Multivalent and multispecific nanoparticle platforms and methods

Abstract

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 bioactive 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] Their combination.

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

[0069] In one respect, the antibody or a fragment thereof includes an antibody-drug conjugate.

[0070] In one respect, the antigen is associated with vaccine-preventable and / or vaccine-treatable conditions.

[0071] In one respect, the antigen is associated with: infectious agents, including viruses, bacteria, parasites, fungi, or yeast; cancers, including solid and liquid cancers; or immune diseases, including autoimmune diseases.

[0072] In one aspect, the detectable portion includes fluorescent proteins such as GFP, EGFP, auroral, and / or flavin-based fluorescent proteins such as LOV proteins (such as iLOV).

[0073] In one respect, the agent may include small molecules, peptides, lipids, carbohydrates, or toxins.

[0074] In one 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 combined with one or more complete nanocage monomers, self-assemble to form a nanocage.

[0075] In one aspect, the nanocage monomer is selected from ferritin, deferroferritin, encapsulated proteins, SOR, dioxetine synthase, pyruvate dehydrogenase, carboxyl group, fornix protein, GroEL, heat shock protein, E2P, MS2 capsid protein, fragments thereof, and variants thereof.

[0076] In one respect, the nanocage monomer is deferroferritin.

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

[0078] In one aspect, the "N" region of deferroferritin 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 with, or is composed of, the following sequences:

[0079] MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEW.

[0080] In one aspect, the "C" region of deferroferritin 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 with, or is composed of, the following sequences:

[0081] GKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD.

[0082] In one aspect, the fusion protein also includes a linker between the nanocage monomer subunit and the bioactive portion.

[0083] In one respect, the connector is flexible or rigid and comprises about 1 to about 30 amino acid residues, such as about 8 to about 16 amino acid residues.

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

[0085] In one aspect, the connector 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 with, or is composed of, the following sequences:

[0086] ASTASSASSGGGGGGSGGSGGSGGS.

[0087] In one respect, the fusion protein also includes a C-terminal linker.

[0088] In one aspect, the C-terminal connector 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 with, or is composed of, the following sequences:

[0089] GGSGGSGGSGGSGGGASGGS.

[0090] According to one aspect, the present invention provides a fusion protein pair of the fusion proteins described herein, wherein the fusion protein pair self-assembles to form a nanocage monomer, wherein the first nanocage monomer subunit and the second nanocage monomer subunit are fused to different bioactive moieties.

[0091] According to one aspect, the present invention provides a nanocage 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.

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

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

[0094] In one respect, approximately 20% to approximately 80% of the nanocage monomer comprises the fusion protein or fusion protein pair described herein.

[0095] In one respect, the fusion protein comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different bioactive components.

[0096] In one aspect, the nanocage comprises at least one complete nanocage monomer, which is optionally fused to a bioactive portion that may be the same as or different from the bioactive portion described herein.

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

[0098] In one aspect, the nanocage includes the first fusion protein, the second fusion protein, and the third fusion protein described herein, and at least one intact nanocage monomer optionally fused to a bioactive portion, wherein the bioactive portions of the first fusion protein, the second fusion protein, and the third fusion protein, as well as the bioactive portion of the intact nanocage monomer, are all distinct from each other.

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

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

[0101] In one aspect, the Fc fragment includes one or more mutations, such as LS, YTE, LALA, and / or LALAP, which regulate the half-life of the fusion protein, for example, from minutes or hours to days, weeks, or months.

[0102] In one 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 one respect, the nanocage comprises or is composed of the following four fusion proteins:

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

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

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

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

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

[0109] In one 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 with 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 lowercase letters:

[0110]

[0111]

[0112] In one respect, the nanocage carries cargo molecules, such as pharmaceuticals, diagnostic agents, and / or imaging agents.

[0113] In one respect, the cargo molecule does not fuse to the fusion protein and is contained within the nanocage.

[0114] In one respect, the cargo molecule is a protein and is fused to the fusion protein such that the cargo molecule is contained within the nanocage.

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

[0116] In one respect, the cargo molecule is contained within to provide T-cell epitopes, but optionally not B-cell epitopes.

[0117] In one aspect, the cargo molecule is fused to the fusion protein and contained therein to provide T-cell epitopes, but optionally does not provide B-cell epitopes.

[0118] In one respect, the cargo molecules are small molecules, radioactive isotopes, or magnetic particles.

[0119] In one respect, the nanocage also includes antigens on its surface.

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

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

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

[0123] According to one aspect, the present invention provides a nucleic acid molecule that encodes the fusion protein or fusion protein pair described herein.

[0124] According to one aspect, the present invention provides a carrier comprising the nucleic acid molecules described herein.

[0125] According to one aspect, the present invention provides a host cell that includes the vector described herein and produces the fusion protein or fusion protein pair described herein.

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

[0127] According to one aspect, the present invention provides a method for treating and / or preventing diseases or conditions, the method comprising administering the nanocage or vaccine described herein.

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

[0129] According to one aspect, the present invention provides a method for diagnostic imaging, the method comprising: administering a subject, tissue, or sample a nanocage as described herein, wherein the nanocage includes diagnostic markers, such as fluorescent proteins or magnetic imaging portions; and imaging the subject, tissue, or sample.

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

[0131] According to one aspect, the present invention provides the use of the nanocage or vaccine described herein for the treatment and / or prevention of diseases or conditions.

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

[0133] According to one aspect, the present invention provides the use of the nanocage described herein for diagnostic imaging of a subject, tissue, or sample, wherein the nanocage includes diagnostic markers, such as fluorescent proteins or magnetic imaging portions, and images the subject, tissue, or sample.

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

[0135] According to one aspect, the present invention provides the nanocages or vaccines described herein for immunizing subjects.

[0136] According to one aspect, the present invention provides the nanocages or vaccines described herein for the treatment and / or prevention of diseases or conditions.

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

[0138] According to one aspect, the present invention provides a nanocage as described herein for diagnostic imaging of a subject, tissue, or sample, wherein the nanocage includes diagnostic markers, such as fluorescent proteins or magnetic imaging portions, and images the subject, tissue, or sample.

[0139] According to one aspect, the present invention provides the fusion proteins, fusion protein pairs or nanocages described herein for use as research tools, such as in FACS or ELISA.

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

[0141] Each fusion protein includes a ferritin light chain and a Fab fragment.

[0142] Each Fab fragment can specifically bind to an antigen.

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

[0144] The plurality of fusion proteins includes at least 12 fusion proteins.

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

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

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

[0148] In one respect, the Fab fragments of the multiple fusion proteins are capable of specifically binding to the same antigen.

[0149] In one respect, the nanocage does not contain any ferritin heavy chains.

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

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

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

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

[0154] In one respect, the nanocage is capable of neutralizing the infectious agent with neutralizing activity at least 100, 150, 200, 250, 300, 350, 400, 450, or 500 times that of the control.

[0155] In one respect, the control group includes a full-length form of neutralizing antibody.

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

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

[0158] Each of the first fusion proteins comprises a nanocage monomer or its subunit and a Fab fragment capable of specifically binding to the antigen, and

[0159] Each of the second fusion proteins comprises a nanocage monomer or its subunit and an Fc fragment.

[0160] In one aspect, the nanocage monomer is selected from ferritin, deferroferritin, encapsulated proteins, thiooxygenase reductase (SOR), dioxetine synthase, pyruvate dehydrogenase, carboxyl group, fornix protein, GroEL, heat shock protein, E2P, MS2 capsid protein, fragments thereof, and variants thereof.

[0161] In one respect, the nanocage monomer is deferroferritin or ferritin.

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

[0163] In one respect, the nanocage monomer does not contain any ferritin heavy chains.

[0164] According to one aspect, the present invention 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 to the first antigen, and

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

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

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

[0169] 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.

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

[0171] According to one aspect, the present invention 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 to the first antigen.

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

[0174] (c) This third fusion protein comprises N-ferritin and an Fc fragment.

[0175] 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-ferritin.

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

[0177] In one aspect, the nanocage also includes multiple fourth fusion proteins, wherein the fourth fusion proteins include 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 respect, the Fab fragment is the Fab fragment of the neutralizing antibody.

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

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

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

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

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

[0184] The nanocage can neutralize 100% of the pseudoviruses in the pseudovirus group, and

[0185] The pseudovirus group includes at least one pseudovirus resistant to a neutralizing antibody corresponding to the Fab fragment for each Fab fragment within the nanocage that can specifically bind to the antigen associated with the virus.

[0186] In one respect, the fake virus group includes at least 10, at least 11, at least 12, at least 13, or at least 14 fake viruses.

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

[0188] The nanocage can be configured with ICs smaller than 1 nm, 500 pM, 250 pM, 100 pM, 50 pM, 10 pM, or 5 pM. 50 Neutralize pseudovirus groups, and

[0189] The pseudovirus group includes at least one pseudovirus resistant to a neutralizing antibody corresponding to the Fab fragment for each Fab fragment within the nanocage that can specifically bind to the antigen associated with the virus.

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

[0191] The nanocage can be used with one or more control ICs 50 IC50 concentrations of up to 1 / 10, 1 / 20, 1 / 30, 1 / 40, 1 / 50, 1 / 60, 1 / 70, 1 / 80, 1 / 90, or 1 / 100 of the molar concentration. 50 Neutralize pseudovirus groups, and

[0192] The pseudovirus group includes at least one pseudovirus resistant to a neutralizing antibody corresponding to the Fab fragment for each Fab fragment within the nanocage that can specifically bind to the antigen associated with the virus.

[0193] In one aspect, the one or more controls include neutralizing antibodies corresponding to the Fab fragment within the nanocage, which is capable of specifically binding to antigens associated with the virus.

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

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

[0196] In one respect, these neutralizing antibodies are IgG antibodies.

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

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

[0199] In one respect, 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 this third fusion protein is the human IgG1 Fc fragment, and

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

[0204] In one respect, 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 this third fusion protein is the human IgG1 Fc fragment, and

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

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

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

[0211] According to one aspect, the present invention provides a method for preparing multi-specific self-assembled nanocages, characterized by a preselection ratio of different specificities, the method comprising the following steps:

[0212] Host cells are co-transfected with one or more expression plasmids containing multiple polynucleotides, each polynucleotide encoding a fusion protein.

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

[0214] The co-transfection step includes: co-transfecting the polynucleotide at a ratio based on the preselected ratio;

[0215] Obtain the polypeptide produced by the host cell; and

[0216] All the different specific peptides present in the assembled nanocages were purified by affinity selection.

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

[0218] The first fusion protein includes a first nanocage monomer subunit, and

[0219] The second fusion protein includes a second nanocage monomer subunit capable of self-assembling with the first nanocage monomer subunit.

[0220] The novel features of the invention will become apparent to those skilled in the art after reviewing the following detailed description. However, it should be understood that while the detailed description and specific embodiments provided point to certain aspects of the invention, they are provided for illustrative purposes only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art based on the detailed description and appended claims. Attached Figure Description

[0221] The invention will be further understood from the following description with reference to the accompanying drawings, in which:

[0222] Figure 1 A schematic diagram of the self-assembly of a multispecific, multi-affinity antibody (MULTI) platform. Single-chain Fab (light chain (LC) and heavy chain (HC) are light pink and dark pink, respectively) and the single-chain Fc region (green) are linked to the N-terminus of the light chain (gray) of human deferroferrin via GGS-like flexible linkers (dark). The 24 subunits of deferroferrin self-assemble into a 12 nm globular core, which is surrounded by spatially dispersed antibody fragments.

[0223] Figure 2 Characterization of HIV-1 multispecific and multiaffinity antibodies with different titers. (a) Schematic diagram showing different scFab densities on human deferroferrin. Co-transfection of plasmids encoding scFab-human deferroferrin with different ratios of uncoupled deferroferrin produced 5-valent (deep yellow), 12-valent (black), 19-valent (blue), and 24-valent (red) scFab, as confirmed by early elution volumes and lower amounts of uncoupled deferroferrin in size exclusion chromatography and SDS-PAGE. The figure shows negatively stained electron micrographs of samples with the lowest (20%) titer and the highest (100%) titer. (b) Affinity effects of five bNAb on the 5-PsV group (PVO.04, JRCSF, BG505 T332N, THRO4156.18, and t278-50). IC50 was omitted in the following cases due to neutralizing resistance. 50 Fold-up analysis: N49P7-t278-50, VRC01-T278-50, and 10-1074-THRO4156.18. The fold-up in potency was calculated using the parental IgG IC50 value. 50 (nM) divided by the multispecific multi-affinity antibody IC 50 (nM).

[0224] Figure 3. Design, assembly, and biophysical characterization of the 32-N and 32-I multispecific multi-affinity antibodies. (a) Schematic diagram of the human ferritin segmentation design that facilitates heterodimerization of scFab-human ferritin subunits. The resulting halves, termed N-ferritin and C-ferritin, span residues 1 to 95 and 95 to 175, respectively. scFc is attached to the N-terminus of the N-ferritin half, while N49P7, iMab (also referred to herein as ibalizimab), and 10E8v4 are attached to the N-terminus of the C-ferritin half. Heterodimerization of the segmented halves drives the self-assembly of the different antibody fragments, resulting in the formation of single human ferritin subunits with two cargo molecules. These constructs are further combined with PGDM1400 scFab attached to the full-length human ferritin subunit, leading to the assembly of nanoparticles displaying a mixture of 32 scFab / scFc units on the surface of the multispecific multi-affinity antibody. The figure shows negatively stained electron micrographs, a schematic diagram of the scFab / scFc 32-N / 32-I design, and the specific composition of the 32-N and 32-I multispecific multi-affinity antibodies. Purification of the four-component multispecific multi-affinity antibody, based on heterooligomerization necessary for self-assembly, can be achieved in two steps: protein A (Fc binding) and protein L (PGDM1400 binding). (b) Size exclusion chromatography coupled with multi-angle light scattering for the 24-mer PGDM1400 multispecific multi-affinity antibody (black), the 32-N multispecific multi-affinity antibody (deep magenta), and the 32-I multispecific multi-affinity antibody (blue). The molar mass of each elution peak (below the UV absorbance line) indicates that the sample is monodisperse, and the 32-N / 32-I multispecific multi-affinity antibody is significantly larger than the 24-mer form due to the additional antibody fragment in this design. (c) T cells of 32-N / 32-I multispecific multi-affinity antibody, 12-mer multispecific multi-affinity antibody, parental IgG, and N6 / PGDM1400x10E8v4 trispecific antibody. m Temperature and T agg Temperature comparison. (d) Concentration-response curves of binding of 32-N and 32-I multispecific and multi-affinity antibodies to multiple epitopes. The PGDM1400 binding site, N49P7 binding site, and 10E8 binding site are shown in red, blue, and pink, respectively, on the surface of HIV Env (grey). iMab binding was assessed using soluble CD4, and the functional binding of Fc to human FcRn was tested by measuring binding at pH 7.5 and pH 5.6. BG505 SOSIP.664_D368R trimer and 93TH057 gp120 monomer were selected as epitope-specific ligands for PGDM1400 and N49P7, respectively.

[0225] Figure 4 The impact of split design on the biophysical and functional properties of multispecific and multi-affinity antibodies. Comparison of 12-mer multispecific and multi-affinity antibodies composed of 6 copies of PGDM1400 and 6 copies of Fc with full-length deferroferritin subunits (left panel) or ferritin hemiunits (Fc attached to the N-ferritin hemiunit and PGDM1400 linked to the C-ferritin hemiunit, right panel). a) Biomembrane interference technique (BLI) concentration-response curves of BG505.664 binding to the 12-mer multispecific and multi-affinity antibody loaded onto an anti-hIgG Fc capture (AHC) biosensor. b) Relationship between centroidal mean fluorescence (BCM) and static light scattering (SLS) at 266 nm and temperature. T m and T agg c) Neutralization test of BG505T332NPsV. (Indicated by yellow line)

[0226] Figure 5 Multispecific multi-affinity antibody affinity purification protocol. Sequential affinity purification of proteins A and L. Binding to protein A enriches multispecific multi-affinity antibodies with Fc (green), while binding to protein L enriches multispecific multi-affinity antibodies with κ chain FabPGDM1400 (blue). A point mutation from alanine to proline at position 12 of the κ chain of iMab is introduced to disrupt binding to protein L. 75 The complementarity of the two halves of human deferroferritin ensures the presence of N49P7 / iMab scFab (orange) and 10E8 scFab (pink) fused to Cferritin during the protein A purification step. Gel filtration is performed to separate any aggregated material.

[0227] Figure 6 .32-N multispecific multi-affinity antibodies showed the least batch-to-batch variation. a) SEC chromatogram. b) Relationship between BCM and SLS at 266 nm and temperature. Thermal transition temperature (T m and T agg c) Concentration-response curve of 93TH057 gp120 binding to N49P7 within 32-N. d) Neutralization distribution of 32-N to the 4-PsV group, which was selected to include a PsV resistant to each Fab in a multispecific multi-affinity antibody.

[0228] Figure 7Thermal stability analysis. Relationship between BCM (top image) and SLS (bottom image) at 266 nm for 32-N and 32-I multispecific multi-affinity antibodies, their respective 12-mer multispecific multi-affinity antibodies, parental IgG, and N6 / PGDM1400x10E8 trispecific antibody versus temperature. Thermal transition temperature (T0). m and T agg (Used as yellow lines)

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

[0230] Figure 9 Neutralizing properties of .32-N and .32-I multispecific multi-affinity antibodies against 14 pseudoviruses. Width values ​​and IC50 values ​​for multispecific multi-affinity antibodies (red rhombuses), parental bNAb (black circles), IgG combination (black triangles), and N6 / PGDM1400x10E8v4 trispecific antibody (black squares). 50 Median (μg / mL). The relative concentrations of each parental antibody in the IgG mixture were the same as those in the multispecific multi-affinity antibody samples (i.e., 66% PGDM1400, 17% N49P7 / iMab, and 17% 10E8v4). The 14-PsV group was selected based on susceptibility and resistance to parental IgG.

[0231] Figure 10 Neutralizing properties of .32-N and .32-I multispecific multi-affinity antibodies against 14 pseudoviruses. Width and IC50 of multispecific multi-affinity antibodies (red rhombuses), parental bNAb (black circles), IgG combination (66% PGDM1400, 17% N49P7 / iMab and 17% 10E8v4, black triangles), and N6 / PGDM1400x10E8v4 trispecific antibody (black squares). 50 Median (nM).

[0232] Figure 11Immunogenicity and exposure to multispecific multi-affinity antibodies in mice. Five male C57BL / 6 mice per group were used to assess the circulation of anti-drug antibodies and multispecific multi-affinity antibodies in the blood following subcutaneous administration of 5 mg / kg of alternative multispecific multi-affinity antibodies and Fc-modified multispecific multi-affinity antibodies (LALAP mutation to disrupt Fc receptor binding). Reference sample HpFerritin-PfCSP malaria peptide and parental mouse IgG1 and IgG2a isotypes were used for immunogenicity and exposure comparison, respectively. Detailed Implementation

[0233] definition

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

[0235] It should also be understood that the terminology used herein is not intended to limit the invention, but is merely for the purpose of describing particular aspects. Numerous patent applications, patents, and publications are cited herein to aid in understanding the described aspects. The full text of each of these references is incorporated herein by reference.

[0236] In understanding the scope of this application, the articles “a,” “an,” “the,” and “the” are intended to indicate the presence of one or more elements. Additionally, as used herein, the term “comprising” and its derivatives are open-ended terms that specify the presence of the stated feature, element, component, group, whole, and / or step, but do not exclude the presence of other unmentioned features, elements, components, groups, wholes, and / or steps. The foregoing also applies to words with similar meanings, such as the terms “comprising,” “having,” and their derivatives.

[0237] It should be understood that any aspect described as “comprising” certain components may also be “composed of these components” or “substantially composed of these components,” where “composed of these components” has a closed or limiting meaning, and “substantially composed of these components” means including the specified components but excluding components other than materials present as impurities, unavoidable materials present as a result of the methods used to provide these components, and components added for purposes other than achieving the technical effects of the invention. For example, compositions defined using the phrase “substantially composed of” encompass any known and acceptable additives, excipients, diluents, and carriers, etc. Typically, a composition substantially composed of a group of components will contain less than 5% by weight, typically less than 3% by weight, more typically less than 1% by weight, and even more typically less than 0.1% by weight of unspecified components.

[0238] It should be understood that any component defined as included herein may be expressly excluded from the claimed invention by way of incidental conditions or negative limitations. For example, in some aspects, the nanocages and / or fusion proteins described herein may not include ferritin heavy chains and / or may not include iron-binding components.

[0239] Furthermore, all ranges given in this document include the endpoints of the ranges as well as any intermediate range points, whether or not explicitly stated.

[0240] As used herein, degree terms such as “substantially,” “about,” and “approximately” refer to a reasonable amount of deviation from the modified term that will not significantly alter the final result. If this deviation does not negate the meaning of the word it modifies, these degree terms should be understood to include at least ±5% of the deviation from the modified term.

[0241] It should also be understood that all base 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. While suitable methods and materials are described below, methods and materials similar to or equivalent to those described and materials herein may be used in practice and testing of this disclosure. The abbreviation “eg” originates from the Latin *exempli gratia* and is used herein to indicate a non-limiting example. Therefore, the abbreviation “eg” is synonymous with the term “for example.” The word “or” is intended to include “and” unless the context clearly indicates otherwise.

[0242] The terms “protein nanoparticle,” “nanocage,” and “multispecific, multi-affinity antibody” are used interchangeably herein and refer to a multi-subunit protein polyhedral structure. Each subunit or nanocage monomer is composed of a protein or polypeptide (e.g., a glycosylated polypeptide) and optionally consists of one or more of the following features: nucleic acid, prosthetic group, organic and inorganic compound. 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), and encapsulated protein nanoparticles (see, e.g., Sutter et al., Nature Struct. and Mol. Biol., 15:939-947, 2008, incorporated herein by reference), thiooxygenase reductase (SOR) nanoparticles (see, e.g., Urich et al., Science, 311:996-1000, 2006, incorporated herein by reference), dioxetine 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, deferferritin, encapsulated proteins, SOR, dioxetine synthase, and pyruvate dehydrogenase are monomeric proteins that self-assemble into globular protein complexes, which in some cases consist of 24, 60, 24, 60, and 60 protein subunits, respectively. Ferritin and deferroferritin are generally referred to interchangeably herein and are understood to apply to the fusion proteins, nanocages, and methods described herein. The use of carboxyl bodies, fornix proteins, GroEL, heat shock proteins, E2P, and MS2 shell proteins that also produce nanocages is considered herein. Furthermore, fully or partially synthesized self-assembling monomers are also anticipated for use herein.

[0243] It should be understood that each nanocage monomer can be divided into two or more subunits that will self-assemble into a functional nanocage monomer. For example, ferritin or deferroferritin can be divided into N and C subunits, for example, by substantially splitting full-length ferritin in half, such that each subunit can individually bind a different bioactive moiety for subsequent self-assembly into a nanocage monomer, which in turn self-assembles into a nanocage. "Functional nanocage monomer" means a nanocage monomer capable of self-assembling with other such monomers into a nanocage as described herein.

[0244] The terms "ferritin" and "deferroferritin" are used interchangeably herein and generally refer to polypeptides (e.g., ferritin chains) capable of assembling into ferritin complexes typically comprising 24 protein subunits. It should be understood that ferritin can be derived from any species. Typically, ferritin is human ferritin. In some embodiments, the ferritin is wild-type ferritin. For example, ferritin may be wild-type human ferritin. In some embodiments, ferritin light chains are used as nanocage monomers, and / or subunits of the ferritin light chains are used as nanocage monomer subunits. In some embodiments, the assembled nanocages do not include any ferritin heavy chains or other ferritin components capable of binding iron.

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

[0246] As used herein, the term "multivalent" refers to the characteristic of having at least two binding sites, and having a binding partner, such as an antigen or receptor (e.g., an Fc receptor), that can bind to the at least two binding sites. The binding partners that can bind to the at least two binding sites may 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 a pathogen, such as a viral pathogen, or against an antigen-specific cellular component associated with a pathological condition. Vaccines may include polynucleotides (e.g., nucleic acids encoding a disclosed antigen), peptides or polypeptides (e.g., a disclosed antigen), viruses, cells, or one or more cellular components. In one specific, non-limiting example, a vaccine induces an immune response that, compared to a control, reduces the severity of symptoms associated with malaria infection and / or reduces the parasite load. In another, non-limiting example, a vaccine induces an immune response that, compared to a control, reduces and / or prevents malaria or HIV infection.

[0248] As used herein, the term "antibody," also known in the art as "immunoglobulin" (Ig), refers to a protein constructed from paired heavy and light polypeptide chains; multiple Ig isotypes exist, including IgA, IgD, IgE, IgG (such as IgG1, IgG2, IgG3, and IgG4), and IgM. It should be understood that antibodies can originate from any species, including humans, mice, rats, monkeys, llamas, or sharks. When antibodies fold correctly, each chain folds into multiple distinct globular domains linked by a more linear polypeptide sequence. For example, the immunoglobulin light chain folds into a variable domain (V... L ) and a constant structural domain (C L The heavy chain folds into a variable structural domain (V). H ) and three constant structural domains (C H C H2 C H3 ). Heavy chain variable structural domains and light chain variable structural domains (V H and V L The interaction of these domains leads to the formation of the antigen-binding region (Fv). Each domain has an established structure familiar to those skilled in the art.

[0249] The variable regions of the light and heavy chains are responsible for binding to target antigens and thus exhibit significant sequence diversity among antibodies. Constant regions exhibit lower sequence diversity and are responsible for binding to many native proteins to trigger important immune events. The variable regions of an antibody contain the molecule's antigen-binding determinants and thus determine the antibody's specificity for its target antigen. Most sequence variability occurs in the six hypervariable regions, three each on the variable heavy and light chains; these hypervariable regions combine to form antigen-binding sites and aid in the binding and recognition of antigenic determinants. The specificity and affinity of an antibody for its antigen are determined by the structure of the hypervariable regions and the size, shape, and chemical properties of the surface they present to the antigen.

[0250] As used herein, "antibody fragment" may include any suitable antigen-binding antibody fragment known in the art. Antibody fragments may be naturally occurring antibody fragments or may be obtained by manipulating naturally occurring antibodies or by using recombinant methods. For example, antibody fragments may include, but are not limited to, Fv, single-chain Fv (scFv; composed of V... L and V H (molecules composed of linker peptides), Fc, single-chain Fc, Fab, single-chain Fab, F(ab')2, single-domain antibody (sdAb; composed of a single V L or V H (the constituent fragments) and any of these multivalent forms.

[0251] As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA technology. The term should also be interpreted as meaning an antibody produced by synthesizing a DNA molecule encoding an antibody, wherein the DNA molecule expresses an antibody protein or an amino acid sequence indicating the antibody, wherein the DNA or amino acid sequence is obtained using DNA or amino acid sequence synthesis techniques available in the art and known to the public.

[0252] The term "epitaph" refers to an antigenic determinant. An epitope is a specific antigenic (i.e., that which elicits a specific immune response) chemical group or peptide sequence on a molecule. Antibodies specifically bind to (e.g., on a polypeptide) a specific antigenic epitope. Epitopes can be formed from sequential amino acids or from discontinuous amino acids juxtaposed through the ternary folding of a protein. Epitopes formed from sequential amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed from ternary folds are generally lost upon treatment with denaturing solvents. Epitopes typically comprise at least 3, more commonly at least 5, about 9, about 11, or about 8 to about 12 amino acids in a distinctive spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and 2D nuclear magnetic resonance. See, for example, 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 may involve antibody production or activation of specific immune-competent cells or both. Those skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA, including nucleotide sequences or partial nucleotide sequences encoding proteins that elicit an immune response, therefore encodes the term "antigen" as used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is apparent that the aspects described herein include, but are not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences can be arranged in various combinations to elicit the desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded entirely by a "gene." It is clear that antigens can be synthetic or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, cells, or biological fluids.

[0254] Therefore, the compositions described herein are suitable for protecting or treating vertebrate subjects against a variety of disease states, such as viral, bacterial, fungal, or parasitic infections, cancer, and autoimmune diseases. It should be understood that these specific 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 those skilled in the art. Antigens can be modified live 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 similar products, or allergens. Antigen moieties can also be subunits of proteins, peptides, polysaccharides, or similar products. Antigens can also be genetic antigens, i.e., DNA or RNA that elicits an immune response.

[0256] Representatives of usable antigens 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; tumor antigens that can be used in prophylactic or therapeutic vaccines; and allergens. In one embodiment, the antigen comprises virus-like particles (VLPs) derived from various viruses such as influenza virus, HIV, RSV, Newcastle disease virus (NDV), etc. 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, the entire contents of which are incorporated herein by reference. In another embodiment, the antigen comprises a chimeric VLP. A “chimeric VLP” refers to a VLP containing proteins or portions thereof from at least two different sources (organisms). Typically, one of the proteins originates from a virus that can drive the host cell to form a VLP. Therefore, in one embodiment, the chimeric VLP includes the RSV M protein. In another embodiment, the chimeric VLP includes the NDV M protein. In yet another embodiment, the chimeric VLP includes the influenza virus M protein.

[0257] Viral or bacterial products can be components produced by enzyme digestion of an organism, or components of an organism produced by recombinant DNA technology known to those skilled in the art.

[0258] Some specific examples of antigens are derived from hepatitis A, B, C, D, and E viruses; human immunodeficiency virus (HIV); herpesviruses 1, 2, 6, and 7; cytomegalovirus; varicella-zoster virus; papillomavirus; Epstein-Barr virus; parainfluenza virus; adenovirus; Bunyavirus (e.g., hantavirus); Coxsackievirus; pituitary RNA virus; rotavirus; respiratory syncytial virus; rhinovirus; rubella virus; papillomavirus; mumps virus; measles virus; poliovirus (multiple types); adenovirus (multiple types); parainfluenza virus (multiple types); avian influenza or pandemic influenza virus. Antigens of viral infections caused by various types of viruses, including seasonal influenza virus, shipping fever virus, western and eastern equine encephalomyelitis virus, Japanese encephalomyelitis virus, Russian spring and summer encephalomyelitis virus, swine fever virus, Newcastle disease virus, fowlpox virus, rabies virus, feline and canine distemper virus, slow encephalitis virus, Rous sarcoma virus (RSV), papillomaviruses, parvoviruses, parvoviruses, poxviruses (e.g., smallpox or cowpox virus), reoviridae (e.g., rotavirus), retroviridae (HTLV-I, HTLV-II, lentiviruses), and leptoviridae (rubella virus). Viruses belonging to these families can cause a variety of diseases or symptoms, including but not limited to: arthritis, bronchiolitis, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, Japanese encephalitis, Argentine hemorrhagic fever, chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt lymphoma, chickenpox, hemorrhagic fever, measles, mumps, parainfluenza, rabies, the common cold, poliomyelitis, leukemia, rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's disease, warts), and viremia.

[0259] Antigens can also originate from bacterial and fungal infections, such as those caused by Mycobacteria (which cause TB and leprosy), Streptococcus pneumoniae, aerobic Gram-negative bacilli, Mycoplasma, Staphylococcus infections, Streptococcus infections, Salmonella and Chlamydia, Bordetella pertussis, Leptospira pomona, and Treponema hemorrhagicum. Specific implementation plans include Salmonella paratyphi (S. paratyphi) and Salmonella diphtheriae (C. diphtheriae), Clostridium tetani (C. tetani), Clostridium botulinum (C. botulinum), Clostridium perfringens (C. perfringens), Clostridium feseri and other gas gangrene bacteria, B. anthracis, Yersinia pestis (P. pestis), Pasteurella multocida (P. multocida), Neisseria meningitidis, Neisseria gonorrheae, Haemophilus influenzae, Actinomyces (e.g., Nocardia), Acinetobacter, Bacillus (e.g., Bacillus anthrax), and Bacteroides (e.g., Bacteroides fragilis). fragilis), Blastodea dermatitidis, Bordetella, Borrelia (e.g., Borrelia burgdorferi), Brucella, Candida, Campylobacter, Chlamydia, Coccidioides, Corynebacterium (e.g., Corynebacterium diptheriae), Cryptococcus, Dermatocycoses, Escherichia coli (E. coli).*Escherichia coli* (e.g., enterotoxigenic *Escherichia coli* and enterohemorrhagic *Escherichia coli*), *Enterobacter* (e.g., *Enterobacter aerogenes*), *Enterobacteriaceae* (*Klebsiella*), *Salmonella* (e.g., *Salmonella typhi*, *Salmonella enteritidis*), *Serratia*, *Yersinia*, *Shigella*), *Erysipelothrix*, *Haemophilus* (e.g., *Haemophilus influenzae*), *Helicobacter*, *Legionella* (e.g., *Legionella pneumophila*). The following bacteria are listed: *Pneumophila*, *Leptospira*, *Listeria* (e.g., *Listeria monocytogenes*), *Mycoplasma*, *Mycobacterium* (e.g., *Mycobacterium leprae* and *Mycobacterium tuberculosis*), *Vibrio* (e.g., *Vibrio cholerae*), *Pasteurella*, *Proteus*, and *Pseudomonas* (e.g., *Pseudomonas aeruginosa*). (e.g., species of *Aeruginosa*), Rickettsiaceae, Spirochetes (e.g., species of *Treponema*, *Leptospira*, and *Borrelia*), Shigella species, Neisseria meningitidis, *Streptococcus pneumoniae*, *Streptococcus* (e.g., *Streptococcus pneumoniae* and types I, II, and III), *Ureaplasma urealyticum*, *Treponema pollidum*, etc.; *Staphylococcus aureus*, *Plasmodium* species (*Pl. falciparum*, *Pl. vivax*).The study included inactivated cells of various fungi, including *Aspergillus* species, *Candida albicans*, *Pasteurella haemolytica*, *Corynebacterium diphtheriae* toxoid, *Neisseria meningitidis* polysaccharide, *Bordetella pertussis*, *Streptococcus pneumoniae* polysaccharide, *Clostridium tetani* toxoid, *Mycobacterium bovis*, *Salmonella typhi*, *Cryptococcus neoformans*, and *Aspergillus* species.

[0260] Antigens can also originate from parasitic malaria, leishmaniasis, trypanosomiasis, toxoplasmosis, schistosomiasis, filariasis, amoebiasis, babesiosis, coccidiosis, cryptosporidiosis, binuclear amoebiasis, trypanosomiasis, ectoparasites, giardiasis, helminthiasis, Theileria, trichomoniasis, and spore-forming organisms (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, mites, eye infections, intestinal diseases (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS-related), malaria, pregnancy complications, and toxoplasmosis.

[0261] Tumor-associated antigens applicable to the compositions described herein include mutant and non-mutant molecules that can indicate a single tumor type, be shared by several tumor types, and / or be specifically expressed or overexpressed in tumor cells compared to normal cells. In addition to proteins and glycoproteins, tumor-specific expression patterns of carbohydrates, gangliosides, glycolipids, and mucins are also described. Exemplary tumor-associated antigens for use in the cancer vaccines of this invention include protein products of oncogenes, tumor suppressor genes, and other genes with tumor cell-specific mutations or rearrangements, reactivated embryonic gene products, carcinoembryonic antigens, tissue-specific (but not tumor-specific) differentiation antigens, growth factor receptors, cell surface glycosidic residues, exogenous viral proteins, and many other endogenous proteins. Specific implementation schemes for 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-ab1 oncogenes, as well as CDK4, MUM1, caspase 8, and β-catenin; overexpressed antigens, such as galactagogue 4, galactagogue 9, carbonic anhydrase, aldolase A, PRAME, Her2 / neu, ErbB-2, and KSA; oncoemulsification antigens, such as alpha-fetoprotein (AFP) and human chorionic gonadotropin (hCG); autoantigens such as carcinoembryonic antigen (CEA), and melanocyte differentiation antigens such as Mart 1 / Melan A, gp100, gp75, tyrosinase, TRP1, and TRP2; prostate-associated antigens, such as PSA, PAP, PSMA, PSM-P1, and PSM-P2; and reactivated embryonic gene products, such as MAGE 1, MAGE3, MAGE 4, GAGE ​​1, and GAGE ​​2. 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. Tumor-associated antigens included in this article are also whole-cell and tumor cell lysates and their immunogenic fractions, as well as immunoglobulin idiotypes expressed on monoclonal proliferation of B lymphocytes for anti-B-cell lymphoma. Tumor-associated antigens and their respective tumor cell targets include, for example, cytokeratins as cancer antigens, particularly cytokeratins 8, 18, and 19. Epithelial membrane antigen (EMA), EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6, human embryonic antigen (HEA-125), human milk fat globule, MBR1, MBR8, Ber-EP4, 17-1A, C26, and T16 are also known cancer antigens.Desmin and muscle-specific actin are antigens of myogenic sarcomas. Placental alkaline phosphatase, β-human chorionic gonadotropin, and alpha-fetoprotein are antigens of trophoblastic and germ cell tumors. Prostate-specific antigen (PSA) is an antigen of prostate cancer and carcinoembryonic antigen (CEA) of colonic adenocarcinoma. HMB-45 is an antigen of melanoma. In cervical cancer, useful antigens can be encoded by human papillomavirus (HPV). Chromogranin-A and synaptic vesicle proteins are antigens of neuroendocrine and neuroectodermal tumors. Of particular interest are aggressive tumors that form solid masses with necrotic areas. The lysis of these necrotic cells is a rich source of antigens for antigen-presenting cells, thus this treatment may have advantageous use in combination with conventional chemotherapy and / or radiotherapy. Antigens can be derived from any tumor or malignant cell lineage.

[0262] Antigens can also originate from common allergens that cause allergies. Allergens include organic or inorganic materials of various man-made or natural origins, such as plant materials, metals, ingredients in cosmetics or detergents, latex, etc. Suitable allergens for use in the compositions and methods described herein may include, but are not limited to, pollen, animal dander, grass, mold, dust, antibiotics, irritating insect venom, and various environmental (including chemical and metal) drug and food allergens. Common tree allergens include pollen from poplar, ash, birch, maple, oak, elm, hickory, and pecan trees; common plant allergens include allergens from rye, hogweed, longleaf plantain, sorrel, and quinoa; plant contact allergens include allergens from poison oak, poison ivy, and nettle; common grass allergens include allergens from cat's tail grass, rock grass, bermudagrass, fescue, and Kentucky bluegrass; 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; epidermal allergens can be obtained from indoor or organic dust (usually from fungi) and insects such as house mites (dermalphagoides). Common food allergens include milk and cheese (dairy products), eggs, wheat, nuts (such as peanuts), seafood (such as shellfish), peas, beans, 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 perfumes; common drug allergens include local anesthetics and salicylates; antibiotic allergens include penicillin and sulfonamides; common insect allergens include bee, wasp, and ant venom and cockroach cup allergens. The well-characterized allergens include, but are not limited to, the major and occult epitopes of Der pI allergen (Hoyne et al., 1994, Immunology, 83:190-195), bee venom phospholipase A2 (PLA; Akdis et al., 1996, J. Clin. Invest., 98:1676-1683), birch pollen allergen Bet v1 (Bauer et al., 1997, Clin. Exp. Immunol., 107:536-541), and multi-epitope recombinant grass allergen rKBG8.3 (Cao et al., 1997, Immunology, 90:46-51).These and other suitable allergens are commercially available and / or can be readily prepared as extracts using known techniques.

[0263] Antigens can be in the form of purified or partially purified antigens and can be derived from any of the aforementioned antigens, antigenic peptides, proteins known and available in the art, and other antigens that can be identified using conventional techniques. Antigens are typically in a form in which their toxic or toxic properties have been reduced or destroyed, and when introduced in an appropriate form, will induce an immune response against a specific microorganism, extract, or microbial product used in the antigen formulation, or, in the case of an allergen, will help alleviate symptoms of an allergic reaction caused by a specific 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 or viral toxoids, multiple tumor antigens, multiple allergens, or any combination of the aforementioned products can be combined with adjuvant compositions to produce multivalent antigen compositions and / or vaccines. In the compositions described herein, the antigen can be an antigen encapsulated in, adsorbed onto, or mixed with the vesicular component of the composition.

[0264] In one embodiment, suitable antigens for use in the compositions described herein include antigens with poor immunogenicity, such as malaria antigen, dengue antigen, and HIV antigen, or antigens designed to confer immunity against pandemic diseases, such as influenza antigen. Any combination of such antigens described herein or known herein is intended for use in the fusion proteins, fusion protein pairs, and nanocages described herein.

[0265] "Encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide such as a gene, cDNA, or mRNA, which serves as a template for the synthesis of other polymers and macromolecules having a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the resulting biological properties during biological processes. Therefore, if the transcription and translation of mRNA corresponding to a gene produces a protein in a cell or other biological system, then that gene encodes that protein. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is typically provided in the sequence listing, and the non-coding strand, which serves as a template for gene or cDNA transcription, can be referred to as encoding the protein or other product of that gene or cDNA.

[0266] As used in this article, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0267] "Separated" means altered or removed from its natural state. For example, nucleic acids or peptides naturally present in living organisms are not "separated," but the same nucleic acids or peptides that are partially or completely separated from their natural counterparts are "separated." Separated nucleic acids or proteins can exist in a substantially purified form or can exist in non-natural environments, such as host cells.

[0268] Unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. A nucleotide sequence encoding a protein or RNA phrase may also include introns to such an extent that a nucleotide sequence encoding a protein may contain introns in some forms.

[0269] As used herein, the term "modulation" refers to a detectable increase or decrease in response levels in a subject compared to response levels in subjects without treatment or the compound, and / or compared to response levels in other subjects who are otherwise identical but untreated. The term encompasses perturbing and / or influencing natural signals or responses that mediate a beneficial therapeutic response in a subject (typically a human).

[0270] The term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence, resulting in the latter's expression. For example, when a first nucleic acid sequence has a functional relationship with a second nucleic acid sequence, the first and second nucleic acid sequences are operably linked. Similarly, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. Typically, operably linked DNA sequences are contiguous and, in cases where two protein-coding regions need to be linked, are within the same reading frame.

[0271] "Parenteral" administration of immunogenic compositions includes techniques such as subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion.

[0272] As used herein, the term "polynucleotide" is defined as a nucleotide chain. Furthermore, nucleic acids are polymers of nucleotides. Therefore, as used herein, nucleic acids and polynucleotides are interchangeable. Those skilled in the art have general knowledge that nucleic acids are polynucleotides and can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including but not limited to recombinant methods (i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using common cloning techniques and PCR, etc.) and synthetic methods.

[0273] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds composed of amino acid residues covalently linked 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 form the sequence of a protein or peptide. A polypeptide includes any peptide or protein comprising two or more amino acids linked together by peptide bonds. As used herein, the term refers not only to short chains (also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers) but also to long chains (commonly referred to in the art as proteins, of which many types exist). “Polypeptide” includes, for example, bioactive fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, etc. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0274] As used herein with respect to antibodies, the term "specific binding" refers to an antibody that recognizes a specific antigen but substantially does not recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, this cross-species reactivity itself does not change the antibody's classification as specific. In another instance, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, this cross-reactivity itself does not change the antibody's classification as specific. In some cases, the term "specific binding" can be used to refer to the interaction of an antibody, protein, or peptide with a second chemical substance, meaning that the interaction depends on the presence of a specific structure on that chemical substance (e.g., an antigenic determinant or epitope); for example, the antibody recognizes and binds to a specific protein structure rather than a protein in general. If an antibody is specific to epitope "A," then in a reaction containing labeled "A" and an antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody.

[0275] The terms “therapeutic effective amount,” “effective amount,” or “sufficient amount” refer to an amount that, when administered to a subject (including mammals, such as humans), is sufficient to achieve the desired outcome, such as an amount that effectively elicits a protective immune response. The effective amount of the compounds described herein may vary depending on factors such as the immunogen, the subject's age, sex, and weight. As understood by those skilled in the art, dosages or treatment regimens can be adjusted to provide an optimal therapeutic response. For example, administering a therapeutically effective amount of the fusion protein described herein may be sufficient in some respects to enhance immunity against pathogens such as Plasmodium or HIV. In other respects, administering a therapeutically effective amount of the fusion protein described herein may be sufficient to treat diseases or conditions such as cancer, HIV, malaria, or autoimmune diseases. In still other respects, administering a therapeutically effective amount of the fusion protein described herein may be sufficient to act as an adjuvant to increase the effectiveness of a vaccine. In yet another respect, administering a therapeutically effective amount of the fusion protein described herein may be sufficient to prevent contraction of a disease or infection.

[0276] Furthermore, treatment regimens administering a therapeutically effective amount to the subject may consist of a single dose or alternatively include a series of administrations. The duration of treatment depends on a variety of factors, such as the immunogen, the subject's age, the drug concentration, the patient's responsiveness to the drug, or combinations thereof. It should also be understood that the effective dose of the drug used for treatment may be increased or decreased during a particular treatment regimen. This can result in dose variations, which become apparent by standard diagnostic assays known in the art. In some respects, the fusion proteins described herein may be administered before, during, or after treatment of a target disease or condition (such as malaria, HIV, or cancer) with conventional therapies. For example, the fusion proteins described herein may be specifically used in combination with immunotherapy to treat cancer.

[0277] As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process of transferring or introducing exogenous nucleic acids into host cells. “Transfected,” “transformed,” or “transduced” cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. These cells include primary subject cells and their progeny.

[0278] As used in this article, the phrase “under transcriptional control” or “operably linked” refers to the promoter being in the correct position and orientation relative to the polynucleotide to control the initiation of transcription and the expression of the polynucleotide performed by RNA polymerase.

[0279] A “vector” is a composition of substances comprising isolated nucleic acids and capable of delivering those isolated nucleic acids into the cell. Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term “vector” includes autonomously replicating plasmids or viruses. The term should also be understood to include non-plasmid and non-viral compounds that facilitate nucleic acid transfer into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, etc.

[0280] As used in this article, the term "subject" refers to any member of the animal kingdom, typically a mammal. The term "mammal" refers to any animal classified as a mammal, including humans, other higher primates, livestock and farm animals, as well as zoo animals, sporting animals, or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. Typically, humans are considered a mammal.

[0281] "Combined" administration with one or more other therapeutic agents includes simultaneous (parallel) administration and administration in any order.

[0282] The term "pharmaceutical acceptable" means that a compound or combination of compounds is compatible with the rest of the ingredients in a pharmaceutical preparation and is generally safe to administer 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, antimicrobial agents, antifungal agents, isotonic agents, and / or absorption delay agents. The use of pharmaceutically acceptable carriers is well-known.

[0284] The term "adjuvant" refers to a compound or mixture present in a vaccine that enhances the immune response to an antigen present in the vaccine. For example, an adjuvant can enhance the immune response to a peptide present in a vaccine as considered herein, or to an immunogenic fragment or variant thereof as considered herein. Adjuvants can serve as tissue reservoirs for the slow release of antigens or as nonspecific activators of the lymphoid system that enhance the immune response. Examples of adjuvants that may be used include MPL-TDM adjuvant (monophosphoryl ester A / synthetic trehalose mycobacterium ester, available, for example, from GSK Biologics). Another suitable adjuvant is the immunostimulatory adjuvant AS021 / AS02 (GSK). These immunostimulatory adjuvants are formulated to produce a strong T-cell response and include QS-21, a saponin from the quillayus saponaria, a TL4 ligand, monophosphoryl ester A, and a lipid or liposome carrier. Other adjuvants include, but are not limited to, nonionic block copolymer adjuvants (e.g., CRL 1005), aluminum phosphate (e.g., AlPO4), R-848 (Th1-like adjuvant), imiquimod, PAM3CYS, poly(I:C), loxoribin, BCG (Bacillus Calmette-Guérin) and Corynebacterium parvum, CpG oligodeoxynucleotides (ODN), cholera toxin-derived antigens (e.g., CTA 1-DD), lipopolysaccharide adjuvants, complete Freund's adjuvants, incomplete Freund's adjuvants, saponins, mineral gels such as aluminum hydroxide, surfactants such as lysophosphatidylcholine, prolindic polyols, polyanionic, peptides, aqueous emulsions of oils or hydrocarbons (e.g., MF59 or Montanide ISA 720 available from Novartis vaccines), keyhole hemocyanin, and dinitrophenol.

[0285] A “variant” is a bioactive fusion protein, antibody, or fragment thereof that has a sequence different from the comparative sequence due to the insertion, deletion, modification, and / or substitution of one or more amino acid residues in the comparative sequence. Variants typically have less than 100% sequence identity compared to the comparative sequence. However, generally, bioactive variants will have an amino acid sequence identity of at least about 70% compared to the comparative sequence, such as at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. Variants include peptide fragments of at least 10 amino acids that retain a certain level of bioactivity of the comparative sequence. Variants also include polypeptides in which one or more amino acid residues are added to the N or C terminus or interior of the contrast sequence. Variants also include polypeptides in which multiple amino acid residues are missing and optionally substituted with one or more amino acid residues. Variants may also be covalently modified, for example by partial substitution with a non-naturally occurring amino acid or by modifying amino acid residues to produce a non-naturally occurring amino acid.

[0286] "Percentage amino acid sequence identity" is defined herein as the percentage of amino acid residues in a candidate sequence that are identical to residues in a target sequence (e.g., the polypeptide of this invention) after sequence alignment and, where necessary, nicking to achieve maximum percentage sequence identity, without regard to any conserved substitutions as part of sequence identity. N-terminal, C-terminal, or internal extensions, deletions, or insertions in the candidate sequence should not be interpreted as affecting sequence identity or homology. The methods and computer programs used for alignment are well known in the art, such as "BLAST".

[0287] "Active" or "active" herein refers to the biological and / or immunological activity of the fusion protein described herein, where "biological" activity refers to the biological function (inhibition or stimulation) induced by the fusion protein.

[0288] The fusion proteins described herein may include modifications. Such modifications include, but are not limited to, conjugation with effector molecules such as antimalarial drugs or adjuvants. Modifications also include, but are not limited to, conjugation with a detectable reporter moiety. Modifications that extend half-life (e.g., PEGylation) are also included. Protein and non-protein drugs may be conjugated to the fusion protein by methods known in the art. Conjugation methods include direct linking, linker linking via covalent linkage, and specific binding of paired members (e.g., avidin-biotin). These methods include, for example, those described in Greenfield et al., Cancer Research, 50:6600-6607, 1990, which is incorporated herein by reference; and those described in 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 protein

[0290] This article describes fusion proteins. Fusion proteins include a first nanocage monomer subunit linked to a bioactive moiety. The fusion protein self-assembles with a protein including a second nanocage monomer subunit to form a nanocage monomer. Multiple such fusion protein pairs self-assemble to form nanocages. Thus, the bioactive moiety can modify the inner surface of the assembled nanocage, the outer surface of the assembled nanocage, or both.

[0291] The bioactive part can be any part that can be part of a fusion protein, and is typically a protein. Common bioactive parts include: antibodies or fragments thereof, antigens, detectable parts, pharmaceuticals, diagnostic agents, or combinations thereof.

[0292] When the bioactive part is an antibody and its fragments, it may include one or both chains of, for example, an Fc fragment. As will be understood, the Fc fragment may be derived from any type of antibody, but is typically a gG1 Fc fragment. The Fc fragment may also include one or more mutations that regulate the half-life of the fusion protein and / or the resulting nanocage comprising the fusion protein assembly, such as LS, YTE, LALA, and / or LALAP. For example, the half-life may be measured in minutes, days, weeks, or even months.

[0293] Furthermore, other alternatives to the fusion proteins and nanocages described herein are envisioned, including Fc sequence modification and the addition of other agents (e.g., human serum albumin peptide sequences), which allow for variations in bioavailability and will be understood by those skilled in the art. Additionally, the fusion proteins and nanocages described herein can be modulated by adjusting the sequence or by adding other agents to attenuate their immunogenicity and anti-drug responses (therapeutic, e.g., by matching the sequence to the host, or by adding immunosuppressive therapies [such as, for example, methotrexate, a major strategy for reducing the incidence of FVIII inhibitors when infliximab is administered for the treatment of rheumatoid arthritis or to induce tolerance in newborns (see: DiMichele DM, Hoots WK, Pipe SW, Rivard GE, Santagostino E, International workshop on immunetolerance induction: consensus recommendations, Haemophilia, 2007, 13:1-22, the full text of which is incorporated herein by reference]), or to enhance immune responses (e.g., bacterial sequences for vaccines).

[0294] In other respects, when the biologically active portion is an antibody or a fragment thereof, it may include, for example, a heavy chain and / or light chain of a Fab fragment. The antibody or fragment thereof may include, for example, a scFab fragment, a scFv fragment, or a sdAb fragment. It should be understood that any antibody or fragment thereof may be used in the fusion protein described herein.

[0295] Typically, the fusion proteins described herein are associated with Fab light chains and / or heavy chains, which can be produced separately from or continuously with the fusion protein.

[0296] In cases where the antibody or its fragment comprises two chains, such as a first and second chain in the case of an Fc fragment, or a heavy and light chain, these 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 appropriately. Although it should be understood that the length of this linker will vary depending on the sequence of the nanocage monomer and the bioactive moiety, as well as the three-dimensional conformation of the fusion protein, the linker is usually long enough to impart some flexibility to the fusion protein. Therefore, the linker is typically about 1 to about 30 amino acid residues, such as 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 about 8 to about 16 amino acid residues, such as 8, 10 or 12 amino acid residues.

[0297] The adapter can be any amino acid sequence, and in a typical example, the adapter includes GGS repeats, and more generally, the adapter includes about 2, 3, 4, 5, or 6 GGS repeats, such as about 4 GGS repeats. In some specific aspects, the adapter 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 with, or is composed of, the following sequences:

[0298] GGSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGTATAGASSGSGSSGSSSSGGTG.

[0299] In some typical aspects, the antibody or a fragment thereof specifically binds to an antigen associated with a condition that the antibody can prevent and / or treat. For example, the antigen bound to the antibody or a fragment thereof may be associated with: infectious agents, including viruses (e.g., HIV (including HIV-1), influenza virus, RSV, rotavirus), bacteria (e.g., TB, Clostridium difficile), parasites (e.g., malaria), fungi, or yeast; cancers (e.g., CD19, CD22, CD79, BCMA, or CD20), including solid and liquid cancers; or immune diseases, including autoimmune diseases. Typically, the antigen is associated with HIV-1, and the antibody or a fragment thereof includes, for example, ibalizumab-A12P, 10E8, 10E8.v4, N49P7, PGDM1400, 10-1074, VRC01, or combinations thereof.

[0300] In a specific example, 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 with, or is composed of, 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] Their combination.

[0344] In a further aspect, the antibody or a fragment thereof may be conjugated to or associated with other parts, such as antigens, detectable parts (e.g., small molecules, fluorescent molecules, radioisotopes, or magnetic particles), pharmaceuticals, diagnostic agents, or combinations thereof, and may include, for example, antibody-drug conjugates.

[0345] The biologically active portion of this is the aspect of the antigen, which may be associated with, for example, a disease that can be prevented and / or treated by a vaccine. In such cases, the antigen may be associated with, for example, infectious agents, including viruses, bacteria, parasites, fungi, or yeasts; cancers, including solid and liquid cancers; or immune diseases, including autoimmune diseases.

[0346] The bioactive portion is the detectable portion, which may include fluorescent proteins such as GFP, EGFP, auroral, and / or flavin-based fluorescent proteins such as LOV proteins (such as iLOV).

[0347] The bioactive portion is the pharmaceutical aspect, which may include, for example, small molecules, peptides, lipids, carbohydrates, or toxins.

[0348] In some typical aspects, the nanocages assembled by the fusion proteins described herein comprise approximately 3 to approximately 100 nanocage monomers, such as approximately 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, 9 4, 96, or 98 to approximately 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. Nanocage monomers can be any known natural, synthetic, or partially synthetic nanocage monomer, and in some respects selected from ferritin, deferriferritin, encapsulated proteins, SOR, dioxetine synthase, pyruvate dehydrogenase, carboxyl bodies, fornix proteins, GroEL, heat shock proteins, E2P, MS2 capsid proteins, fragments thereof, and variants thereof. Typically, nanocage monomers are ferritin or deferriferritin.

[0349] When aferritin is chosen as the nanocage monomer, the first and second nanocage monomer subunits typically interchangeably include the "N" and "C" regions of aferritin. It should be understood that other nanocage monomers can be divided into two subunits, very similar to aferritin as described herein, such that the subunits can self-assemble and are individually adapted to fuse with the bioactive moiety.

[0350] Typically, the "N" region of deferroferritin comprises, or consists of, sequences that have at least 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with, or are composed of, the following sequences:

[0351] MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEW.

[0352] Typically, the "C" region of deferroferritin includes, or is composed of, sequences that have at least 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with, or are composed of, the following sequences:

[0353] GKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD.

[0354] In some respects, the fusion proteins described herein also include a linker between the nanocage monomer subunits and the bioactive moiety, very similar to the linker described above. Again, this linker can be flexible or rigid, but it is typically flexible to allow the bioactive moiety to retain its activity and the nanocage monomer subunits to retain their self-assembly properties. While it should be understood that the length of this linker will vary depending on the sequence of the nanocage monomers and the bioactive moiety, as well as the three-dimensional conformation of the fusion protein, it is generally long enough to impart some flexibility to the fusion protein. Therefore, the linker is typically about 1 to about 30 amino acid residues, such as 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 about 8 to about 16 amino acid residues, such as 8, 10 or 12 amino acid residues.

[0355] The adapter can be any amino acid sequence, and in a typical example, the adapter includes GGS repeats, and more generally, the adapter includes about 2, 3, 4, 5, or 6 GGS repeats, such as about 4 GGS repeats. In some specific aspects, the adapter 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 with, or is composed of, the following sequences:

[0356] ASTASSASSGGGGGGSGGSGGSGGS.

[0357] Similarly, the fusion protein may also include a C-terminal adapter for improving one or more properties of the fusion protein. In some aspects, the adapter includes GGS repeats, and more generally, the adapter includes about 2, 3, 4, 5, or 6 GGS repeats, such as about 4 GGS repeats. In some specific aspects, the C-terminal adapter 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 with, or is composed of, the following sequences:

[0358] GGSGGSGGSGGSGGGASGGS.

[0359] This article also describes fusion protein pairs of the aforementioned fusion proteins, wherein the fusion protein pairs self-assemble to form nanocage monomers, wherein a first nanocage monomer subunit and a second nanocage monomer subunit are fused to different bioactive moieties. This provides multivalence and / or multispecificity to the individual nanocage monomers assembled from this subunit pair.

[0360] In some respects, the fusion protein may also include an antigen. These respects are clearly described in International Patent Application No. WO 2019 / 023812, the entire contents of which are incorporated herein by reference. In summary, in these respects, the antigen has at least a first antibody-binding epitope and a second antibody-binding epitope; and an antibody or a fragment thereof specific to at least the first antigenic epitope. Binding of the antibody or fragment thereof to the first antigenic epitope presents a second antigenic epitope for binding to the antigen-binding portion, and / or the first antibody-binding epitope binds to the antibody or fragment thereof, wherein, in the case of the antibody or fragment thereof, the binding presents the second antibody-binding epitope.

[0361] In other respects, antibodies or fragments thereof can target any antigen, such as those listed above. Typically, antigens originate from cancer or infectious agents, such as hepatitis A, B, C, HIV, mycobacteria, malaria pathogens, SARS pathogens, herpesviruses, influenza viruses, polioviruses, or bacterial pathogens such as Chlamydia and mycobacteria, or from self-reactive B cells or any T cells used for co-recruitment and cytotoxic killing.

[0362] The fusion proteins described herein can alternatively be used as therapeutic or diagnostic agents. Therefore, in some respects, antibodies or fragments thereof may be specific to, for example, tumor antigens or autoantigens.

[0363] A substantially identical sequence may include one or more conserved amino acid mutations. It is known in the art that one or more conserved amino acid mutations on a reference sequence can produce a mutant peptide that does not substantially alter the physiological, chemical, or functional properties of the reference sequence; in this case, the reference sequence and the mutant sequence will be considered “substantially identical” polypeptides. Conserved amino acid mutations may include the insertion, deletion, or substitution of amino acids; “conserved amino acid substitution” is defined herein as the substitution of an amino acid residue by another amino acid residue having similar chemical properties (e.g., size, charge, or polarity).

[0364] In a non-limiting example, a conserved mutation can be an amino acid substitution. Such a conserved amino acid substitution can be performed by replacing a basic, neutral, hydrophobic, or acidic amino acid with another amino acid from the same group. The term "basic amino acid" refers to a hydrophilic amino acid with a side chain pK value greater than 7 that is normally 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 known as "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 those 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 (Gln or Q). The term "hydrophobic amino acid" (also known as "nonpolar amino acid") refers to amino acids that exhibit hydrophobicity greater than zero according to the Eisenberg Normalized Consensus Hydrophobicity Scale (1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (Ile or I), phenylalanine (Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G).

[0365] "Acidic amino acids" refer to hydrophilic amino acids with a side chain pK value of less than 7, which are usually 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 between two sequences; it is determined by calculating the percentage of identical residues when the two sequences are aligned to obtain the maximum correspondence between residue 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 limit itself, sequence identity can be calculated using software such as the NCBI BLAST2 service maintained by the Swiss Institute of Bioinformatics (also available at ca.expasy.org / tools / blast / ), BLAST-P, Blast-N, or FASTA-N, or any other suitable software known in the art.

[0367] The substantially identical sequence of the present invention may have at least 85% identity; in another example, the substantially identical sequence may be a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between therewith) identity at the amino acid level with the sequence described herein. In some specific aspects, the substantially identical sequence retains the activity and specificity of the reference sequence. In a non-limiting embodiment, differences in sequence identity may be due to conserved amino acid mutations.

[0368] The peptides or fusion proteins of the present invention may also include additional sequences to aid in their expression, detection, or purification. Any such sequences or tags known to those skilled in the art may be used. For example, and not to be limiting, the fusion protein may include a targeting or signaling sequence (e.g., but not limited to ompA), a detection tag, exemplary tag boxes including the 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, polyglutamate 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; purification tags (e.g., but not limited to His5 or His6), or combinations thereof.

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

[0370] More specifically, the tag cassette may include an extracellular component capable of specifically binding to an antibody with high affinity or high affinity. In a single-chain fusion protein structure, the tag cassette may be located immediately adjacent to the amino terminus of the linker region, (b) inserted between and connecting linker modules, (c) immediately adjacent to the carboxyl terminus 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 terminus of the single-chain fusion protein. In some embodiments, one or more linking amino acids may be disposed between and connecting the hydrophobic portion of the tag cassette, or between and connecting the linker region, or between and connecting the linker module, or between and connecting the binding domain.

[0371] This article also covers isolated or purified fusion proteins, peptides, or fragments thereof immobilized on a surface using various methods; for example, and not wishing to be limiting, the peptide may be linked or coupled to the surface via 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, beads (such as magnetic-based beads or agarose-based beads or other chromatography resins), glass, a thin film, or any other useful surface.

[0372] In other respects, 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 conjugation, chelation, etc.). The cargo molecule can be any type of molecule, such as a therapeutic agent or a diagnostic agent. For example, and not wishing to be limited in any way, the therapeutic agent can be a radioisotope that can be used for radioimmunotherapy; a toxin, such as an immunotoxin; a cytokine, such as an immune cytokine; a cytotoxic agent; an apoptosis inducer; an enzyme; an anticancer antibody for immunotherapy; or any other suitable therapeutic molecule known in the art. Alternatively, the diagnostic agent may include, but is by no means limited to: a radioisotope, paramagnetically labeled 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 reagent detectable by imaging methods. In specific, non-limiting examples, the fusion protein may be linked to a fluorescent agent such as FITC, or genetically fused to enhanced green fluorescent protein (EGFP).

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

[0374] The fusion proteins described in this article specifically bind to their targets. "Antibody specificity" refers to the selective recognition of a specific epitope of an antigen by an antibody. The antibody specificity of the antibodies or fragments described in this article can be determined based on affinity and / or cohesion. This is determined by the dissociation equilibrium constant (Ka) between the antigen and antibody. D Affinity, expressed as a measure of the binding strength between an antigenic determinant (epitope) and an antibody binding site, is a metric for the strength of the binding between an antibody and its antigen. Antibodies are typically expressed in terms of 10-1. -5 M to 10 -11 M of K D Combine. Anything greater than 10 -4 M of K D This K is generally considered to indicate nonspecific binding. D The smaller the value, the stronger the binding strength between the antigenic determinant and the antibody binding site. In some respects, the antibodies described herein have a value of less than 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 of K D .

[0375] This paper also describes nanocages comprising at least one fusion protein as described herein and at least one second nanocage monomer subunit that self-assembles with the fusion protein to form a nanocage monomer. Furthermore, this paper describes fusion protein pairs, wherein the fusion protein pairs self-assemble to form nanocage monomers, and wherein the first and second nanocage monomer subunits are fused to different bioactive moieties.

[0376] It should be understood that the nanocage can be self-assembled from: multiple identical fusion proteins, multiple different fusion proteins (and thus the nanocage is multivalent and / or multispecific), combinations of fusion proteins and wild-type proteins, and any combination thereof. For example, the nanocage can be internally and / or externally modified by combining at least one fusion protein described herein with at least one anticancer antibody for immunotherapy. In some typical aspects, approximately 20% to approximately 80% of the nanocage monomer comprises the fusion protein described herein. Given the modular solution described herein, the nanocage can theoretically comprise up to twice the number of bioactive moieties in the monomers within the nanocage, since each nanocage monomer can be divided into two subunits, each of which can independently bind a different bioactive moiety. It should be understood that this modularity can be used to achieve any desired ratio of bioactive moieties as described herein in specific examples, where, in some specific examples, the four different bioactive moieties are 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 bioactive moieties. Thus, the nanocage can be multivalent and / or multispecific, and its extent can be relatively easily controlled.

[0377] In some respects, the nanocages described herein may also include at least one complete nanocage monomer, which may optionally be fused to a bioactive portion that is the same as or different from the bioactive portion connected to the nanocage monomer subunits described herein.

[0378] In a typical embodiment, the nanocages described herein include a first fusion protein, a second fusion protein, and a third fusion protein, as well as at least one intact nanocage monomer optionally fused to a bioactive portion, wherein the bioactive portions of the first fusion protein, the second fusion protein, and the third fusion protein and the bioactive portion of the intact nanocage monomer are all distinct from each other.

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

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

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

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

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

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

[0385] In some respects, the nanocages described herein comprise, or consist of, sequences having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with 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 lowercase letters:

[0386]

[0387]

[0388]

[0389] It should be understood that the nanocages described herein are typically hollow, thus capable of carrying cargo molecules such as pharmaceuticals, diagnostic agents, and / or imaging agents. Typically, the cargo molecule is not fused to the fusion protein and is contained within the nanocage; however, the cargo molecule may alternatively be a protein and fused to the fusion protein, such that the cargo molecule is contained within the nanocage.

[0390] In some respects, the cargo molecule is included internally to provide T-cell epitopes, but optionally not to provide B-cell epitopes. Alternatively, the cargo molecule is fused to the fusion protein and included internally to provide T-cell epitopes, but optionally not to provide B-cell epitopes.

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

[0392] In addition, nanocages may also include antigens on the surface, which can be expressed as fusion proteins with nanocage monomers.

[0393] This document also describes vaccines comprising the nanocages described herein, and compositions comprising the nanocages, such as therapeutic or prophylactic compositions. This document further describes relevant methods and uses for treating and / or preventing diseases or conditions, wherein such methods or uses include administering the nanocages, vaccines, or compositions described herein to a subject in need. The nanocages can be used to treat any disease or condition for which bioactive therapies, or more specifically, antibody therapies, are available, but for example, the disease or condition is typically cancer, infectious diseases such as HIV, malaria, influenza, RSV, rotavirus, or autoimmune diseases.

[0394] This article also describes nucleic acid molecules encoding the fusion proteins and peptides described herein, as well as vectors comprising these nucleic acid molecules and host cells comprising these vectors.

[0395] The polynucleotide encoding the fusion protein described herein includes a polynucleotide having a nucleic acid sequence substantially identical to that of the polynucleotide of the present invention. A “substantially identical” nucleic acid sequence is defined herein as a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% identity with another nucleic acid sequence when two sequences are optimally aligned (with appropriate nucleotide insertions or deletions) and compared to determine an exact nucleotide match between the two sequences.

[0396] Suitable sources of polynucleotides encoding antibody fragments include any cell expressing full-length antibodies, such as hybridomas and spleen cells. As mentioned above, the fragment itself can be used as an antibody equivalent, or can be reconstituted into an equivalent. The DNA deletions and recombinations 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 as described below. Another source of DNA is single-chain antibodies generated from phage display libraries, as known in the art.

[0397] Additionally, this document provides expression vectors comprising the polynucleotide sequence described above, which is operatively linked to an expression sequence, a promoter sequence, and an enhancer sequence. Various expression vectors have been developed for the efficient synthesis of antibody peptides in prokaryotic (such as bacterial) and eukaryotic systems (including, but not limited to, yeast and mammalian cell culture systems). The vectors of this invention may comprise fragments of chromosomal, non-chromosomal, and synthetic DNA sequences.

[0398] Any suitable expression vector can be used. For example, prokaryotic cloning vectors include plasmids from *E. coli*, such as colEl, 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 yeast is the 2μ plasmid. Suitable vectors for expression in mammalian cells include known derivatives of SV-40, adenoviruses, 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] Other 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; Kaufhiann and Sharp, "Amplification And Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene", J. Mol. Biol, 159:601-621, 1982; Kaufhiann 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 cited herein).

[0400] This expression vector typically contains at least one expression control sequence operatively linked to the DNA sequence or fragment to be expressed. This control sequence is inserted into the vector to control and regulate the expression of the cloned DNA sequence. Examples of useful expression control sequences are the lac system, trp system, tac system, trc system, the major operon and promoter regions of λ phage, the control region of fd coat proteins, yeast glycolysis promoters (e.g., promoters of 3-phosphoglycerate kinase), yeast acid phosphatase promoters (e.g., Pho5), yeast α-mating factor promoters, and promoters derived from polyomas, adenoviruses, retroviruses, and simian viruses (e.g., early and late promoters of SV40), as well as other sequences known to control gene expression in prokaryotic or eukaryotic cells and their viruses or combinations thereof.

[0401] This article also describes recombinant host cells comprising the expression vector as described above. The fusion proteins described herein can be expressed in cell lines rather than hybridomas. Nucleic acids comprising sequences encoding polypeptides according to the invention 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 suitable as hosts for expression are well-known in the art and include many unproliferating cell lines, such as, but not limited to, Chinese hamster ovary (CHO) cells, young hamster kidney (BHK) cells, and many others. Other suitable eukaryotic cells include yeast and other fungi. Useful prokaryotic hosts include, for example, *Escherichia coli* (such as *E. coli* SG-936, *E. coli* HB 101, *E. coli* W3110, *E. coli* X1776, *E. coli* X2282, *E. coli* DHI, and *E. coli* MRC1), *Pseudomonas*, *Bacillus* (such as *Bacillus subtilis*), and *Streptomyces*.

[0403] These recombinant host cells can be used to generate fusion proteins by culturing cells under conditions that allow peptide expression and purifying the peptide from the host cells or the culture medium surrounding the host cells. The targeted secretion of the expressed peptide in recombinant host cells can be facilitated by inserting a sequence encoding a signal peptide or secretion precursor 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, all of which are incorporated herein by reference). These secretion precursor peptide elements can be derived from prokaryotic or eukaryotic sequences. Thus, appropriately, secretion precursor peptides are used; secretion precursor peptides are amino acids linked to the N-terminus of the peptide to guide the peptide's migration from the host cell cytosol and secretion into the culture medium.

[0404] The fusion proteins described herein can be fused to additional amino acid residues. For example, such amino acid residues could be peptide tags that facilitate separation. Other amino acid residues are also envisioned for targeting antibodies to specific organs or tissues.

[0405] It should be understood that Fab nanocages can be generated through co-transfection of HC ferritin and LC. Alternatively, single-stranded Fab-ferritin nanocages that only require transfection of one plasmid can be used, such as... Figure 1As shown. This can be achieved via linkers of varying lengths (e.g., 60 or 70 amino acids) between LC and HC. When using single-chain Fab, this ensures that the heavy and light chains are paired. Tags (e.g., Flag, HA, myc, His6x, Strep, etc.) can also be added to the N-terminus of the construct or within the linker to facilitate purification as described above. Furthermore, when co-transfecting different Fab-nanoparticle plasmids, a tagging system can be used to ensure the presence of many different Fabs on the same nanoparticle via tandem / additive affinity chromatography steps. This provides multispecificity for the nanoparticles. If desired, protease sites (e.g., TEV, 3C, etc.) can be inserted to cleave the linker and tag after expression and / or purification. An example of such constructs is scFab 10E8 for broad-spectrum anti-HIV neutralization:

[0406] YELTQETGVSVALGRTVTITCRGDSLRSHYASWYQKKPGQAPILLFYGKNNRPSGVPDRFSGSASGNRASLTISGAQAEDDAEYYCSSRDKSGSRLSVFGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAAS SYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECGGSSGSGSTGENLYFQGSAGTTGTSASTSGYPYDVPDYAGGGGSAGGTATLEVLFQGPSSSGSSSSGGTGEVQLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAAPVEGRFT ISRLNSINFLYLEMNNLRMEDSGLYFCARTGKYYDFWSGYPPGEEYFQDWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCSRGGGGGSGGSGGS GGSMSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD

[0407] In another aspect, this article describes a method for vaccinating subjects by administering a therapeutically effective amount of the fusion protein described herein to mammals in need (typically juvenile, immature, or newborn mammals). "Therapeutically effective amount" refers to the amount that effectively produces the desired therapeutic effect, such as providing a protective immune response against the target antigen.

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

[0409] It should be understood that the fusion proteins described herein, when used in mammals for preventative or therapeutic purposes, will be administered in the form of compositions that additionally include pharmaceutically acceptable carriers. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate-buffered saline, dextran, glycerol, ethanol, etc., and combinations thereof. Pharmaceutically acceptable carriers may also include small amounts of excipients such as wetting agents or emulsifiers, preservatives, or buffers, wherein such excipients prolong the shelf life of the bound protein or improve its effectiveness. As is well known in the art, injectable compositions can be formulated to provide rapid, sustained, or delayed release of the active ingredient upon administration to mammals.

[0410] While human antibodies are specifically designed for administration to humans, they can also be administered to other mammals. As used herein, the term "mammal" is intended to include, but is not limited to, humans, laboratory animals, domestic pets, and farm animals.

[0411] This document also includes a kit for vaccination, comprising a therapeutically or prophylactically effective amount of the fusion protein described herein. The kit may also include, for example, any suitable adjuvant. The kit includes instructions for use.

[0412] The foregoing disclosure describes the invention in its entirety. A more complete understanding can be obtained by referring to the following specific embodiments. Unless otherwise stated, these embodiments are provided for illustrative purposes only and are not intended to be limiting. Therefore, the invention should not in any way be construed as limited to the following embodiments, but should be understood to cover any and all variations that become apparent from the teachings provided herein.

[0413] The following examples do not include detailed descriptions of conventional methods, such as methods for constructing vectors and plasmids, inserting genes encoding polypeptides into such vectors and plasmids, or introducing plasmids into host cells. These methods are well known to those skilled in the art and have been described in numerous publications, including Sambrook, J., Fritsch, EF., and Maniatis, T., *Molecular Cloning: A Laboratory Manual*, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, which are incorporated herein by reference.

[0414] Without further description, it is believed that those skilled in the art can use the foregoing description and the following illustrative examples to prepare and utilize the compounds of the present invention and to implement the claimed methods. Therefore, the following working examples specifically point to typical aspects of the invention and should not be construed as limiting the remainder of this 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. 37–39Similar to the combination of different drugs in HIV-1 treatment regimens, this observation suggests that successful anti-HIV-1 antibody therapy should include bNAb-specific combinations. Therefore, bispecific antibodies against Env have recently been developed. 40–42 or trispecific 43–45 Studies of different forms of antibody-like molecules. Another consideration is the amount of antibody required for in vivo efficacy. In fact, studies using structure-guided design or bioinformatics methods (such as VRC01) are being conducted. 46 10E8 47,48 and NIH45-46 49 Extensive work has been done to modify bNAbs to improve their potency, but so far, there has been no complete success. For bispecific and trispecific antibodies targeting multiple epitopes in Env, potency is often limited by the potency of their parent mAb. Therefore, while neutralization width has been significantly improved to date, 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 significant impact on its efficacy. 50–52 This effect, commonly referred to as affinity enhancement (epistem affinity enhancement), is a property of IgM antibodies in nature that compensates for their typically low affinity. Therefore, the addition of the μ-tail of IgM to the constant region of IgG has been investigated to produce dodecyl IgM-like molecules with improved biological activity. 53,54 Engineering has produced a variety of non-natural antibody forms at a speed far exceeding that of evolution, overcoming the limitation of IgG's bivalent nature. Some of these designs include Fab tandem fusions in a linear head-to-tail manner. 55 Tandem dual antibody combination (Tamdabs) 56 Or a di-diabody fused with the CH3 group of IgG. 57 Additional IgG 58–60 and the use of polymerized stents (e.g., p53) 61 Leucine zipper spiral 62 Streptomycin 63 Bacillus RNase - Bacillus RNase inhibitor protein module 64 and self-assemble into pentamer form 65 Furthermore, it can be further engineered into a decavalent. 66 The Escherichia coli Vero cytotoxin B subunit). These antibody architectures present different challenges for their successful development as therapeutics. Multimeric antibody forms that depend on antibody variable fragments (Fv) are often associated with low stability and the resulting high tendency to aggregate. 67Furthermore, the dissociation of the dimerizing modules, determined by the affinity constant of the complex, can limit the long-term stability of the molecule in vivo. Additionally, the titers achieved with most of the aforementioned antibody forms are typically at most 3-5 valences, thus excluding combinations of high affinity and multispecificity.

[0422] This article describes a multispecific multi-affinity antibody (multabody) platform that, in some respects, utilizes deferroferritin as a modular subunit for the polymerization of up to 32 antibody fragments (antigen-binding fragments [Fab] and crystallizable fragments [Fc]) in a single molecule. Using this method, we efficiently combined four different specificities into a single molecule, including the Fab portions of three of the best anti-HIV-1 bNAbs and the crystallizable fragment (Fc) from IgG1, to confer multispecificity, high affinity, effector function, and extended serum half-life to the molecule. Compared to combinations of their individual parental antibodies or IgGs, the resulting multispecific multi-affinity antibodies exhibited panvironic neutralizing width and significantly higher neutralizing potency. Notably, the multispecific multi-affinity antibodies showed a significantly higher mean IC50 against a group of 14 pseudoviruses (PsVs) compared to anti-HIV trispecific N6 / PGDM1400x10E8 antibodies or mixtures made from the best-known bNAbs. 50 The median values ​​are one and two orders of magnitude lower in mass and molar concentration, respectively. The multispecific, multi-affinity antibody design described in this paper represents a robust and powerful plug-and-play platform for polymerizing antibodies to enhance their therapeutic properties in inhibiting HIV-1 infection.

[0423] Materials and methods

[0424] Expression and purification of multispecific, multi-affinity antibodies based on Fab-only deferroferrin. Genes encoding the human deferroferrin light chain and the scFab-human deferroferrin fusion protein were synthesized and cloned into the pHLsec expression vector using GeneArt (Life Technologies). 200 mL of HEK293F cells (Thermo Fisher Scientifics) were cultured at 0.8 × 10⁻⁶ cells / mL. 6Cells were seeded at a density of 1 / mL in Freestyle expression medium and incubated in a MultitronPro shaker (Infors HT) at 37°C, 8% CO2, and 70% humidity at 125 rpm. Within 24 hours post-inoculation, cells were transiently transfected with 50 μg of filtered DNA pre-incubated at room temperature (RT) for 10 min with the transfection reagent FectoPRO (Polyplus Transfections) at a 1:1 ratio. Plasmids encoding scFab-human deferroferritin and human deferroferritin were mixed at ratios of 1:4, 1:1, 4:1, and 1:0 to obtain nanoparticles with 20%, 50%, 80%, and 100% scFab titers, respectively. After 6–7 days, the cell suspension was harvested by centrifugation at 5000 × g for 15 min, and the supernatant was filtered through a 0.22 μm Steritop filter (EMD Millipore). The nanoparticles were purified by affinity chromatography of Fab followed by washing and elution. The fractions containing proteins were combined, concentrated, and loaded into a Superose 6 10 / 300GL size exclusion column (GE Heathcare) in 20 mM sodium phosphate (pH 8.0) and 150 mM NaCl.

[0425] Design, expression, and purification of 32-N and 32-I multispecific and multi-affinity antibodies. Genes encoding scFab and scFc fragments linked to half-ferritin were generated using the KOD-plus mutagenesis kit (Toyobo, Osaka, Japan) by deleting residues 1-95 (C-ferritin) and 95-175 (N-ferritin) of the human deferroferritin light chain. Furthermore, using the same mutagenesis kit, site-directed mutagenesis was performed to disrupt the binding specificity of protein L to iMab-C-ferritin by mutating alanine 12 to a proline residue in the antibody light chain. 7532-N multispecific multi-affinity 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 deferroferritin:Fc-human deferroferritin:N49P7scFab-C ferritin:10E8 scFab-C ferritin. For 32-I multispecific multi-affinity antibody, iMabscFab-C ferritin was used instead of N49P7 scFab-C ferritin. The DNA mixture was filtered before addition to cell culture and incubated at room temperature with 60 μL LectoPRO. The multispecific multi-affinity antibody was first purified by affinity chromatography using a HiTrap Protein A HP column (GE Healthcare) with 20 mM Tris pH 8.0, 3 M MgCl2, and 10% glycerol elution buffer. After medium replacement using a PD-10 desalting column (GE Healthcare), the multispecific multi-affinity antibody was further purified by a second affinity chromatography using a HiTrap Protein L column (GE Healthcare). The protein-containing fraction was concentrated and further purified by gel filtration on a Superose 610 / 300GL column (GE Healthcare).

[0426] Negative staining electron microscopy examination. 3 μL of a multispecific, multi-affinity antibody at a concentration of approximately 0.02 mg / mL was added to a carbon-coated copper grid, incubated for 30 seconds, and stained with 3 μL of 2% uranyl formate. Excess dye was immediately removed from the grid using Whatman No. 1 filter paper, and another 3 μL of 2% uranyl formate was added, incubated for 20 seconds. The grid was then 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).

[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 multi-affinity antibody or control samples (HD37IgG1, HD37IgG2a, and hpFerritin-PfCSP malaria peptide) in 200 μL of PBS (pH 7.5) solution was administered. Blood samples were collected at multiple time points, and circulating antibody and ADA levels in serum samples were evaluated by ELISA. In short, 96-well Pierce nickel-coated plates (Thermo Fisher) were used with 50 μL of 0.5 μg / mL His... 6x The labeled antigen hCD19 was used as a coating, and the specific concentration of circulating HD37 was determined using a reagent-specific standard curve with IgG and multispecific multi-affinity antibodies. In the anti-drug antibody assay, Nunc MaxiSorp plates (Biolegend) were coated with 12-mer HD37 scFab multispecific multi-affinity antibody or hpFerritinPfCSP malaria peptide. HRP-Protein A (Invitrogen) was used as a second molecule, and the chemiluminescent signal was quantified using a Synergy Neo2 multimodal analyzer (Biotek Instruments).

[0432] result

[0433] Multispecific, multi-affinity antibodies can neutralize HIV-1 with 500 times greater potency than the gold standard IgG. The strong self-assembly properties of human deferroprotein light chains were used to polymerize Fab onto the surface of hollow, spherical protein cages formed by 24 monomers. In fact, deferroprotein self-assembles into a 12nm diameter structure composed of 24 identical polypeptides and readily undergoes gene fusion. 70 Each deferroferritin subunit has its N-terminus pointing outwards from the spherical cage, thus enabling gene fusion of the target protein. To maintain all properties of the IgG molecule, including high thermal stability and correct chain pairing, we generated fusions of deferroferritin with single-chain Fab (scFab) and single-chain Fc (scFc) fragments. Upon folding, the deferroferritin subunits act as the basic building blocks driving the polymerization of 24 proteins fused to their N-termini. Figure 1 Importantly, the presence of multiple specificities (e.g., Fab and Fc) on the same molecule can be achieved and controlled by co-transfecting selected proportions of DNA encoding each component and by selecting a strict affinity purification protocol that selects all specificities (e.g., affinity chromatography for protein L and protein A combinations selecting Fab and Fc, respectively).

[0434] First, we investigated the effect of the pleiotropic titer of HIV-1 bNAb displayed on our novel multispecific and multi-affinity antibody platform on its ability to block viral infection, and compared it with the display of standard bivalent IgG with the same bNAb. bNAb groups with different specificities to Env were selected and co-transfected with scFab-human deferroferrin-encoding plasmids at different ratios of uncoupled deferroferrin, causing their scFab to polymerize at different densities. Figure 2 a). Multispecific, multi-affinity antibodies are assembled into monodisperse, well-formed spherical particles, which have uninterrupted dense rings and regularly spaced protruding Fabs. Figure 2 a). Notably, compared to the IgG forms of the five pseudovirus groups, PGDM1400 is the most potent anti-HIV bNAb (IC) agent described to date. 50 Median (0.003 μg / mL) as a 24-mer multispecific multi-affinity antibody showed 100 to 500-fold neutralization (improvement factor of IC50). 50 (multiples of the median value of nM) Figure 2 (b) Compared to its IgG, bNAb 10-1074, as a multispecific and multi-affinity antibody, also showed a significant improvement in neutralizing efficacy, while bNAb 10E8, N49P7, and VRC01, although still effective, did not show the same enhancement.

[0435] Deferroferritin modification resulted in efficient heterooligomerization of the 32-mer multispecific multi-affinity antibody. Secondly, we sought to improve the width of the exceptionally efficient 24-mer PGDM1400 multispecific multi-affinity antibody by conferring molecular multispecificity. To this end, in addition to the Fc fragment of the human IgG1 isotype, we combined the PGDM1400 Fab with the near-pan-neutralizing antibody 10E8v4 (a modified 10E8 with improved solubility). 71 The Fab combination of N49P7 and Fc. To achieve this level of four-component (three Fabs and one Fc) heterooligomerization, we split the human deferroferritin structure into two subunits (N-ferritin and C-ferritin) and attached Fabs to the N-terminus of each of the two halves respectively. Figure 3a The complementation of the split aferroferrin halves leads to self-association of the two halves, and thus a very efficient heterodimerization process for the fusion protein. Importantly, no significant differences were observed in the biophysical and functional properties of multispecific and multi-affinity antibodies assembled with split and full-length aferroferrin. Figure 4 This design allows for a simple two-step purification procedure to select for four different types of multispecific and multi-affinity antibodies. Figure 5 It also showed high batch-to-batch uniformity. Figure 6Furthermore, splitting the deferroferritin into two halves allows for the inclusion of additional Fab / Fc fragments, up to 32 components per molecule, compared to the standard deferroferritin base material (Figure 3b). The eight additional sites (increasing from the conventional 24 to 32 in our engineered platform) play a significant role in providing multispecificity without sacrificing most of the potency increases observed in the 24-mer PGDM1400 multispecific multi-affinity antibody. Therefore, the multispecific multi-affinity antibody 32-N was designed to yield 16 copies of PGDM1400, 8 copies of Fc, 4 copies of 10E8v4, and 4 copies of N49P7 via co-transfection of plasmids encoding scFab and scFc in a 4:2:1:1 ratio. Figure 3a The multispecific, multi-affinity antibody 32-N forms highly modified and homogeneous particles (Fig. 3b) and shows T-cell affinity with the corresponding IgG molecule. m and T agg The unfolding and aggregation transition temperatures are similarly distributed, and also as previously reported for IgG. 72 (Figure 3c and) Figure 7 Kinetic experiments were used to demonstrate that each component of the multispecific, multi-affinity antibody (PDGM1400, N49P7, 10E8v4, and the Fc fragment) can bind to its epitope by binding to epitope-specific molecules (BG505 SOSIP D368R, 93TH057 gp120, MPER peptide, and human FcRn, respectively). Figure 3d Individual IgG molecules do not have the ability to bind to all antigens. Figure 8 The ability of 32-N to bind to multiple antigens suggests that the geometry conferred by the oligomeric form of multispecific and multi-affinity antibodies does not negatively affect antigen binding, presumably because the binding interface is outward-facing.

[0436] To investigate whether it is possible to design multispecific and multi-affinity antibodies that cross-target HIV Env and T-cell receptor CD4, we used iMab (a CD4-directed post-attachment inhibitor proven to effectively eradicate HIV). 68,69 ) replaced N49P7. A multispecific, multi-affinity antibody containing PDGM1400, iMab, 10E8v4, and Fc fragments (referred to as 32-I) exhibits similar homogeneity, thermostability, and multispecificity to 32-N (Figure 3). Figure 7 and Figure 8 This highlights the powerful plug-and-play nature of multispecific and multi-affinity antibody platforms that allow for easy exchange of antibody sequences to alter specificity.

[0437] HIV-1 multispecific multi-affinity antibodies exhibited anomalous pan-neutralizing activity and potency. In the 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 antidrug antibody profile of the multispecific multi-affinity antibody were similar to those of the corresponding IgG. We then examined the in vivo toxicity, immunogenicity, and bioavailability of the multispecific multi-affinity antibody after subcutaneous administration of 5 mg / kg to mice. To evaluate our novel platform technology, we used a species-matched alternative multispecific multi-affinity antibody composed of mouse Fab and mouse Fc (IgG2a isotype) fused to mouse aferroferrin subunits, the opposite of the fully human component used for HIV-1 multispecific multi-affinity antibodies intended for human use. The Fab specificity selected for this alternative multispecific multi-affinity antibody is the Fab specificity that does not bind endogenous mouse proteins, similar to HIV-1 human mAbs that do not bind endogenous human proteins. Administration of the multispecific multi-affinity antibody was well-tolerated, with no observed weight loss or signs of toxicity. The alternative multispecific multi-affinity antibody did not induce a significant immunogenic response in mice; the level of the anti-drug antibody (ADA) detected after 14 days was negligible compared to both the alternative multispecific multi-affinity antibody and its sequencing-matched IgG2a. Figure 11 b). This contrasts with highly immunogenic particles displaying malaria cyclosporin (CSP) on the surface of Helicobacter pylori ferritin, which were used as a positive control for immunogenicity. Furthermore, the alternative multispecific multi-affinity antibody showed similar in vivo exposure days to parental IgG1 and IgG2a molecules within a similar range. Figure 11 b). By introducing LALAP mutations that have been reported to silence Fc effector function. 74 This can prolong serum half-life. In summary, these data demonstrate the feasibility of tuning the bioavailability properties of our multispecific, multi-affinity antibody platform and provide an exciting set of initial in vivo validation data for development.

[0443] References

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Claims

1. A fusion protein, said fusion protein comprising: The first nanocage monomer subunit of the nanocage monomer; and The bioactive portion connected to the first nanocage monomer subunit; The fusion protein therein self-assembles with a protein including a second nanocage monomer subunit to form a nanocage monomer.

2. The fusion protein according to claim 1, wherein the bioactive portion modifies the inner and / or outer surface of the assembled nanocage.

3. The fusion protein according to claim 1 or 2, wherein the bioactive portion comprises: Antibody or fragment thereof, antigen, detectable part, drug, diagnostic agent or combination thereof.

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

5. The fusion protein according to claim 4, wherein the Fc fragment is an IgG1 Fc fragment.

6. The fusion protein according to claim 4 or 5, wherein the Fc fragment comprises one or more mutations, such as LS, YTE, LALA and / or LALAP, said one or more mutations regulating the half-life of the fusion protein, for example from minutes or hours to days, weeks or months.

7. The fusion protein of claim 3, wherein the antibody or a 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 the heavy chain and / or light chain of the 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