a biologically active variant will have an amino acid sequence having at least about 70% amino acid sequence identity compared to the comparison sequence, for example 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 a certain level of biological activity of the comparison sequence. A variant also includes a polypeptide in which one or more amino acid residues are added at the N or C terminus or internally of the comparison sequence. A variant also includes a polypeptide in which a number of amino acid residues are deleted and optionally replaced by one or more amino acid residues. A variant can also be covalently modified, for example by substitution with a moiety of a non-naturally occurring amino acid or by modification of an amino acid residue to produce a non-naturally occurring amino acid.

[0286] "Percent amino acid sequence identity" is defined herein as the percentage of amino acid residues in a candidate sequence that are identical with the residues in the target sequence (e.g., a polypeptide of the application), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. N-terminal, C-terminal or internal extensions, deletions or insertions of the candidate sequence are not to be construed as affecting sequence identity or homology. Methods and computer programs for alignment are well known in the art, e.g., "BLAST".

[0287] "Active" or "activity" herein refers to the biological and / or immunological activity of the fusion proteins described herein, wherein "biological" activity refers to a biological function (inhibition or stimulation) caused by the fusion protein.

[0288] The fusion proteins described herein can include modifications. Such modifications include, but are not limited to, conjugation to an effector molecule such as an anti-malarial drug or an adjuvant. Modifications also include, but are not limited to, conjugation to a detectable reporter moiety. Modifications to extend half-life (e.g., pegylation) are also included. Proteins and non-protein drugs can be conjugated to the fusion proteins by methods known in the art. Conjugation methods include direct linkage, linkage through a covalently attached linker, and specific binding pairs of members (e.g., avidin-biotin). These methods include, for example, those described by Greenfield et al., Cancer Research, 50:6600-6607, 1990, which is incorporated herein by reference; and Amon et al., Adv. Exp. Med. Biol., 303:79-90, 1991 and Kiseleva et al., Mol. Biol. (USSR), 25:508-514, 1991, both of which are incorporated herein by reference.

[0289] Fusion proteins

[0290] Described herein are fusion proteins. The fusion proteins include a first nanocage monomer subunit linked to a biologically active moiety. The fusion proteins self-assemble with proteins including a second nanocage monomer subunit to form nanocage monomers. Multiple such fusion proteins self-assemble to form nanocages. As such, the biologically active moiety can modify the interior surface of the assembled nanocage, the exterior surface of the assembled nanocage, or both.

[0291] The biologically active moiety can be any moiety capable of being part of a fusion protein, and is typically a protein. Typically, the biologically active moiety includes an antibody or fragment thereof, an antigen, a detectable moiety, a pharmaceutical agent, a diagnostic agent, or combinations thereof.

[0292] When the biologically active moiety is an antibody and fragments thereof, one or both chains can be included, such as an Fc fragment. As will be appreciated, the Fc fragment can be derived from any type of antibody, but is typically a gG1 Fc fragment. The Fc fragment can also include one or more mutations that modulate the half-life of the fusion protein and / or the resulting assembled nanocage including the fusion protein, such as LS, YTE, LALA, and / or LALAP. For example, the half-life can be in units of minutes, days, weeks, or even months.

[0293] In addition, other alternatives in the fusion proteins and nanocages described herein are contemplated, including Fc sequence modifications and addition of other agents (e.g., human serum albumin peptide sequences), which allow for changes in bioavailability and will be understood by the skilled artisan. In addition, the fusion proteins and nanocages described herein can be modulated by sequences or by addition of other agents to attenuate their immunogenicity and anti-drug responses (therapeutically, e.g., matching sequences to the host, or addition of immunosuppressive therapies [such as, for example, methotrexate, a primary strategy to reduce the incidence of FVIII inhibitors when administering enlimomab for the treatment of rheumatoid arthritis or to induce tolerance in neonates (see: DiMichele DM, Hoots WK, Pipe SW, Rivard GE, Santagostino E, International workshop on immune tolerance induction: consensus recommendations, Haemophilia, 2007, 13: 1-22, which is incorporated by reference herein in its entirety)] or to enhance the immune response (e.g., bacterial sequences for vaccines).

[0294] In other aspects, when the biologically active moiety is an antibody or fragment thereof, the heavy and / or light chain can include, for example, a Fab fragment. The antibody or fragment thereof can include, for example, a scFab fragment, a scFv fragment, or a sdAb fragment. It will be appreciated that any antibody or fragment thereof can be used in the fusion proteins described herein.

[0295] Generally, the fusion proteins described herein are associated with a Fab light and / or heavy chain, which can be produced separately or sequentially from the fusion protein.

[0296] In the case where the antibody or fragment thereof comprises two chains, such as the first and second chains in the case of an Fc fragment, or such as a heavy chain and a light chain, the two chains are optionally separated by a linker. The linker can be flexible or rigid, but it is typically flexible to allow the chains to fold properly. While it will be appreciated that the length of the linker will vary depending on the sequences of the nanocage monomer and the biologically active moiety, as well as the three-dimensional conformation of the fusion protein, the linker is typically long enough to impart some flexibility to the fusion protein. Thus, the linker is typically from about 1 to about 30 amino acid residues, such as from 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, or 29 to about 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 amino acid residues, such as from about 8 to about 16 amino acid residues, such as 8, 10, or 12 amino acid residues.

[0297] The linker can be any amino acid sequence, and in one typical example, the linker comprises GGS repeats, and more typically, the linker comprises about 2, 3, 4, 5, or 6 GGS repeats, such as about 4 GGS repeats. In some particular aspects, the linker comprises or consists of a sequence having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the following sequence:

[0298] GGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGTATAGASSGSGSSGSSSSGGTG.

[0299] In some typical aspects, the antibody or fragment thereof specifically binds to an antigen associated with an antibody preventable and / or antibody treatable condition. For example, the antigen to which the antibody or fragment thereof binds can be associated with an infectious agent, including a virus (e.g., HIV (including HIV-1), influenza virus, RSV, rotavirus), a bacterium (e.g., TB, C. difficile), a parasite (e.g., malaria), a fungus, or a yeast; a cancer (e.g., CD19, CD22, CD79, BCMA, or CD20), including a solid cancer and a liquid cancer; or an immune disease, including an autoimmune disease. Typically, the antigen is associated with HIV-1, and the antibody or fragment thereof comprises, for example, ibalizumab-A12P, 10E8, 10E8.v4, N49P7, PGDM1400, 10-1074, VRC01, or a combination thereof.

[0300] In particular examples, the antibody or fragment thereof comprises or consists of a sequence having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identity to one or more of the following sequences:

[0301] Fc chain 1:

[0302] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

[0303] GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK

[0304] AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK;

[0305] Fc chain 2:

[0306] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

[0307] GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK

[0308] AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK;

[0309] Ibalizumab-A12P light chain:

[0310] DIVMTQSPDSLPVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWAST

[0311] RESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIKRTVAAPS

[0312] VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0313] Ibalizumab-A12P heavy chain:

[0314] QVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGT

[0315] DYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQG

[0316] TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGSR;

[0317] 10E8.v4 light chain:

[0318] SELTQDPAVSVALKQTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDR

[0319] FSGSASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVLSQPKAAPSVTL

[0320] FPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC;

[0321] 10E8.v4 heavy chain:

[0322] EVRLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEG

[0323] WSVDYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGE

[0324] EYFQDWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS

[0325] GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCSR;

[0326] N49P7 light chain:

[0327] QSALTQPRSVSASPGQSVTISCTGTHNLVSWCQHQPGRAPKLLIYDFNKRPSGVPDRFSG

[0328] SGSGGTASLTITGLQDDDDAEYFCWAYEAFGGGTKLTVLGQPKAAPSVTLFPPSSEELQ

[0329] ANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC;

[0330] N49P7 heavy chain:

[0331] ADLVQSGAVVKKPGDSVRISCEAQGYRFPDYIIHWIRRAPGQGPEWMGWMNPMGGQV

[0332] NIPWKFQGRVSMTRDTSIETAFLDLRGLKSDDTAVYYDRSNGSGKRFESSNWFLDLWG

[0333] RGTAVTIQSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDSR;

[0334] PGDM1400 light chain:

[0335] DFVLTQSPHSLSVTPGESASISCKSSHSLIHGDRNNYLAWYVQKPGRSPQLLIYLASSRAS

[0336] GVPDRFSGSGSDKDFTLKISRVETEDVGTYYCMQGRESPWTFGQGTKVDIKRTVAAPSV

[0337] FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0338] PGDM1400 Heavy Chain:

[0339] QAQLVQSGPEVRKPGTSVKVSCKAPGNTLKTYDLHWVRSVPGQGLQWMGWISHEGDK

[0340] KVIVERFKAKVTIDWDRSTNTAYLQLSGLTSGDTAVYYCAKGSKHRLRDYALYDDDGA

[0341] LNWAVDVDYLSNLEFWGQGTAVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY

[0342] FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDSR; or

[0343] combinations thereof.

[0344] In further aspects, the antibody or fragment thereof is coupled to or associated with an additional moiety, such as an antigen, a detectable moiety (e.g., a small molecule, a fluorescent molecule, a radioisotope, or a magnetic particle), a pharmaceutical agent, a diagnostic agent, or a combination thereof, and can include, for example, an antibody drug conjugate.

[0345] In aspects where the biologically active moiety is an antigen, the antigen can be associated with, for example, a vaccine-preventable condition and / or a vaccine-treatable condition. In such cases, the antigen can be associated with, for example: an infectious agent, including a virus, a bacterium, a parasite, a fungus, or a yeast; a cancer, including a solid cancer and a liquid cancer; or an immune disease, including an autoimmune disease.

[0346] In aspects where the biologically active moiety is a detectable moiety, the detectable moiety can include a fluorescent protein, such as GFP, EGFP, violacein, and / or a flavin-based fluorescent protein such as a LOV protein (such as iLOV).

[0347] In aspects where the biologically active moiety is an agent, the agent can include, for example, a small molecule, a peptide, a lipid, a carbohydrate, or a toxin.

[0348] In some exemplary aspects, the nanocage assembled from the fusion protein described herein includes about 3 to about 100 nanocage monomers, such as 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 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, such as 24, 32, or 60 nanocage monomers. The nanocage monomers can be any known natural, synthetic, or partially synthetic nanocage monomer, and in some aspects are selected from ferritin, apoferritin, encapsulin, SOR, lumazine synthase, pyruvate dehydrogenase, carboxysome, vault protein, GroEL, heat shock protein, E2P, MS2 coat protein, fragments thereof, and variants thereof. Typically, the nanocage monomers are ferritin or apoferritin.

[0349] When apoferritin is selected as the nanocage monomer, typically the first nanocage monomer subunit and the second nanocage monomer subunit interchangeably include the “N” region and the “C” region of apoferritin. It will be appreciated that other nanocage monomers can be divided into two subunits very similar to apoferritin as described herein, such that the subunits can self-assemble and are each suitable for fusion to a biologically active moiety.

[0350] Typically, the “N” region of apoferritin includes or consists of a sequence having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the following sequence:

[0351] MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEW.

[0352] Typically, the "C" region of an apo-transferrin includes or consists of a sequence having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the following sequence:

[0353] GKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD.

[0354] In some aspects, the fusion proteins described herein further include a linker between the nanocage monomer subunit and the biologically active moiety, much like the linkers described above. Again, this linker can be flexible or rigid, but it is typically flexible to allow the biologically active moiety to retain activity and allow the nanocage monomer subunit to retain self-assembly properties. While it will be appreciated that the length of this linker will vary depending on the sequences of the nanocage monomer and the biologically active moiety as well as the three-dimensional conformation of the fusion protein, the linker is typically long enough to impart some flexibility to the fusion protein. Thus, the linker is typically from about 1 to about 30 amino acid residues, such as from 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, or 29 to about 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 amino acid residues, such as from about 8 to about 16 amino acid residues, such as 8, 10, or 12 amino acid residues.

[0355] The linker can be any amino acid sequence, and in one typical example, the linker includes GGS repeats, and more typically, the linker includes about 2, 3, 4, 5, or 6 GGS repeats, such as about 4 GGS repeats. In some particular aspects, the linker includes or consists of a sequence having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the following sequence:

[0356] ASTASSASSGGGGGGSGGSGGSGGS.

[0357] Similarly, the fusion protein can also include a C-terminal linker for improving one or more attributes of the fusion protein. In some aspects, the linker includes GGS repeats, and more generally, the linker includes about 2, 3, 4, 5, or 6 GGS repeats, such as about 4 GGS repeats. In some particular aspects, the C-terminal linker includes or consists of a sequence having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the following sequence:

[0358] GGSGGSGGSGGSGGGASGGS.

[0359] Also described herein are fusion protein pairs of the above-described fusion proteins, wherein the fusion protein pairs self-assemble to form a nanocage monomer, wherein the first and second nanocage monomer subunits are fused to different biologically active moieties. This provides multivalency and / or multispecificity to the individual nanocage monomer assembled from the pair of subunits.

[0360] In certain aspects, the fusion protein can also include an antigen. These aspects are explicitly described in International Patent Application No. WO 2019 / 023812, which is incorporated by reference herein in its entirety. Briefly, in these aspects, the antigen has at least a first and a second antibody binding epitope; and an antibody or fragment thereof specific for at least a first antigen epitope. Binding of the antibody or fragment thereof to the first antigen epitope presents for binding the second antigen epitope of the antigen binding moiety, and / or the first antibody binding epitope is bound by the antibody or fragment thereof, and wherein said binding presents said second antibody binding epitope in the context of the antibody or fragment thereof.

[0361] In other aspects, the antibody or fragment thereof can be directed against any antigen, such as those listed above. Typically, the antigen is derived from a cancer or infectious agent, such as hepatitis A, B, or C, HIV, mycobacteria, malaria pathogen, SARS pathogen, herpes virus, influenza virus, polio virus, or from a bacterial pathogen such as Chlamydia and mycobacteria, or from an autoreactive B cell or any T cell for co-recruitment and cytotoxic killing.

[0362] The fusion proteins described herein can alternatively be used as therapeutic or diagnostic agents. Thus, in some aspects, the antibody or fragment thereof can be specific for, for example, a tumor antigen or an autoantigen.

[0363] Substantially identical sequences can 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 can result in a mutant peptide that is not substantially altered in physiologic, chemical, or functional properties compared to the reference sequence; in such cases, the reference sequence and the mutant sequence will be considered to be "substantially identical" polypeptides. Conservative amino acid mutations can include insertions, deletions, or substitutions of amino acids; a "conservative amino acid substitution" is defined herein as a substitution of an amino acid residue by another amino acid residue with similar chemical properties (e.g., size, charge, or polarity).

[0364] In non-limiting examples, a conservative mutation can be an amino acid substitution. Such conservative amino acid substitutions can substitute a basic, neutral, hydrophobic, or acidic amino acid with another amino acid in 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 referred to as a "polar amino acid") refers to a hydrophilic amino acid with a side chain that is uncharged at physiological pH, but which has at least one bond in which an electron pair shared by two atoms is held more tightly by one of the two 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 (Gin or Q). The term "hydrophobic amino acid" (also referred to as a "non-polar amino acid") refers to an amino acid that exhibits a hydrophobicity greater than zero according to the Eisenberg normalized consensus hydrophobicity scale (1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (lie 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).

[0365] An "acidic amino acid" refers to a hydrophilic amino acid with a side chain pK value 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).

[0366] Sequence identity is used to assess the similarity of two sequences; sequence identity is determined by calculating the percentage of residues that are identical when the two sequences are aligned to give the maximum correspondence between residues positions. Any known method can be used to calculate sequence identity; for example, computer software can be used to calculate sequence identity. Without wishing to be limiting, sequence identity can be calculated by software such as the NCBI BLAST2 service maintained by the Swiss Institute of Bioinformatics (also found at ca.expasy.org / tools / blast / ), BLAST-P, Blast-N, or FASTA-N, or by any other suitable software known in the art.

[0367] Substantially identical sequences of the application can have at least 85% identity; in another example, substantially identical sequences can be sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity at the amino acid level to the sequences described herein. In some particular aspects, substantially identical sequences retain the activity and specificity of the reference sequence. In non-limiting embodiments, differences in sequence identity can be due to conservative amino acid mutations.

[0368] The polypeptides or fusion proteins of the application can also include additional sequences to aid in their expression, detection, or purification. Any such sequences or tags known to those skilled in the art can be used. For example, and without wishing to be limiting, the fusion protein can include a targeting sequence or signal sequence (for example, but not limited to ompA), a detection tag, exemplary tag cassettes include Strep tag or any variant thereof; see, for example, U.S. Patent No. 7,981,632, His tag, Flag tag having the sequence motif DYKDDDDK, Xpress tag, Avi tag, Calmodulin tag, Polyglutamic acid tag, HA tag, Myc tag, Nus tag, S tag, SBP tag, Softag 1, Softag 3, V5 tag, CREB-binding protein (CBP), glutathione S-transferase (GST), maltose binding protein (MBP), green fluorescent protein (GFP), thioredoxin tag, or any combination thereof; a purification tag (for example, but not limited to His5 or His6), or combinations thereof.

[0369] In another example, the additional sequence can be a biotin recognition site, such as described in Cronan et al. in WO 95 / 04069 or Voges et al. in WO / 2004 / 076670. As also known to those skilled in the art, linker sequences can be used in conjunction with the additional sequence or tag.

[0370] More specifically, the tag cassette can include an extracellular component that can specifically bind to an antibody with high affinity or avidity. In a single-chain fusion protein structure, the tag cassette can be located (a) immediately adjacent to the amino-terminal end of the linker region, (b) inserted between and connecting the linker modules, (c) immediately adjacent to the carboxy-terminal end of the binding domain, (d) inserted between and connecting the binding domain (e.g., scFv or scFab) and the effector domain, (e) inserted between and connecting the binding domain subunits, or (f) at the amino-terminal end of the single-chain fusion protein. In certain embodiments, one or more linking amino acids can be disposed between and connecting the tag cassette and the hydrophobic moiety, or between and connecting the tag cassette and the linker region, or between and connecting the tag cassette and the linker module, or between and connecting the tag cassette and the binding domain.

[0371] Also encompassed herein are isolated or purified fusion proteins, polypeptides, or fragments thereof immobilized on a surface using various methods; for example, and without wishing to be limiting, the polypeptide can be attached or coupled to the surface by His tag coupling, biotin binding, covalent binding, adsorption, etc. The solid surface can be any suitable surface, such as, 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 a magnetic-based bead or an agarose-based bead or other chromatography resin), glass, a thin film, or any other useful surface.

[0372] 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 techniques, chemical coupling, chelation, etc.). The cargo molecule can be any type of molecule, such as a therapeutic or diagnostic agent. For example, and without wishing to be limiting in any way, the therapeutic agent can be a radioisotope, which can be used in radioimmunotherapy; a toxin, such as an immunotoxin; a cytokine, such as an immunocytokine; a cytotoxin; an apoptosis inducer; an enzyme; an anti-cancer antibody for immunotherapy; or any other suitable therapeutic molecule known in the art. Alternatively, a diagnostic agent can include, but is in no way limited to: a radioisotope, a paramagnetic label such as gadolinium or iron oxide, a fluorescent group, a near-infrared (NIR) fluorescent dye or dye such as Cy3, Cy5.5, Alexa680, Dylight680, or Dylight800, an affinity label fused to a detectable protein-based molecule (e.g., biotin, avidin, etc.), or any other suitable agent that can be detected by an imaging method. In a specific, non-limiting example, the fusion protein can be linked to a fluorescent agent such as FITC, or genetically fused to an enhanced green fluorescent protein (EGFP).

[0373] In some aspects, the cargo molecule is a protein and is fused to the fusion protein, such that the cargo molecule is contained inside the nanocage. In other aspects, the cargo molecule is not fused to the fusion protein and is contained inside the nanocage. The cargo molecule is typically a protein, a small molecule, a radioisotope, or a magnetic particle.

[0374] The fusion proteins described herein specifically bind to their target. "Antibody specificity" refers to the selective recognition of a particular epitope of an antigen by an antibody, the antibody specificity of the antibodies or fragments described herein can be determined based on affinity and / or avidity. Affinity, as represented by the dissociation equilibrium constant (Kd) between an antigen and an antibody, measures the strength of binding 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 typically bind with Kd of 10 D M to 10 -5 M. Any Kd greater than 10 -11 M is generally considered to indicate non-specific binding. The smaller the Kd value, the stronger the binding strength between the antigenic determinant and the antibody binding site. In some aspects, the antibodies described herein have a Kd of less than 10 D M, 10 -4 M, 10 D M, 10 D M, 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 M's K D .

[0375] Also described herein are nanocages comprising at least one fusion protein described herein and at least one second nanocage monomer subunit that self-assembles with the fusion protein to form a nanocage monomer. Further, described herein are pairs of fusion proteins, wherein the pair of fusion proteins self-assemble to form a nanocage monomer, and wherein the first and second nanocage monomer subunits are fused to different biologically active moieties.

[0376] It will be appreciated that the nanocage can be self-assembled from a plurality of identical fusion proteins, a plurality of different fusion proteins (and thus the nanocage is multivalent and / or multispecific), a combination of fusion proteins and wild-type proteins, and any combination thereof. For example, the nanocage can be internally and / or externally modified by at least one fusion protein described herein in combination with at least one anti-cancer antibody for immunotherapy. In some typical aspects, about 20% to about 80% of the nanocage monomers comprise a fusion protein described herein. In view of the modular solution described herein, the nanocage can theoretically comprise up to twice as many biologically active moieties as monomers in the nanocage, as each nanocage monomer can be divided into two subunits, each of which can independently bind a different biologically active moiety. It will be appreciated that any desired ratio of biologically active moieties as described herein in specific examples can be achieved with this modularity, in some specific examples, four different biologically active moieties in a 4:2:1:1 ratio. For example, the nanocage described herein can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different biologically active moieties. As such, the nanocage can be multivalent and / or multispecific, and the degree thereof can be relatively easily controlled.

[0377] In some aspects, the nanocage described herein can also comprise at least one complete nanocage monomer, optionally fused to a biologically active moiety that can be the same or different from the biologically active moieties described herein that are attached to the nanocage monomer subunits.

[0378] In typical aspects, the nanocage described herein comprises a first fusion protein, a second fusion protein, and a third fusion protein, and at least one complete nanocage monomer optionally fused to a biologically active moiety, wherein the biologically active moiety of the first fusion protein, the second fusion protein, and the third fusion protein and the biologically active moiety of the complete nanocage monomer are each different from one another.

[0379] More typically, the first fusion protein, the second fusion protein, and the third fusion protein each comprise an antibody or fragment thereof fused to an N ferritin or a C ferritin, wherein at least one of the first fusion protein, the second fusion protein, and the third fusion protein is fused to an N ferritin, and at least one of the first fusion protein, the second fusion protein, and the third fusion protein is fused to a C ferritin. For example, the antibody or fragment thereof of the first fusion protein is typically an Fc fragment; the second fusion protein and the third fusion protein typically each comprise an antibody or fragment thereof specific for a different antigen of a virus, such as HIV, or one of the second fusion protein and the third fusion protein comprises an antibody or fragment thereof specific for an antigen of a virus, such as HIV, and the third fusion protein comprises an antibody or fragment thereof specific for a different antigen, such as the CD4 receptor; and the complete nanocage monomer is fused to a biologically active moiety specific for another different antigen, optionally a different antigen of the same virus, such as HIV.

[0380] In some aspects, the antibody or fragment thereof of the second fusion protein is N49P7 or iMab A12P; and the antibody or fragment thereof of the third fusion protein is 10E8v4. In a typical aspect, the nanocage described herein comprises the following four fusion proteins, optionally in a 4:2:1:1 ratio:

[0381] a. PGDM1400 (optionally scPGDM1400) fused to full length ferritin;

[0382] b. Fc (optionally scFc) fused to N ferritin;

[0383] c. N49P7 or iMab A12P (optionally scN49P7 or sciMab A12P) fused to C ferritin; and

[0384] d. 10E8v4 (optionally sc10E8v4) fused to C ferritin.

[0385] In some aspects, the nanocage described herein comprises or consists of a sequence having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to one or more of the following sequences, wherein ferritin subunits are shown in bold, linkers are underlined, light chains are shown in italics, and heavy chains are shown in lower case:

[0386]

[0387]

[0388]

[0389] It will be appreciated that typically the nanocage described herein is hollow, and thus is able to carry a cargo molecule such as a pharmaceutical agent, a diagnostic agent and / or an imaging agent. Typically, the cargo molecule is not fused to the fusion protein and is contained inside the nanocage, however, the cargo molecule can alternatively be a protein and be fused to the fusion protein such that the cargo molecule is contained inside the nanocage.

[0390] In some aspects, the cargo molecule is contained inside to provide a T cell epitope, but optionally not a B cell epitope. Alternatively, the cargo molecule is fused to the fusion protein and contained inside to provide a T cell epitope, but optionally not a B cell epitope.

[0391] The cargo molecule can be a fluorescent protein such as GFP, EGFP, sapphyrin and / or a flavin-based fluorescent protein such as a LOV protein (such as iLOV), and / or the cargo molecule can be a small molecule, a radioisotope or a magnetic particle.

[0392] In addition, the nanocage can also include an antigen on the surface, which can be expressed as a fusion protein with the nanocage monomer.

[0393] Also described herein are vaccines comprising the nanocage described herein, and compositions comprising the nanocage, such as a therapeutic composition or a prophylactic composition. Also described herein are related methods and uses for treating and / or preventing a disease or disorder, wherein the method or use comprises administering to a subject in need thereof the nanocage, vaccine or composition described herein. The nanocage can be used for treating any disease or disorder for which a biologically active therapy, or more specifically an antibody therapy, is available, but for example, the disease or disorder is typically a cancer, an infectious disease such as HIV, malaria, influenza, RSV, rotavirus or an autoimmune disease.

[0394] Also described herein are nucleic acid molecules encoding the fusion proteins and polypeptides described herein, as well as vectors comprising the nucleic acid molecules and host cells comprising the vectors.

[0395] Polynucleotides encoding the fusion proteins described herein include polynucleotides having a nucleic acid sequence that is essentially identical to the nucleic acid sequence of a polynucleotide of the present application. An "essentially identical" nucleic acid sequence is defined herein as a sequence that is 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% identical to another nucleic acid sequence when the two sequences are optimally aligned (with appropriate nucleotide insertions or deletions) and compared to determine exact matches of nucleotides between the two sequences.

[0396] Suitable sources of polynucleotides encoding antibody fragments include any cell expressing a full-length antibody, such as hybridomas and splenocytes. As described above, the fragment can be used as an antibody equivalent per se, or can be recombined into an equivalent. DNA deletions and recombination described in this section can be performed by known methods, such as those described in the section entitled "Functional Equivalents of Antibodies" in the published patent application listed above, and / or other standard DNA recombination techniques such as those described below. Another source of DNA is single chain antibodies produced from phage display libraries, as known in the art.

[0397] In addition, provided herein are expression vectors comprising a polynucleotide sequence as described above operably linked to an expression sequence, a promoter sequence, and an enhancer sequence. Various expression vectors have been developed for the efficient synthesis of antibody polypeptides in prokaryotic (such as bacteria) and eukaryotic systems, including but not limited to yeast and mammalian cell culture systems. The vectors of the present application can include fragments of chromosomal, non-chromosomal, and synthetic DNA sequences.

[0398] Any suitable expression vector can be used. For example, prokaryotic cloning vectors include plasmids of E. coli such as col El, pCRl, pBR322, pMB9, pUC, pKSM, and RP4. Prokaryotic vectors also include derivatives of phage DNA, such as M13 and other filamentous single-stranded DNA phages. An example of a vector for use in yeast is the 2μ plasmid. Suitable vectors for expression in mammalian cells include well-known derivatives of SV-40, adenovirus, DNA sequences derived from retroviruses, and shuttle vectors derived from combinations of functional mammalian vectors such as those described above, as well as functional plasmids and phage DNA.

[0399] Additional eukaryotic expression vectors are known in the art (e.g., P J. Southern and P. Berg, J. Mol. Appl. Genet, 1 :327-341, 1982; Subramani et al., Mol. Cell. Biol, 1 :854-864, 1981 ; Kaufmann and Sharp, "Amplification And Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene", J. Mol. Biol, 159:601-621, 1982; Kaufmann and 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 and Chasin, Proc. Nat'l Acad. Sci USA, 77:4216-4220, 1980, all of which are incorporated herein by reference).

[0400] The expression vector typically comprises at least one expression control sequence operably linked to the DNA sequence or fragment to be expressed. The control sequences are inserted into the vector in order to control and regulate the expression of cloned DNA sequences. Examples of useful expression control sequences are the lac system, the trp system, the tac system, the trc system, the major operator and promoter regions of phage lambda, the control regions of fd coat proteins, the glycolytic enzymes promoter (for example, the 3-phosphoglycerate kinase promoter) of yeast, the promoters of the yeast acid phosphatase gene (for example, Pho5), the promoters of the yeast alpha-mating factors, and promoters derived from retroviruses, adenoviruses, vaccinia virus, and simian virus (for example, the early and late promoters of SV40), as well as other sequences known to control the expression of genes in prokaryotic or eukaryotic cells and their viruses, or combinations thereof.

[0401] Also described herein are recombinant host cells comprising the expression vectors as described herein. The fusion proteins described herein can be expressed in cell lines rather than in hybridomas. Nucleic acids comprising sequences encoding polypeptides according to the present application can be used to transform suitable mammalian host cells.

[0402] Particularly preferred cell lines are selected based on high level expression of the target protein, constitutive expression, and minimal contamination from host proteins. Mammalian cell lines that can be used as hosts for expression are well known in the art and include a number of immortal cell lines such as, but not limited to, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, and many others. Other 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 DH1, and E. coli MRC1), Pseudomonas, Bacillus (such as Bacillus subtilis), and Streptomyces.

[0403] These recombinant host cells can be used to produce the fusion proteins by culturing the cells under conditions permitting expression of the polypeptide and purifying the polypeptide from the host cells or the culture medium surrounding the host cells. Targeted secretion of the expressed polypeptide in the recombinant host cell can be facilitated by inserting a sequence encoding a signal peptide or secretory leader peptide at the 5' end of the target gene encoding the antibody (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, each of which is incorporated herein by reference). These secretory leader peptide elements can be derived from prokaryotic sequences or eukaryotic sequences. Thus, as appropriate, a secretory leader peptide is used, which is an amino acid joined to the N-terminus of the polypeptide to direct the polypeptide out of the host cell cytosol and into the culture medium.

[0404] The fusion proteins described herein can be fused to additional amino acid residues. For example, such amino acid residues can be a peptide tag that facilitates isolation. Other amino acid residues that are used to target the antibody to a particular organ or tissue are also contemplated.

[0405] It will be appreciated that the Fab nanocage can be produced by co-transfection of the HC ferritin and LC. Alternatively, single chain Fab-ferritin nanocages that only require transfection of one plasmid can be used, such as Figure 1as shown. This can be achieved by linkers of different lengths (e.g., 60 or 70 amino acids) between the LC and HC. When using single chain Fabs, one can ensure that the heavy and light chains are paired. One can also add tags (e.g., Flag, HA, myc, His6x, Strep, etc.) at the N-terminus of the construct or within the linker to ease purification as described above. Furthermore, when co-transfecting different Fab-nanoparticle plasmids, one can use a tag system to ensure that many different Fabs are present on the same nanoparticle by tandem / additive affinity chromatography steps. This provides multispecificity to the nanoparticle. If desired, protease sites (e.g., TEV, 3C, etc.) can be inserted to cleave the linker and tags after expression and / or purification. An example of such a construct is for the anti-HIV broadly neutralizing scFab 10E8:

[0406] YELTQETGVSVALGRTVTITCRGDSLRSHYASWYQKKPGQAPILLFYGKNNRPSGVPDRFSGSASGNRASLTISGAQAEDDAEYYCSSRDKSGSRLSVFGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECGGSSGSGSGSTGENLYFQGSAGTTGTSASTSGYPYDVPDYAGGGGSAGGTATLEVLFQGPSSGSSSSGGTGEVQLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAAPVEGRFTISRLNSINFLYLEMNNLRMEDSGLYFCARTGKYYDFWSGYPPGEEYFQDWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCSRGGGGGSGGSGGSGGSMSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD

[0407] In another aspect, described herein are methods of vaccinating a subject by administering to a mammal (typically a young, immature, or neonatal mammal) in need thereof a therapeutically effective amount of a fusion protein described herein. A "therapeutically effective amount" refers to an amount effective to produce a desired therapeutic effect, such as providing a protective immune response against a target antigen.

[0408] Any suitable method or route can be used to administer the fusion proteins and vaccines described herein. Routes of administration include, for example, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration.

[0409] It is understood that the fusion proteins described herein, when used for prophylactic or therapeutic purposes in a mammal, will be administered in the form of a composition comprising a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and combinations thereof. Pharmaceutically acceptable carriers can further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, such agents and substances being relatively non-toxic and non-pyrogenic to the subject mammal. Injection compositions can be formulated to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal, as is well known in the art.

[0410] While human antibodies are particularly suitable for administration to humans, they can also be administered to other mammals. The term "mammal" as used herein is intended to include, but is not limited to, humans, laboratory animals, household pets and farm animals.

[0411] Also included herein are kits for vaccination, the kits comprising a therapeutically effective amount or a prophylactically effective amount of a fusion protein described herein. The kits can further comprise, for example, any suitable adjuvant. The kits include instructions.

[0412] The foregoing disclosure describes the application in general terms. A more complete understanding can be obtained by reference to the following specific examples. These examples are provided only for purposes of illustration and are not intended to limit the application in any way. The application thus is not to be construed as being limited to the following examples, but is to be accorded the full scope consistent with the teachings herein.

[0413] The following examples do not include detailed descriptions of conventional methods such as those 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 ordinary skill in the art and are described in numerous publications including Sambrook, J., Fritsch, E.F., and Maniatis, T., 1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., published by Cold Spring Harbor Laboratory Press, which is incorporated herein by reference.

[0414] Without further description, it is believed that one of ordinary skill in the art, utilizing the foregoing description and the following illustrative examples, can make and utilize the compounds of the present application and practice the claimed methods. The following working examples specifically point out typical aspects of the application and are not intended to limit the remainder of the disclosure in any way.

[0415] Example

[0416] Example 1

[0417] Introduction

[0418] Despite 30 years of effort, there remains no effective vaccine or treatment for human immunodeficiency virus type 1 (HIV-1). However, an exciting fact in this endeavor is that a small percentage of HIV-1-infected individuals have developed antibodies with excellent neutralizing potency against circulating HIV-1 isolates. This progress has been made since the discovery of the first-generation broad-spectrum neutralizing antibody (bNAb) 2F5. 1 4E10 2,3 2G12 4 and b12 5,6 Since then, due to Env-specific single B cell sorting 7–9 Antibody cloning and high-throughput neutralization assay 10–13 New technologies and recent proteomics deconvolution techniques 14 With the implementation of [the program / initiative], the catalog of bNAbs has significantly increased. Dozens of HIV bNAbs have now been described, targeting six conserved sites on the HIV trimer envelope (Env), including the V1 / V2 loop at the trimer apex, the V3 ring glycosyl group, the CD4 binding site (CD4bs), the gp120-g41 interface, the fusion peptide, and the juxtamembrane external region (MPER). 7,9,19,10,12–18 .

[0419] Interest in bNAb as a therapeutic molecule in combating HIV-1 stems from studies in macaques. 20–24 and humanized mice 25–28 Some potent antiviral activities were observed in challenge experiments, and bNAb achieved a reduction in viremia in infected individuals when administered therapeutically. 29–33 Furthermore, antibodies offer key advantages over oral antiretroviral therapy (ART): they have a longer circulating half-life and can form immune complexes that enhance the host's immunity against the virus. These observations have led to clinical evaluation of antibody therapy as a substitute for or supplement to conventional immunology in providing protection against HIV-1 through passive administration of bNAb, and have contributed to efforts to control and / or clear HIV-1 in infected individuals.

[0420] One of the main limitations of bNAb clinical application is the rapid selection of neutralizing viral populations. 30–32,34,35 RNA viruses, such as HIV, exhibit abnormal genetic diversity. 36 This allows the virus to develop resistant mutations to evade mAb recognition. However, mutations that eliminate binding to certain bNAbs have a significant adverse impact on viral fitness. 37–39. Similar to the combination of different drugs in HIV-1 treatment regimens, this observation suggests that successful anti-HIV-1 antibody therapy should include a combination of bNAb specificities. Thus, there has been recent interest in developing different formats of antibody-like molecules that are bispecific 40–42 or trispecific 43–45 for Env. An additional consideration is the amount of antibody required for in vivo efficacy. Indeed, there has been a significant amount of work in engineering bNAb for improved potency using structure-guided design or bioinformatics approaches (such as VRC01 46 , 10E8 47,48 , and NIH45-46 49 ) but so far has not been completely successful. For bispecific and trispecific antibodies that target multiple epitopes in Env, potency is often limited by that of their parent mAbs. Thus, to date, while there has been a significant improvement in neutralization breadth, the antiviral potency remains relatively weak 40,43,44,45 .

[0421] The ability of an antibody to interact with more than one epitope on the same virus has a tremendous impact on its potency 50–52 . This effect, often referred to as avidity (apparent affinity enhancement), is a property of IgM antibodies in nature to compensate for their generally low affinity. Thus, the mu tail of IgM has been added to the constant region of IgG to generate dodeca-valent IgM-like molecules with improved biological activity 53,54 . Engineered to produce a variety of non-natural antibody formats far beyond the speed of evolution, the limitations of IgG bivalency have been overcome. Some of these designs include Fab tandem fusion in a linear head-to-tail fashion 55 , tandem diabody combinations (Tamdabs) 56 or diabodies fused to the CH3 of IgG 57 , additional IgG 58–60 and the use of multimerization scaffolds (e.g. p53 61 , leucine zipper helices 62 , streptavidin 63 , barnase-barnase inhibitor protein modules 64 , and self-assembling into pentameric formats 65 and further engineered into dodecameric 66 Escherichia coli Vero cytotoxin B subunit). These antibody architectures face different challenges for their successful development as therapeutics. Multimeric antibody formats that rely on the variable fragment (Fv) of the antibody are often associated with low stability and consequent high aggregation propensity 67Furthermore, dissociation of the dimerization module, determined by the affinity constant of the complex, can limit the long-term stability of the molecule in vivo. In addition, the valency achieved with most of the antibody formats described above is usually at most 3-5 valencies, thus excluding the combination of high avidity and multispecificity.

[0422] Described herein is a multispecific multibody platform that, in some aspects, uses apoferritin protomers as modular subunits for the multimerization of up to 32 antibody fragments (antigen-binding fragments [Fabs] and a crystallizable fragment [Fc]) in a single molecule. Using this approach, we efficiently combined four different specificities into a single molecule, including Fab portions of three of the best anti-HIV-1b NAbs and a crystallizable fragment from IgG1 (Fc) to endow the molecule with multispecificity, high avidity, and effector function and prolonged serum half-life. The resulting multispecific multibody displayed a pan-viral neutralization breadth and significantly higher neutralization potency compared to the combination of their individual parental antibodies or IgGs. Strikingly, the multispecific multibody had a mean IC 50 The median was 1 and 2 orders of magnitude lower in mass and molarity, respectively. The multispecific multibody design described herein represents a robust and powerful plug-and-play platform for multimerizing antibodies to enhance their therapeutic properties for inhibiting HIV-1 infection.

[0423] Materials and Methods

[0424] Expression and purification of the Fab-based apoferritin multispecific multibody. The gene encoding the human apoferritin light chain and the scFab-human apoferritin fusion protein was synthesized and cloned into the pHLsec expression vector by GeneArt (Life Technologies). Two hundred mL of HEK293F cells (Thermo Fisher Scientifics) were transfected with 1.5 mg of the plasmid using the calcium phosphate method (Life Technologies) and incubated for 6 days at 37°C in a 5% CO2 incubator. The supernatant was clarified by centrifugation at 3000 x g for 10 min and filtered through a 0.22 pm filter. The protein was purified from the supernatant using a HiTrap Q HP 5 mL column (GE Healthcare) and eluted with a linear gradient of 0-1 M NaCl in 20 mM Tris-HCl, pH 8.0. Fractions containing the protein were pooled and concentrated using an Amicon Ultra 15 mL centrifugal filter unit with a 30 kDa MWCO (Millipore). The protein was further purified by size-exclusion chromatography using a HiLoad 16 / 600 Superdex 200 pg column (GE Healthcare) and eluted with PBS. Fractions containing the protein were pooled and concentrated using an Amicon Ultra 15 mL centrifugal filter unit with a 30 kDa MWCO (Millipore). The protein was stored at 4°C in PBS. 6Cells / mL were seeded in Freestyle expression medium and incubated at 37°C, 8% C02 and 70% humidity in a Multitron Pro shaker (Infors HT) at 125 rpm shaking. Within 24 hours after seeding, cells were transiently transfected with 50 pg filtered DNA pre-incubated at a 1 : 1 ratio with transfection reagent FectoPRO (Polyplus Transfections) for 10 min at room temperature (RT). Plasmids encoding scFab-human apoferritin and human apoferritin were mixed at a 1 :4, 1 : 1, 4: 1 and 1 :0 ratio to obtain nanoparticles with 20%, 50%, 80% and 100% scFab potency, respectively. After 6-7 days, cell suspensions were harvested by centrifugation at 5000 x g for 15 min and supernatants were filtered with a 0.22 pm Steritop filter (EMD Millipore). Nanoparticles were purified by affinity chromatography on Fabs and washed eluted. Protein containing fractions were pooled, concentrated and loaded on a Superose 6 10 / 300 GL size exclusion chromatography column (GE Heathcare) in 20 mM sodium phosphate pH 8.0, 150 mM NaCl.

[0425] 32- Design, expression and purification of N and I multispecific, multivalent antibodies. Genes encoding scFab and scFc fragments linked to half-ferritin were generated by deletion of residues 1 to 95 (C-ferritin) and 95 to 175 (N-ferritin) of the light chain of human apoferritin using the KOD-plus mutagenesis kit (Toyobo, Osaka, Japan). In addition, the binding specificity of protein L to iMab-C-ferritin was disrupted by site-directed mutagenesis of alanine 12 to a proline residue in the light chain of the antibody using the same mutagenesis kit 75The 32-N multispecific multimeric antibody was transiently transfected into HEK 293F cells by mixing 66 μg of plasmid PGDM1400 with a 4:2:1:1 mixture of scFab-human aphaferretin:Fc-human aphaferretin:N49P7 scFab-C ferritin:10E8 scFab-C ferritin. For the 32-I multispecific multimeric antibody, the iMab scFab-C ferritin plasmid was used in place of the N49P7 scFab-C ferritin plasmid. The DNA mixture was filtered and incubated with 60 μL of FectoPRO at room temperature prior to addition to the cell culture. The multispecific multimeric antibody was purified by affinity chromatography using a HiTrap Protien A HP column (GE Healthcare) with 20 mM Tris pH 8.0, 3 M MgCl2, and 10% glycerol elution buffer first. The multispecific multimeric antibody was further purified by a second affinity chromatography using a HiTrap Protien L column (GE Healthcare) after a buffer exchange using a PD-10 desalting column (GE Healthcare). The protein containing fractions were concentrated and further purified by gel filtration on a Superose 610 / 300 GL column (GE Healthcare).

[0426] Negative stain electron microscopy. 3 μL of multispecific multimeric antibody at a concentration of approximately 0.02 mg / mL was added to a carbon coated copper grid, left for 30 seconds, and stained with 3 μL of 2% uranyl formate. Excess stain was immediately removed from the grid with Whatman No. 1 filter paper and an additional 3 μL of 2% uranyl formate was added, left for 20 seconds. The grid was imaged using a field emission FEI Tecnai F20 electron microscope operating at 200 kV and equipped with an Orius charge-coupled device (CCD) camera (Gatan Inc.).

[0427] Thin-layer interference of biomembranes. Binding kinetics were measured using an Octet RED96 BLI system (Pall ForteBio) in PBS pH 7.4, 0.01% BSA, and 0.002% Tween. A unique His-labeled ligand for each multispecific multi-affinity antibody fraction was selected and loaded onto a Ni-NTA biosensor to achieve a signal response of 0.8 nm. Binding rates were measured by transferring the loaded biosensor to wells containing serial dilutions of the multispecific multi-affinity antibody (50-25-12.5-6.25-3.1-1.5 nM) and wells containing buffer, respectively. Dissociation rates were measured by immersing the biosensor in wells containing buffer. Both steps lasted 180 seconds. To achieve selective binding with PGDM1400, a D368R mutation was introduced into the CD4bs of the BG5050 SOSIP.664 trimer, thus disrupting the binding of N49P7 to this antigen. Similarly, gp120 subunit 93TH057, the MPER peptide fused with mVenus, soluble CD4, and hFcRn complexed with β2-microglobulin were generated as the sole ligands for N49P7, 10E8, iMab, and Fc, respectively. The ability to recycle endosomally was tested by measuring the binding of multispecific, multi-affinity antibodies to the hFcRnβ2-microglobulin complex at physiological (7.5) and acidic (5.6) pH values.

[0428] Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) was performed. MiniDAWN TREOS and Optilab T-rEX refractometers (Wyatt) were coupled with an Agilent Technologies 1260 Infinity II HPLC system. 50 μg of 24-mer PGDM1400scFab multispecific multi-affinity antibody, multispecific multi-affinity antibody 32-N, and multispecific multi-affinity antibody 32-I were loaded onto a Superose 6 10 / 300 column (GE Healthcare) in 20 mM sodium phosphate (pH 8.0) and 150 mM NaCl. Data collection and analysis were performed using ASTRA software (Wyatt).

[0429] Dematuration and aggregation temperature measurements. The dematuration temperatures (T1) of multispecific multi-affinity antibodies, parental IgG, 12-mer homologous oligomers Fab and Fc, and N6 / PGDM1400x10E8 trispecific antibodies were determined using the UNit system (Unchained Labs). m ) and aggregation temperature (T agg ). T m It was obtained by measuring the average fluorescence at the centroid, while T aggThis is the temperature at which static light scattering increased by 50% relative to the baseline at a wavelength of 266 nm. The sample was concentrated to 1.0 mg / mL and increased in a 1 °C gradient from 25 °C to 95 °C. The mean and standard error of three independent measurements were calculated using UNit analysis software.

[0430] Virus production and TZM-bl neutralization test. This was achieved using HIV-1 subtype B backbone NL4-3.Luc.R. - The E (AIDS Research and Reference Reagents Program (ARRRP)) and plasmids encoding the full-length Env clone were co-transfected into 293T cells to produce groups of 14 HIV-1 pseudoviruses as described above. 73 HIV isolates X2988, ZM106.9, and 3817 were provided by the HIV Vaccines for Development Collaboration (CAVD), SF162 by JL Nieva (Biofisika Institute), and pCNE8, 1632, THRO, 278, ZM197, JRCSF, t257, Du422, and BG505 by NIH ARRRP. Neutralization was determined in a single-cycle neutralization assay using the standard TZM-bl neutralization assay. Briefly, antibodies and antibody-based particles were incubated with 10–15% tissue culture infectious doses of pseudovirus at 37°C for 1 hour, followed by incubation with TZM-bl cells for 44–72 hours. Viral neutralization was monitored by adding Britelite Plus reagent (PerkinElmer) to cells and measuring the relative optical units (RLU) luminescence using a Synergy Neo2 multimodal analyzer (Biotek Instruments).

[0431] Pharmacokinetic and immunogenicity studies were conducted. In vivo studies were performed using 20g male C57BL / 6 mice. Alternative multispecific and multi-affinity antibodies were used, consisting of scFab and scFc fragments of mouse HD37 IgG2a fused to the N-terminus of the mouse aferroferrin light chain. HD37 scFab-mFerritin, Fc-mFerritin, and mFerritin were transfected and purified at a 2:1:1 ratio as described above. L35A, L234A, and P329G mutations were introduced into the mouse IgG2a Fc construct to silence the effector function of the multispecific and multi-affinity antibody. 74A single subcutaneous injection of 5 mg / kg of multispecific polyaffinity antibody or control samples (HD37 IgGl, HD37 IgG2a and hpFerritin-PfCSP malaria peptide) in 200 μL PBS (pH 7.5) solution. Blood samples were collected at multiple time points and serum samples were evaluated for circulating antibody and ADA levels by ELISA. Briefly, 96-well Pierce nickel-coated plates (Thermo Fisher) were coated with 50 μL of 0.5 μg / mL His 6x Labeled antigen hCD19 was coated to determine circulating HD37-specific concentrations using reagent-specific standard curves for IgG and multispecific polyaffinity antibodies. In anti-drug antibody assays, Nunc MaxiSorp plates (Biolegend) were coated with 12-mer HD37 scFab multispecific polyaffinity antibody or with hpFerritin PfCSP malaria peptide. HRP-Protein A (Invitrogen) was used as secondary molecule and chemiluminescent signal was quantified using a Synergy Neo2 multimode plate reader (Biotek Instruments).

[0432] Results

[0433] Multispecific polyaffinity antibodies can neutralize HIV-1 with 500-fold higher potency than the gold standard IgG. The strong self-assembly properties of the human apo ferritin light chain were used to multimerize Fab onto the surface of a hollow spherical protein cage formed by 24 monomers. Indeed, apo ferritin self-assembles into a 12 nm diameter structure composed of 24 identical polypeptides and is amenable to genetic fusion 70 The N-terminus of each apo ferritin subunit points to the outside of the spherical cage, thus enabling genetic fusion of a protein of interest. To preserve all the properties of the IgG molecule, including high thermal stability and correct chain pairing, we generated fusions of apo ferritin with single-chain Fab (scFab) and single-chain Fc (scFc) fragments. Upon folding, the apo ferritin subunit acts as a base material driving the multimerization of the 24 proteins fused to its N-terminus Figure 1 Importantly, the presentation of multiple specificities (e.g., Fab and Fc) on the same molecule can be achieved and controlled by co-transfecting selected ratios of DNA encoding the individual components and a strict affinity purification protocol for all specificities (e.g., protein L combined with protein A affinity chromatography to select Fab and Fc, respectively).

[0434] First, we investigated the impact of the multivalency of HIV-1 bNAb displayed on our novel platform of multispecific polyaffinity antibodies on their ability to block viral infection and compared it to the standard bivalent IgG display of the same bNAb. A panel of bNAb with different specificity against Env was chosen and their scFabs were multimerized at different densities by co-transfecting scFab-human apoferritin-encoding plasmids with different ratios of uncomplexed apoferritin Figure 2 a) Multispecific polyaffinity antibodies assemble into monodisperse, well-formed spherical particles with uninterrupted dense rings and regularly spaced Fab protrusions Figure 2 a) Remarkably, PGDM1400, the most potent anti-HIV bNAb described to date (IC 50 median = 0.003 pg / mL) as a 24-mer multispecific polyaffinity antibody displays a 100- to 500-fold neutralization (fold improvement of IC 50 (nM) median) compared to its IgG form against a panel of five pseudoviruses Figure 2 b) In comparison to its IgG, bNAb 10-1074 also displays a significant improvement of neutralization potency as a multispecific polyaffinity antibody, while bNAb 10E8, N49P7 and VRC01, although still potent, do not display the same enhancement.

[0435] Apoferritin engineering leads to efficient hetero-oligomerization of 32-mer multispecific polyaffinity antibodies. Second, we tried to improve the width of the exceptionally potent 24-mer PGDM1400 multispecific polyaffinity antibody by conferring multispecificity to the molecule. To do so, we combined PGDM1400 Fabs with the Fabs of the near-pan neutralizing antibodies 10E8v4 (modified 10E8 71 ) and N49P7 in addition to the Fc fragment of the human IgGl isotype. To achieve this level of four-component (three Fabs and one Fc) hetero-oligomerization, we split the human apoferritin structure into two subunits (N- and C-apoferritin) and linked the Fabs to the N-termini of the two halves Figure 3a ) respectively. The complementarity of the split apoferritin leads to the self-association of the two halves and thus to a very efficient heterodimerization process of the fusion protein. Importantly, no significant differences between the biophysical and functional properties of the multispecific polyaffinity antibodies assembled with split and full-length apoferritin were observed Figure 4 ) This design allows a simple two-step purification procedure to select multispecific polyaffinity antibodies with four different specificities Figure 5 ) and displays a high batch-to-batch homogeneity Figure 6). In addition, splitting the apoferritin into two halves allows for the accommodation of additional Fab / Fc fragments, up to 32 components per molecule (Figure 3b). The 8 additional positions (from 24 to 32 in our engineered platform) play an important role in providing multispecificity without sacrificing the large part of the potency increase observed in the 24-mer PGDM1400 multispecific polyaffinity antibody. Thus, the multispecific polyaffinity antibody 32-N was designed to be obtained in a 4:2:1:1 ratio of 16 copies of PGDM1400, 8 copies of Fc, 4 copies of 10E8v4 and 4 copies of N49P7 Figure 3a ) by co-transfection of scFab and scFc plasmids. Multispecific polyaffinity antibody 32-N forms highly modified and uniform particles (Figure 3b) and displays T m and T agg distributions similar to those of the corresponding IgG molecules, and also unfolds and aggregates with similar transition temperatures as previously reported for IgGs 72 (Figure 3c and Figure 7 ). Binding kinetics experiments were used to demonstrate that each component of the multispecific polyaffinity antibody (PDGM1400, N49P7, 10E8v4 and Fc fragments) is able to engage its epitope by binding to epitope-specific molecules (BG505 SOSIP D368R, 93TH057 gp120, MPER peptide and human FcRn, respectively) Figure 3d ). None of the IgG molecules alone has the ability to bind all antigens Figure 8 ). The ability of 32-N to engage its multiple antigens indicates that the geometry conferred by the oligomeric form of the multispecific polyaffinity antibody does not negatively affect antigen binding, presumably because the binding interfaces are outward facing.

[0436] To explore whether it is also possible to design a multispecific polyaffinity antibody that cross-targets HIV Env and the T cell receptor CD4, we replaced N49P7 with iMab, a CD4-directed post-attachment inhibitor that has proven effective in eradicating HIV 68,69 ). The multispecific polyaffinity antibody containing PDGM1400, iMab, 10E8v4 and Fc fragments (called 32-I) displays similar uniformity, thermal stability and multispecificity as 32-N (Figure 3, Figure 7 and Figure 8 ), highlighting the powerful plug-and-play nature of the multispecific polyaffinity antibody platform that allows for easy exchange of antibody sequences to change specificity.

[0437] HIV-1 multispecific polyaffinity antibodies display exceptional pan-neutralizing activity and potency. In a standardized in vitro TZM-bl neutralization assay 73In this study, the neutralizing potency and width of multispecific multi-affinity antibodies 32-N and 32-I against 14 pseudoviruses were evaluated. The 14 PsV groups were designed to include low-sensitivity PsVs, with each evaluated bNAb containing at least one resistant PsV. The IC50 of the multispecific multi-affinity antibodies was measured. 50 The values ​​and widths were compared with: (i) individual IgGs, (ii) mixtures of IgGs containing the same relative amounts of each IgG present in multispecific and multi-affinity antibodies, and (iii) N6 / PGDM1400x10E8 trispecific antibodies. 43 Both 32-N and 32-I multispecific multi-affinity antibodies showed 100% width for this group, IC50. 50 The median values ​​were 0.0093 μg / mL (4 pM) and 0.0085 μg / mL (3.5 pM), respectively. Figure 9 and Figure 10 Complete viral coverage was also achieved using an IgG mixture and a trispecific antibody. However, the potency of the mixture or trispecific antibody combination against PsV in all tests was similar to that of the optimal mAb when tested alone (Table 1). Thus, the IC50 with the IgG mixture and trispecific antibody... 50 Compared to the values, the IC50 of multispecific and multi-affinity antibodies, expressed in μg / mL and nM, is... 50 The median values ​​decreased by more than 90% and 99% respectively. Figure 9 and Figure 10 (Tables 1 and 2).

[0438] Table 1.

[0439]

[0440] Table 2.

[0441]

[0442] The in vivo pharmacokinetics and anti-drug antibody profile of the multispecific multivalent antibody were similar to the corresponding IgG. We next tested the in vivo toxicity, immunogenicity and bioavailability of the multispecific multivalent antibody after subcutaneous administration of 5 mg / kg in mice. To assess our new platform technology, we used a species-matched surrogate multispecific multivalent antibody consisting of a mouse Fab fused to a mouse Fc (IgG2a isotype) in contrast to the fully human components of the HIV-1 multispecific multivalent antibody for human purposes. The Fab specificity chosen for this surrogate multispecific multivalent antibody was a Fab specificity that does not bind to endogenous mouse proteins, similar to the HIV-1 human mAbs that do not bind to endogenous human proteins. The multispecific multivalent antibody administration was well tolerated, no decrease in body weight was seen, no signs of toxicity were visible. The surrogate multispecific multivalent antibody did not induce a significant immunogenicity response in mice; the levels of anti-drug antibodies (ADA) detected 14 days after administration were negligible for both the surrogate multispecific multivalent antibody and its sequenced matched IgG2a Figure 11 b). This is in contrast to the highly immunogenic particles displaying the malaria circumsporozoite protein (CSP) on the surface of Helicobacter Pylori ferritin (hpFerritin) used as immunogenic positive control. In addition, the surrogate multispecific multivalent antibody showed a similar in vivo exposure days in proximity to the parental IgGl and IgG2a molecules Figure 11 b) by introducing the LALAP mutation reported to silence Fc effector functions 74 Prolonged serum half-life. In summary, these data demonstrate the feasibility of tuning the bioavailability properties of our multispecific multivalent antibody platform and provide an exciting initial in vivo proof-of-concept data set for development.

[0443] References

[0444] 1. Conley, A.J. et al. Neutralization of divergent human immunodeficiency virus type 1 variants and primary isolates by IAM-41-2F5, an anti-gp41 human monoclonal antibody. Proc. Natl. Acad. Sci., 2006, doi: 10.1073 / pnas.91.8.3348

[0445] 2. Stiegler, G. et al. A Potent Cross-Clade Neutralizing Human Monoclonal Antibody against a Novel Epitope on gp41 of Human Immunodeficiency Virus Type 1. AIDS Res. Hum. Retroviruses, 2002, doi:10.1089 / 08892220152741450

[0446] 3. Zwick, M. B. et al. Broadly Neutralizing Antibodies Targeted to the Membrane- Proximal External Region of Human Immunodeficiency Virus Type 1 Glycoprotein gp41. J. Virol., 2002, doi:10.1128 / jvi.75.22.10892-10905.2001

[0447] 4. BUCHACHER, A. et al. Generation of Human Monoclonal Antibodies against HIV-1 Proteins; Electrofusion and Epstein-Barr Virus Transformation for Peripheral Blood Lymphocyte Immortalization. AIDS Res. Hum. Retroviruses, 2009, doi:10.1089 / aid.1994.10.359

[0448] 5. Barbas, C. F. et al. Recombinant human Fab fragments neutralize human type 1 immunodeficiency virus in vitro. Proc. Natl. Acad. Sci. U. S. A., 1992

[0449] 6. Burton, D. R. et al. Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody. Science, 80-, 1994, doi:10.1126 / science.7973652.

[0450] 7. Wu, X. et al. Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science, 80-, 2010, doi:10.1126 / science.1187659.

[0451] 8. Scheid, J. F. et al. Broad diversity of neutralizing antibodies isolated from memory B cells in HIV-infected individuals. Nature, 2009, doi:10.1038 / nature07930.

[0452] 9. Sok, D. et al. A Recombinant HIV Envelope Trimer Selects for Quaternary Dependent Antibodies Targeting the Trimer Apex. AIDS Res. Hum. Retroviruses, 2014, doi:10.1089 / aid.2014.5002.abstract.

[0453] 10. 1089 / aid.2014.5002.abstract.

[0454] 10. Walker, L. M. et al. Broad and Potent Neutralizing Antibodies from an African Donor Reveal a New HIV-1 Vaccine Target TL-326. Science, 80-, 2009, doi:10.1126 / science.1178746.

[0455] 11. Walker, L. M. et al. Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature, doi:10.1038 / nature10373 (2011)

[0456] 12. Doria-Rose, N. A. et al. Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies. Nature, doi:10.1038 / nature13036 (2014)

[0457] 13. Huang, J. et al. Broad and potent neutralization of HIV-1 by a gp41-specific human antibody. Nature, doi:10.1038 / nature11544 (2012)

[0458] 14. Sajadi, M. M. et al. Identification of Near-Pan-neutralizing Antibodies against HIV-1 by Deconvolution of Plasma Humoral Responses. Cell, doi:10.1016 / j.cell.2018.03.061 (2018)

[0459] 15. Huang, J. et al. Identification of a CD4-Binding-Site Antibody to HIV that Evolved Near-Pan Neutralization Breadth. Immunity, doi:10.1016 / j.immuni.2016.10.027 (2016)

[0460] 16. Pejchal, R. et al. A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield. Science, doi:10.1126 / science.1213256 (2011)

[0461] 17. Blattner, C. et al. Structural delineation of a quaternary, cleavage-dependent epitope at the gp41-gp120 interface on intact HIV-1 env trimers. Immunity, 2014, doi: 10.1016 / j.immuni.2014.04.008

[0462] 18. Mouquet, H. et al. Complex-type N-glycan recognition by potent broadly neutralizing HIV antibodies. Proc. Natl. Acad. Sci., 2012, doi: 10.1073 / pnas.1217207109

[0463] 19. Scheid, J. F. et al. Sequence and Structural Convergence of Broad and Potent HIV Antibodies That Mimic CD4 Binding. Science, 80-, 2011, doi: 10.1126 / science.1207227

[0464] 20. Baba, T. W. et al. Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection. Nat. Med., 2000, doi: 10.1038 / 72309

[0465] 21. Hessell, A. J. et al. Effective, low-titer antibody protection against low-dose repeated mucosal SHIV challenge in macaques. Nat. Med., 2009, doi: 10.1038 / nm.1974

[0466] 22. Hessell, A.J. et al. Broadly neutralizing human anti-HIV antibody 2G12 is effective in protection against mucosal SHIV challenge even at low serum neutralizing titers. PLoS Pathog., 2009, doi:10.1371 / journal.ppat.1000433

[0467] 23. Hofmann-Lehmann, R. et al. Postnatal pre-and postexposure passive immunization strategies: Protection of neonatal macaques against oral simian-human immunodeficiency virus challenge. J. Med. Primatol., 2002, doi:10.1034 / j.1600-0684.2002.01014.x

[0468] 24. Hofmann-Lehmann, R. et al. Postnatal Passive Immunization of Neonatal Macaques with a Triple Combination of Human Monoclonal Antibodies against Oral Simian-Human Immunodeficiency Virus Challenge. J. Virol., 2002, doi:10.1128 / jvi.75.16.7470-7480.2001

[0469] 25. van der Velden, Y.U. et al. Short Communication: Protective Efficacy of Broadly Neutralizing Antibody PGDM1400 Against HIV-1 Challenge in Humanized Mice. AIDS Res. Hum. Retroviruses, 2018, doi:10.1089 / aid.2018.0114

[0470] 26. Klein, F. et al. HIV therapy by a combination of broadly neutralizing antibodies in humanized mice. Nature, 2012, doi: 10.1038 / naturel 1604

[0471] 27. Deruaz, M. et al. Protection of humanized mice from repeated intravaginal HIV challenge by passive immunization: A model for studying the efficacy of neutralizing antibodies in vivo. J. Infect. Dis., 2016, doi: 10.1093 / infdis / jiw203

[0472] 28. Horwitz, J. A. et al. HIV-1 suppression and durable control by combining single broadly neutralizing antibodies and antiretroviral drugs in humanized mice. Proc. Natl. Acad. Sci., 2013, doi: 10.1073 / pnas.1315295110

[0473] 29. Mehandru, S. et al. Adjunctive Passive Immunotherapy in Human Immunodeficiency Virus Type 1 -Infected Individuals Treated with Antiviral Therapy during Acute and Early Infection. J. Virol., 2007, doi: 10.1128 / jvi.01340-07

[0474] 30. Caskey, M. et al. Viraemia suppressed in HIV-1 -infected humans by broadly neutralizing antibody 3BNC117. Nature, 2015, doi: 10.1038 / naturel 441 1

[0475] 31. Caskey, M. et al. Antibody 10-1074 suppresses viremia in HIV-1 -infected individuals. Nat. Med. doi:10.1038 / nm.4268 (2017)

[0476] 32. Lynch, R. M. et al. Virologic effects of broadly neutralizing antibody VRC01 administration during chronic HIV-1 infection. Sci. Transl. Med. doi:10.1126 / scitranslmed.aad5752 (2015)

[0477] 33. Ledgerwood, J. E. et al. Safety, pharmacokinetics and neutralization of the broadly neutralizing HIV-1 human monoclonal antibody VRC01 in healthy adults. Clin. Exp. Immunol. doi:10.1111 / cei.12692 (2015)

[0478] 34. Toma, J. et al. Loss of Asparagine-Linked Glycosylation Sites in Variable Region 5 of Human Immunodeficiency Virus Type 1 Envelope Is Associated with Resistance to CD4 Antibody Ibalizumab. J. Virol. doi:10.1128 / jvi.02237-10 (2011)

[0479] 35. Jacobson, J. M. et al. Antiviral Activity of Single-Dose PRO 140, a CCR5 Monoclonal Antibody, in HIV-Infected Adults. J. Infect. Dis. doi:10.1086 / 592169 (2008)

[0480] 36. Onafuwa-Nuga, A. & Telesnitsky, A. The Remarkable Frequency of Human Immunodeficiency Virus Type 1 Genetic Recombination. Microbiol. Mol. Biol. Rev., 2009. doi:10.1128 / mmbr.00012-09

[0481] 37. Sather, D. N. et al. Broadly Neutralizing Antibodies Developed by an HIV-Positive Elite Neutralizer Exact a Replication Fitness Cost on the Contemporaneous Virus. J. Virol., 2012. doi:10.1128 / JVI.01893-12

[0482] 38. Lynch, R. M. et al. HIV-1 Fitness Cost Associated with Escape from the VRC01 Class of CD4 Binding Site Neutralizing Antibodies. J. Virol., 2015. doi:10.1128 / jvi.03608-14

[0483] 39. Pietzsch, J. et al. Human anti-HIV-neutralizing antibodies frequently target a conserved epitope essential for viral fitness. J. Exp. Med., 2010. doi:10.1084 / jem.20101176

[0484] 40. Asokan, M. et al. Bispecific Antibodies Targeting Different Epitopes on the HIV-1 Envelope Exhibit Broad and Potent Neutralization. J. Virol., 2015. doi:10.1128 / jvi.02097-15

[0485] 41. Bournazos, S., Gazumyan, A., Seaman, M. S., Nussenzweig, M. C. & Ravetch, J. V. Bispecific Anti-HIV-1 Antibodies with Enhanced Breadth and Potency. Cell, 2016, doi:10.1016 / j.cell.2016.04.050

[0486] 42. Huang, Y. et al. Engineered Bispecific Antibodies with Exquisite HIV-1-Neutralizing Activity. Cell, 2016, doi:10.1016 / j.cell.2016.05.024

[0487] 43. Xu, L. et al. Trispecific broadly neutralizing HIV antibodies mediate potent SHIV protection in macaques. Science, 80-, 2017, doi:10.1126 / science.aan8630

[0488] 44. Steinhardt, J. J. et al. Rational design of a trispecific antibody targeting the HIV-1 Env with elevated anti-viral activity. Nat. Commun., 2018, doi:10.1038 / s41467-018-03335-4

[0489] 45. Khan, S. N. et al. Targeting the HIV-1 Spike and Coreceptor with Bi- and Trispecific Antibodies for Single-Component Broad Inhibition of Entry. J. Virol., 2018, doi:10.1128 / jvi.00384-18

[0490] 46. Rudicell, R. S. et al. Enhanced Potency of a Broadly Neutralizing HIV-1 Antibody In Vitro Improves Protection against Lentiviral Infection In Vivo. J. Virol., 2014, doi:10.1128 / JVI.02213-14

[0491] 47. Kwon, Y. D. et al. Surface-Matrix Screening Identifies Semi-specific Interactions that Improve Potency of a Near Pan-reactive HIV-1-Neutralizing Antibody. Cell Rep., 2018, doi:10.1016 / j.celrep.2018.01.023

[0492] 48. Rujas, E. et al. Functional Optimization of Broadly Neutralizing HIV-1 Antibody 10E8 by Promotion of Membrane Interactions. J. Virol., 2018, doi:10.1128 / jvi.02249-17

[0493] 49. Diskin, R. et al. Increasing the potency and breadth of an HIV antibody by using structure-based rational design. Science, 80-., 2011, doi:10.1126 / science.1213782

[0494] 50. Wu, H. et al. Ultra-potent antibodies against respiratory syncytial virus: Effects of binding kinetics and binding valence on viral neutralization. J. Mol. Biol., 2005, doi:10.1016 / j.jmb.2005.04.049

[0495] 51. Icenogle, J., et al., Neutralization of poliovirus by a monoclonal antibody: Kinetics and stoichiometry. Virology, 1983, doi:10.1016 / 0042-6822(83)90154-X.

[0496] 52. Cavacini, L. A., Emes, C. L., Power, J., Duval, M. & Posner, M. R. Effect of antibody valency on interaction with cell-surface expressed HIV-1 and viral neutralization. J. Immunol., 1994

[0497] 53. Smith, R. I., Coloma, M. J. & Morrison, S. L. Addition of a mu-tailpiece to IgG results in polymeric antibodies with enhanced effector functions including complement-mediated cytolysis by IgG4. J. Immunol., 1995

[0498] 54. Olafsen, T., Rasmussen, B. I., Norderhaug, L., Bruland, O., Sandlie, I. & Sandlie, I. IgM secretory tailpiece drives multimerisation of bivalent scFv fragments in eukaryotic cells. Immunotechnology, 1998, doi:10.1016 / S1380-2933(98)00014-1

[0499] 55. Miller, K., et al., Design, Construction, and In Vitro Analyses of Multivalent Antibodies. J. Immunol., 2003, doi:10.4049 / jimmunol.170.9.4854

[0500] ​56. Kipriyanov, S. M. et al. Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics. J. Mol. Biol. doi:10.1006 / jmbi.1999.3156 (1999)

[0501] 57. Lu, D. et al. Di-diabody: A novel tetravalent bispecific antibody molecule by design. J. Immunol. Methods doi:10.1016 / S0022-1759(03)00251-5 (2003)

[0502] 58. Wu, C. et al. Simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin. Nat. Biotechnol. doi:10.1038 / nbt1345 (2007)

[0503] 59. Klein, C., Schaefer, W. & Regula, J. T. The use of CrossMAb technology for the generation of bi- and multispecific antibodies. mAbs doi:10.1080 / 19420862.2016.1197457 (2016)

[0504] 60. Steinmetz, A. et al. CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications. mAbs doi:10.1080 / 19420862.2016.1162932 (2016)

[0505] 61. Kubetzko, S., Balic, E., Waibel, R., Zangemeister-Wittke, U. & Pluckthun, A. PEGylation and multimerization of the anti-p185HER-2 single chain Fv fragment 4D5: Effects on tumor targeting. J. Biol. Chem., 2006, doi:10.1074 / jbc.M604127200

[0506] 62. Pack, P., K., Zahn, R. & Pluckthun, A. Tetravalent Miniantibodies with High Avidity Assembling in Escherichia coli. Journal of Molecular Biology, 1995, doi:10.1006 / jmbi.1994.0062

[0507] 63. Kipriyanov, S. M. et al. Affinity enhancement of a recombinant antibody: formation of complexes with multiple valency by a single-chain Fv fragment - core streptavidin fusion. Protein Eng. Des. Sel., 2007, doi:10.1093 / protein / 9.2.203

[0508] 64. Deyev, S. M., Waibel, R., Lebedenko, E. N., Schubiger, A. P. & Pluckthun, A. Design of multivalent complexes using the barnase · barstar module. Nat. Biotechnol., 2003, doi:10.1038 / nbt916

[0509] 65. Zhang, J. et al. Pentamerization of single-domain antibodies from phage libraries: A novel strategy for the rapid generation of high-avidity antibody reagents. J. Mol. Biol. 2004, doi:10.1016 / j.jmb.2003.09.034

[0510] 66. Stone, E. et al. The assembly of single domain antibodies into bispecific decavalent molecules. J. Immunol. Methods 2007, doi:10.1016 / j.jim.2006.10.006

[0511] 67. A. & Pluckthun, A. Different equilibrium stability behavior of scFv fragments: Identification, classification, and improvement by protein engineering. Biochemistry 1999, doi:10.1021 / bi9902079

[0512] 68. Jacobson, J. M. et al. Safety, pharmacokinetics, and antiretroviral activity of multiple doses of ibalizumab (formerly TNX-355), an anti-CD4 monoclonal antibody, in human immunodeficiency virus type 1 -infected adults. Antimicrob. Agents Chemother. 2009, doi:10.1128 / AAC.00942-08

[0513] 69. Kuritzkes, D. R. et al. Antiretroviral Activity of the Anti-CD4 Monoclonal Antibody TNX-355 in Patients Infected with HIV Type 1. J. Infect. Dis. 2004, doi:10.1086 / 380802

[0514] 70. Lawson, D. M. et al. Solving the structure of human H ferritin by genetically engineering intermolecular crystal contacts. Nature 1991, doi:10.1038 / 349541a0

[0515] 71. Kwon, Y. D. et al. Optimization of the Solubility of HIV-1-Neutralizing Antibody 10E8 through Somatic Variation and Structure-Based Design. J. Virol. 2016, doi:10.1128 / jvi.03246-15 72. Goto, Y., Ichimura, N. & Hamaguchi, K. Effects of Ammonium Sulfate on the Unfolding and Refolding of the Variable and Constant Fragments of an Immunoglobulin Light Chain. Biochemistry 1988, doi:10.1021 / bi00405a043

[0516] 73. Montefiori, D. C. Measuring HIV neutralization in a luciferase reporter gene assay. Methods Mol. Biol. 2009, doi:10.1007 / 978-1-59745-170-3_26

[0517] 74. Schlothauer, T. et al. Novel human IgGl and IgG4 Fc-engineered antibodies with completely abolished immune effector functions. Protein Eng. Des. Sel., 2016, doi:10.1093 / protein / gzw040

[0518] 75. Graille, M. et al. Complex between Peptostreptococcus magnus protein Land a human antibody reveals structural convergence in the interaction modes of Fab binding proteins. Structure, 2001, doi:10.1016 / S0969-2126(01)00630-X.

Claims

1. A fusion protein comprising: a first nanocage monomer subunit of a nanocage monomer; and a biologically active moiety linked to the first nanocage monomer subunit; wherein the fusion protein self-assembles with a protein comprising a second nanocage monomer subunit to form a nanocage monomer.

2. The fusion protein of claim 1, wherein the biologically active moiety modifies an inner surface and / or an outer surface of the assembled nanocage.

3. The fusion protein of claim 1 or 2, wherein the biologically active moiety comprises: an antibody or fragment thereof, an antigen, a detectable moiety, a pharmaceutical agent, a diagnostic agent, or a combination thereof.

4. The fusion protein of claim 3, wherein the antibody or fragment thereof comprises an Fc fragment.

5. The fusion protein of claim 4, wherein the Fc fragment is an IgGl Fc fragment.

6. The fusion protein of claim 4 or 5, wherein the Fc fragment comprises one or more mutations, e.g., LS, YTE, LALA, and / or LALAP, that modulate the half-life of the fusion protein, e.g., from minutes or hours to days, weeks, or months.

7. The fusion protein of claim 3, wherein the antibody or fragment thereof comprises a Fab fragment.

8. The fusion protein of claim 3, wherein the antibody or fragment thereof comprises a scFab fragment, a scFv fragment, or a sdAb fragment.

9. The fusion protein of claim 3, wherein the antibody or fragment thereof comprises a heavy chain and / or a light chain of a Fab fragment.

10. The fusion protein of claim 9, wherein the antibody or fragment thereof comprises both a light chain and a heavy chain, or in the case of an Fc fragment, a first chain and a second chain, optionally separated by a linker.

Images