Multivalent and multispecific nanoparticle platforms and methods

Abstract

To provide nanoparticle subunit fusion proteins, vaccines, prophylactics, and therapeutics comprising nanoparticles, and related compositions and methods.SOLUTION: The fusion protein comprises: a first nanocage monomer subunit of a nanocage monomer; and a biologically active moiety linked to the first nanocage monomer subunit; wherein the fusion protein self-assembles with a protein comprising a second nanocage monomer subunit to form a nanocage monomer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to nanoparticles, particularly to nanoparticle-subunit fusion proteins, nanoparticle-containing vaccines, prophylactics, and therapeutics, and related compositions and methods. [Background technology]

[0002] Nanoparticles have contributed to advances in a variety of areas: their use has the potential to provide targeted delivery, enable ordered microarrays for catalytic processes, controlled release, and enable the engineering of caged microenvironments.

[0003] Protein self-assembly is an attractive method for the production of nanoparticles containing sensitive and metastable proteins. Indeed, self-assembled nanoparticles form under physiological conditions through noncovalent interactions, reliably resulting in uniform and often symmetric nanocapsules or nanocages. Self-assembled protein nanoparticles possess three distinct surfaces: exterior, interior, and intersubunit surfaces, all of which can be tailored to carry added functionality.

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

[0005] There is a need for improved compositions and methods involving nanocages. Summary of the Invention

[0006] In aspects, described herein are fusion proteins and self-assembled nanocages, as well as related compositions and methods, that allow for the presentation and coordination of multiple cargoes, e.g., multiple copies of the same and / or different cargoes, on a single nanoparticle. In some embodiments, the fusion proteins, nanocages, compositions, and methods of the present disclosure allow for control over the ratio of different cargo molecules, e.g., optimizing the self-assembled nanocage for specific therapeutic and / or prophylactic purposes.

[0007] According to one aspect, a first nanocage monomer subunit of the nanocage monomer; and a bioactive moiety linked to the first nanocage monomer subunit; A fusion protein comprising: The fusion protein self-assembles with a protein containing a second nanocage monomer subunit to form a nanocage monomer.

[0008] In some embodiments, bioactive moieties decorate the interior and / or exterior surfaces of the organized nanocages.

[0009] In some embodiments, the biologically active moiety comprises an antibody or fragment thereof, an antigen, a detectable moiety, a pharmaceutical agent, a diagnostic agent, or a combination thereof.

[0010] In certain embodiments, the antibody or fragment thereof comprises an Fc fragment.

[0011] In one embodiment, the Fc fragment is an IgG1 Fc fragment.

[0012] In certain embodiments, the Fc fragment comprises one or more mutations, such as 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.

[0013] In some embodiments, the antibody or fragment thereof comprises a Fab fragment.

[0014] In certain embodiments, the antibody or fragment thereof comprises an scFab fragment, an scFv fragment, or an sdAb fragment.

[0015] In certain embodiments, the antibody or fragment thereof comprises the heavy and / or light chain of a Fab fragment.

[0016] In certain embodiments, the antibody or fragment thereof comprises both a light chain and a heavy chain, or in the case of an Fc fragment, a first and second chain, optionally separated by a linker.

[0017] In some embodiments, the linker comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following: [ka]

[0018] In one embodiment, the fusion protein is in combination with a separately produced Fab light chain and / or heavy chain.

[0019] In some embodiments, the antibody or fragment thereof specifically binds to an antigen associated with an antibody-preventable and / or antibody-treatable condition.

[0020] In some embodiments, 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, including solid and liquid cancers (e.g., CD19, CD22, CD79, BCMA, or CD20); or an immune disease, including an autoimmune disease.

[0021] In some embodiments, the antigen is related to 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.

[0022] In some embodiments, the antibody or fragment thereof comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to: Fc chain 1: [ka] Fc chain 2: [ka] Ibalizumab-A12P light chain: [ka] Ibalizumab-A12P heavy chain: [ka] 10E8.v4 light chain: [ka] 10E8.v4 heavy chain: [ka] N49P7 light chain: [ka] N49P7 heavy chain: [ka] PGDM1400 light chain: [ka] PGDM1400 heavy chain: [ka] or A combination of them.

[0023] In some embodiments, the antibody or fragment thereof is conjugated or associated with a further moiety, such as an antigen, a detectable moiety (e.g., a small molecule, fluorescent molecule, radioisotope, or magnetic particle), a pharmaceutical agent, a diagnostic agent, or a combination thereof.

[0024] In some embodiments, the antibody or fragment thereof comprises an antibody-drug conjugate.

[0025] In some embodiments, the antigen is associated with a vaccine-preventable and / or vaccine-treatable condition.

[0026] In some embodiments, the antigen is associated with an infectious agent, including a virus, bacterium, parasite, fungus, or yeast; a cancer, including solid and liquid cancers; or an immune disease, including an autoimmune disease.

[0027] In some embodiments, the detectable moiety comprises a flavin-based fluorescent protein, such as a fluorescent protein, such as GFP, EGFP, ametrine, and / or an LOV protein, such as iLOV.

[0028] In some embodiments, the pharmaceutical agent comprises a small molecule, peptide, lipid, carbohydrate, or toxin.

[0029] In some embodiments, about 3 to about 100 nanocage monomers, e.g., 24, 32, or 60 monomers, or about 4 to about 200 nanocage monomer subunits, e.g., 4, 6, 8, 10, 12, 14, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 or more, optionally in combination with one or more whole nanocage monomers, self-assemble to form a nanocage.

[0030] In certain embodiments, the nanocage monomer is selected from ferritin, apoferritin, encapsulin, SOR, lumazine synthase, pyruvate dehydrogenase, carboxysome, vault protein, GroEL, heat shock protein, E2P, MS2 coat protein, fragments thereof, and variants thereof.

[0031] In some embodiments, the nanocage monomer is apoferritin.

[0032] In some embodiments, the first and second nanocage monomer subunits interchangeably comprise the "N" and "C" regions of apoferritin.

[0033] In some embodiments, the "N" region of apoferritin comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following: [ka]

[0034] In some embodiments, the "C" region of apoferritin comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following: [ka]

[0035] In some embodiments, the fusion protein further comprises a linker between the nanocage monomer subunit and the biologically active moiety.

[0036] In one embodiment, the linker is flexible or rigid and comprises from about 1 to about 30 amino acid residues, for example, from about 8 to about 16 amino acid residues.

[0037] In some embodiments, the linker comprises GGS repeats, for example, 1, 2, 3, 4 or more GGS repeats.

[0038] In some embodiments, the linker comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following: [ka]

[0039] In certain embodiments, the fusion protein further comprises a C-terminal linker.

[0040] In some embodiments, the C-terminal linker comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following: [ka]

[0041] According to one aspect, there is provided a pair of fusion proteins as described herein, which self-assemble to form a nanocage monomer, and wherein the first and second nanocage monomer subunits are fused to different biologically active moieties.

[0042] According to certain embodiments, a nanocage is provided that includes 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.

[0043] According to certain embodiments, there is provided a nanocage comprising at least one pair described herein.

[0044] In some aspects, each nanocage monomer comprises a fusion protein or pair described herein.

[0045] In some embodiments, between about 20% and about 80% of the nanocage monomers comprise a fusion protein or pair described herein.

[0046] In some embodiments, the fusion protein comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different biologically active moieties.

[0047] In some embodiments, the nanocage comprises at least one whole nanocage monomer, optionally fused to a bioactive moiety, which may be the same as or different from a bioactive moiety described herein.

[0048] In some embodiments, the nanocages are multivalent and / or multispecific.

[0049] In one aspect, the nanocage comprises first, second, and third fusion proteins described herein and at least one whole nanocage monomer, optionally fused to a biologically active moiety, wherein the biologically active moieties of the first, second, and third fusion proteins and the biologically active moiety of the whole nanocage monomer are all different from one another.

[0050] In one embodiment, the first, second, and third fusion proteins each comprise an antibody or fragment thereof fused to N- 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.

[0051] In one embodiment, the antibody or fragment thereof of the first fusion protein is an Fc fragment; the second and third fusion proteins each comprise an antibody or fragment thereof specific for a different antigen of a virus, such as HIV, or one of the second and third fusion proteins comprises an antibody or fragment thereof specific for an antigen of a virus, such as HIV, and the third fusion protein comprises an antibody or fragment thereof specific for a different antigen, such as the CD4 receptor; and the entire nanocage monomer is optionally fused to a biologically active portion specific for another, different antigen of the same virus, such as HIV.

[0052] In certain embodiments, the Fc fragment comprises one or more mutations, such as 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.

[0053] In one embodiment, the antibody or fragment thereof of the second fusion protein is N49P7 or iMab A12P; and the antibody or fragment thereof of the third fusion protein is 10E8v4.

[0054] In one embodiment, the nanocage comprises or consists of the following four fusion proteins: a. PGDM1400 fused to full-length ferritin (possibly scPGDM1400); bFc (optionally scFc) fused to N-ferritin; N49P7 or iMab A12P (optionally scN49P7 or sciMab A12P) fused to cC-ferritin; and 10E8v4 (occasionally sc10E8v4) fused to dC-ferritin.

[0055] In some embodiments, the nanocages comprise a:b:c:d in a ratio of 4:2:1:1.

[0056] In some embodiments, the nanocage comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to one or more of the following sequences, where ferritin subunits are in bold, linkers are underlined, light chains are in italics, and heavy chains are in lowercase: a.PGDM1400-hFerr: [ka] b.Fc-N-hFerr LS [ka] c1.N49P7-C-hFerr [ka] c2. Ibalizumab-A12P-C-hFerr [ka] or d.10E8.v4-C-hFerr [ka]

[0057] In some embodiments, the nanocages carry cargo molecules such as pharmaceuticals, diagnostic agents, and / or imaging agents.

[0058] In some embodiments, the cargo molecule is not fused to a fusion protein but is contained within the nanocage.

[0059] In some embodiments, the cargo molecule is a protein and is fused to a fusion protein such that the cargo molecule is contained within the nanocage.

[0060] In some embodiments, the cargo molecule is a flavin-based fluorescent protein, such as a fluorescent protein, such as GFP, EGFP, ametrine, and / or an LOV protein, such as iLOV.

[0061] In some embodiments, a cargo molecule may be included to provide a T cell epitope, but not a B cell epitope.

[0062] In one embodiment, the cargo molecule is fused to and contained within the fusion protein to provide a T cell epitope, but may not provide a B cell epitope.

[0063] In some embodiments, the cargo molecule is a small molecule, a radioisotope, or a magnetic particle.

[0064] In some embodiments, the nanocage further comprises an antigen on the surface.

[0065] In some embodiments, the antigen is expressed as a fusion protein with the nanocage monomer.

[0066] According to certain aspects, there is provided a vaccine comprising the nanocages described herein.

[0067] In accordance with certain aspects, there are provided therapeutic or prophylactic compositions comprising the nanocages described herein.

[0068] In one aspect, there is provided a nucleic acid molecule encoding a fusion protein or pair described herein.

[0069] In accordance with one aspect, a vector is provided that includes a nucleic acid molecule described herein.

[0070] In one aspect, a host cell is provided that comprises a vector described herein and produces a fusion protein or pair described herein.

[0071] According to certain aspects, methods of immunizing a subject are provided, the methods comprising administering a nanocage or vaccine described herein.

[0072] According to certain aspects, methods are provided for treating and / or preventing a disease or condition, the methods comprising administering a nanocage or vaccine described herein.

[0073] In some embodiments, the disease or condition is cancer, an infectious disease such as HIV, malaria, influenza, RSV, rotavirus, or an autoimmune disease.

[0074] According to certain aspects, methods for diagnostic imaging are provided, the methods comprising administering to a subject, tissue, or sample a nanocage as described herein, wherein the nanocage comprises a diagnostic label, such as a fluorescent protein or a magnetic imaging moiety, and imaging the subject, tissue, or sample.

[0075] In accordance with certain aspects, there is provided a use of a nanocage or vaccine described herein for immunizing a subject.

[0076] According to certain aspects, there is provided a use of a nanocage or vaccine described herein for treating and / or preventing a disease or condition.

[0077] In some embodiments, the disease or condition is cancer, an infectious disease such as HIV, malaria, influenza, RSV, rotavirus, or an autoimmune disease.

[0078] According to certain aspects, there is provided a use of the nanocages described herein for diagnostic imaging of a subject, tissue, or sample, wherein the nanocage comprises a diagnostic label, such as a fluorescent protein or a magnetic imaging moiety, to image the subject, tissue, or sample.

[0079] In one aspect, there is provided the use of a fusion protein, pair, or nanocage described herein as a research tool, such as in FACS or ELISA.

[0080] In accordance with certain aspects, there is provided a nanocage or vaccine described herein for use in immunizing a subject.

[0081] According to certain aspects, there is provided a nanocage or vaccine as described herein for use in treating and / or preventing a disease or condition.

[0082] In some embodiments, the disease or condition is cancer, an infectious disease such as HIV, malaria, influenza, RSV, rotavirus, or an autoimmune disease.

[0083] In accordance with certain aspects, there is provided a nanocage as described herein for use in diagnostic imaging of a subject, tissue, or sample, wherein the nanocage comprises a diagnostic label, such as a fluorescent protein or a magnetic imaging moiety, to image the subject, tissue, or sample.

[0084] In certain aspects, there is provided a fusion protein, pair, or nanocage described herein for use as a research tool, such as in FACS or ELISA.

[0085] According to one aspect, a nanocage is provided comprising a plurality of fusion proteins, Each fusion protein comprises a ferritin light chain and a Fab fragment, Each Fab fragment is capable of specifically binding to an antigen, Each Fab fragment decorates the outer surface of the nanocage, The plurality comprises at least 12 fusion proteins.

[0086] In some embodiments, the plurality comprises at least 19 fusion proteins.

[0087] In some embodiments, the plurality comprises at least 24 fusion proteins.

[0088] In one embodiment, the plurality is 24 fusion proteins.

[0089] In certain embodiments, the Fab fragments of the fusion proteins are capable of specifically binding to the same antigen.

[0090] In some embodiments, the nanocage does not include any ferritin heavy chains.

[0091] In one embodiment, the Fab fragment is a Fab fragment of a neutralizing antibody.

[0092] In some embodiments, the antigen is associated with an infectious agent.

[0093] In some embodiments, the infectious agent is a virus.

[0094] In some embodiments, the virus is human immunodeficiency virus (HIV).

[0095] In certain embodiments, the nanocages can neutralize an infectious agent with at least 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, or 500-fold greater neutralizing activity compared to a control.

[0096] In some embodiments, the control comprises a full-length version of a neutralizing antibody.

[0097] In some embodiments, the neutralizing antibody is an IgG antibody.

[0098] According to one aspect, there is provided a nanocage comprising a plurality of first fusion proteins and a plurality of second fusion proteins, each first fusion protein comprises a nanocage monomer or a subunit thereof and a Fab fragment capable of specifically binding to an antigen; Each second fusion protein comprises a nanocage monomer or a subunit thereof and an Fc fragment.

[0099] In certain embodiments, the nanocage monomer is selected from ferritin, apoferritin, encapsulin, sulfur oxygen reductase (SOR), lumazine synthase, pyruvate dehydrogenase, carboxysome, vault protein, GroEL, heat shock protein, E2P, MS2 coat protein, fragments thereof, and variants thereof.

[0100] In some embodiments, the nanocage monomer is apoferritin or ferritin.

[0101] In some embodiments, the nanocage monomer is a ferritin light chain.

[0102] In some embodiments, the nanocage monomer does not contain any ferritin heavy chains.

[0103] According to one aspect, there is provided a nanocage comprising a plurality of first fusion proteins and a plurality of second fusion proteins, (a)(i) a first fusion protein comprising a ferritin light chain and a Fab fragment capable of specifically binding to a first antigen; (ii) the second fusion protein comprises a ferritin light chain and a Fab fragment capable of specifically binding to a second antigen; or (b)(i) a first fusion protein comprising N-ferritin and a Fab fragment capable of specifically binding to a first antigen; (ii) a second fusion protein comprising C-ferritin and a Fab fragment capable of specifically binding to a second antigen; Within each fusion protein, the Fab fragment is fused to the N-terminus of ferritin light chain, N-ferritin, or C-ferritin; The first antigen is different from the second antigen.

[0104] According to one aspect, there is provided a nanocage comprising a plurality of first fusion proteins, a plurality of second fusion proteins, and a plurality of third fusion proteins; (a) a first fusion protein comprising a ferritin light chain and a Fab fragment capable of specifically binding to a first antigen; (b) a second fusion protein comprising C-ferritin and a Fab fragment capable of specifically binding to a second antigen; (c) a third fusion protein comprising N-ferritin and an Fc fragment; Within each fusion protein, the Fab or Fc fragment is fused to the N-terminus of ferritin light chain, C-ferritin, or N-ferritin; The first antigen is different from the second antigen.

[0105] In some embodiments, the nanocage further comprises a plurality of fourth fusion proteins, the fourth fusion proteins comprising C-ferritin and a Fab fragment capable of specifically binding to a third antigen, the third antigen being different from the first and second antigens.

[0106] In one embodiment, the Fab fragment is a Fab fragment of a neutralizing antibody.

[0107] In some embodiments, the first and second antigens are each associated with an infectious agent.

[0108] In some embodiments, the first and second antigens are associated with the same infectious agent.

[0109] In some embodiments, the infectious agent is a virus.

[0110] In some embodiments, the virus is human immunodeficiency virus (HIV).

[0111] In one embodiment, the first and second antigens are each associated with a virus; The nanocages were able to neutralize 100% of the pseudoviruses in the panel of pseudoviruses. The panel of pseudoviruses includes, for each Fab fragment within the nanocage that can specifically bind to an antigen associated with the virus, at least one pseudovirus that is resistant to the neutralizing antibody corresponding to that Fab fragment.

[0112] In certain embodiments, the panel of pseudoviruses includes at least 10, at least 11, at least 12, at least 13, or at least 14 pseudoviruses.

[0113] In one embodiment, the first and second antigens are each associated with a virus; The nanocages have an IC of less than 1 nM, less than 500 pM, less than 250 pM, less than 100 pM, less than 50 pM, less than 10 pM, or less than 5 pM. 50 can neutralize a panel of pseudoviruses, The panel of pseudoviruses includes, for each Fab fragment within the nanocage that can specifically bind to an antigen associated with the virus, at least one pseudovirus that is resistant to the neutralizing antibody corresponding to that Fab fragment.

[0114] In one embodiment, the first and second antigens are each associated with a virus; The nanocages contain one or more control ICs. 50 an IC at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold lower than (molar concentration) 50 can neutralize a panel of pseudoviruses, The panel of pseudoviruses includes, for each Fab fragment within the nanocage that can specifically bind to an antigen associated with the virus, at least one pseudovirus that is resistant to the neutralizing antibody corresponding to that Fab fragment.

[0115] In one embodiment, the one or more controls comprise a neutralizing antibody corresponding to a Fab fragment within the nanocage, the Fab fragment being capable of specifically binding to an antigen associated with the virus.

[0116] In some embodiments, the neutralizing antibody is an IgG antibody.

[0117] In one embodiment, the one or more controls comprise a cocktail of neutralizing antibodies, where for each Fab fragment within the nanocage capable of specifically binding to an antigen associated with the virus, the cocktail comprises a neutralizing antibody corresponding to that Fab fragment.

[0118] In some embodiments, the neutralizing antibody is an IgG antibody.

[0119] In certain aspects, the one or more controls comprise one or more multispecific antibodies, wherein the one or more multispecific antibodies are capable of collectively binding to the first and second antigens, and optionally a third antigen.

[0120] In certain embodiments, the one or more controls comprise a trispecific antibody capable of specifically binding to the first, second, and third antigens.

[0121] In one embodiment, the first, second, and third antigens are associated with HIV-1; the Fab fragment of the first fusion protein is PDGM1400 Fab; the Fab fragment of the second fusion protein is 10E8v4 Fab; the Fc fragment of the third fusion protein is a human IgG1 Fc fragment; The Fab fragment of the fourth fusion protein is N49P7 Fab.

[0122] In one embodiment, the first and second antigens are associated with HIV-1 and the third antigen is associated with CD4; the Fab fragment of the first fusion protein is PDGM1400 Fab; the Fab fragment of the second fusion protein is 10E8v4 Fab; the Fc fragment of the third fusion protein is a human IgG1 Fc fragment; The Fab fragment of the fourth fusion protein is an iMab Fab.

[0123] In accordance with certain aspects, there are provided therapeutic or prophylactic compositions comprising the nanocages described herein.

[0124] According to certain aspects, methods are provided for treating or preventing a disease or condition, the methods comprising administering to a subject in need thereof a nanocage or composition described herein.

[0125] According to certain aspects, there is provided a method of making multispecific self-assembled nanocages characterized by a preselected ratio of different specificities, the method comprising the steps of: co-transfecting a host cell with one or more expression plasmids comprising multiple polynucleotides each encoding a fusion protein; Each fusion protein comprises (i) a nanocage monomer or a subunit thereof, and (ii) an antibody or antibody fragment of a given specificity; The step of co-transfecting comprises co-transfecting the polynucleotides in a ratio based on a preselected ratio; Obtaining the polypeptide produced by the host cell; and Purifying the polypeptides by affinity selection against all the different specificities present in the assembled nanocages.

[0126] In one embodiment, the plurality of polynucleotides comprises at least one polynucleotide encoding a first fusion protein and at least one polynucleotide encoding a second fusion protein; the first fusion protein comprises a first nanocage monomer subunit; The second fusion protein comprises a second nanocage monomer subunit capable of self-assembling with the first nanocage monomer subunit.

[0127] The novel features of the present invention will become apparent to those skilled in the art upon examination of the following detailed description of the invention. It should be understood, however, that the detailed description of the invention and the specific examples presented, while indicating certain particular aspects of the invention, 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 from the detailed description of the invention and the claims that follow.

[0128] The invention will be better understood from the following description taken in conjunction with the drawings. [Brief explanation of the drawings]

[0129] [Figure 1] Schematic of the self-assembly of the multispecific, multi-affinity antiBODY (multabody) platform. A single-chain Fab (light chain (LC) and heavy chain (HC) in light and dark pink, respectively) and a single-chain Fc region (green) are linked to the N-terminus of the light chain of human apoferritin (gray) via a GGS-like flexible linker (dark). The 24 subunits of apoferritin self-assemble into a 12 nm spherical core surrounded by spatially dispersed antibody fragments. [Figure 2a] Characterization of HIV-1 multabodies with different valencies. Schematic diagram of different Fab densities displayed on human apoferritin. Cotransfection of a plasmid encoding scFab-human apoferritin with unconjugated apoferritin at different ratios resulted in scFab valencies of 5 (dark yellow), 12 (black), 19 (blue), and 24 (red), as confirmed by earlier elution volumes and less unconjugated apoferritin in size-exclusion chromatography and SDS-PAGE, respectively. Negative-stain electron micrographs of samples with the lowest (20%) and highest (100%) valencies are shown. [Figure 2b] Characterization of HIV-1 multabodies with different valencies. Avidity effect of five bNAbs for neutralization against a panel of five PsVs (PVO.04, JRCSF, BG505 T332N, THRO4156.18, and t278-50). Fold-increase in IC50 analysis was omitted in the following cases due to neutralization resistance: N49P7-t278-50, VRC01-T278-50, and 10-1074-THRO4156.18. Fold-increase in potency was calculated as the IC50 (nM) of the parental IgG divided by the IC50 (nM) of the multabody. [Figure 3a]Design, assembly, and biophysical characterization of 32-N and 32-I multabodies. Schematic diagram of the design of a split human apoferritin fragment to promote heterodimerization of the scFab-human apoferritin subunit. The resulting two halves, designated N-ferritin and C-ferritin, span residues 1–95 and 95–175, respectively. An scFc fragment was linked to the N-terminus of the N-ferritin half, while N49P7, iMab (also referred to herein as ibalizumab), and 10E8v4 were linked to the N-terminus of the C-ferritin half. Heterodimerization of the split halves drives self-assembly of the different antibody fragments, resulting in the formation of a single human apoferritin subunit bearing two cargoes. Further combination of these constructs with PGDM1400 scFab linked to intact human apoferritin subunits resulted in nanoparticle assemblies displaying a mixture of 32 scFab / scFc species on the surface of multabodies. Negative-stain electron micrographs, model depictions of the scFab / scFc 32-N / 32-I design, and the specific composition of the 32-N and 32-I multabodies are shown. Based on the hetero-oligomerization required to drive self-assembly, purification of the four-component multabodies can be achieved by a two-step purification: Protein A (Fc binding) and Protein L (PGDM1400 binding). [Figure 3b] Design, assembly, and biophysical characterization of 32-N and 32-I multabodies. Multi-angle light scattering and in-line size-exclusion chromatography of 24-mer PGDM1400 multabodies (black), 32-N multabodies (dark magenta), and 32-I multabodies (blue). The molar mass of each elution peak (line below UV absorbance) indicates that the samples are monodisperse and that the 32-N / 32-I multabodies are significantly larger than the 24-mer format due to the additional antibody fragment in this design. [Figure 3c]Design, assembly, and biophysical characterization of 32-N and 32-I multabodies. Comparison of Tm and Tagg temperatures of 32-N / 32-I multabodies, 12-mer multabodies, parental IgG, and N6 / PGDM1400x10E8v4 trispecific antibody. [Figure 3d] Design, assembly, and biophysical characterization of 32-N and 32-I multabodies. Concentration-response curves for binding of 32-N and 32-I multabodies to multiple epitopes. The binding sites of PGDM1400, N49P7, and 10E8 are colored red, blue, and pink, respectively, on the surface representation of HIV Env (gray). iMab binding was assessed using soluble CD4, while functional binding of Fc to human FcRn was tested by measuring binding at pH 7.5 and pH 5.6. BG505 SOSIP.664_D368R trimer and 93TH057 gp120 monomer were selected as epitope-specific ligands for PGDM1400 and N49P7, respectively. [Figure 4a] Effect of the segmentation design on the biophysical and functional properties of multabodies. Comparison of 12-mer multabodies composed of six copies of PGDM1400 and six copies of Fc multimerized with the intact apoferritin subunit (left panel) or half-ferritin (Fc bound to the N-ferritin half and PGDM1400 bound to the C-ferritin half; right panel). Biolayer interferometry (BLI) concentration-response curves for the binding of BG505.664 to 12-mer multabodies loaded onto an anti-hIgG Fc capture (AHC) biosensor. [Figure 4b]Effect of the split design on the biophysical and functional properties of multabodies. Comparison of 12-mer multabodies composed of six copies of PGDM1400 and six copies of Fc multimerized with the intact apoferritin subunit (left panel) or half-ferritin (Fc bound to the N-ferritin half and PGDM1400 bound to the C-ferritin half; right panel). Bent-centered mean fluorescence (BCM) and static light scattering (SLS) at 266 nm versus temperature plots. Tm and Tag are indicated by yellow lines. [Figure 4c] Effect of the split design on the biophysical and functional properties of multabodies. Comparison of 12-meric multabodies composed of six copies of PGDM1400 and six copies of Fc multimerized with the intact apoferritin subunit (left panel) or half-ferritin (Fc bound to the N-ferritin half and PGDM1400 bound to the C-ferritin half, right panel). Neutralization assay against BG505 T332N PsV. [Figure 5] Multabody affinity purification scheme. Sequential affinity purification of Protein A and Protein L. Protein A binding enriches multabodies with Fc (green), while Protein L enriches multabodies with the kappa chain Fab PGDM1400 (blue). An alanine-to-proline point mutation at position 12 of the iMab kappa chain disrupted binding to Protein L. Complementarity of the two halves of human apoferritin ensures the presence of N49P7 / iMab (orange) and 10E8 scFab (pink) (fused to C-ferritin) during the Protein A purification step. Gel filtration is performed to separate any aggregated material. [Figure 6a] Batch-to-batch variation of 32-N multabody is minimal. SEC chromatogram. [Figure 6b]Batch-to-batch variation of 32-N multabody is minimal. BCM and SLS at 266 nm versus temperature plot. Thermal transition temperatures (Tm and Tagg) are shown by the yellow lines. [Figure 6c] Batch-to-batch variation of the 32-N multabody is minimal. Concentration-response curve for binding of N49P7 to 93TH057 gp120 in 32-N. [Figure 6d] Batch-to-batch variation of the 32-N multabody is minimal. Neutralization profile of 32-N against a panel of four PsVs selected to include one PsV resistant to each Fab in the multabody. [Figure 7] Thermal stability analysis. BCM (upper panel) and SLS (lower panel) at 266 nm versus temperature plots for 32-N and 32-I multabodies, their individual 12-mer multabodies, parental IgG, and N6 / PGDM1400x10E8 trispecific antibody. Thermal transition temperatures (Tm and Tagg) are indicated by yellow lines. [Figure 8] Binding profiles of bNAbs PGDM1400, 10E8v4, N49P7, and iMab. BLI response curves of IgG binding to 93TH057 gp120, BG505 SOSIP.664_D368R, MPER-mVenus, and CD4 immobilized on Ni-NTA biosensors. [Figure 9]Neutralization properties of 32-N and 32-I multabodies against a panel of 14 pseudoviruses. Breadth and median IC50 values ​​(μg / mL) for multabodies (red diamonds), parental bNAbs (black circles), IgG combinations (black triangles), and the N6 / PGDM1400x10E8v4 trispecific antibody (black squares). The IgG cocktail contained the same relative amounts of each parental antibody as the multabody samples (i.e., 66% PGDM1400, 17% N49P7 / iMab, and 17% 10E8v4). The 14-PsV panel was selected based on sensitivity and resistance to the parental IgGs. [Figure 10] Neutralization properties of 32-N and 32-I multabodies against a panel of 14 pseudoviruses. Breadth and median IC50 (nM) of multabodies (red diamonds), parental bNAb (black circles), IgG combinations (66% PGDM1400, 17% N497 / iMab, and 17% 10E8v4; black triangles), and N6 / PGDM1400x10E8v4 trispecific antibody (black squares). [Figure 11] Immunogenicity and exposure of multabodies in mice. Five male C57BL / 6 mice per group were used to assess circulating anti-drug antibodies and multabodies in the blood after subcutaneous administration of 5 mg / kg of murine surrogate multabody and Fc-modified multabody (LALAP mutation to disrupt Fc receptor binding). Reference samples Hp ferritin malaria PfCSP peptide and parental murine IgG1 and IgG2a isotypes were used for immunogenicity and exposure comparisons, respectively.

[0130] Detailed Description of Certain Embodiments definition Unless otherwise explained, 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 belongs. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, (published by Oxford University Press), 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, (published by Blackwell Science Ltd.), 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, (published by VCH Publishers, Inc.), 1995 (ISBN 1-56081-569-8). Although any methods and materials similar or equivalent to those described herein can be used in carrying out the tests of the present invention, exemplary materials and methods are described herein. In describing and claiming the present invention, the following terminology is used.

[0131] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. Numerous patent applications, patents, and publications are referenced herein to aid in the understanding of the described aspects. Each of these references is incorporated herein by reference in its entirety.

[0132] In understanding the scope of this application, the articles "a," "an," "the," and "said" are intended to mean that there are one or more elements. Furthermore, the term "comprising," and its derivatives, as used herein, are intended to be open-ended terms that specify the presence of stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other, unstated features, elements, components, groups, integers, and / or steps. The above also applies to words of similar meaning, such as the terms "including," "having," and their derivatives.

[0133] Any embodiment described as "comprising" certain components may also be "consist of" or "consist essentially of," with "consisting of" having a closed-ended or restrictive meaning, and "consisting essentially of" being understood to mean including the specified components but excluding other components, excluding materials present as impurities, unavoidable materials present as a result of the process used to provide the components, and components added for purposes other than achieving the technical effects of the invention. For example, a composition defined using the phrase "consisting essentially of" encompasses any known acceptable additives, excipients, diluents, carriers, etc. Typically, a composition consisting essentially of a set of components contains less than 5% by weight, typically less than 3% by weight, more typically less than 1% by weight, and even more typically less than 0.1% by weight of unspecified component(s).

[0134] It will be understood that any component defined herein as included may be expressly excluded from the claimed invention by condition or negative limitation. For example, in some embodiments, the nanocages and / or fusion proteins described herein may exclude ferritin heavy chains and / or may exclude iron-binding components.

[0135] Moreover, all ranges given herein include the range endpoints and any intermediate range points, whether or not expressly stated.

[0136] Terms of degree, such as "substantially," "about," and "approximately," as used herein, refer to a reasonable amount of deviation from the modified term so that the end result does not change significantly. These terms of degree should be interpreted as including a deviation of at least ±5% of the modified term if this deviation does not negate the meaning of the word it modifies.

[0137] Furthermore, it should 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 are provided for illustrative purposes. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "eg" is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example." The word "or" is intended to include "and" unless the context clearly dictates otherwise.

[0138] The terms "protein nanoparticle," "nanocage," and "multabody" are used interchangeably herein to refer to multi-subunit, protein-based, polyhedral-shaped structures. Each subunit or nanocage monomer is composed of a protein or polypeptide (e.g., a glycosylated polypeptide), and optionally one or more of the following: nucleic acids, prosthetic groups, organic and inorganic compounds. Non-limiting examples of protein nanoparticles include ferritin nanoparticles (see, e.g., Zhang, Y. Int. J. Mol. Sci., 12:5406-5421, 2011, which is incorporated herein by reference), encapsulin nanoparticles (see, e.g., Sutter et al., Nature Struct., and Mol. Biol., 15:939-947, 2008, which is incorporated herein by reference), sulfur oxygenase reductase (SOR) nanoparticles (see, e.g., Urich et al., Science, 311:996-1000, 2006, which is incorporated herein by reference), lumazine synthase nanoparticles (see, e.g., Zhang et al., J. Mol. Biol., 306:1099-1114, 2001), or pyruvate dehydrogenase nanoparticles (see, e.g., Izard et al., PNAS 96:1240-1245, 1999). Ferritin, apoferritin, encapsulin, SOR, lumazine synthase, and pyruvate dehydrogenase are monomeric proteins that in some cases self-assemble into globular protein complexes consisting of 24, 60, 24, 60, and 60 protein subunits, respectively. Ferritin and apoferritin are generally referred to interchangeably herein, and it is understood that both are suitable for use in the fusion proteins, nanocages, and methods described herein. Carboxysomes, vault proteins, GroEL, heat shock proteins, E2P, and MS2 coat proteins also generate nanocages contemplated for use herein. Additionally, fully or partially synthetic self-assembling monomers are also contemplated for use herein.

[0139] It will be understood that each nanocage monomer can be split into two or more subunits that self-assemble into functional nanocage monomers. For example, ferritin or apoferritin can be split into N- and C-subunits, e.g., N- and C-subunits obtained by essentially splitting full-length ferritin in half, such that each subunit can be separately bound to a different bioactive moiety for subsequent self-assembly into nanocage monomers and then into nanocages. By "functional nanocage monomer," it is intended that the nanocage monomer can self-assemble with other such monomers into the nanocage described herein.

[0140] The terms "ferritin" and "apoferritin" are used interchangeably herein and generally refer to polypeptides (e.g., ferritin chains) that can assemble into ferritin complexes, which typically contain 24 protein subunits. It will be understood that the ferritin can be derived from any species. Typically, the ferritin is human ferritin. In some embodiments, the ferritin is wild-type ferritin. For example, the ferritin may be wild-type human ferritin. In some embodiments, ferritin light chains are used as nanocage monomers, and / or subunits of ferritin light chains are used as nanocage monomer subunits. In some embodiments, the assembled nanocages do not contain ferritin heavy chains or other ferritin components capable of binding iron.

[0141] The term "multispecificity," as used herein, refers to the characteristic of having at least two binding sites capable of binding to at least two different binding partners, e.g., antigens or receptors (e.g., Fc receptors). For example, a nanocage comprising at least two Fab fragments, each of which binds to a different antigen, is "multispecific." As a further example, a nanocage comprising an Fc fragment (capable of binding to an Fc receptor) and a Fab fragment (capable of binding to an antigen) is "multispecific."

[0142] The term "multivalent," as used herein, refers to the characteristic of having at least two binding sites capable of binding to a binding partner, e.g., an antigen or a receptor (e.g., an Fc receptor). The binding partners capable of binding to the at least two binding sites can be the same or different.

[0143] A "vaccine" is a pharmaceutical composition that induces a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine induces an antigen-specific immune response against an antigen of a pathogen (e.g., a viral pathogen) or a cellular component that correlates with a pathological symptom. A vaccine can comprise a polynucleotide (e.g., a nucleic acid encoding a disclosed antigen), a peptide or polypeptide (e.g., a disclosed antigen), a virus, a cell, or one or more cellular components. In one specific, non-limiting example, a vaccine induces an immune response that reduces the severity of symptoms associated with malaria infection and / or reduces parasite load compared to a control. In another non-limiting example, a vaccine induces an immune response that reduces and / or prevents malaria or HIV infection compared to a control.

[0144] As used herein, the term "antibody" (also referred to in the art as "immunoglobulin" (Ig)) refers to a protein constructed from paired heavy and light polypeptide chains; various Ig isotypes exist, including IgA, IgD, IgE, IgG, e.g., IgG1, IgG2, IgG3, and IgG4, and IgM. It will be understood that antibodies may be derived from any species, including human, mouse, rat, monkey, llama, or shark. When antibodies are properly folded, each chain folds into several distinct globular domains linked by more linear polypeptide sequences. For example, immunoglobulin light chains are variable (V L ) and constant (CL) domains, while the heavy chain folds into variable (V H ) and three steady-state (C H , C H2 , C H3) domains. The heavy and light chain variable domains (V H and V L The interaction of these domains results in the formation of an antigen-binding domain (Fv). Each domain has a well-defined structure known to those skilled in the art.

[0145] The light and heavy chain variable regions are involved in binding to target antigens and can therefore exhibit significant sequence diversity among antibodies. The constant regions exhibit less sequence diversity and are involved in binding several natural proteins to trigger important immunological events. The variable regions of an antibody contain the antigen-binding determinants of the molecule and therefore determine the specificity of the antibody for its target antigen. The majority of sequence variability occurs in six hypervariable regions, three per variable heavy chain and three per variable light chain; the hypervariable regions combine to form the antigen-binding site and contribute to binding and recognition of antigenic determinants. The specificity and affinity of an antibody for an antigen are determined by the structure of the hypervariable regions and the size, shape, and chemistry of the surface they present to the antigen.

[0146] "Antibody fragments" as referred to herein may include any suitable antigen-binding antibody fragment known in the art. Antibody fragments may be naturally occurring antibody fragments or may be obtained by manipulation of naturally occurring antibodies or by using recombinant methods. For example, antibody fragments may include Fv, single chain Fv (scFv; V linked with a peptide linker), and the like. L and V H Fc, single-chain Fc, Fab, single-chain Fab, F(ab')2, single-domain antibody (sdAb; a molecule consisting of a single V L or V H (fragment consisting of) and multivalent presentation bodies of any of these.

[0147] As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA technology. This term should also be taken to mean an antibody produced by synthesis of a DNA molecule encoding the antibody, which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, where the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.

[0148] The term "epitope" refers to an antigenic determinant. An epitope is a specific chemical group or peptide sequence of a molecule that is antigenic, i.e., that elicits a specific immune response. An antibody specifically binds to a specific antigenic epitope of, for example, a polypeptide. Epitopes can be formed from both contiguous or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are usually retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are usually lost upon treatment with denaturing solvents. An epitope typically contains at least three, more usually at least five, about nine, about eleven, or about eight to about twelve amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, "Epitope Mapping Protocols," Methods in Molecular Biology, Vol. 66, edited by Glenn E. Morris (1996).

[0149] The term "antigen," as used herein, is defined as a molecule that elicits an immune response. This immune response may include antibody production, activation of specific immunocompetent cells, or both. Those skilled in the art will understand that any macromolecule, including virtually any protein or peptide, can act as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response encodes an "antigen," as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the embodiments described herein include, but are not limited to, the use of partial nucleotide sequences of two or more genes, and that these nucleotide sequences can be arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be synthesized or derived from a biological sample. Such a biological sample may include, but is not limited to, a tissue sample, a cell, or a biological fluid.

[0150] Thus, the compositions described herein may be suitable for the protection or treatment of vertebrate subjects against a variety of disease states, such as, for example, viral, bacterial, fungal, or parasitic infections, cancer, and autoimmune disorders, etc. It should be recognized that these specific disease states are mentioned by way of example only and are not intended to be limiting.

[0151] Suitable antigens useful in combination with the compositions described herein include any of the antigens defined herein. Antigens are commercially available or can be produced by those skilled in the art. Antigens can be either modified live or dead 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. The antigenic moiety can also be a subunit of a protein, peptide, polysaccharide, or similar product. Antigens can also be genetic antigens, i.e., DNA or RNA that elicit an immune response.

[0152] Representative antigens that can be used include, but are not limited to, natural, recombinant, or synthetic products derived from viruses, bacteria, fungi, parasites, and other infectious agents, as well as autoimmune disease, hormone, or tumor antigens that can be used in prophylactic or therapeutic vaccines and allergens. In one embodiment, the antigen comprises a virus-like particle (VLP) derived from various viruses, such as influenza, HIV, RSV, Newcastle disease virus (NDV), etc. See PCT / US2006 / 40862, PCT / US2004 / 022001, US 11 / 582,540, US 60 / 799,343, US 60 / 817,402, US 60 / 859,240, all of which are incorporated herein by reference in their entireties. In another embodiment, the antigen comprises a chimeric VLP. A "chimeric VLP" refers to a VLP comprising proteins or portions thereof from at least two different sources (organisms). Usually, one protein is derived from a virus that can drive the formation of VLPs from host cells. Thus, in one embodiment, the chimeric VLP comprises the RSV M protein. In another embodiment, the chimeric VLP comprises the NDV M protein. In another embodiment, the chimeric VLP comprises the influenza virus M protein.

[0153] Viral or bacterial products may be components of an organism produced by enzymatic cleavage, or may be components of an organism produced by recombinant DNA techniques well known to those skilled in the art.

[0154] Some specific examples of antigens include hepatitis viruses A, B, C, D, and E3, human immunodeficiency virus (HIV), herpesvirus 1, 2, 6, and 7, cytomegalovirus, varicella zoster, papillomavirus, Epstein-Barr virus, parainfluenza virus, adenovirus, bunyaviruses (e.g., hantavirus), coxsackievirus, picoma virus, rotavirus, respiratory syncytial viruses, rhinovirus, rubella virus, papovavirus, mumps virus, measles virus, poliovirus(es), adenovirus(es), parainfluenza virus(es), avian or pandemic influenza(es), seasonal influenza, shipping fever virus, Western and Eastern equine encephalomyelitis (WEEE), and avian influenza (AVA). and viruses such as B. encephalomyelitis, Japanese B. encephalomyelitis, Russian Spring / Summer encephalomyelitis, hog cholera virus, Newcastle disease virus, fowl pox, rabies, feline and canine distemper, slow brain viruses, Rous sarcoma virus (RSV), Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (e.g., smallpox or vaccinia), Reoviridae (e.g., rotavirus), Retroviridae (HTLV-I, HTLV-II, lentivirus), and Togaviridae (e.g., rubivirus).Viruses within 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 B encephalitis, Junin, chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's lymphoma, chickenpox, hemorrhagic fever, measles, mumps, parainfluenza, rabies, the common cold, polio, leukemia, rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia.

[0155] Antigens can also be derived from bacterial and fungal infections, such as those caused by mycobacteria that cause TB and leprosy, pneumocci, aerobic gram-negative bacilli, mycoplasmas, staphylococcal infections, streptococcal infections, salmonella and chlamydia, B. pertussis, Leptospira pomona and icterohaemorrhagiae. Specific embodiments include S. paratyphi A and B, C. diphtheriae, C. tetani, C. botulinum, C. perfringens, C. feseri and other gas gangrene bacteria, B. anthracis, P. pestis, P. multocida, Neisseria meningitidis, N. gonorrheae, Hemophilus influenzae, Actinomyces (e.g., Nocardia), Acinetobacter, Bacillaceae (e.g., Bacillus anthracis), and other bacteria. anthrasis), Bacteroides (e.g., Bacteroides fragilis), Blastomycosis, Bordetella, Borrelia (e.g., Borrelia burgdorferi), Brucella, Candida, Campylobacter, Chlamydia, Coccidioides, Corynebacterium (e.g., Corynebacterium diptheriae), Cryptococcus, Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli),coli), Enterobacter (e.g., Enterobacter aerogenes), Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, Salmonella enteritidis, Serratia, Yersinia, Shigella), Erysipelothrix, Haemophilus (e.g., Haemophilus influenzae type b), Helicobacter, Legionella (e.g., Legionella pneumophila), Leptospira, Listeria (e.g., Listeria monocytogenes), Mycoplasma, Mycobacterium (e.g., Mycobacterium leprae), and Mycobacterium tuberculosis tuberculosis), Vibrio (e.g., Vibrio cholerae, Pasteurellaceae, Proteus, Pseudomonas (e.g., Pseudomonas aeruginosa), Rickettsiaceae, Spirochetes (e.g., Treponema spp., Leptospira spp., Borrelia spp.), Shigella spp., Meningiococcus, Pneumococcus and Streptococcus (e.g., Streptococcus pneumoniae and Groups A, B, and C Streptococci), Ureaplasma, Treponema pallidum pollidum), etc.; Staphylococcus aureus, Plasmodium sp. (Plasmodium falciparum, Plasmodium vivax, etc.), Aspergillus sp., Candida albicans, Pasteurella haemolytica, Corynebacterium diptheriae toxoid, Meningococcal polysaccharide, Bordetella pertussis, Streptococcus pneumoniae (pneumococcus) polysaccharide, Clostridium tetani toxoid, Mycobacterium bovis, Salmonella typhi killed cells, Cryptococcus neoformans, and Aspergillus.

[0156] Antigens may also be derived from parasitic malaria, leishmania, trypanosoma, toxoplasma, schistosomiasis, filariasis malaria, amebia, babesia, coccidia, cryptosporidium, dientamoebia, dourine, ectoparasites, giardia, helminthia, theileria, trichomonas, and sporozoans (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, scrub typhus, eye infections, intestinal diseases (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS-related), malaria, pregnancy complications, and toxoplasmosis.

[0157] Tumor-associated antigens suitable for use in the compositions described herein include both mutated and non-mutated molecules that may represent a single tumor type, may be shared among several types of tumors, and / or may be exclusively 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 have also been demonstrated. Exemplary tumor-associated antigens for use in the subject cancer vaccines include the protein products of oncogenes, tumor suppressor genes, and other genes with mutations or rearrangements unique to tumor cells, reactivated embryonic gene products, carcinoembryonic antigens, tissue-specific (but not tumor-specific) differentiation antigens, growth factor receptors, cell surface carbohydrate residues, foreign viral proteins, and some other self-proteins. Specific embodiments of tumor-associated antigens include, for example, the Ras p21 proto-oncogene, the tumor suppressor p53, and the protein products of the HER-2 / neu and BCR-ab1 oncogenes, as well as mutant antigens such as CDK4, MUM1, caspase 8, and beta-catenin; overexpressed antigens such as galectin 4, galectin 9, carbonic anhydrase, aldolase A, PRAME, Her2 / neu, ErbB-2, and KSA, oncofetal antigens such as alpha-fetoprotein (AFP) and human chorionic gonadotropin (hCG); self-antigens such as carcinoembryonic antigen (CEA) and melanocyte differentiation antigens, e.g., Mart 1 / Melan A, gp100, gp75, tyrosinase, TRP1, and TRP2; prostate-associated antigens such as PSA, PAP, PSMA, PSM-P1, and PSM-P2; MAGE 1, MAGE 3, MAGE 4, GAGE ​​1, GAGE ​​2, GAGE ​​3, GAGE ​​4, GAGE ​​5, GAGE ​​6, GAGE ​​7, GAGE ​​8, GAGE ​​9, GAGE ​​10, GAGE ​​11, GAGE ​​12, GAGE ​​13, GAGE ​​14, GAGE ​​15, GAGE ​​16, GAGE ​​17, GAGE ​​18, GAGE ​​19, GAGE ​​20, GAGE ​​21, GAGE ​​22, GAGE ​​23, GAGE ​​24, GAGE ​​25, GAGE ​​26, GAGE ​​27, GAGE ​​28, GAGE ​​29, GAGE ​​30, GAGE ​​31, GAGE ​​32, GAGE ​​33, GAGE ​​34, GAGE ​​35, GAGE ​​36, GAGE ​​37, GAGE ​​38, GAGE ​​39, GAGE These include reactivated embryonic gene products such as BAGE and 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 globoH; and glycoproteins such as Tn, Thompson-Freidenreich antigen (TF), and sTn.Also included herein as tumor-associated antigens are whole cells and tumor cell lysates and their immunogenic portions, as well as immunoglobulin idiotypes expressed in monoclonal proliferation of B lymphocytes for use against B-cell lymphomas. Tumor-associated antigens and their respective tumor cell targets include, for example, cytokeratins, particularly cytokeratins 8, 18, and 19, as antigens for carcinomas. Other known carcinoma antigens include 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. Desmin and muscle-specific actin are antigens for myogenic sarcomas. Placental alkaline phosphatase, β-human chorionic gonadotropin, and α-fetoprotein are antigens of trophoblastic and germ cell tumors. Prostate-specific antigen is an antigen of prostate carcinoma and a carcinoembryonic antigen of colon adenocarcinoma. HMB-45 is an antigen of melanoma. In cervical cancer, useful antigens may be encoded by human papillomavirus. Chromagranin-A and synaptophysin are antigens of neuroendocrine and neuroectodermal tumors. Of particular interest are invasive tumors that form solid tumor masses with necrotic areas. Lysates of such necrotic cells are a rich source of antigens for antigen-presenting cells, and therefore, targeted therapy may find advantageous use in combination with conventional chemotherapy and / or radiation therapy. Antigens can be derived from any tumor or malignant cell line.

[0158] Antigens may also be derived from common allergens that cause allergies. Allergens include organic or inorganic materials derived from various artificial or natural sources, such as plant materials, metals, ingredients in cosmetics or detergents, and latex. Classes of allergens suitable for use in the compositions and methods described herein may include, but are not limited to, pollen, animal dander, grasses, mold, dust, antibiotics, stinging insect venom, and various environmental (including chemical and metal) drugs and food allergens. Common tree allergens include pollen from cottonwood, poplar, ash, birch, maple, oak, elm, hickory, and pecan trees; common plant allergens include those from rye, ragweed, English plantain, sorrel-dock, and pigweed; plant contact allergens include poison oak, poison sumac, and poison oak. Common grass allergens include those from ivy, and nettle; common grass allergens include Timothy, Johnson, Bermuda, fescue, and bluegrass allergens; common allergens can also be derived 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 derived from house or organic dust (typically fungal in origin) and house dust mites (dermalphagoides pterosinyssis) or from animal sources such as feathers and cat and dog dander; common food allergens include milk and cheese (dairy), eggs, wheat, tree nuts (e.g., peanuts), seafood (e.g., shellfish), pea, bean, and gluten allergens;Common environmental allergens include metal (nickel and gold), chemicals (formaldehyde, trinitrophenol, and turpentine), latex, rubber, textiles (cotton or wool), burlap, hair dye, cosmetics, detergents, and fragrance allergens; common drug allergens include local anesthetic allergens and salicylate allergens; antibiotic allergens include penicillin and sulfonamide allergens; and common insect allergens include bee, wasp, and ant venom, and cockroach calyx allergens. Particularly well-characterized allergens include, but are not limited to, the major and cryptic 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 v 1 (Bauer et al. (1997) Clin. Exp. Immunol. 107, 536-541), and the multiepitope recombinant grass allergen rKBG 8.3 (Cao et al. (1997) Immunology, 90, 46-51). These and other suitable allergens are commercially available and / or can be readily prepared as extracts according to known techniques.

[0159] Antigens may be in the form of purified or partially purified antigens and may be derived from any of the antigens described above, antigenic peptides, proteins known and available in the art, and others that can be identified using conventional techniques. Antigens are typically in a form in which their toxicity or virulence properties have been reduced or destroyed, and when introduced into an appropriate medium, they induce an immune response against the specific microorganism, extract, or microbial product used to prepare the antigen, or, in the case of allergens, they help alleviate the symptoms of allergy caused by the 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 an adjuvant composition to create a multivalent antigenic composition and / or vaccine. In the compositions described herein, the antigen can be entrapped in, adsorbed to, or in admixture with the vesicle component of the composition.

[0160] In one embodiment, antigens suitable for use with the compositions described herein include antigens with low immunogenicity, such as malaria antigens, dengue antigens, and HIV antigens, or antigens intended to confer immunity against pandemic diseases, such as influenza antigens. Any such antigen combinations described herein or known are contemplated for use in the fusion proteins, fusion protein pairs, and nanocages described herein.

[0161] "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, which has either a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and which acts as a template for the synthesis of other polymers and macromolecules in biological processes, and the biological properties that result therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0162] The term "expression," as used herein, is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0163] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0164] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include introns, to the extent that a nucleotide sequence that encodes a protein may, in some versions, contain intron(s).

[0165] The term "modulate," as used herein, means to mediate a detectable increase or decrease in the level of a response in a subject compared to the level of the response in the subject in the absence of a treatment or compound, and / or compared to the level of the response in an otherwise identical but untreated subject. This term encompasses perturbing and / or affecting a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, typically a human.

[0166] The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to link two protein-coding regions, in the same reading frame.

[0167] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, or infusion techniques.

[0168] The term "polynucleotide," as used herein, is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Therefore, as used herein, nucleic acid and polynucleotide are interchangeable. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide that can be hydrolyzed into monomeric "nucleotides." The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques, PCR, etc., and by synthetic means.

[0169] 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, with no limit on the maximum number of amino acids that a protein or peptide sequence can contain. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, many of which are commonly referred to in the art as proteins. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins, among others. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0170] The term "specifically binds" as used herein with respect to antibodies refers to an antibody that recognizes a specific antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, such cross-species reactivity does not, in itself, change the specific classification of the antibody. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not, in itself, change the specific classification of the antibody. In some instances, the terms "specific binding" or "specifically binds" can be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species, meaning that the interaction is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, antibodies generally recognize and bind to specific protein structures rather than proteins. If an antibody is specific for epitope "A," in a reaction involving labeled "A" and the antibody, the presence of a molecule containing epitope A (i.e., free, unlabeled A) will reduce the amount of labeled A bound to the antibody.

[0171] The terms "therapeutically effective amount," "effective amount," or "sufficient amount" refer to an amount sufficient to achieve a desired result, e.g., an amount effective to elicit a protective immune response, when administered to a subject, including a mammal, e.g., a human. An effective amount of a compound described herein may vary depending on factors such as the immunogen, age, sex, and weight of the subject. As will be appreciated by one of skill in the art, dosages or treatment regimens can be adjusted to provide an optimal therapeutic response. For example, administration of a therapeutically effective amount of a fusion protein described herein is, in embodiments, sufficient to increase immunity to a pathogen (e.g., Plasmodium or HIV). In other embodiments, administration of a therapeutically effective amount of a fusion protein described herein is sufficient to treat a disease or condition, such as cancer, HIV, malaria, or an autoimmune disease. In yet other embodiments, administration of a therapeutically effective amount of a fusion protein described herein is sufficient to act as an adjuvant to enhance the effectiveness of a vaccine. In still other embodiments, administration of a therapeutically effective amount of a fusion protein described herein is sufficient to prevent the acquisition of a disease or infection.

[0172] Furthermore, a treatment regimen for a subject with a therapeutically effective amount may consist of a single administration or, alternatively, may include a series of applications. The length of the treatment period depends on various factors, such as the immunogen, the subject's age, the drug concentration, the patient's responsiveness to the drug, or a combination thereof. It will also be understood that the effective dosage of the drug used for treatment may increase or decrease over the course of a particular treatment regimen. Changes in dosage may occur and be revealed by standard diagnostic assays known in the art. In embodiments, the fusion proteins described herein may be administered before, during, or after treatment with conventional therapies for the disease or disorder in question, such as malaria, HIV, or cancer. For example, the fusion proteins described herein may find particular use in combination with immunotherapy to treat cancer.

[0173] The terms "transfected" or "transformed" or "transduced," as used herein, refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0174] The phrases "under transcriptional control" or "operably linked," as used herein, mean that the promoter is in the correct location and orientation relative to the polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0175] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or virus. This term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

[0176] The term "subject," as used herein, refers to any member of the animal kingdom, typically a mammal. The term "mammal" refers to any animal classified as a mammal, including humans, other higher primates, domestic and farm animals, and zoo, sport, or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. Typically, the mammal is a human.

[0177] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0178] The term "pharmaceutically acceptable" means that the compound or combination of compounds is compatible with the other ingredients of the formulation for pharmaceutical use and is generally safe for administration to humans according to established government standards, including those promulgated by the U.S. Food and Drug Administration.

[0179] The term "pharmaceutically acceptable carrier" includes, but is not limited to, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and / or absorption delaying agents, etc. The use of pharmaceutically acceptable carriers is well known.

[0180] 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 may enhance the immune response to a polypeptide present in a vaccine contemplated herein, or an immunogenic fragment or variant thereof contemplated herein. An adjuvant may also act as a tissue depot that slowly releases antigens and as a lymphoid activator that nonspecifically enhances the immune response. An example of an adjuvant that can be used is MPL-TDM adjuvant (monophosphoryl lipid A / synthetic trehalose dicorynomycolate, e.g., available from GSK Biologics). Another suitable adjuvant is the immunostimulatory adjuvant AS021 / AS02 (GSK). These immunostimulatory adjuvants are formulated to provide a strong T cell response and contain QS-21, a saponin from Quillaya saponaria, TL4 ligand, and monophosphoryl lipid A together in a lipid or liposome carrier. Other adjuvants include non-ionic block copolymer adjuvants (e.g., CRL1005), aluminum phosphate (e.g., AIPO.sub.4), R-848 (a Th1-like adjuvant), imiquimod, PAM3CYS, poly(I:C), loxoribine, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum, CpG oligodeoxynucleotides (ODN), cholera toxin-derived antigens (e.g., CTA 1-DD), lipopolysaccharide adjuvants, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface-active substances such as lysolecithin, Pluronic® polyols, polyanions, peptides, oil-in-water or hydrocarbon emulsions (e.g., MF59 or Montanide ISA available from Novartis Vaccines). 720), keyhole limpet hemocyanin, and dinitrophenol.

[0181] A "variant" is a biologically active fusion protein, antibody, or fragment thereof that has an amino acid sequence that differs from a comparator sequence by the insertion, deletion, modification, and / or substitution of one or more amino acid residues in the comparison sequence. Variants generally have less than 100% sequence identity with the comparison sequence. However, typically, a biologically active variant will have an amino acid sequence that has at least about 70% amino acid sequence identity with the comparison sequence, e.g., 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 some level of biological activity of the comparator sequence. Variants also include polypeptides in which one or more amino acid residues have been added to the N-terminus or C-terminus of the comparison sequence, or within the comparison sequence. Variants also include polypeptides in which some amino acid residues have been deleted and, optionally, replaced with one or more amino acid residues. Variants can also be covalently modified, for example, by substitution with a moiety other than a naturally occurring amino acid, or by modifying an amino acid residue to generate a non-naturally occurring amino acid.

[0182] "Percent 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 sequence of interest, such as a polypeptide of the present invention, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Neither N-terminal, C-terminal, nor internal extensions, deletions, nor insertions into the candidate sequence should be construed as affecting sequence identity or homology. Methods and computer programs for alignment are well known in the art, such as "BLAST."

[0183] For purposes of this specification, "active" or "activity" refers to the biological and / or immunological activity of the fusion proteins described herein, and "biological" activity refers to a biological function (either inhibitory or stimulatory) caused by the fusion protein.

[0184] The fusion proteins described herein may contain modifications. Such modifications include, but are not limited to, conjugation to an effector molecule, such as an antimalarial agent or an adjuvant. Modifications further include, but are not limited to, conjugation to a detectable reporter moiety. Modifications that extend half-life (e.g., pegylation) are also included. Proteins and non-protein agents can be conjugated to fusion proteins by methods known in the art. Conjugation methods include direct linkage, linkage via a covalently attached linker, and specific binding pair members (e.g., avidin-biotin). Such methods include, for example, those described by Greenfield et al., Cancer Research 50, 6600-6607 (1990), which is incorporated herein by reference, as well as those described by Amon et al., Adv. Exp. Med. Biol. 303, 79-90 (1991) and Kiseleva et al., Mol. Biol. (USSR) 25, 508-514 (1991), both of which are incorporated herein by reference.

[0185] fusion proteins Fusion proteins are described herein. The fusion protein comprises a first nanocage monomer subunit of a nanocage monomer linked to a biologically active moiety. The fusion protein self-assembles with a protein comprising a second nanocage monomer subunit to form a nanocage monomer. Pairs of multiple such fusion proteins self-assemble to form nanocages. In this manner, the biologically active moiety can decorate the inner surface of the assembled nanocage, the outer surface of the assembled nanocage, or both.

[0186] The biologically active moiety can be any moiety that can be part of a fusion protein, and is typically a protein. Typically, the biologically active moiety includes an antibody or fragment thereof, an antigen, a detectable moiety, a pharmaceutical, a diagnostic agent, or a combination thereof.

[0187] When the biologically active moiety is an antibody or fragment thereof, it may, for example, comprise one or both chains of the Fc fragment. The Fc fragment may be derived from any type of antibody, as will be understood, but is typically a gG1 Fc fragment. The Fc fragment may further comprise one or more mutations, such as LS, YTE, LALA, and / or LALAP, that modulate the half-life of the fusion protein and / or the resulting assembled nanocages containing the fusion protein. For example, the half-life may be on the scale of minutes, days, weeks, or even months.

[0188] Additionally, other substitutions in the fusion proteins and nanocages described herein are contemplated, including modifications of the Fc sequence and addition of other agents (e.g., human serum albumin peptide sequences), which allow for changes in bioavailability and will be understood by those skilled in the art. Furthermore, the fusion proteins and nanocages described herein can be modulated by sequencing or adding other agents to attenuate immunogenicity and anti-drug responses (therapeutic, e.g., matching sequences to the host, or administering immunosuppressive therapy [e.g., methotrexate when administering infliximab to treat rheumatoid arthritis or neonatal tolerance induction, which is a major strategy in reducing the incidence of inhibitors to FVIII (reviewed in DiMichele DM, Hoots WK, Pipe SW, Rivard GE, Santagostino E. International workshop on immune tolerance induction: consensus recommendations Haemophilia. 2007;13:1-22, incorporated herein by reference in its entirety]) or to enhance immune responses (e.g., bacterial sequences to vaccines).

[0189] In other aspects, when the biologically active moiety is an antibody or fragment thereof, the biologically active moiety can comprise, for example, the heavy and / or light chain of a Fab fragment. The antibody or fragment thereof can comprise, for example, an scFab fragment, an scFv fragment, or an sdAb fragment. It will be understood that any antibody or fragment thereof can be used in the fusion proteins described herein.

[0190] Generally, the fusion proteins described herein are associated with a Fab light chain and / or a heavy chain, which may be produced separately or adjacent to the fusion protein.

[0191] An antibody or fragment thereof comprises two chains, e.g., a first and second chain in the case of an Fc fragment, or a heavy chain and a light chain, and the two chains are optionally separated by a linker. The linker can be flexible or rigid, but is typically flexible to allow the chains to fold appropriately. The linker is generally long enough to provide some flexibility to the fusion protein, although it will be understood that the length of the linker will vary depending on the sequence of the nanocage monomer and the biologically active moiety and the three-dimensional conformation of the fusion protein. Thus, the linker is typically from about 1 to about 30 amino acid residues, e.g., from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues, e.g., from about 8 to about 16 amino acid residues, e.g., 8, 10, or 12 amino acid residues.

[0192] The linker can be of any amino acid sequence, and in one typical example, the linker comprises GGS repeats, more typically the linker comprises about 2, 3, 4, 5, or 6 GGS repeats, e.g., about 4 GGS repeats. In certain embodiments, the linker comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following: [ka]

[0193] In typical embodiments, the antibody or fragment thereof specifically binds to an antigen associated with an antibody-preventable and / or antibody-treatable condition. For example, the antigen to which the antibody or fragment thereof binds can be associated with an infectious agent, including a virus (e.g., HIV, including HIV-1, influenza, RSV, rotavirus), a bacterium (e.g., TB, C. difficile), a parasite (e.g., malaria), a fungus or yeast, a cancer, including solid and liquid cancers (e.g., CD19, CD22, CD79, BCMA, or CD20), or an immune disease, including an autoimmune disease. Typically, 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.

[0194] In certain embodiments, the antibody or fragment thereof comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to one or more of the following sequences: Fc chain 1: [ka] Fc chain 2: [ka] Ibalizumab-A12P light chain: [ka] Ibalizumab-A12P heavy chain: [ka] 10E8.v4 light chain: [ka] 10E8.v4 heavy chain: [ka] N49P7 light chain: [ka] N49P7 heavy chain: [ka] PGDM1400 light chain: [ka] PGDM1400 heavy chain: [ka] or A combination of them.

[0195] In further embodiments, the antibody or fragment thereof may be conjugated or associated with a further moiety, such as an antigen, a detectable moiety (e.g., a small molecule, fluorescent molecule, radioisotope, or magnetic particle), a pharmaceutical agent, a diagnostic agent, or a combination thereof, and may include, for example, an antibody-drug conjugate.

[0196] In embodiments in which the biologically active moiety is an antigen, the antigen can be associated with, for example, a vaccine-preventable and / or vaccine-treatable condition. In such cases, the antigen can be associated with, for example, an infectious agent, including a virus, bacterium, parasite, fungus, or yeast; a cancer, including solid and liquid cancers; or an immune disorder, including an autoimmune disease.

[0197] In embodiments where the biologically active moiety is a detectable moiety, the detectable moiety may comprise a fluorescent protein such as GFP, EGFP, ametrine, and / or a flavin-based fluorescent protein such as an LOV protein such as iLOV.

[0198] In aspects where the biologically active moiety is a pharmaceutical agent, the pharmaceutical agent can include, for example, a small molecule, a peptide, a lipid, a carbohydrate, or a toxin.

[0199] In exemplary embodiments, nanocages assembled from the fusion proteins described herein comprise from about 3 to about 100 nanocage monomers, e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 55, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, or 98 to about 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 55, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or 100 nanocage monomers, e.g., 24, 32, or 60 monomers. The nanocage monomer may be any known nanocage monomer, natural, synthetic, or partially synthetic, and in embodiments is selected from ferritin, apoferritin, encapsulin, SOR, lumazine synthase, pyruvate dehydrogenase, carboxysome, vault protein, GroEL, heat shock proteins, E2P, MS2 coat protein, fragments thereof, and variants thereof. Typically, the nanocage monomer is ferritin or apoferritin.

[0200] When apoferritin is selected as the nanocage monomer, the first and second nanocage monomer subunits typically interchangeably comprise the "N" and "C" regions of apoferritin. It will be appreciated that other nanocage monomers can be split into binary subunits similar to the apoferritin described herein, such that the subunits are susceptible to self-assembly and fusion with respective biologically active moieties.

[0201] Typically, the "N" region of apoferritin comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to: [ka]

[0202] Typically, the "C" region of apoferritin comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following: [ka]

[0203] In some embodiments, the fusion proteins described herein further comprise a linker between the nanocage monomer subunit and the biologically active moiety, similar to the linkers described above. The linker can be flexible or rigid, but is typically flexible to allow the biologically active moiety to retain its activity and to allow the pair of nanocage monomer subunits to retain their self-assembly properties. The linker is generally long enough to provide some flexibility to the fusion protein, although it will be understood that the length of the linker will vary depending on the sequences of the nanocage monomer and the biologically active moiety and the three-dimensional conformation of the fusion protein. Thus, the linker is typically from about 1 to about 30 amino acid residues, e.g., from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues, e.g., from about 8 to about 16 amino acid residues, e.g., 8, 10, or 12 amino acid residues.

[0204] The linker can be of any amino acid sequence, and in one typical example, the linker comprises GGS repeats, more typically the linker comprises about 2, 3, 4, 5, or 6 GGS repeats, e.g., about 4 GGS repeats. In certain embodiments, the linker comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following: [ka]

[0205] Similarly, the fusion protein may further comprise a C-terminal linker to improve one or more attributes of the fusion protein. In embodiments, the C-terminal linker comprises GGS repeats, more typically the linker comprises about 2, 3, 4, 5, or 6 GGS repeats, e.g., about 4 GGS repeats. In particular embodiments, the C-terminal linker comprises or consists of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following: [ka]

[0206] Also described herein are pairs of the above fusion proteins, which self-assemble to form a nanocage monomer, with the first and second nanocage monomer subunits fused to different biologically active moieties, providing multivalency and / or multispecificity to a single nanocage monomer assembled from the pair of subunits.

[0207] In certain embodiments, the fusion protein may further comprise an antigen. Such embodiments are explicitly described in International Patent Application No. WO2019 / 023812, which is incorporated herein by reference in its entirety. Briefly, in such embodiments, the antigen has at least a first and a second antibody-binding epitope; and an antibody or fragment thereof specific for at least the first antigen epitope. Binding of the antibody or fragment thereof to the first antigen epitope presents the second antigen epitope for binding to the antigen-binding moiety, and / or the first antibody-binding epitope binds to the antibody or fragment thereof, such binding presenting the second antibody-binding epitope in the context of the antibody or fragment thereof.

[0208] In other embodiments, the antibody or fragment thereof may be directed against any antigen, such as those listed above. Typically, the antigen is derived from a cancer or an infectious agent, such as hepatitis A, B, C, HIV, mycobacteria, malaria pathogens, SARS pathogens, herpes viruses, influenza viruses, polioviruses, or bacterial pathogens such as chlamydia and mycobacteria, or autoreactive B cells or any T cells for co-recruitment and cytotoxic killing.

[0209] The fusion proteins described herein may alternatively find use as therapeutic or diagnostic agents. Thus, the antibody or fragment thereof in embodiments may be specific for, for example, a tumor antigen or an autoantigen.

[0210] A substantially identical sequence may contain one or more conservative amino acid mutations. It is known in the art that one or more conservative amino acid mutations to a reference sequence can result in a variant peptide that does not have a substantial change in physiological, chemical, or functional properties compared to the reference sequence; in such cases, the reference sequence and the variant sequence would be considered "substantially identical" polypeptides. Conservative amino acid mutations can include amino acid additions, deletions, or substitutions; a conservative amino acid substitution is defined herein as the substitution of an amino acid residue for another amino acid residue having similar chemical properties (e.g., size, charge, or polarity).

[0211] In a non-limiting example, a conservative mutation can be an amino acid substitution. Such a conservative amino acid substitution can replace a basic, neutral, hydrophobic, or acidic amino acid with another from the same group. The term "basic amino acid" refers to a hydrophilic amino acid with a side chain pK value greater than 7, which is typically positively charged at physiological pH. Basic amino acids include histidine (His or H), arginine (Arg or R), and lysine (Lys or K). The term "neutral amino acid" (also "polar amino acid") refers to a hydrophilic amino acid that is uncharged at physiological pH but has a side chain with at least one bond in which the electron pair commonly shared by two atoms is held more closely by one of the atoms. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine ​​(Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and glutamine (Gln or Q). The term "hydrophobic amino acid" (also "nonpolar amino acid") is meant to include amino acids that exhibit a hydrophobicity greater than zero according to Eisenberg's 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).

[0212] "Acidic amino acid" refers to a hydrophilic amino acid having a side chain pK value of less than 7, which are typically negatively charged at physiological pH. Acidic amino acids include glutamic acid (Glu or E) and aspartic acid (Asp or D).

[0213] Sequence identity is used to evaluate the similarity of two sequences; it is determined by calculating the percentage of residues that are the same when the two sequences are aligned for maximum correspondence between residue positions.Sequence identity can be calculated using any known method; for example, computer software is available for calculating sequence identity.Without intending to be limited, sequence identity can be calculated by software such as the NCBI BLAST2 service, BLAST-P, Blast-N, or FASTA-N, which is maintained by the Swiss Institute of Bioinformatics (and can be found at ca.expasy.org / tools / blast / ), or any other suitable software known in the art.

[0214] Substantially identical sequences of the present invention can be at least 85% identical; in another example, a substantially identical sequence can be at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% (or any percentage therebetween) identical at the amino acid level to a sequence described herein. In certain aspects, a substantially identical sequence retains the activity and specificity of the reference sequence. In non-limiting embodiments, differences in sequence identity can be due to conservative amino acid mutation(s).

[0215] The polypeptides or fusion proteins of the present invention may also contain additional sequences to aid in their expression, detection, or purification. Any such sequences or tags known to those skilled in the art can be used. For example, and without wishing to be limiting, the fusion protein may contain exemplary tag cassettes including a targeting or signal sequence (such as, but not limited to, ompA), a detection tag, a Strep tag, or any variant thereof; see, e.g., U.S. Pat. No. 7,981,632, a His tag, a Flag tag with the sequence motif DYKDDDDK, an Xpress tag, an Avi tag, a calmodulin tag, a polyglutamate tag, an HA tag, a Myc tag, a Nus tag, an S tag, an SBP tag, Softag 1, Softag 3, a V5 tag, a CREB-binding protein (CBP), a glutathione S-transferase (GST), a maltose-binding protein (MBP), a green fluorescent protein (GFP), a thioredoxin tag, or any combination thereof; a purification tag (such as, but not limited to, His5 or His6), or a combination thereof.

[0216] 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 is known to those skilled in the art, linker sequences may be used in combination with additional sequences or tags.

[0217] More specifically, the tag cassette can comprise an extracellular moiety capable of specifically binding to an antibody with high affinity or avidity. Within a single-chain fusion protein structure, the tag cassette can be positioned (a) immediately amino terminal to the connector region, (b) interposed between and connecting linker modules, (c) immediately carboxy terminal to the binding domain, (d) interposed between binding domains (e.g., scFv or scFab) and connected to the effector domain, (e) interposed between and connecting subunits of the binding domain, or (f) at the amino terminus of the single-chain fusion protein. In certain embodiments, one or more junction amino acids can be positioned between the tag cassette and the hydrophobic moiety, connecting the tag cassette and the hydrophobic moiety, between the tag cassette and the connector region, connecting the tag cassette and the connector region, between the tag cassette and the linker module, connecting the tag cassette and the linker module, or between the tag cassette and the binding domain, connecting the tag cassette and the binding domain.

[0218] Also encompassed herein are isolated or purified fusion proteins, polypeptides, or fragments thereof, immobilized on a surface using various methodologies; for example, and without wishing to be limiting, polypeptides may be linked or coupled to a surface via His-tag coupling, biotin binding, covalent binding, adsorption, etc. The solid surface may be any suitable surface, for example, but not limited to, the well surface of a microtiter plate, a channel of a surface plasmon resonance (SPR) sensor chip, a membrane, beads (e.g., magnetic-based or sepharose-based beads, or other chromatography resins), glass, film, or any other useful surface.

[0219] In other aspects, the fusion protein can be linked to a cargo molecule; the fusion protein can deliver the cargo molecule to a desired site and can be linked to the cargo molecule using any method known in the art (recombinant technology, chemical conjugation, chelation, etc.). The cargo molecule can be any type of molecule, such as a therapeutic or diagnostic agent. For example, and without wishing to be limiting in any way, the therapeutic agent can be a radioisotope that can be used in radioimmunotherapy; a toxin such as an immunotoxin; a cytokine such as an immunocytokine; a cytotoxin; an apoptosis inducer; an enzyme; an anti-cancer antibody for immunotherapy; or any other suitable therapeutic molecule known in the art. Alternatively, the diagnostic agent may include, but is in no way limited to, a radioisotope, a paramagnetic label such as gadolinium or iron oxide, a fluorophore, a near-infrared (NIR) fluorescent dye or pigment (e.g., Cy3, Cy5.5, Alexa680, Dylight680, or Dylight800), an affinity label (e.g., biotin, avidin, etc.) fused to a detectable protein-based molecule, or any other suitable agent that can be detected by imaging methods. In a specific, non-limiting example, the fusion protein may be linked to a fluorescent agent such as FITC or may be genetically fused to enhanced green fluorescent protein (EGFP).

[0220] In some embodiments, the cargo molecule is a protein and is fused to a fusion protein such that the cargo molecule is contained within the nanocage. In other embodiments, the cargo molecule is not fused to a fusion protein and is contained within the nanocage. The cargo molecule is typically a protein, a small molecule, a radioisotope, or a magnetic particle.

[0221] The fusion proteins described herein specifically bind to their targets. Antibody specificity, which refers to the selective recognition of the antibody or fragment described herein for a particular epitope of an antigen, can be determined based on affinity and / or avidity. Affinity is the equilibrium constant for the dissociation of an antigen with an antibody (K D) and measures the strength of binding between an antigenic determinant (epitope) and the antibody binding site. Avidity is a measure of the strength of binding between an antibody and its antigen. Antibodies typically have a binding affinity of 10 -5 ~10 -11 K of M D Combine with 10 -4 K exceeds M D is generally considered to represent non-specific binding. D The smaller the value of , the stronger the binding strength between the antigenic determinant and the antibody binding site. -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 K less than M D It has.

[0222] Also described herein are nanocages comprising at least one fusion protein described herein and at least one second nanocage monomer subunit that self-assembles with the fusion protein to form a nanocage monomer. Additionally, described herein are pairs of fusion proteins that self-assemble to form a nanocage monomer, wherein the first and second nanocage monomer subunits are fused to different biologically active moieties.

[0223] It will be understood that nanocages can self-assemble from multiple identical fusion proteins, multiple different fusion proteins (thus being multivalent and / or multispecific), combinations of fusion proteins and wild-type proteins, and any combination thereof. For example, nanocages can be decorated internally and / or externally with at least one of the fusion proteins described herein in combination with at least one anti-cancer antibody for immunotherapy. In typical embodiments, about 20% to about 80% of the nanocage monomers contain a fusion protein described herein. Given the modular nature described herein, nanocages can theoretically contain up to twice as many biologically active moieties as there are monomers in the nanocage, since each nanocage monomer can be divided into two subunits, each of which can independently bind a different biologically active moiety. It will be understood that this modularity can be exploited to achieve any desired ratio of the biologically active moieties described herein, specifically a ratio of four different biologically active moieties, such as 4:2:1:1. For example, the nanocages described herein can contain at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different biologically active moieties. In this manner, the nanocages can be multivalent and / or multispecific, the degree of which can be relatively easily controlled.

[0224] In aspects, the nanocages described herein may further comprise at least one entire nanocage monomer optionally fused to a biologically active moiety, which may be the same as or different from the biologically active moiety described herein linked to the nanocage monomer subunit.

[0225] In typical embodiments, the nanocages described herein comprise first, second, and third fusion proteins and at least one whole nanocage monomer optionally fused to a biologically active moiety, wherein the biologically active moieties of the first, second, and third fusion proteins and the biologically active moiety of the whole nanocage monomer are all different from one another.

[0226] More typically, the first, second, and third fusion proteins each comprise an antibody or fragment thereof fused to N- or C-ferritin, with at least one of the first, second, and third fusion proteins fused to N-ferritin and at least one of the first, second, and third fusion proteins 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 each typically comprise an antibody or fragment thereof specific for a different antigen of a virus such as HIV, or one of the second and third fusion proteins comprises an antibody or fragment thereof specific for an antigen of a virus such as HIV, and the third fusion protein comprises an antibody or fragment thereof specific for a different antigen, such as the CD4 receptor; and the entire nanocage monomer is optionally fused to a biologically active moiety specific for another, different antigen of the same virus, such as HIV.

[0227] In embodiments, the antibody or fragment thereof of the second fusion protein is N49P7 or iMab A12P; and the antibody or fragment thereof of the third fusion protein is 10E8v4. In exemplary embodiments, the nanocages described herein comprise the following four fusion proteins, optionally in a ratio of 4:2:1:1: a. PGDM1400 fused to full-length ferritin (possibly scPGDM1400); bFc (optionally scFc) fused to N-ferritin; N49P7 or iMab A12P (optionally scN49P7 or sciMab A12P) fused to cC-ferritin; and 10E8v4 (occasionally sc10E8v4) fused to dC-ferritin.

[0228] In aspects, the nanocages described herein comprise or consist of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to one or more of the following sequences, where ferritin subunits are in bold, linkers are underlined, light chains are in italics, and heavy chains are in lowercase: a.PGDM1400-hFerr: [ka] b.Fc-N-hFerr LS [ka] c1.N49P7-C-hFerr [ka] c2. Ibalizumab-A12P-C-hFerr [ka] or d.10E8.v4-C-hFerr [ka]

[0229] It will be understood that, generally, the nanocages described herein are hollow and, therefore, capable of carrying a cargo molecule, such as a pharmaceutical, diagnostic, and / or imaging agent. Generally, the cargo molecule is not fused to a fusion protein and is contained within the nanocage, although the cargo molecule may alternatively be a protein and be fused to a fusion protein such that the cargo molecule is contained within the nanocage.

[0230] In embodiments, a cargo molecule may be included internally to provide a T cell epitope but not a B cell epitope, or alternatively, the cargo molecule may be fused to a fusion protein and included internally to provide a T cell epitope but not a B cell epitope.

[0231] The cargo molecule may be a fluorescent protein such as GFP, EGFP, ametrine, and / or a flavin-based fluorescent protein such as an LOV protein such as iLOV, and / or the cargo molecule may be a small molecule, a radioisotope, or a magnetic particle.

[0232] Additionally, the nanocage may further comprise an antigen on the surface, which may be expressed as a fusion protein with the nanocage monomer.

[0233] Also described herein are vaccines comprising the nanocages described herein, as well as compositions, e.g., therapeutic or prophylactic compositions, comprising the nanocages. Related methods and uses for treating and / or preventing a disease or condition are also described, where the method or use comprises administering to a subject in need thereof a nanocage, vaccine, or composition described herein. The nanocages can be used to treat any disease or condition for which bioactive therapy, or more specifically antibody therapy, can be used, although, for example, the disease or condition is typically cancer, an infectious disease, e.g., HIV, malaria, influenza, RSV, rotavirus, or an autoimmune disease.

[0234] Also described herein are nucleic acid molecules encoding the fusion proteins and polypeptides described herein, as well as vectors containing the nucleic acid molecules, and host cells containing the vectors.

[0235] Polynucleotides encoding the fusion proteins described herein include polynucleotides having a nucleic acid sequence substantially the same as that of a polynucleotide of the invention. A "substantially the same" nucleic acid sequence is defined herein as a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% identity to another nucleic acid sequence when the two sequences are optimally aligned (with appropriate nucleotide insertions or deletions) and compared to determine the exact nucleotide matches between the two sequences.

[0236] Suitable sources of polynucleotides encoding antibody fragments include any cell that expresses a full-length antibody, such as hybridomas and spleen cells. Fragments may be used alone as antibody equivalents or recombined into such equivalents. The DNA deletions and recombinations described in this section can be performed by known methods, such as those described in the published patent applications listed above in the section entitled "Functional Equivalents of Antibodies," and / or other standard recombinant DNA techniques, such as those described below. Another source of DNA is single-chain antibodies produced from phage display libraries, as known in the art.

[0237] Additionally, an expression vector is provided that comprises the aforementioned polynucleotide sequence operably linked to an expression sequence, a promoter, and an enhancer sequence. A variety of expression vectors have been developed for the efficient synthesis of antibody polypeptides in prokaryotic systems (e.g., bacteria) and eukaryotic systems (including, but not limited to, yeast and mammalian cell culture systems). The vectors of the present invention may contain segments of chromosomal, non-chromosomal, and synthetic DNA sequences.

[0238] Any suitable expression vector can be used. For example, prokaryotic cloning vectors include plasmids derived from Escherichia coli (E. coli), such as colEl, pCRl, pBR322, pMB9, pUC, pKSM, and RP4. Prokaryotic vectors also include derivatives of phage DNA, such as Ml3 and other filamentous single-stranded DNA phages. An example of a vector useful in yeast is the 2μ plasmid. Suitable vectors for expression in mammalian cells include well-known derivatives of DNA sequences derived from SV-40, adenovirus, and retrovirus, as well as shuttle vectors derived from functional mammalian vectors (e.g., those described above) combined with functional plasmids and phage DNA.

[0239] Additional eukaryotic expression vectors are known in the art (e.g., P. J. Southern & P. ​​Berg, J. Mol. Appl. Genet. 1:327-341 (1982); Subramani et al., Mol. Cell. Biol. 1:854-864 (1981); Kaufhiann & Sharp, "Amplification And Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene," J. Mol. Biol. 159:601-621 (1982); Kaufhiann & Sharp, Mol. Cell. Biol. 159:601-664 (1982); Scahill et al., "Expression And Characterization Of The Product Of A Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells," Proc. Nat'l Acad. Sci. USA, 80:4654-4659 (1983); Urlaub & Chasin, Proc. Nat'l Acad. Sci USA, 77:4216-4220, (1980) (all of which are incorporated herein by reference).

[0240] Expression vectors typically contain at least one expression control sequence operably linked to the DNA sequence or fragment to be expressed. Control sequences are inserted into the vector to control and regulate the expression of the cloned DNA sequence. Examples of useful expression control sequences include the lac system, trp system, tac system, trc system, the phage lambda major operator and promoter region, the fd coat protein control region, yeast glycolytic promoters such as the 3-phosphoglycerate kinase promoter, yeast acid phosphatase promoters such as Pho5, the yeast alpha-mating factor promoter, and promoters derived from polyoma, adenovirus, retrovirus, and simian virus, such as the early and late promoters or SV40, as well as other sequences known to control the expression of genes in prokaryotic or eukaryotic cells and viruses, or combinations thereof.

[0241] Recombinant host cells containing the above-described expression vectors are also described herein. The fusion proteins described herein may be expressed in cell lines other than hybridomas. Nucleic acids containing sequences encoding the polypeptides according to the present invention can be used to transform suitable mammalian host cells.

[0242] Particularly preferred cell lines are selected based on high levels of expression, constitutive expression of the protein of interest, and minimal contamination from host proteins. Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines, such as, but not limited to, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, and many others. Additional suitable eukaryotic cells include yeast and other fungi. Useful prokaryotic hosts include, for example, E. coli, such as E. coli SG-936, E. coli HB101, E. coli W3110, E. coli X1776, E. coli X2282, E. coli DHI, and E. coli MRC1, Pseudomonas, Bacillus, such as Bacillus subtilis, and Streptomyces.

[0243] These recombinant host cells can be used to produce fusion proteins by culturing the cells under conditions that allow expression of the polypeptide and purifying the polypeptide from the host cells or the medium surrounding the host cells. Targeting of an expressed polypeptide for secretion in a recombinant host cell can be facilitated by inserting a signal or secretory leader peptide coding sequence (see Shokri et al. (2003) Appl Microbiol Biotechnol. 60(6):654-664; Nielsen et al., Prot. Eng., 10:1-6 (1997); von Heinje et al., Nucl. Acids Res. 14:4683-4690 (1986), all of which are incorporated herein by reference) at the 5' end of the gene encoding the antibody of interest. These secretory leader peptide elements can be derived from either prokaryotic or eukaryotic sequences. Thus, suitably, a secretory leader peptide, which is an amino acid sequence linked to the N-terminus of a polypeptide, is used to direct the translocation of the polypeptide from the host cell cytosol and secretion into the medium.

[0244] The fusion proteins described herein can be fused to additional amino acid residues. Such amino acid residues can be, for example, peptide tags to facilitate isolation. Other amino acid residues for homing antibodies to specific organs or tissues are also contemplated.

[0245] It will be appreciated that Fab-nanocages can be generated by cotransfection of HC-ferritin and LC. Alternatively, single-chain Fab-ferritin nanocages, requiring only one plasmid transfection, can be used, as shown in Figure 1C. This can be done using linkers of different lengths between the LC and HC, e.g., 60 or 70 amino acids. When using single-chain Fabs, pairing of the heavy and light chains can be ensured. Tags (e.g., Flag, HA, myc, His6x, Strep, etc.) can also be added to the N-terminus of the construct or within the linker for ease of purification, as described above. Furthermore, when using a tag system to cotransfect different Fab nanoparticle plasmids, sequential / additional affinity chromatography steps can be used to ensure the presence of many different Fabs on the same nanoparticle. This provides multispecificity to the nanoparticles. Protease sites (e.g., TEV, 3C, etc.) can be inserted, if necessary, to cleave the linker and tag after expression and / or purification. An example of such a construct is for the anti-HIV broadly neutralizing scFab 10E8: [ka]

[0246] In another aspect, described herein are methods of vaccinating a subject by administering to a mammal, typically a young, juvenile, or neonatal mammal, in need thereof a therapeutically effective amount of a fusion protein described herein, where therapeutically effective amount means an amount effective to produce the desired therapeutic effect, such as providing a protective immune response against the antigen.

[0247] Any suitable method or route can be used to administer the fusion proteins and vaccines described herein, including, for example, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration.

[0248] It is understood that when the fusion protein described herein is used in a mammal for prophylactic or therapeutic purposes, it is administered in the form of a composition further comprising a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof. The pharmaceutically acceptable carrier may further contain small amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the binding protein. Injectable compositions can be formulated to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal, as is well known in the art.

[0249] Human antibodies are particularly useful for administration to humans, but can also be administered to other mammals. The term "mammal," as used herein, is intended to include, but is not limited to, humans, laboratory animals, domestic pets, and farm animals.

[0250] Also included herein are vaccination kits comprising a therapeutically or prophylactically effective amount of the fusion proteins described herein. The kits can further comprise, for example, any suitable adjuvant. The kits can also include instructions.

[0251] The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited to the following examples in any way, but rather as embracing any and all variations that become evident as a result of the teachings provided herein.

[0252] The following examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, insert genes encoding polypeptides into such vectors and plasmids, or introduce plasmids into host cells. Such methods are well known to those of skill in the art and are described in many publications, including Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989), Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, incorporated herein by reference.

[0253] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore specifically point out exemplary aspects of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0254] example Example 1 introduction Despite over 30 years of efforts, there are no effective vaccines or cures for human immunodeficiency virus type 1 (HIV-1). However, in this quest, the fact that a small percentage of HIV-1-infected individuals develop antibodies with exceptional neutralizing potency across circulating HIV-1 isolates offers hope. The first-generation broadly neutralizing antibody (bNAb), 2F5, 1 , 4E10 2、3 , 2G12 4 , and b12 5、6 Since the discovery of Env-specific single B cell sorting, a catalog of bNAbs has been developed. 7~9 , antibody cloning and high-throughput neutralization assays 10~13 , and more recently proteome deconvolution 14The number of HIV bNAbs has increased dramatically with the implementation of new technologies. Dozens of HIV bNAbs have now been described that target six conserved sites of the trimeric HIV envelope (Env), including the V1 / V2 loop at the trimer apex, the V3 loop glycan, the CD4 binding site (CD4bs), the gp120-g41 interface, the fusion peptide, and the membrane-proximal external region (MPER). 7、9、19、10、12~18 .

[0255] The interest of bNAbs as therapeutic molecules in the fight against HIV-1 has been fuelled by the development of macaque 20~24 and humanized mice 25~28 This results from the potent antiviral activity observed in several challenge studies in humans and from the reduction in viremia achieved in infected humans when bNAbs are therapeutically infused. 29~33 Furthermore, antibodies have important advantages over oral antiretroviral therapy (ART): they have a longer circulating half-life and can form immune complexes that enhance host immunity to the virus. These findings have led to the clinical evaluation of antibody-based therapies to confer protection against HIV-1 through the passive administration of bNAbs, instead of or in addition to traditional vaccinology, and efforts to control and / or eliminate HIV-1 in infected individuals.

[0256] One of the major limitations to the clinical use of bNAbs is the rapid selection of neutralization-resistant virus populations. 30~32、34、35 RNA viruses such as HIV have extraordinary genetic diversity that allows the virus to generate resistance mutations to escape mAb recognition. 36 However, mutations that abolish binding to certain bNAbs can result in a significant fitness disadvantage for the virus. 37~39 Similar to the combination of different drugs in HIV-1 treatment regimens, this finding suggests that successful antibody-based therapy against HIV-1 should involve a combination of bNAb specificities. Consequently, dual specificities against Env are needed. 40~42 or trispecific 43~45The development of different formats of antibody-like molecules with β-glucan has recently been explored. A further consideration is the amount of antibody required for in vivo efficacy. Indeed, many studies have been conducted to develop bNAbs (e.g., VRC01) to improve their potency using structure-guided design or bioinformatics approaches. 46 , 10E8 47、48 , and NIH45-46 49 Extensive efforts have been made to engineer antibodies against Env, with only moderate success to date. In the case of bispecific and trispecific antibodies targeting multiple epitopes on Env, efficacy is generally limited by the potency of their parent mAbs. As a result, although neutralization breadth has improved significantly, relatively little antiviral efficacy has been achieved to date. 40、43、44、45 .

[0257] Antibody efficacy is greatly influenced by its ability to interact with two or more epitopes on the same virus. 50~52 This effect is commonly known as avidity (apparent enhanced affinity), a property used in nature by IgM antibodies to compensate for their normally low affinity. Therefore, the addition of the IgM mu-tailpiece to the IgG constant region has been explored to generate a dodecavalent IgM-like molecule with improved biological activity. 53、54 Across evolution, various non-natural antibody formats have been engineered to overcome the limitations of IgG bivalency. Some of these designs involve tandem fusions of Fabs in a linear head-to-tail fashion. 55 , Tandem diabody combinations (Tamdabs) 56 , or a diabody fused to the CH3 of IgG (di-diabody) 57 , added IgG 58~60 , and multimerization scaffolds, e.g., p53 61 , leucine zipper helix 62 , streptavidin 63 , barnase-barstar modules 64or the B subunit of Escherichia coli verotoxin, which self-assembles into a pentameric form. 65 can be further engineered to be 10-valent, including the use of 66 These antibody architectures face various challenges for their successful development as therapeutics. Multimeric antibody formats that rely on antibody variable fragments (Fv) often exhibit poor stability and consequently a high tendency to aggregate. 67 Furthermore, dissociation of the dimeric modules, which is determined by the affinity constant of the complex, may limit the long-term stability of the molecule in vivo. Furthermore, a maximum of 3-5 valency is usually achieved in most of the antibody formats mentioned above, thus preventing the combination of high avidity and multispecificity.

[0258] In one embodiment, a multispecific, multi-affinity antiBODY (multabody) platform is described herein that uses apoferritin protomers as modular subunits to multimerize up to 32 antibody fragments (antigen-binding fragments [Fab] and crystallizable fragments [Fc]) within a single molecule. Using this approach, we efficiently combined four different specificities, including the Fab portions of the three best bNAbs against HIV-1 and a crystallizable fragment (Fc) derived from IgG1, into one single molecule to confer multispecificity, high avidity, effector function, and extended serum half-life to the molecule. The resulting multabodies demonstrated pan-viral neutralization breadth and significantly higher neutralization potency compared to their individual parent antibodies or IgG combinations. Remarkably, the mean median IC50 values ​​of multabodies against a panel of 14 pseudoviruses (PsVs) were one and two orders of magnitude lower in mass and molar concentration, respectively, compared to the anti-HIV trispecific N6 / PGDM1400x10E8 antibody or a cocktail made from the currently best-known bNAbs. The multabody design described herein presents a robust and potent plug-and-play platform for multimerizing antibodies to enhance their therapeutic properties for inhibiting HIV-1 infection.

[0259] Materials and Methods Expression and purification of Fab-only apoferritin-based multabodies. Genes encoding the light chain of human apoferritin and scFab-human apoferritin fusions were synthesized and cloned into the pHLsec expression vector by GeneArt (Life Technologies). 200 ml of HEK293F cells (Thermo Fisher Scientifics) were cultured in Freestyle expression medium at 0.8 × 10 6Cells were seeded at a density of 1000 cells / mL and incubated at 125 rpm in a Multitron Pro shaker (Infors HT) at 37°C, 8% CO2, and 70% humidity. Within 24 hours of seeding, cells were transiently transfected with 50 μg of filtered DNA at a 1:1 ratio, preincubated for 10 minutes at room temperature (RT) using the transfection reagent FectoPRO (Polyplus Transfections). The scFab-human apoferritin and human apoferritin-encoding plasmids were mixed at ratios of 1:4, 1:1, 4:1, and 1:0 to obtain nanoparticles with scFab titers of 20%, 50%, 80%, and 100%, 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). Nanoparticles were purified by affinity chromatography against Fab and eluted after washing. Protein-containing fractions were pooled, concentrated, and loaded onto a Superose 6 10 / 300 GL size-exclusion column (GE Heathcare) in 20 mM sodium phosphate (pH 8.0), 150 mM NaCl.

[0260] Design, expression, and purification of 32-N and 32-I multabodies. Genes encoding scFab and scFc fragments linked to half-ferritin were generated by deleting residues 1–95 (C-ferritin) and 95–175 (N-ferritin) of the light chain of human apoferritin using the KOD-Plus mutagenesis kit (Toyobo, Osaka, Japan). Furthermore, the binding specificity of Protein L for iMab-C-ferritin was confirmed by mutating alanine at position 12 of the antibody light chain to proline using the same mutagenesis kit. 75The gene was disrupted by site-directed mutagenesis using 32-N multabodies. Transient transfection of 32-N multabodies in HEK 293F cells was achieved by mixing 66 μg of the following plasmids: PGDM 1400 scFab-human apoferritin, Fc-human apoferritin, N49P7 scFab-C-ferritin, and 10E8 scFab-C-ferritin in a 4:2:1:1 ratio. For the 32-I multabody, the N49P7 scFab-C-ferritin plasmid was replaced with iMab scFab-C-ferritin. The DNA mixture was filtered and incubated with 60 μl of FectoPRO at room temperature before being added to the cell culture. Multabodies were first purified by affinity chromatography using a HiTrap Protein A HP column (GE Healthcare) with an elution buffer of 20 mM Tris (pH 8.0), 3 M MgCl, and 10% glycerol. After buffer exchange using a PD-10 desalting column (GE Healthcare), the multabodies were further purified by a second affinity chromatography using a HiTrap Protein L column (GE Healthcare). Protein-containing fractions were concentrated and further purified by gel filtration on a Superose 6 10 / 300 GL column (GE Healthcare).

[0261] Negative stain electron microscopy. Three microliters of multabody at a concentration of approximately 0.02 mg / mL was applied to a carbon-coated copper grid for 30 seconds and stained with 3 μl of 2% uranyl formate. The excess stain was immediately removed from the grid using Whatman No. 1 filter paper, and an additional 3 μl of 2% uranyl formate was added for 20 seconds. Grids were imaged using a field-emission FEI Tecnai F20 electron microscope operated at 200 kV and equipped with an Orius charge-coupled device (CCD) camera (Gatan Inc.).

[0262] Biolayer interferometry. Binding kinetics measurements were performed using an Octet RED96 BLI system (Pall ForteBio) in PBS (pH 7.4), 0.01% BSA, and 0.002% Tween. Unique His-tagged ligands for each multabody component were selected and loaded onto Ni-NTA biosensors to achieve a signal response of 0.8 nM. Association rates were measured by transferring the loaded biosensors into wells containing serial dilutions of multabodies (50, 25, 12.5, 6.25, 3.1, and 1.5 nM) and buffer-containing wells, respectively. Dissociation rates were measured by immersing the biosensors in buffer-containing wells. The duration of these two steps was 180 s each. To achieve selective binding to PGDM1400, a D368R mutation was introduced into the CD4bs of the BG5050 SOSIP.664 trimer, thereby disrupting N49P7 binding to this antigen. Similarly, hFcRn in complex with the gp120 subunit 93TH057, the MPER peptide fused to mVenus, soluble CD4, and β2-microglobulin were generated as the sole ligands for N49P7, 10E8, iMab, and Fc, respectively. The ability of multabodies to undergo endosomal recycling was tested by measuring their binding to the hFcRn β2-microglobulin complex at physiological pH (7.5) and acidic pH (5.6).

[0263] Multi-angle light scattering and inline size-exclusion chromatography (SEC-MALS). A MiniDAWN TREOS and Optilab T-rEX refractometer (Wyatt) were used inline with an Agilent Technologies 1260 infinity II HPLC. 50 μg of 24-mer PGDM1400 scFab multabody, multabody 32-N, and multabody 32-I were loaded onto a Superose 6 10 / 300 (GE Healthcare) column in 20 mM sodium phosphate pH 8.0, 150 mM NaCl. Data acquisition and analysis were performed using ASTRA software (Wyatt).

[0264] Measurement of melting and aggregation temperatures. The UNit system (Unchained Labs) was used to measure the melting temperatures (T) of multabodies, parental IgG, 12-mer homo-oligomeric Fab and Fc, and N6 / PGDM1400x10E8 trispecific antibody. m ) and agglomeration temperature (T agg ) was determined. m is obtained by measuring the centroid mean fluorescence, while T agg was determined as the temperature at which a 50% increase in static light scattering at a wavelength of 266 nm relative to baseline was observed. Samples were concentrated to 1.0 mg / mL and subjected to a temperature gradient from 25 to 95 °C in 1 °C increments. The mean and standard error of three independent measurements were calculated using UNit analysis software.

[0265] Virus production and TZM-bl neutralization assay. A panel of 14 HIV-1 pseudotyped viruses was tested using the HIV-1 subtype B backbone NL4-3.Luc.R. - The E plasmid (AIDS Research and Reference Reagent Program (ARRRP)) was generated by cotransfection of 293T cells with a plasmid encoding the full-length Env clone as described above. 73HIV isolates X2988, ZM106.9, and 3817 were kindly provided by the Collaboration for AIDS Vaccine Discovery (CAVD), SF162 was provided by JL Nieva (Biofisika Institute), and pCNE8, 1632, THRO, 278, ZM197, JRCSF, t257, Du422, and BG505 were provided by the NIH ARRRP. Neutralization was determined in single-cycle neutralization assays using the standard TZM-bl neutralization assay. Briefly, antibodies and antibody-based particles were incubated with 10–15% tissue culture infectious dose of pseudovirus for 1 h at 37°C, followed by incubation with TZM-bl cells for 44–72 h. Virus neutralization was monitored by adding Britelite plus reagent (PerkinElmer) to the cells and measuring luminescence in relative light units (RLU) using a Synergy Neo2 multimode assay microplate reader (Biotek Instruments).

[0266] Pharmacokinetic and immunogenicity studies. In vivo studies were performed using 20g C57BL / 6 male mice. A surrogate multabody consisting of the scFab and scFc fragments of murine HD37 IgG2a fused to the N-terminus of the light chain of murine apoferritin was used for the study. A 2:1:1 ratio of HD37 scFab-mFerritin:Fc-mFerritin:mFerritin was transfected and purified according to the procedure described above. L35A, L234A, and P329G mutations were introduced into the murine IgG2a Fc construct to silence the effector function of the multabody. 74A single injection of 5 mg / kg multabodies or control samples (HD37 IgG1, HD37 IgG2a, and hpFerritin-PfCSP malaria peptide) in 200 μL of PBS (pH 7.5) was injected subcutaneously. Blood samples were collected at multiple time points, and serum samples were assessed for circulating antibody and ADA levels by ELISA. Briefly, 96-well Pierce nickel-coated plates (Thermo Fisher) were filled with 50 μL of 0.5 μg / ml His 6x Circulating HD37-specific concentrations were determined using reagent-specific standard curves of IgG and multabodies coated with the tagged antigen hCD19. For anti-drug antibody determination, Nunc MaxiSorp plates (Biolegend) were coated with the 12-mer HD37 scFab multabody or the hpFerritin PfCSP malaria peptide. HRP-Protein A (Invitrogen) was used as the secondary molecule, and the chemiluminescent signal was quantified using a Synergy Neo2 Multimode Assay Microplate Reader (Biotek Instruments).

[0267] result Multabodies can neutralize HIV-1 up to 500 times more potently than the gold standard IgG. They used the strong self-assembly properties of the light chain of human apoferritin to multimerize Fab onto the surface of a hollow globular protein cage formed by 24 monomers. Indeed, apoferritin self-assembles into a 12 nm diameter structure composed of 24 identical polypeptides, making it easily amenable to gene fusion. 70The N-terminus of each apoferritin subunit faces the outside of the globular cage and is therefore available for genetic fusion of proteins of interest. To maintain all the properties of IgG molecules, including high thermal stability and correct chain pairing, we created apoferritin fusions to single-chain Fab (scFab) and single-chain Fc (scFc) fragments. Upon folding, the apoferritin subunit acts as a building block to drive multimerization of the 24 proteins fused to its N-terminus (Figure 1). Importantly, the display of multiple specificities on the same molecule (e.g., Fab and Fc) can be achieved and controlled by cotransfection of selected ratios of DNA encoding each component, followed by a stringent affinity purification protocol that selects for all specificities (e.g., Protein L combined with Protein A affinity chromatography selects for Fab and Fc, respectively).

[0268] First, we investigated the impact of multivalency on HIV-1 bNAbs displayed on our novel multabody platform on their ability to block viral infection and compared them with standard bivalent IgG display of the same bNAbs. A panel of bNAbs with different specificities for Env was selected, and their scFabs were multimerized at different densities by cotransfection of a plasmid encoding scFab-human apoferritin with different ratios of unconjugated apoferritin (Fig. 2a). The multabodies organized into monodisperse, well-formed spherical particles with dense, continuous rings and regularly spaced protruding Fabs (Fig. 2a). Remarkably, the most potent anti-HIV bNAb described to date, PGDM1400 (median IC50 = 0.003 μg / mL), exhibits 100-500-fold higher neutralization (IC) as a 24-mer multabody compared to its IgG format against a panel of five pseudoviruses. 50Median (nM) fold improvement (Figure 2b). bNAb 10-1074 also showed a dramatic improvement in neutralization potency as a multabody compared to its IgG, whereas bNAbs 10E8, N49P7, and VRC01, while still effective, did not show the same enhancement.

[0269] Engineering apoferritin results in efficient hetero-oligomerization of 32-mer multabodies. Next, we sought to improve the breadth of the highly potent 24-mer PGDM1400 multabody by conferring multispecificity to the molecule. To this end, we engineered the PGDM1400 Fab in addition to the Fc fragment of the human IgG1 isotype into the near-pan-neutralizing antibody 10E8v4 (a modified 10E8 with improved solubility). 71) and N49P7 Fab. To achieve this level of hetero-oligomerization of four components (three Fabs and one Fc), we split the human apoferritin structure into two subunits (N-ferritin and C-ferritin) and attached a Fab to the N-terminus of each half (Figure 3a). Complementation of the split apoferritin led to self-association of the two halves, resulting in a highly efficient heterodimerization process of the fusion protein. Importantly, no significant differences were observed between the biophysical and functional properties of multabodies assembled with split and full-length apoferritin (Figure 4). This design enabled a simple two-step purification procedure to select multabodies with four distinct specificities (Figure 5) and showed high batch-to-batch uniformity (Figure 6). Furthermore, bisection of apoferritin allowed for the accommodation of additional Fab / Fc fragments, up to 32 components per molecule, compared to the standard apoferritin building block (Figure 3b). The eight extra locations (from the conventional 24 to 32 in our engineered platform) play a key role in providing multispecificity without sacrificing much of the increased potency observed with the 24-mer PGDM1400 multabody. Therefore, we designed multabody 32-N by cotransfection of scFab- and scFc-encoding plasmids at a 4:2:1:1 ratio, respectively, to achieve 16 copies of PGDM1400, 8 copies of Fc, 4 copies of 10E8v4, and 4 copies of N49P7 (Figure 3a). Multabody 32-N formed highly decorated, uniform particles (Figure 3b), with Tm and Tag distributions similar to those of the corresponding IgG molecules and previously reported IgG. 72The unfolding and aggregation transition temperatures were shown for 32-N (Fig. 3c and Fig. 7). Using binding kinetics experiments, we demonstrated that each component of the multabody (PDGM1400, N49P7, 10E8v4, and Fc fragment) was able to engage its epitope by binding to epitope-specific molecules: BG505 SOSIP D368R, 93TH057 gp120, MPER peptide, and human FcRn, respectively (Fig. 3d). None of the individual IgG molecules had the capacity to bind all antigens (Fig. 8). The ability of 32-N to engage multiple antigens indicates that the geometry created by the oligomeric format of the multabody does not adversely affect antigen binding, presumably because the binding interface faces outward.

[0270] To investigate whether a multabody could also be designed that cross-targets HIV Env and the T cell receptor CD4, we replaced N49P7 with iMab, a CD4-directed post-attachment inhibitor that has been shown to effectively eradicate HIV. 68、69 A multabody containing PDGM1400, iMab, 10E8v4, and an Fc fragment (termed 32-I) exhibited similar homogeneity, thermostability, and multispecificity to 32-N (Figures 3, 7, and 8), highlighting the robust plug-and-play nature of the multabody platform, where antibody sequences can be easily swapped to alter specificity.

[0271] HIV-1 multabodies exhibit exceptional pan-neutralizing activity and potency. Standardized in vitro TZM-bl neutralization assay 73 In this study, the neutralization potency and breadth of multabodies 32-N and 32-I were evaluated against a panel of 14 pseudoviruses (PsVs). The 14-PsV panel was designed to include low-susceptibility PsVs with a minimum of one resistant PsV for each bNAb evaluated. The IC of multabodies 50Values ​​and ranges are calculated for (i) each individual IgG, (ii) an IgG cocktail containing the same relative amount of each IgG present in the multabody, and (iii) the N6 / PGDM1400x10E8 trispecific antibody. 43 The 32-N and 32-I multabodies demonstrated 100% coverage against this panel, with median IC50 values ​​of 0.0093 μg / mL (4 pM) and 0.0085 μg / mL (3.5 pM), respectively (Figures 9 and 10). Complete viral coverage was also achieved with the IgG cocktail and trispecific antibody. However, the potency of the combined cocktail or trispecific antibody against all PsVs tested was similar to that of the best mAb tested alone (Table 1). Thus, more than 10- and 100-fold reductions in median IC50 values, calculated in μg / mL and nM, were obtained for the multabodies compared to the IC50 values ​​of the IgG cocktail and trispecific antibody, respectively (Figures 9 and 10, Tables 1 and 2). [Table 1] [Table 2]

[0272] The in vivo pharmacokinetics and anti-drug antibody profile of multabodies are similar to those of the corresponding IgG. Next, we investigated the in vivo toxicity, immunogenicity, and bioavailability of multabodies after subcutaneous administration of 5 mg / kg in mice. To evaluate our novel platform technology, we used a species-matched surrogate multabody consisting of a mouse Fab and a mouse Fc (IgG2a isotype) fused to a mouse apoferritin subunit, as opposed to the all-human components used for HIV-1 multabodies targeted for human use. The Fab specificity selected for this surrogate multabody does not bind to endogenous mouse proteins, similar to the HIV-1 human mAb, which does not bind to endogenous human proteins. Multabody administration was well tolerated, with no weight loss or visible signs of toxicity. The surrogate multabody did not induce a significant immunogenic response in mice; the levels of anti-drug antibodies (ADA) detected after 14 days were negligible for both the surrogate multabody and its sequence-matched IgG2a (Figure 11b). This contrasts with highly immunogenic particles displaying malaria circumsporozoite protein (CSP) on the surface of Helicobacter pylori ferritin (hpFerritin), which was used as a positive immunogenic control. Furthermore, the surrogate multabody exhibited a range of in vivo exposure days similar to those of the parent IgG1 and IgG2a molecules (Figure 11b). The serum half-life was significantly reduced following the introduction of the LALAP mutation, which has been reported to silence Fc effector function. 74 Overall, these data demonstrate that our multabody platform is feasible for tailoring bioavailability properties and provide a promising set of early in vivo validation for its developability.

[0273] References JPEG2026004351000044.jpg92149 JPEG2026004351000045.jpg225149 JPEG2026004351000046.jpg226149 JPEG2026004351000047.jpg226149 JPEG2026004351000048.jpg226149 JPEG2026004351000049.jpg220149 JPEG2026004351000050.jpg21149 [Sequence List Free Text]

[0274] Sequence Listing 1 <223> Linker Sequence Listing 4 <223> Ibalizumab-A12P light chain Sequence Listing 5 <223> Ibalizumab-A12P heavy chain Sequence Listing 6 <223> 10E8.v4 light chain Sequence Listing 7 <223> 10E8.v4 heavy chain Sequence Listing 8 <223> N49P7 light chain Sequence Listing 9 <223> N49P7 heavy chain Sequence Listing 10 <223> PGDM1400 light chain Sequence Listing 11 <223> PGDM1400 heavy chain Sequence Listing 14 <223> Linker Sequence Listing 15 <223> Linker Sequence Listing 16 <223> PGDM1400-hFerr Sequence Listing 17 <223> Fc-N-hFerr LS Sequence Listing 18 <223> N49P7-C-hFerr Sequence Listing 19 <223> Ibalizumab-A12P-C-hFerr Sequence Listing 20 <223> 10E8.v4-C-hFerr Sequence Listing 21 <223> scFab 10E8

Claims

1. a first nanocage monomer subunit of the nanocage monomer; and Biologically active moieties linked to the first nanocage monomer subunit A fusion protein comprising: The above fusion protein, wherein the fusion protein self-assembles with a protein comprising a second nanocage monomer subunit to form a nanocage monomer.

2. The fusion protein of claim 1 , wherein the biologically active moiety decorates the interior and / or exterior surfaces of the assembled nanocages.

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

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

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

6. 6. The fusion protein of claim 4 or 5, wherein the Fc fragment comprises one or more mutations, such as LS, YTE, LALA, and / or LALAP, that modulate 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 fragment thereof comprises a Fab fragment.

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

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

10. 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 and a second chain, optionally separated by a linker.

11. The linker is: 【Chemistry 1】 11. The fusion protein of claim 10, comprising or consisting of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to

12. 10. The fusion protein of claim 9 in association with a separately produced Fab light chain and / or heavy chain.

13. 13. The fusion protein of any one of claims 3 to 12, wherein the antibody or fragment thereof specifically binds to an antigen associated with an antibody-preventable and / or antibody-treatable condition.

14. 14. The fusion protein of claim 13, wherein 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, including solid and liquid cancers (e.g., CD19, CD22, CD79, BCMA, or CD20), or an immune disease, including an autoimmune disease.

15. 15. The fusion protein of claim 14, wherein the antigen is related to HIV-1 and the antibody or fragment thereof comprises, for example, ibalizumab-A12P, 10E8, 10E8.v4, N49P7, PGDM1400, 10-1074, VRC01, or a combination thereof.

16. The antibody or fragment thereof is selected from the group consisting of: Fc chain 1: 【Chemistry 2】 Fc chain 2: 【Transformation 3】 Ibalizumab-A12P light chain: 【Chemistry 4】 Ibalizumab-A12P heavy chain: 【Transformation 5】 10E8. v4 light chain: 【Transformation 6】 10E8. v4 heavy chain: 【Transformation 7】 N49P7 light chain: 【Transformation 8】 N49P7 heavy chain: 【Chemistry 9】 PGDM1400 light chain: 【Chemistry 10】 PGDM1400 heavy chain: 【Chemistry 11】 or combinations of these 16. The fusion protein of claim 15, comprising or consisting of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to

17. 17. The fusion protein of any one of claims 3 to 16, wherein the antibody or fragment thereof is conjugated or associated with a further moiety such as an antigen, a detectable moiety (e.g., a small molecule, a fluorescent molecule, a radioisotope, or a magnetic particle), a pharmaceutical agent, a diagnostic agent, or a combination thereof.

18. The fusion protein of claim 17, wherein the antibody or fragment thereof comprises an antibody-drug conjugate.

19. The fusion protein of claim 3 , wherein the antigen is associated with a vaccine-preventable and / or vaccine-treatable condition.

20. 20. The fusion protein of claim 19, wherein the antigen is associated with an infectious agent, including a virus, bacterium, parasite, fungus, or yeast; a cancer, including solid and liquid cancers; or an immune disease, including an autoimmune disease.

21. 4. The fusion protein of claim 3, wherein the detectable moiety comprises a fluorescent protein such as GFP, EGFP, ametrine, and / or a flavin-based fluorescent protein such as an LOV protein such as iLOV.

22. The fusion protein of claim 3 , wherein the pharmaceutical agent comprises a small molecule, a peptide, a lipid, a carbohydrate, or a toxin.

23. 23. The fusion protein of any one of claims 1 to 22, wherein about 3 to about 100 nanocage monomers, e.g., 24, 32, or 60 monomers, or about 4 to about 200 nanocage monomer subunits, e.g., 4, 6, 8, 10, 12, 14, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 or more, optionally in combination with one or more whole nanocage monomers, self-assemble to form a nanocage.

24. 24. The fusion protein of any one of claims 1 to 23, wherein the nanocage monomer is selected from ferritin, apoferritin, encapsulin, SOR, lumazine synthase, pyruvate dehydrogenase, carboxysome, vault protein, GroEL, heat shock protein, E2P, MS2 coat protein, fragments thereof, and variants thereof.

25. 25. The fusion protein of claim 24, wherein the nanocage monomer is apoferritin.

26. 26. The fusion protein of claim 25, wherein the first and second nanocage monomer subunits interchangeably comprise the "N" and "C" regions of apoferritin.

27. The "N" region of apoferritin is as follows: 【Chemistry 12】 27. The fusion protein of claim 26, comprising or consisting of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to

28. The "C" region of apoferritin is as follows: 【Chemistry 13】 28. The fusion protein of claim 26 or 27, comprising or consisting of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to

29. 29. The fusion protein of any one of claims 1 to 28, further comprising a linker between the nanocage monomer subunit and the biologically active moiety.

30. 30. The fusion protein of claim 29, wherein the linker is flexible or rigid and comprises from about 1 to about 30 amino acid residues, such as from about 8 to about 16 amino acid residues.

31. 31. The fusion protein of claim 29 or 30, wherein the linker comprises a GGS repeat, such as 1, 2, 3, 4 or more GGS repeats.

32. The linker is: 【Chemistry 14】 30. The fusion protein of claim 29, comprising or consisting of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to

33. 33. The fusion protein of any one of claims 1 to 32, further comprising a C-terminal linker.

34. The C-terminal linker is: 【Chemistry 15】 34. The fusion protein of claim 33, comprising or consisting of a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to

35. A fusion protein pair described in any one of claims 1 to 34, wherein the pair self-assembles to form a nanocage monomer, and the first and second nanocage monomer subunits are fused to different biologically active moieties.

36. 35. A nanocage comprising at least one fusion protein of any one of claims 1 to 34 and at least one second nanocage monomer subunit that self-assembles with the fusion protein to form a nanocage monomer.

37. 36. A nanocage comprising at least one pair of the nanocage of claim 35.

38. 38. The nanocage of claim 36 or 37, wherein each nanocage monomer comprises a fusion protein of any one of claims 1 to 34 or a pair of claim 35.

39. 38. The nanocage of claim 36 or 37, wherein about 20% to about 80% of the nanocage monomers comprise the fusion protein of any one of claims 1 to 34 or the pair of claim 35.

40. 40. The nanocage of any one of claims 36-39, comprising at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different biologically active moieties.

41. 41. The nanocage of any one of claims 36 to 40, comprising at least one whole nanocage monomer, optionally fused to a biologically active moiety, which may be the same as or different from the biologically active moiety of any one of claims 1 to 37.

42. 42. The nanocage of any one of claims 36 to 41, wherein the nanocage is multivalent and / or multispecific.

43. 43. A nanocage according to any one of claims 36 to 42, comprising first, second, and third fusion proteins according to any one of claims 1 to 34, and at least one whole nanocage monomer, optionally fused to a biologically active moiety, wherein the biologically active moieties of the first, second, and third fusion proteins and the biologically active moiety of the whole nanocage monomer are all different from each other.

44. 44. The nanocage of claim 43, wherein the first, second, and third fusion proteins each comprise an antibody or fragment thereof fused to N- or C-ferritin, and 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.

45. 45. The nanocage of claim 44, wherein the antibody or fragment thereof of the first fusion protein is an Fc fragment, and the second and third fusion proteins each comprise an antibody or fragment thereof specific for a different antigen of a virus, such as HIV, or wherein one of the second and third fusion proteins comprises an antibody or fragment thereof specific for an antigen of a virus, such as HIV, and the third fusion protein comprises an antibody or fragment thereof specific for a different antigen, such as the CD4 receptor, and the entire nanocage monomer is optionally fused to a biologically active portion specific for another, different antigen of the same virus, such as HIV.

46. 46. ​​The nanocage of claim 45, wherein the Fc fragment comprises one or more mutations, such as 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.

47. 47. The nanocage of claim 45 or 46, wherein the antibody or fragment thereof of the second fusion protein is N49P7 or iMab A12P, and the antibody or fragment thereof of the third fusion protein is 10E8v4.

48. The following four fusion proteins: a. PGDM1400 fused to full-length ferritin (optionally scPGDM1400); b. Fc (optionally scFc) fused to N-ferritin; c. N49P7 or iMab A12P (optionally scN49P7 or sciMab A12P) fused to C-ferritin, and d. 10E8v4 (optionally sc10E8v4) fused to C-ferritin 48. The nanocage of claim 47, comprising:

49. 49. The nanocage of claim 48, comprising a ratio of a:b:c:d of 4:2:1:

1.

50. The following array: a. PGDM1400-hFerrr: 【Chemistry 16】 b. Fc-N-hFerrr LS 【Chemistry 17】 c1. N49P7-C-hFerrr [Chemistry 18] c2. Ibalizumab-A12P-C-hFerr 【Chemistry 19】 or d. 10E8. v4-C-hFerrr 【Chemistry 20】 50. The nanocage of claim 48 or 49, wherein the ferritin subunits are in bold, the linker is underlined, the light chain is in italics, and the heavy chain is in lowercase.

51. 51. The nanocage of any one of claims 36 to 50, carrying a cargo molecule such as a pharmaceutical, diagnostic, and / or imaging agent.

52. 52. The nanocage of claim 51, wherein the cargo molecule is not fused to a fusion protein and is contained within the nanocage.

53. 52. The nanocage of claim 51, wherein the cargo molecule is a protein and is fused to a fusion protein such that the cargo molecule is contained within the nanocage.

54. 54. The nanocage of any one of claims 51 to 53, wherein the cargo molecule is a fluorescent protein such as GFP, EGFP, ametrine, and / or a flavin-based fluorescent protein such as an LOV protein such as iLOV.

55. 55. The nanocage of any one of claims 51 to 54, wherein a cargo molecule is contained therein to provide a T cell epitope, but may not provide a B cell epitope.

56. 55. The nanocage of any one of claims 51 to 54, wherein a cargo molecule is fused to a fusion protein and is included internally to provide a T cell epitope, but may not provide a B cell epitope.

57. 53. The nanocage of claim 51 or 52, wherein the cargo molecule is a small molecule, a radioisotope, or a magnetic particle.

58. 58. The nanocage of any one of claims 36-57, further comprising an antigen on its surface.

59. 59. The nanocage of claim 58, wherein the antigen is expressed as a fusion protein with the nanocage monomer.

60. 60. A vaccine comprising the nanocage of any one of claims 36 to 59.

61. 60. A therapeutic or prophylactic composition comprising the nanocages of any one of claims 36 to 59.

62. A nucleic acid molecule encoding a fusion protein according to any one of claims 1 to 34 or a pair according to claim 35.

63. 63. A vector comprising the nucleic acid molecule of claim 62.

64. A host cell comprising the vector of claim 63 and producing a fusion protein of any one of claims 1 to 34 or a pair of claims 35.

65. 61. A method of immunizing a subject comprising administering the nanocage of any one of claims 36 to 59 or the vaccine of claim 60.

66. 61. A method for treating and / or preventing a disease or condition, comprising administering a nanocage described in any one of claims 36 to 59 or a vaccine described in claim 60.

67. 67. The method of claim 66, wherein the disease or condition is cancer, an infectious disease such as HIV, malaria, influenza, RSV, rotavirus, or an autoimmune disease.

68. 60. A method for diagnostic imaging comprising administering a nanocage described in any one of claims 36 to 59 to a subject, tissue, or sample, wherein the nanocage comprises a diagnostic label such as a fluorescent protein or a magnetic imaging moiety, and imaging the subject, tissue, or sample.

69. 61. Use of the nanocage of any one of claims 36 to 59 or the vaccine of claim 60 for immunizing a subject.

70. 61. Use of a nanocage according to any one of claims 36 to 59 or a vaccine according to claim 60 for treating and / or preventing a disease or condition.

71. 71. The use of claim 70, wherein the disease or condition is cancer, an infectious disease such as HIV, malaria, influenza, RSV, rotavirus, or an autoimmune disease.

72. 60. Use of a nanocage according to any one of claims 36 to 59 for diagnostic imaging of a subject, tissue or sample, wherein the nanocage comprises a diagnostic label such as a fluorescent protein or a magnetic imaging moiety, and the subject, tissue or sample is imaged.

73. 60. Use of a fusion protein according to any one of claims 1 to 34, a pair according to claim 35 or a nanocage according to any one of claims 36 to 59 as a research tool, such as in FACS or ELISA.

74. 61. A nanocage according to any one of claims 36 to 59 or a vaccine according to claim 60 for use in immunising a subject.

75. 61. A nanocage according to any one of claims 36 to 59 or a vaccine according to claim 60 for treating and / or preventing a disease or condition.

76. 71. The nanocage of claim 70, wherein the disease or condition is cancer, an infectious disease such as HIV, malaria, influenza, RSV, rotavirus, or an autoimmune disease.

77. 60. The nanocage of any one of claims 36 to 59 for use in diagnostic imaging of a subject, tissue, or sample, wherein the nanocage comprises a diagnostic label, such as a fluorescent protein or a magnetic imaging moiety, and images the subject, tissue, or sample.

78. A fusion protein according to any one of claims 1 to 34, a pair according to claim 35 or a nanocage according to any one of claims 36 to 59 for use as a research tool, such as in FACS or ELISA.

79. A nanocage comprising a plurality of fusion proteins, Each fusion protein comprises a ferritin light chain and a Fab fragment; each Fab fragment is capable of specifically binding to an antigen; Each Fab fragment decorates the exterior surface of the nanocage; and the plurality comprises at least 12 fusion proteins; The nanocage shown above.

80. 80. The nanocage of claim 79, wherein the plurality comprises at least 19 fusion proteins.

81. 81. The nanocage of claim 80, wherein the plurality comprises at least 24 fusion proteins.

82. 83. The nanocage of claim 82, wherein the plurality is 24 fusion proteins.

83. 83. The nanocage of any one of claims 79-82, wherein the Fab fragments of multiple fusion proteins are capable of specifically binding to the same antigen.

84. 84. The nanocage of any one of claims 79-83, wherein the nanocage does not contain any ferritin heavy chains.

85. 85. The nanocage of any one of claims 79 to 84, wherein the Fab fragment is a Fab fragment of a neutralizing antibody.

86. 86. The nanocage of any one of claims 79-85, wherein the antigen is associated with an infectious agent.

87. 87. The nanocage of claim 86, wherein the infectious agent is a virus.

88. 88. The nanocage of claim 87, wherein the virus is human immunodeficiency virus (HIV).

89. 89. The nanocage of any one of claims 85-88, capable of neutralizing an infectious agent with at least 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, or 500-fold greater neutralizing activity compared to a control.

90. 90. The nanocage of claim 89, wherein the control comprises a full-length version of a neutralizing antibody.

91. 91. The nanocage of claim 90, wherein the neutralizing antibody is an IgG antibody.

92. A nanocage comprising a plurality of first fusion proteins and a plurality of second fusion proteins, Each first fusion protein comprises a nanocage monomer or a subunit thereof and a Fab fragment capable of specifically binding to an antigen; and Each second fusion protein comprises a nanocage monomer or a subunit thereof and an Fc fragment; The nanocage shown above.

93. 93. The nanocage of claim 92, wherein the nanocage monomer is selected from ferritin, apoferritin, encapsulin, sulfur oxygen reductase (SOR), lumazine synthase, pyruvate dehydrogenase, carboxysome, vault protein, GroEL, heat shock protein, E2P, MS2 coat protein, fragments thereof, and variants thereof.

94. 94. The nanocage of claim 93, wherein the nanocage monomer is apoferritin or ferritin.

95. 94. The nanocage of claim 93, wherein the nanocage monomer is a ferritin light chain.

96. 96. The nanocage of claim 94 or 95, wherein the nanocage monomer does not contain any ferritin heavy chains.

97. A nanocage comprising a plurality of first fusion proteins and a plurality of second fusion proteins, (a)(i) a first fusion protein comprising a ferritin light chain and a Fab fragment capable of specifically binding to a first antigen; and (ii) the second fusion protein comprises a ferritin light chain and a Fab fragment capable of specifically binding to a second antigen; or (b)(i) a first fusion protein comprises N-ferritin and a Fab fragment capable of specifically binding to a first antigen; and (ii) a second fusion protein comprising C-ferritin and a Fab fragment capable of specifically binding to a second antigen; Within each fusion protein, the Fab fragment is fused to the N-terminus of a ferritin light chain, N-ferritin, or C-ferritin; and the first antigen is different from the second antigen; The nanocage shown above.

98. A nanocage comprising a plurality of first fusion proteins, a plurality of second fusion proteins, and a plurality of third fusion proteins, (a) a first fusion protein comprising a ferritin light chain and a Fab fragment capable of specifically binding to a first antigen; (b) a second fusion protein comprising C-ferritin and a Fab fragment capable of specifically binding to a second antigen; and (c) a third fusion protein comprising N-ferritin and an Fc fragment; Within each fusion protein, the Fab or Fc fragment is fused to the N-terminus of a ferritin light chain, C-ferritin, or N-ferritin; and the first antigen is different from the second antigen; The nanocage shown above.

99. 99. The nanocage of claim 97 or 98, further comprising a plurality of fourth fusion proteins, the fourth fusion proteins comprising C-ferritin and a Fab fragment capable of specifically binding to a third antigen, the third antigen being different from the first and second antigens.

100. 100. The nanocage of claim 97, 98, or 99, wherein the Fab fragment is a Fab fragment of a neutralizing antibody.

101. 101. The nanocage of any one of claims 97-100, wherein the first and second antigens are each associated with an infectious agent.

102. 102. The nanocage of claim 101, wherein the first and second antigens are associated with the same infectious agent.

103. 103. The nanocage of claim 101 or 102, wherein the infectious agent is a virus.

104. 104. The nanocage of claim 103, wherein the virus is human immunodeficiency virus (HIV).

105. the first and second antigens are each associated with a virus; The nanocages are capable of neutralizing 100% of the pseudoviruses in the panel of pseudoviruses; The panel of pseudoviruses includes, for each Fab fragment in the nanocage capable of specifically binding to an antigen associated with the virus, at least one pseudovirus that is resistant to a neutralizing antibody corresponding to that Fab fragment.

101. The nanocage of claim 100.

106. The nanocage of claim 105, wherein the panel of pseudoviruses comprises at least 10, at least 11, at least 12, at least 13, or at least 14 pseudoviruses.

107. the first and second antigens are each associated with a virus; The nanocages have an IC of less than 1 nM, less than 500 pM, less than 250 pM, less than 100 pM, less than 50 pM, less than 10 pM, or less than 5 pM. ５０ can neutralize a panel of pseudoviruses, The panel of pseudoviruses includes, for each Fab fragment in the nanocage capable of specifically binding to an antigen associated with the virus, at least one pseudovirus that is resistant to a neutralizing antibody corresponding to that Fab fragment.

107. The nanocage of any one of claims 100, 105, or 106.

108. the first and second antigens are each associated with a virus; The nanocages are then transferred to one or more control ICs. ５０ (molar concentration) at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold lower ５０ and capable of neutralizing a panel of pseudoviruses having The panel of pseudoviruses includes, for each Fab fragment in the nanocage capable of specifically binding to an antigen associated with the virus, at least one pseudovirus that is resistant to a neutralizing antibody corresponding to that Fab fragment.

108. The nanocage of any one of claims 100 and 105-107.

109. 109. The nanocage of claim 108, wherein the one or more controls comprise a neutralizing antibody corresponding to a Fab fragment within the nanocage, the Fab fragment being capable of specifically binding to an antigen associated with the virus.

110. 110. The nanocage of claim 109, wherein the neutralizing antibody is an IgG antibody.

111. 111. The nanocage of claim 108, 109, or 110, wherein the one or more controls comprise a cocktail of neutralizing antibodies, the cocktail comprising, for each Fab fragment in the nanocage capable of specifically binding to an antigen associated with the virus, a neutralizing antibody corresponding to that Fab fragment.

112. 112. The nanocage of claim 111, wherein the neutralizing antibody is an IgG antibody.

113. 113. The nanocage of any one of claims 108-112, wherein the one or more controls comprise one or more multispecific antibodies, wherein the one or more multispecific antibodies are capable of collectively binding to the first and second antigens, and optionally a third antigen.

114. 114. The nanocage of claim 113, wherein the one or more controls comprise a trispecific antibody capable of specifically binding to the first, second, and third antigens.

115. the first, second, and third antigens are associated with HIV-1; the Fab fragment of the first fusion protein is PDGM1400 Fab; the Fab fragment of the second fusion protein is 10E8v4 Fab; the Fc fragment of the third fusion protein is a human IgG1 Fc fragment; and the Fab fragment of the fourth fusion protein is N49P7 Fab; 115. The nanocage of any one of claims 99 to 114.

116. the first and second antigens are associated with HIV-1, and the third antigen is associated with CD4; the Fab fragment of the first fusion protein is PDGM1400 Fab; the Fab fragment of the second fusion protein is 10E8v4 Fab; the Fc fragment of the third fusion protein is a human IgG1 Fc fragment; and the Fab fragment of the fourth fusion protein is an iMab Fab; 115. The nanocage of any one of claims 99 to 114.

117. 117. A therapeutic or prophylactic composition comprising the nanocage of any one of claims 79 to 116.

118. A method for treating or preventing a disease or condition, comprising administering a nanocage described in any one of claims 79 to 116 or a composition described in claim 117 to a subject in need thereof.

119. 1. A method for making multispecific self-assembled nanocages characterized by a preselected ratio of different specificities, comprising: co-transfecting a host cell with one or more expression plasmids comprising multiple polynucleotides each encoding a fusion protein; wherein each fusion protein comprises (i) a nanocage monomer or a subunit thereof, and (ii) an antibody or antibody fragment of a given specificity; the step of co-transfecting comprises co-transfecting the polynucleotides in a ratio based on a preselected ratio; Obtaining the polypeptide produced by the host cell; and Purifying the polypeptides by affinity selection against all the different specificities present in the assembled nanocages. The above method, comprising:

120. the plurality of polynucleotides comprises at least one polynucleotide encoding a first fusion protein and at least one polynucleotide encoding a second fusion protein; the first fusion protein comprises a first nanocage monomer subunit; the second fusion protein comprises a second nanocage monomer subunit capable of self-assembly with the first nanocage monomer subunit; 120. The method of claim 119.

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