Multi-valent and multi-specific nanoparticle platforms and methods

Abstract

A fusion protein comprising: a first nanocage monomer subunit of a nanocage monomer; and a bioactive moiety attached to the first nanocage monomeric subunit; wherein the fusion protein is self-assembled with a protein comprising a second nanocage monomer subunit to form a nanocage monomer.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202511333631.X (22) Application Date 2020.07.31 (30) Priority Data 62 / 881,899 2019.08.01 US (62) Divisional Application Data 202080069559.3 2020.07.31 (71) Applicant: Sick Children's Hospital Address: Ontario, Canada Applicant: University of Toronto Management Board (72) Inventors: Jean-Philippe Julien, Edierne Luhas, Diaz, Bessin, Treynor, Zhao Tiantian (74) Patent Agency: Shenzhen Eagle Wing Intellectual Property Agency Co., Ltd. 44658 Patent Attorneys: Wang Yijin, Huang Xing'er (51) Int.Cl. C07K 19 / 00 (2006.01) C07K 16 / 114 (2026.01) (54) Title of Invention: Multivalent and Multispecific Nanoparticle Platform and Method (57) Abstract: A fusion protein comprising: a first nanocage monomer subunit of a nanocage monomer; and a bioactive portion connected 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. Claims 1 page Description 49 pages Sequence Listing (Electronic Publication) Figures 11 pages CN 121449754 A 2026.02.03 CN 1 21 44 97 54 A 1. A fusion protein comprising: a first nanocage monomer subunit of a nanocage monomer; and a bioactive portion connected to the first nanocage monomer subunit; wherein the fusion protein self-assembles with a protein comprising a second nanocage monomer subunit to form a nanocage monomer. 2. The fusion protein of claim 1, wherein the bioactive portion modifies the inner and / or outer surface of the assembled nanocage. 3. The fusion protein of claim 1 or 2, wherein the bioactive portion comprises: an antibody or a fragment thereof, an antigen, a detectable portion, a pharmaceutical agent, a diagnostic agent, or a combination thereof. 4. The fusion protein of claim 3, wherein the antibody or a fragment thereof comprises an Fc fragment. 5. The fusion protein of claim 4, wherein the Fc fragment is an IgG1 Fc fragment. 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, said one or more mutations regulating the half-life of the fusion protein, for example, from minutes or hours to days, weeks, or months. 7. The fusion protein of claim 3, wherein the antibody or a fragment thereof comprises a Fab fragment.8. The fusion protein of claim 3, wherein the antibody or fragment thereof comprises a scFab fragment, a scFv fragment, or a sdAb fragment. 9. The fusion protein of claim 3, wherein the antibody or fragment thereof comprises a heavy chain and / or a light chain of a Fab fragment. 10. The fusion protein of claim 9, wherein the antibody or fragment thereof comprises both a light chain and a heavy chain, or, in the case of an Fc fragment, comprises a first chain and a second chain optionally separated by a linker. Claims 1 / 1 page 2 CN 121449754 A Multivalent and Multispecific Nanoparticle Platform and Method Technical Field

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

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

[0003] Protein self-assembly is an attractive method for fabricating nanoparticles containing sensitive and metastable proteins. In fact, self-assembled nanoparticles are formed under physiological conditions through non-covalent interactions and reliably produce uniform and generally symmetrical nanocapsules or nanocages. Self-assembled protein nanoparticles have three distinct surfaces: an outer surface, an inner surface, and an intersubunit surface, all of which can be tuned to increase their functionality.

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

[0005] However, there is still a need for improved compositions and methods involving nanocages. Summary of the Invention

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

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

[0008] a first nanocage monomer subunit; and

[0009] a bioactive portion connected to the first nanocage monomer subunit;

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

[0011] In one aspect, the bioactive portion modifies the inner and / or outer surfaces of the assembled nanocage.

[0012] In one aspect, the bioactive portion comprises: an antibody or a fragment thereof, an antigen, a detectable portion, a pharmaceutical agent, a diagnostic agent, or a combination thereof.

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

[0014] In one aspect, the Fc fragment is an IgG1 Fc fragment.

[0015] In one aspect, the Fc fragment comprises one or more mutations, such as LS, YTE, LALA, and / or LALAP, said one or more mutations regulating the half-life of the fusion protein, for example, from minutes or hours to days, weeks, or months.

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

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

[0018] In one aspect, the antibody or a fragment thereof comprises the heavy chain and / or light chain of a Fab fragment. Specification 1 / 49 page 3 CN 121449754 A

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

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

[0021] GGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGTATAGASSGSGSSGSSSS GGTG.

[0022] In one aspect, the fusion protein associates with separately generated Fab light chains and / or heavy chains.

[0023] In one aspect, the antibody or a fragment thereof specifically binds to an antigen associated with a disease that the antibody can prevent and / or a disease that the antibody can treat.

[0024] In one aspect, the antigen is associated with: infectious agents, including viruses (e.g., HIV (including HIV-1), influenza virus, RSV, rotavirus), bacteria (e.g., TB, Clostridium difficile), parasites (e.g., malaria), fungi, or yeast; cancers (e.g., CD19, CD22, CD79, BCMA, or CD20), including solid and liquid cancers; or immune diseases, including autoimmune diseases.

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

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

[0027] Fc chain 1:

[0028] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

[0029] GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK

[0030] AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK;

[0031] Fc chain 2:

[0032] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

[0033] GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK

[0034] AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK; Ibalizumab-A12P light chain:

[0035] DIVMTQSPDSLPVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWAST

[0036] RESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIKRTVAAPS

[0037] VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0038] Ibalizumab-A12P heavy chain:

[0039] QVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGT

[0040] DYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQG

[0041] TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGSR; 10E8.v4 light chain:

[0042] SELTQDPAVSVALKQTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDR

[0043] FSGSASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVLSQPKAAPSVTL

[0044] FPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQW Specification Page 2 / 49 4 CN 121449754 A KSHRSYSCQVTHEGSTVEKTVAPTEC;

[0045] 10E8.v4 heavy chain:

[0046] EVRLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEG

[0047] WSVDYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGE

[0048] EYFQDWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS

[0049] GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCSR;

[0050] N49P7 light chain:

[0051] QSALTQPRSVSASPGQSVTISCTGTHNLVSWCQHQPGRAPKLLIYDFNKRPSGVPDRFSG

[0052] SGSGGTASLTITGLQDDDDAEYFCWAYEAFGGGTKLTVLGQPKAAPSVTLFPPSSEELQ

[0053] ANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTEC;

[0054] N49P7 heavy chain:

[0055] ADLVQSGAVVKKPGDSVRISCEAQGYRFPDYIIHWIRRAPGQGPEWMGWMNPMGGQV

[0056] NIPWKFQGRVSMTRDTSIETAFLDLRGLKSDDTAVYYDRSNGSGKRFESSNWFLDLWG

[0057] RGTAVTIQSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDSR;

[0058] PGDM1400 light chain:

[0059] DFVLTQSPHSLSVTPGESASISCKSSHSLIHGDRNNYLAWYVQKPGRSPQLLIYLASSRAS

[0060] GVPDRFSGSGSDKDFTLKISRVETEDVGTYYCMQGRESPWTFGQGTKVDIKRTVAAPSV

[0061] FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0062] PGDM1400 heavy chain:

[0063] QAQLVQSGPEVRKPGTSVKVSCKAPNTLKTYDLHWVRSVPGQGLQWMGWISHEGDK

[0064] KVIVERFKAKVTIDWDRSTNTAYLQLSGLTSGDTAVYYCAKGSKHRLRDYALYDDDGA

[0065] LNWAVDVDYLSNLEFWGQGTAVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY

[0066] FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDSR; or

[0067] combinations thereof.

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

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

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

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

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

[0073] In one aspect, the agent includes small molecules, peptides, lipids, carbohydrates, or toxins.

[0074] In one aspect, about 3 to about 100 nanocage monomers, such as 24, 32, or 60 monomers, or about 4 to about 200 nanocage monomer subunits, such as 4, 6, 8, 10, 12, 14, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or more subunits, optionally combined with one or more intact nanocage monomers, self-assemble to form a nanocage.

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

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

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

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

[0079] MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLK MQNQRGGRALFQDIKKPAEDEW.

[0080] In one aspect, the "C" region of deferroferritin comprises, or consists of, a sequence having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with, the following sequence:

[0081] GKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGP EAGLGEYLFERLTLRHD.

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

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

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

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

[0086] ASTASSASSGGGGGGSGGSGGSGGS.

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

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

[0089] GGSGGSGGSGGSGGGASGGS.

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

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

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

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

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

[0095] In one aspect, the fusion protein comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different bioactive moieties. Specification 4 / 49 pages 6 CN 121449754 A

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

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

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

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

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

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

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

[0103] In one aspect, the nanocage comprises or is composed of four fusion proteins:

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

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

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

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

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

[0109] In one aspect, the nanocage comprises, or is composed of, a sequence having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with one or more of the following sequences,Ferritin subunits are shown in bold, linkers are underlined, light chains are shown in italics, and heavy chains are shown in lowercase letters: Specification 5 / 49 page 7 CN 121449754 A

[0110] Specification 6 / 49 page 8 CN 121449754 A

[0111]

[0112] In one aspect, the nanocage carries a cargo molecule, such as a pharmaceutical, diagnostic, and / or imaging agent.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0136] According to one aspect, the present invention provides the nanocages or vaccines described herein for treating and / or preventing a disease or condition.

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

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

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

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

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

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

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

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

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

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

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

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

[0149] In one aspect, the nanocage does not include any ferritin heavy chains.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0167] (b)(i) the first fusion protein comprises N-ferritin and a Fab fragment capable of specifically binding to a first antigen, and the specification page 9 / 49 11 CN 121449754 A

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0184] wherein the nanocage is capable of neutralizing 100% of the pseudoviruses in the pseudovirus group, and

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

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

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

[0188] wherein the nanocage is capable of neutralizing the pseudovirus group with an IC50 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, and

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

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

[0191] wherein the nanocage is capable of neutralizing a group of pseudoviruses with an IC50 of up to 1 / 10, up to 1 / 20, up to 1 / 30, up to 1 / 40, up to 1 / 50, up to 1 / 60, up to 1 / 70, up to 1 / 80, up to 1 / 90, or up to 1 / 100 of one or more controls, and

[0192] wherein for each Fab fragment within the nanocage capable of specifically binding to the antigen associated with the virus, the group of pseudoviruses comprises at least one pseudovirus resistant to a neutralizing antibody corresponding to the Fab fragment.

[0193] In one aspect, 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 the antigen associated with the virus. Specification 10 / 49 pages 12 CN 121449754 A

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

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

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

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

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

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

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

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

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

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

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

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

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

[0207] the Fc fragment of the third fusion protein is a human IgG1 Fc fragment, and

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

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

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

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

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

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

[0214] wherein the co-transfection step comprises: co-transfecting the polynucleotide at a ratio based on the preselection ratio;

[0215] obtaining a polypeptide produced by the host cell; and

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

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

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

[0219] wherein the second fusion protein includes a second nanocage monomer subunit capable of self-assembling with the first nanocage monomer subunit. Specification 11 / 49 pages 13 CN 121449754 A

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

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

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

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

[0224] Figure 3. Design, assembly, and biophysical characterization of the 32-N multispecific multi-affinity antibody and the 32-I multispecific multi-affinity antibody. (a) Schematic diagram of the human deferroferrin split design that facilitates the heterodimerization of the scFab-human deferroferrin subunit. The resulting halves, called N-ferritin and C-ferritin, span residues 1 to 95 and residues 95 to 175, respectively. scFc is attached to the N-terminus of the N-ferritin hemisphere, while N49P7, iMab (also referred to as ibalizimab in this paper), and 10E8v4 are attached to the N-terminus of the C-ferritin hemisphere. Heterodimerization of the split halves drives the self-assembly of the different antibody fragments, resulting in the formation of a single human aferritin subunit with two cargo molecules. These constructs are further combined with PGDM1400 scFab attached to the full-length human aferritin subunit, causing the assembly of nanoparticles displaying a mixture of 32 scFab / scFc units on the surface of the multispecific multi-affinity antibody. The figure shows a negative-stained electron micrograph, a schematic diagram of the scFab / scFc 32-N / 32-I design, and the specific composition of the 32-N and 32-I multispecific multi-affinity antibodies. Based on the heterooligomerization necessary to drive self-assembly, purification of the multispecific multi-affinity antibody with four components can be achieved through a two-step purification process: protein A (Fc binding) and protein L (PGDM1400 binding). (b) Size exclusion chromatography of 24-mer PGDM1400 multispecific multi-affinity antibody (black), 32-N multispecific multi-affinity antibody (deep magenta), and 32-I multispecific multi-affinity antibody (blue).Combined with multi-angle light scattering. The molar mass of each elution peak (below the UV absorbance line) shows that the sample is monodisperse, and due to the additional antibody fragment in this design, the 32-N / 32-I multispecific multi-affinity antibody is significantly larger than the 24-mer form of the multispecific multi-affinity antibody. (c) Comparison of Tm and Tagg temperatures for 32-N / 32-I multispecific multi-affinity antibody, 12-mer multispecific multi-affinity antibody, parental IgG, and N6 / PGDM1400x10E8v4 trispecific antibody. (d) Concentration-response curves of binding of 32-N and 32-I multispecific multi-affinity antibodies to multiple epitopes. PGDM1400 binding site specification 12 / 49 pages 14 CN 121449754 A. The N49P7 binding site and the 10E8 binding site are represented in red, blue, and pink, respectively, in the surface schematic diagram of HIV Env (grey). The binding of iMab was assessed using soluble CD4, and the functional binding of Fc to human FcRn was tested by measuring the binding at pH 7.5 and pH 5.6. The BG505 SOSIP.664_D368R trimer and 93TH057 gp120 monomer were selected as epitope-specific ligands for PGDM1400 and N49P7, respectively.

[0225] Figure 4. Effect of split design on the biophysical and functional properties of multispecific and multi-affinity antibodies. Comparison of 12-mer multispecific and multi-affinity antibodies composed of 6 copies of PGDM1400 and 6 copies of Fc with polymerization of the full-length deferroferritin subunit (left figure) or ferritin hemisphere (Fc attached to the N ferritin hemisphere and PGDM1400 linked to the C ferritin hemisphere, right figure). a) Biomembrane Interference Technology (BLI) concentration-response curve of BG505.664 binding to a 12-mer multispecific multi-affinity antibody loaded onto an anti-hIgG Fc capture (AHC) biosensor. b) Centroidal mean fluorescence (BCM) and static light scattering (SLS) at 266 nm versus temperature. Tm and Tagg are indicated by yellow lines. c) Neutralization assay for BG505T332N PsV.

[0226] Figure 5. Multispecific multi-affinity antibody affinity purification protocol. Sequential affinity purification of protein A and protein L. Binding to protein A enriches multispecific multi-affinity antibody with Fc (green), while binding to protein L enriches multispecific multi-affinity antibody with κ chain Fab PGDM1400 (blue). A point mutation of alanine to proline at position 12 of the κ chain of iMab was introduced to disrupt binding to protein L. The complementarity of the two halves of human deferroferritin ensures the presence of N49P7 / iMab scFab (orange) and 10E8 scFab (pink) during protein A purification steps (fused to Cferritin). Performing coagulation...Gel filtration was used to separate any aggregated material.

[0227] Figure 6. Minimal batch-to-batch variation of the 32-N multispecific multi-affinity antibody. a) SEC chromatogram. b) BCM and SLS at 266 nm versus temperature. Thermal transition temperatures (Tm and Tagg) are indicated by yellow lines. c) Concentration-response curve of 93TH057 gp120 binding to N49P7 within 32-N. d) Neutralization distribution of 32-N to the 4-PsV group, which was selected to include a PsV resistant to each Fab in the multispecific multi-affinity antibody.

[0228] Figure 7. Thermal stability analysis. Figure 8. Relationship between BCM (top) and SLS (bottom) at 266 nm for 32-N and 32-I multispecific multi-affinity antibodies, their respective 12-mer multispecific multi-affinity antibodies, parental IgG and N6 / PGDM1400x10E8 trispecific antibody and temperature. Thermal transition temperatures (Tm and Tagg) are indicated by yellow lines.

[0229] Figure 8. Binding characteristics of bNAb 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.

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

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

[0232] Figure 11. Immunogenicity and exposure to multispecific multi-affinity antibodies in mice. Five male C57BL / 6 mice per group were used to evaluate the subcutaneous administration of 5 mg / kg mice to alternative multispecific multi-affinity antibodies and Fc-modified multispecific antibodies.Page 13 / 49 of the document, CN 121449754 A. Circulation of anti-drug antibodies and multispecific multi-affinity antibodies in the blood following the presence of a multi-affinity antibody (LALAP mutation to disrupt Fc receptor binding). Reference sample HpFerritin-PfCSP malaria peptide and parental mouse IgG1 and parental mouse IgG2a isotypes were used for immunogenicity and exposure comparison, respectively. Detailed Description

[0233] Definitions

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

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

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

[0237] It should be understood that any aspect described as “comprising” certain components may also be “consisting of these components” or “substantially composed of these components,” where “consisting of these components” has a closed or limiting meaning, and “substantially composed of these components” means…"These components constitute" means that the specified components are included but not included except for materials present as impurities, unavoidable materials present as a result of the methods used to provide these components, and components added for purposes other than achieving the technical effects of the invention. For example, a composition defined using the phrase "consistently of" covers any known acceptable additives, excipients, diluents, and carriers, etc. Generally, a composition consisting essentially of a set of components will contain less than 5% by weight, typically less than 3% by weight, more typically less than 1% by weight, and even more typically less than 0.1% by weight of unspecified components.

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

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

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

[0241] It should also be understood that all base sizes or amino acid sizes given for nucleic acids or polypeptides, and all molecular weights or molecular mass values ​​as described on page 14 / 49 of the specification, CN 121449754 A, are approximate values ​​and are provided for descriptive purposes. While suitable methods and materials are described below, methods and materials similar to or equivalent to those described and materials herein may be used in practice and testing of this disclosure. The abbreviation “e.g.” originates from the Latin *exempli gratia* and is used herein to indicate non-limiting examples. Therefore, the abbreviation “eg” is synonymous with the term “for example.” The word “or” is intended to include “and” unless the context clearly indicates otherwise.

[0242] The terms “protein nanoparticle,” “nanocage,” and “multispecific, multi-affinity antibody” are used interchangeably herein and refer to a multi-subunit protein polyhedral structure. Each subunit or nanocage monomer is composed of a protein or polypeptide (e.g., a glycosylated polypeptide) and optionally consists of one or more of the following features: nucleic acid, prosthetic group, organic and inorganic compound. Non-limiting examples of protein nanoparticles include ferritin nanoparticles (see, e.g., Zhang, Y., Int. J. Mol. Sci., 12:5406–5421, 2011, incorporated herein by reference), and encapsulated protein nanoparticles (see, e.g., Sutter et al., Nature).Struct. and Mol. Biol., 15:939–947, 2008, incorporated herein by reference), thiooxygenase reductase (SOR) nanoparticles (see, e.g., Urich et al., Science, 311:996–1000, 2006, incorporated herein by reference), dioxetine synthase (see, e.g., Zhang et al., J. Mol. Biol., 306:1099–1114, 2001) or pyruvate dehydrogenase nanoparticles (see, e.g., Izard et al., PNAS, 96:1240–1245, 1999, incorporated herein by reference). Ferritin, deferroferritin, encapsulated proteins, SOR, dioxetine synthase, and pyruvate dehydrogenase are monomeric proteins that self-assemble into globular protein complexes, which in some cases consist of 24, 60, 24, 60, and 60 protein subunits, respectively. Ferritin and deferroferritin are generally referred to interchangeably herein and are understood to apply to the fusion proteins, nanocages, and methods described herein. Carboxyl bodies, fornix proteins, GroEL, heat shock proteins, E2P, and MS2 shell proteins that also produce nanocages are considered herein. In addition, fully or partially synthesized self-assembling monomers are also intended for use herein.

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

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

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

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

[0247] A "vaccine" is a pharmaceutical composition that induces a prophylactic or therapeutic immune response in a subject. In some cases, the specification is on page 15 / 49 of CN 121449754 A. In this context, the immune response is a protective immune response. Typically, a vaccine induces an antigen-specific immune response against a pathogen, such as a viral pathogen, or against an antigen-specific cellular component associated with a pathological condition. Vaccines may include polynucleotides (e.g., nucleic acids encoding a disclosed antigen), peptides or polypeptides (e.g., disclosed antigens), viruses, cells, or one or more cellular components. In one specific, non-limiting example, a vaccine induces an immune response that, compared to a control, reduces the severity of symptoms associated with malaria infection and / or reduces the parasite load. In another, non-limiting example, a vaccine induces an immune response that, compared to a control, reduces and / or prevents malaria or HIV infection.

[0248] The term “antibody” as used herein, also referred to in the art as “immunoglobulin” (Ig), refers to a protein constructed from paired heavy and light polypeptide chains; multiple Ig isotypes exist, including IgA, IgD, IgE, IgG (e.g., IgG1, IgG2, ...). IgG3 and IgG4) and IgM. It should be understood that antibodies can be derived from any species, including humans, mice, rats, monkeys, llamas, or sharks. When antibodies fold correctly, each chain folds into multiple distinct globular domains linked by a more linear polypeptide sequence. For example, the immunoglobulin light chain folds into a variable domain (VL) and a constant domain (CL), while the heavy chain folds into a variable domain (VH) and three constant domains (CH, CH2, CH3). The interaction between the heavy chain variable domain and the light chain variable domain (VH and VL) results in the formation of an antigen-binding region (Fv). Each domain has an established structure familiar to those skilled in the art.

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

[0250] As referred to herein, “antibody fragment” may include any suitable antigen-binding antibody fragment known in the art. Antibody fragments may be naturally occurring antibody fragments or may be obtained by manipulating naturally occurring antibodies or by using recombinant methods. For example, antibody fragments may include, but are not limited to, Fv, single-chain Fv (scFv; a molecule consisting of VL and VH linked to linking peptides), Fc, single-chain Fc, Fab, single-chain Fab, F(ab')2, single-domain antibody (sdAb; a fragment consisting of a single VL or VH), and multivalent forms of any of these.

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

[0252] The term “epitope” refers to an antigenic determinant. An epitope is a specific antigenic (i.e., triggering a specific immune response) chemical group or peptide sequence on a molecule. An antibody specifically binds to (e.g., on a polypeptide) a specific antigenic epitope. Epitopes can be formed from consecutive amino acids or from discontinuous amino acids juxtaposed by the ternary folding of a protein. Epitopes formed from consecutive amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed from ternary folding are generally lost upon treatment with denaturing solvents. Epitopes typically comprise at least 3, more typically at least 5, about 9, about 11, or about 8 to about 12 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography and 2D nuclear magnetic resonance. See, for example, Methods in Molecular Biology, Vol. 66, “Epitope Mapping Protocols,” ed. Glenn E. Morris, 1996.

[0253] As used herein, the term “antigen” is defined as a molecule that elicits an immune response. Such an immune response may involve antibody production or activation of specific immune competent cells or both. Those skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens may be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA including nucleotide sequences or portions of nucleotide sequences encoding proteins that elicit an immune response thus encodes the term “antigen” as used herein. Furthermore, those skilled in the art will understand that an antigen does not necessarily have to be derived solely from the whole genome.Long nucleotide sequences are encoded. It is obvious that the aspects described herein include, but are not limited to, the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences can be arranged in various combinations to elicit the desired immune response. Furthermore, those skilled in the art will understand that antigens do not necessarily need to be encoded by “genes” at all. It is clear that antigens can be synthetic or derived from biological samples. Such biological samples can include, but are not limited to, tissue samples, cells, or biological fluids.

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

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

[0256] Representatives of antigens that may be used include, but are not limited to, natural, recombinant, or synthetic products derived from viruses, bacteria, fungi, parasites, and other infectious agents other than autoimmune diseases; hormones; or tumor antigens that may be used in prophylactic or therapeutic vaccines; and allergens. In one embodiment, the antigen comprises virus-like particles (VLPs) from various viruses such as influenza virus, HIV, RSV, Newcastle disease virus (NDV), etc. See PCT / US2006 / 40862, PCT / US2004 / 022001, U.S. Patent Serial No. 11 / 582,540, U.S. Patent Serial No. 60 / 799,343, U.S. Patent Serial No. 60 / 817,402, and U.S. Patent Serial No. 60 / 859,240, the entire contents of which are incorporated herein by reference. In another embodiment, the antigen comprises chimeric VLPs. A “chimeric VLP” means a VLP containing proteins or portions thereof from at least two different sources (organisms). Typically, one of these proteins originates from a virus that drives the host cell to form a VLP. Therefore, in one embodiment, the chimeric VLP includes the RSV M protein. In another embodiment, the chimeric VLP includes the NDV M protein. In yet another embodiment, the chimeric VLP includes the influenza virus M protein.

[0257] The viral or bacterial product may be a component produced by enzymatic cleavage of an organism, or it may be a component of an organism produced by recombinant DNA technology well known to those skilled in the art.

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

[0259] Antigens can also be derived from bacterial and fungal infections, such as those from Mycobacteria (which cause TB and leprosy), Streptococcus pneumoniae, aerobic Gram-negative bacilli, Mycoplasma, Staphylococcus infections, Streptococcus infections, Salmonella and Chlamydia, Bordetella pertussis, Leptospira pomona, and Treponema hemorrhagicum. Specific implementation schemes include Salmonella paratyphi A and B, Corynebacterium diphtheriae, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Clostridium feseri and other gas gangrene bacteria, and Bacillus anthracis.*Is*, *P. pestis*, *P. multocida*, *Neisseria meningitidis*, *N. gonorrhea*, *Haemophilus influenzae*, *Actinomyces* (e.g., *Norcardia*), *Acinetobacter*, *Bacillus* (e.g., *Bacillus anthrax*), *Bacteroides* (e.g., *Bacteroides fragilis*), *Blastomyces dermatitidis*, *Bordetella*, *Borrelia* (e.g., *Borrelia borrell*). The following bacteria are listed: *Brucella*, *Candida*, *Campylobacter*, *Chlamydia*, *Coccidioides*, *Corynebacterium* (e.g., *Corynebacterium diptheriae*), *Cryptococcus*, *Dermatocycoses*, *Escherichia coli* (e.g., enterotoxigenic *Escherichia coli* and enterohemorrhagic *Escherichia coli*), *Enterobacter* (e.g., *Enterobacter aerogenes*), *Klebsiella*, *Salmonella* (e.g., *Salmonella typhi*, *Salmonella enteritidis*). enteritidis), Serratia, Yersinia, Shigella, Erysipelothrix, Haemophilus (e.g., Haemophilus influenzae), Helicobacter, Legionella (e.g., Legionella pneumophila), Leptospira, Listeria (e.g., Listeria monocytogenes).Mycoplasma, Mycobacterium (e.g., Mycobacterium leprae and Mycobacterium tuberculosis), Vibrio (e.g., Vibrio cholerae), Pasteurella, Proteus, Pseudomonas (e.g., Pseudomonas aeruginosa), Rickettsia, Spirochetes (e.g., species of Treponema, Leptospira, and Borrelia), Shigella, Neisseria meningitidis, and Streptococcus pneumoniae Streptococcus (e.g., Streptococcus pneumoniae, as well as Streptococcus types 1, 2, and 3), Ureaplasma urealyticum, Treponema pollidum, etc.; Staphylococcus aureus, Plasmodium species (Pl. falciparum, Pl. vivax, etc.), Aspergillus species, Candida albicans, Pasteurella haemolytica, Corynebacterium diphtheriae toxoid, Neisseria meningitidis polysaccharide, Bordetella pertussis, Streptococcus pneumoniae (pneumococcus) polysaccharide, Clostridium tetani toxoid, Mycobacterium bovis, and the instructions on pages 18 / 49 of 20 CN. 121449754 A Inactivated cells of Salmonella Typhi, Cryptococcus neoformans, and Aspergillus.

[0260] The antigen may also be derived from parasitic malaria, leishmaniasis, trypanosomiasis, toxoplasmosis, schistosomiasis, filariasis, amebiasis, babesiosis, coccidiosis, cryptosporidiosis, binuclear amoebiasis, trypanosomiasis, ectoparasites, giardiasis, helminthiasis, Theileriasis, trichomoniasis, and spore-forming organisms (e.g., Plasmodium vivax, Plasmodium falciparum, Plasmodium malariae, Plasmodium knowlesi, and Plasmodium ovale).These parasites can cause a variety of diseases or symptoms, including but not limited to: scabies, mites, eye infections, intestinal diseases (e.g., dysentery, giardiasis), liver diseases, lung diseases, opportunistic infections (e.g., AIDS-related), malaria, pregnancy complications, and toxoplasmosis.

[0261] Tumor-associated antigens suitable for the compositions described herein include mutant and non-mutant molecules that can indicate a single tumor type, be shared by several tumor types, and / or be specifically expressed or overexpressed in tumor cells compared to normal cells. In addition to proteins and glycoproteins, tumor-specific expression patterns of carbohydrates, gangliosides, glycolipids, and mucins are also described. Exemplary tumor-associated antigens for the cancer vaccines of the present invention include protein products of oncogenes, tumor suppressor genes, and other genes with mutations or rearrangements specific to tumor cells, reactivated embryonic gene products, carcinoembryonic antigens, tissue-specific (but not tumor-specific) differentiation antigens, growth factor receptors, cell surface glycosidic residues, exogenous viral proteins, and many other endogenous proteins. Specific implementation schemes for tumor-associated antigens include, for example: mutated antigens, such as protein products of the Ras p21 proto-oncogene, the tumor suppressor gene p53, and the HER-2 / neu and BCR-ab1 oncogenes, as well as CDK4, MUM1, caspase 8, and β-catenin; overexpressed antigens, such as galactagogue 4, galactagogue 9, carbonic anhydrase, aldolase A, PRAME, Her2 / neu, ErbB-2, and KSA; oncoemulsification antigens, such as alpha-fetoprotein (AFP) and human chorionic gonadotropin (hCG); autoantigens such as carcinoembryonic antigen (CEA), and melanocyte differentiation antigens such as Mart 1 / Melan A, gp100, gp75, tyrosinase, TRP1, and TRP2; prostate-associated antigens, such as PSA, PAP, PSMA, PSM-P1, and PSM-P2; and reactivated embryonic gene products, such as MAGE 1, MAGE 3, MAGE 4, GAGE ​​1, and GAGE ​​2. 2. BAGE, RAGE, and other cancer testis antigens, such as NY-ESO1, SSX2, and SCP1; mucins, such as Muc-1 and Muc-2; gangliosides such as GM2, GD2, and GD3; neutral glycolipids and glycoproteins such as Lewis(y) and globo-H; and glycoproteins such as Tn, Thompson-Freidenreich antigen (TF), and sTn. Tumor-associated antigens included in this article are also whole-cell and tumor cell lysates and their immunogenic fractions, as well as immunoglobulin idiotypes expressed on monoclonal proliferation of B lymphocytes for anti-B-cell lymphoma. Tumor-associated antigens and their respective tumor cell targets include, for example, cytokeratins as cancer antigens, particularly cytokeratins 8, 18, and 19. Epithelial membrane antigens.EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6, human embryonic antigen (HEA-125), human milk fat globules, MBR1, MBR8, Ber-EP4, 17-1A, C26, and T16 are also known cancer antigens. Desmin and muscle-specific actin are antigens of myogenic sarcoma. Placental alkaline phosphatase, β-human chorionic gonadotropin, and alpha-fetoprotein are antigens of trophoblastic and germ cell tumors. Prostate-specific antigen is an antigen of prostate cancer and carcinoembryonic antigen of colonic adenocarcinoma. HMB-45 is an antigen of melanoma. In cervical cancer, useful antigens can be encoded by human papillomavirus. Chromogranin-A and synaptic vesicle proteins are antigens of neuroendocrine and neuroectodermal tumors. Of particular interest are aggressive tumors that form solid masses with necrotic areas. The lysis of these necrotic cells is a rich source of antigens for antigen-presenting cells, thus this treatment may have advantageous use in combination with conventional chemotherapy and / or radiotherapy. The antigens may be derived from any tumor or malignant cell line.

[0262] The antigens may also be derived from common allergens that cause allergies. Allergens include organic or inorganic materials of various man-made or natural origins, such as plant materials, metals, ingredients in cosmetics or detergents, latex, etc. Suitable allergens for use in the compositions and methods described herein may include, but are not limited to, pollen, animal dander, grass, mold, dust, antibiotics, stinging insect venom, and various environmental (including chemical and metal) drug and food allergens. Common tree allergens (Instruction manual, 19 / 49 pages, 21 CN 121449754 A) include pollen from cotton poplar, poplar, ash, birch, maple, oak, elm, hickory, and American pecan; common plant allergens include allergens from rye, hogweed, longleaf plantain, sorrel, and quinoa; plant contact allergens include allergens from poison oak, poison ivy, and nettle; common grass allergens include allergens from cat's tail grass, rock grass, bermudagrass, fescue, and Kentucky bluegrass; common allergens can also be obtained from molds or fungi, such as Alternaria, Fusarium, Hormodendrum, Aspergillus, Micropolyspora, Mucor, and thermophilic actinomycetes. Actinomycetes); penicillin and tetracycline are common antibiotic allergens; epidermal allergens can be obtained from indoor or organic dust (usually from fungi), from insects such as house mites (dermalphagoides pterosinyssis), or from animal sources such as feathers and cat and dog dander; commonFood allergens include milk and cheese (dairy products), eggs, wheat, nuts (such as peanuts), seafood (such as shellfish), peas, beans, and gluten allergens; common environmental allergens include metals (nickel and gold), chemicals (formaldehyde, trinitrophenol, and turpentine), latex, rubber, fibers (cotton or wool), burlap, hair dyes, cosmetics, detergents, and perfume allergens; common drug allergens include local anesthetics and salicylates; antibiotic allergens include penicillin and sulfonamide allergens; common insect allergens include bee, wasp, and ant venom, as well as cockroach cup allergens. The particularly well-characterized allergens include, but are not limited to, the major and occult epitopes of Der pI allergen (Hoyne et al., 1994, Immunology, 83:190-195), bee venom phospholipase A2 (PLA; Akdis et al., 1996, J. Clin. Invest., 98:1676-1683), birch pollen allergen Bet v 1 (Bauer et al., 1997, Clin. Exp. Immunol., 107:536-541), and multi-epitope recombinant grass allergen rKBG8.3 (Cao et al., 1997, Immunology, 90:46-51). These and other suitable allergens are commercially available and / or can be readily prepared as extracts using known techniques.

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

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

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

[0266] The term “expression” as used herein is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter. Specification 20 / 49 pages 22 CN 121449754 A

[0267] “Separated” means altered or removed from its native state. For example, nucleic acids or peptides that are naturally present in living organisms are not “separated,” but the same nucleic acids or peptides that are partially or completely separated from their native coexisting substances are “separated.” Separated nucleic acids or proteins may exist in a substantially purified form or may exist in a non-native environment, such as a host cell.

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

[0269] As used herein, the term “regulation” refers to a detectable increase or decrease in the level of response in a subject compared to the level of response in a subject without treatment or a compound, and / or compared to the level of response in other subjects who are identical but untreated. This term covers perturbing and / or affecting natural signals or responses, thereby mediating a beneficial therapeutic response in a subject (typically a human).

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

[0271] “Parenteral” administration of the immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion techniques.

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

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

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

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

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

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

[0278] As used herein, the phrase “under transcriptional control” or “operably linked” means that a promoter is in the correct position and orientation relative to a polynucleotide to control the initiation of transcription and the expression of the polynucleotide, performed by an RNA polymerase.

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

[0280] As used herein, the term “subject” means any member of the animal kingdom, typically a mammal. The term “mammal” means any animal classified as a mammal, including humans, other higher primates, livestock and farm animals, as well as zoo animals, sporting animals, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Typically, a mammal is a human.

[0281] Dosing in “combination” with one or more other therapeutic agents includes simultaneous (parallel) and sequential dosing in any order.

[0282] The term “pharmaceuticalally acceptable” means a compound or combination of compounds that is compatible with the remaining components of a pharmaceutical preparation and is generally safe for human administration according to established government standards, including those published by the U.S. Food and Drug Administration.

[0283] The term “pharmaceuticalally acceptable carrier” includes, but is not limited to, solvents, dispersion media, coatings, antimicrobial agents, antifungal agents, isotonic agents, and / or absorption delay agents. The use of pharmaceutically acceptable carriers is well known.

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

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

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

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

[0288] The fusion protein described herein may include modifications. Such modifications include, but are not limited to, conjugation with effector molecules such as antimalarial drugs or adjuvants. Modifications also include, but are not limited to, conjugation with a detectable reporter moiety. Modifications that extend half-life (e.g., PEGylation) are also included. Protein and non-protein drugs may be conjugated to the fusion protein by methods known in the art. Coupling methods include direct linking, linker linking via covalent linkage, and specific binding of paired members (e.g., avidin-biotin). These methods include, for example, the methods described in Greenfield et al., Cancer Research, 50:6600-6607, 1990, which are incorporated herein by reference; and the methods described in Amon et al., Adv. Exp. Med. Biol., 303: 79-90, 1991, and Kiseleva et al., Mol. Biol. (USSR), 25:508-514, 1991, both of which are incorporated herein by reference.

[0289] Fusion Proteins

[0290] Fusion proteins are described herein. Fusion proteins comprise a first nanocage monomer subunit of a nanocage monomer linked to a bioactive portion. The fusion protein self-assembles with a protein comprising a second nanocage monomer subunit to form a nanocage.Page 23 / 49, 25 CN 121449754 A Monomer. Multiple such fusion protein pairs self-assemble to form nanocages. Thus, the bioactive portion can modify the inner surface of the assembled nanocage, the outer surface of the assembled nanocage, or both.

[0291] The bioactive portion can be any part that can be part of a fusion protein and is typically a protein. Typically, the bioactive portion includes: an antibody or a fragment thereof, an antigen, a detectable portion, a pharmaceutical agent, a diagnostic agent, or a combination thereof.

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

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

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

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

[0296] In cases where the antibody or its fragment comprises two chains, such as a first and second chain in the case of an Fc fragment, or a heavy and light chain, these two chains are optionally separated by a linker. The linker can be flexible or rigid, but it is typically flexible to allow the chains to fold appropriately. Although it should be understood that the length of this linker will vary depending on the sequence of the nanocage monomer and the bioactive moiety, as well as the three-dimensional conformation of the fusion protein, the linker is usually long enough to impart some flexibility to the fusion protein. Therefore, the linker is typically about 1 to about 30 amino acid residues, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid residues, such as about 8 to about 16 amino acid residues, such as 8, 10 or 12 amino acid residues.

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

[0298] GGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGTATAGASSGSGSSGSSSS GGTG.

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

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

[0301] Fc chain 1:

[0302] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

[0303] GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK

[0304] AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK; Fc chain 2:

[0306] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

[0307] GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK

[0308] AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK;

[0309] Ibalizumab‑A12P light chain:

[0310] DIVMTQSPDSLPVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWAST

[0311] RESGVPDRFSGSSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIKRTVAAPS

[0312] VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0313] Ibalizumab‑A12P heavy chain:

[0314] QVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGT

[0315] DYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQG

[0316] TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGSR;

[0317] 10E8.v4 light chain:

[0318] SELTQDPAVSVALKQTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDR

[0319] FSGSASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVLSQPKAAPSVTL

[0320] FPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC;

[0321] 10E8.v4 heavy chain:

[0322] EVRLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEG

[0323] WSVDYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWSGYPPGE

[0324] EYFQDWGQGTLVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS

[0325] GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCSR;

[0326] N49P7 light chain:

[0327] QSALTQPRSVSASPGQSVTISCTGTHNLVSWCQHQPGRAPKLLIYDFNKRPSGVPDRFSG DESCRIPTION Page 25 / 49 27 CN 121449754 A

[0328] SGSGGTASLTITGLQDDDDAEYFCWAYEAFGGGTKLTVLGQPKAAPSVTLFPPSSEELQ

[0329] ANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC;

[0330] N49P7 heavy chain:

[0331] ADLVQSGAVVKKPGDSVRISCEAQGYRFPDYIIHWIRRAPGQGPEWMGWMNPMGGQV

[0332] NIPWKFQGRVSMTRDTSIETAFLDLRGLKSDDTAVYYDRSNGSGKRFESSNWFLDLWG

[0333] RGTAVTIQSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDSR;

[0334] PGDM1400 light chain:

[0335] DFVLTQSPHSLSVTPGESASISCKSSHSLIHGDRNNYLAWYVQKPGRSPQLLIYLASSRAS

[0336] GVPDRFSGSGSDKDFTLKISRVETEDVGTYYCMQGRESPWTFGQGTKVDIKRTVAAPSV

[0337] FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0338] PGDM1400 heavy chain:

[0339] QAQLVQSGPEVRKPGTSVKVSCKAPNTLKTYDLHWVRSVPGQGLQWMGWISHEGDK

[0340] KVIVERFKAKVTIDWDRSTNTAYLQLSGLTSGDTAVYYCAKGSKHRLRDYALYDDDGA

[0341] LNWAVDVDYLSNLEFWGQGTAVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY

[0342] FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDSR; or

[0343] combinations thereof.

[0344] In a further aspect, the antibody or a fragment thereof is conjugated to or associated with another part, such as an antigen, a detectable part (e.g., a small molecule, a fluorescent molecule, a radioisotope, or a magnetic particle), a pharmaceutical agent, a diagnostic agent, or a combination thereof, and may include, for example, an antibody-drug conjugate.

[0345] In which the bioactive portion is an aspect of an antigen, the antigen may be associated with, for example, a vaccine-preventable condition and / or a vaccine-treatable condition. In such cases, the antigen may be associated with, for example: infectious agents, including viruses, bacteria, parasites, fungi, or yeasts; cancers, including solid and liquid cancers; or immune diseases, including autoimmune diseases.

[0346] In which the bioactive portion is an aspect of a detectable portion, the detectable portion may include fluorescent proteins, such as GFP, EGFP, amphotericin B, and / or flavin-based fluorescent proteins such as LOV proteins (such as iLOV).

[0347] In which the bioactive portion is an aspect of a pharmaceutical agent, the pharmaceutical agent may include, for example, small molecules, peptides, lipids, carbohydrates, or toxins.

[0348] In some typical aspects, the nanocages assembled by the fusion proteins described herein comprise about 3 to about 100 nanocage monomers, such as about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 55, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, or 98 to about 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 55, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or 100 nanocage monomers, such as 24, 32, or 60 nanocage monomers. The nanocage monomers can be any known natural, synthetic, or partially synthetic nanocage monomer, and in some respects selected from ferritin, deferroferritin, encapsulated proteins, SOR, dioxetine synthase, pyruvate dehydrogenase, carboxyl groups, fornix proteins, GroEL, heat shock proteins, E2P, MS2 capsid proteins, fragments thereof, and variants thereof. Typically, the nanocage monomer is ferritin or deferroferritin. (Instructions 26 / 49, page 28, CN 121449754 A, white.)

[0349] When deferroferritin is selected as the nanocage monomer, the first and second nanocage monomer subunits typically interchangeably comprise the “N” and “C” regions of the deferroferritin. It should be understood that other nanocage monomers can be divided into two subunits, very similar to deferroferritin as described herein, such that the subunits are self-assembly and adapted to fuse with the bioactive moiety, respectively.

[0350] Typically, the “N” region of deferroferritin comprises a sequence having at least 70% (e.g., at least 75%, 80%) of the following sequence.The sequence having at least 70% (such as at least 75%, 80%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with, or consisting of:

[0351] MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLK MQNQRGGRALFQDIKKPAEDEW.

[0352] Typically, the “C” region of deferroferrin includes, or consists of, a sequence having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with, or consisting of:

[0353] GKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGP EAGLGEYLFERLTLRHD.

[0354] In some aspects, the fusion proteins described herein also include a linker between the nanocage monomer subunits and the bioactive portion, very similar to the linker described above. Again, this linker can be flexible or rigid, but it is typically flexible to allow the bioactive portion to retain its activity and the nanocage monomer subunits to retain their self-assembly properties. While it should be understood that the length of this linker will vary depending on the sequence of the nanocage monomers and the bioactive portion, as well as the three-dimensional conformation of the fusion protein, the linker is typically long enough to impart some flexibility to the fusion protein. Therefore, the linker is typically about 1 to about 30 amino acid residues, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid residues, such as about 8 to about 16 amino acid residues, such as 8, 10 or 12 amino acid residues.

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

[0356] ASTASSASSGGGGGGSGGSGGSGGS.

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

[0358] GGSGGSGGSGGSGGGASGGS.

[0359] Fusion protein pairs of the above-described fusion proteins are also described herein, wherein the fusion protein pairs self-assemble to form nanocage monomers, wherein a first nanocage monomer subunit and a second nanocage monomer subunit are fused to different biologically active moieties. This provides multivalent and / or multispecificity to the single nanocage monomer assembled by the subunit pair.

[0360] In some aspects, the fusion protein may also include an antigen. These aspects are clearly described in International Patent Application No. WO 2019 / 023812, the entire contents of which are incorporated herein by reference. In summary, in these aspects, the antigen has at least a first antibody-binding epitope and a second antibody-binding epitope; and an antibody or a fragment thereof specific to at least the first antigenic epitope, or the fragment thereof described on pages 27 / 49 of the specification, CN 121449754 A. Binding of the antibody or a fragment thereof to the first antigenic epitope presents a second antigenic epitope for binding to the antigen-binding portion, and / or the first antibody-binding epitope binds to the antibody or a fragment thereof, wherein, in the case of the antibody or a fragment thereof, the binding presents the second antibody-binding epitope.

[0361] In other aspects, the antibody or a fragment thereof may target any antigen, such as those listed above. Typically, antigens are derived from cancer or infectious agents, such as hepatitis A, B, C, HIV, mycobacteria, malaria pathogens, SARS pathogens, herpesviruses, influenza viruses, polioviruses, or bacterial pathogens such as Chlamydia and mycobacteria, or from self-reactive B cells or any T cells used for co-recruitment and cytotoxic killing.

[0362] The fusion proteins described herein may alternatively be used as therapeutic or diagnostic agents. Thus, in some aspects, antibodies or fragments thereof may be specific to, for example, tumor antigens or self-antigens.

[0363] substantially identical sequences may include one or more conserved amino acid mutations. It is known in the art that one or more conserved amino acid mutations on a reference sequence may produce mutant peptides that are not substantially altered in physiological, chemical, or functional properties compared to the reference sequence; in such cases, the reference sequence and the mutant sequence will be considered “substantially identical” polypeptides. Conserved amino acid mutations may include the insertion, deletion, or substitution of amino acids; “conserved amino acid substitution” is defined herein.This involves replacing an amino acid residue with another amino acid residue that has similar chemical properties (e.g., size, charge, or polarity).

[0364] In a non-limiting example, a conserved mutation can be an amino acid substitution. Such a conserved amino acid substitution can be the substitution of a basic, neutral, hydrophobic, or acidic amino acid with another amino acid from the same group. The term "basic amino acid" refers to a hydrophilic amino acid that has a side chain pK value greater than 7 and is normally positively charged at physiological pH. Basic amino acids include histidine (His or H), arginine (Arg or R), and lysine (Lys or K). The term "neutral amino acid" (also known as "polar amino acid") refers to a hydrophilic amino acid that has a side chain that is uncharged at physiological pH, but has at least one bond in which an electron pair shared by two atoms is held more tightly by one of the two atoms. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine ​​(Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and glutamine (Gln or Q). The term "hydrophobic amino acid" (also known as "nonpolar amino acid") refers to amino acids that exhibit hydrophobicity greater than zero according to the Eisenberg Consensus Hydrophobicity Scale (1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (Ile or I), phenylalanine (Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G).

[0365] "Acidic amino acid" refers to hydrophilic amino acids that have a side chain pK value of less than 7 and are generally negatively charged at physiological pH. Acidic amino acids include glutamic acid (Glu or E) and aspartic acid (Asp or D).

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

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

[0368] The polypeptides or fusion proteins of the present invention may also include additional sequences to aid in their expression, detection, or purification. Specification 28 / 49 pages 30 CN 121449754 A Any such sequences or tags known to those skilled in the art may be used. For example, and not wishing to be limiting, the fusion protein may include a targeting sequence or signaling sequence (e.g., but not limited to ompA), a detection tag, exemplary tag boxes including a Strep tag or any variant thereof; see, for example, U.S. Patent No. 7,981,632, a His tag, a Flag tag having the sequence motif DYKDDDDK, an Xpress tag, an Avi tag, a calmodulin tag, a polyglutamate tag, a HA tag, a Myc tag, a Nus tag, an S tag, an SBP tag, a Softag 1, a Softag 3, a V5 tag, a CREB-binding protein (CBP), a glutathione S-transferase (GST), a maltose-binding protein (MBP), a green fluorescent protein (GFP), a thioredoxin tag, or any combination thereof; a purification tag (e.g., but not limited to His5 or His6), or a combination thereof.

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

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

[0371] This document also covers isolated or purified fusion proteins, peptides, or the like immobilized on surfaces using various methods.The peptide may be a fragment; for example, and not intended to be limited, the peptide may be linked or coupled to a surface by His tag coupling, biotin binding, covalent binding, adsorption, etc. The solid surface may be any suitable surface, such as, but not limited to, the well surface of a microtiter plate, the channel of a surface plasmon resonance (SPR) sensor chip, a membrane, beads (such as magnetic-based beads or agarose-based beads or other chromatography resins), glass, thin film, or any other useful surface.

[0372] In other aspects, the fusion protein may be linked to a cargo molecule; the fusion protein may deliver the cargo molecule to a desired site and may be linked to the cargo molecule using any method known in the art (recombinant technology, chemical coupling, chelation, etc.). The cargo molecule may be any type of molecule, such as a therapeutic agent or diagnostic agent. For example, and not intended to be limited in any way, the therapeutic agent may be a radioisotope that can be used for radioimmunotherapy; a toxin, such as an immunotoxin; a cytokine, such as an immune cytokine; a cytotoxic agent; an apoptosis inducer; an enzyme; an anticancer antibody for immunotherapy; or any other suitable therapeutic molecule known in the art. Alternatively, diagnostic agents may include, but are by no means limited to: radioactive isotopes, paramagnetic labels such as gadolinium or iron oxide, fluorescent groups, near-infrared (NIR) fluorescent dyes or dyes (such as Cy3, Cy5.5, Alexa680, Dylight680, or Dylight800), affinity labels fused to detectable protein-based molecules (e.g., biotin, avidin, etc.), or any other suitable reagent detectable by imaging methods. In specific, non-limiting examples, the fusion protein may be linked to a fluorescent agent such as FITC, or genetically fused to enhanced green fluorescent protein (EGFP).

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

[0374] The fusion proteins described herein specifically bind to their targets. "Antibody specificity" refers to the selective recognition of a specific epitope of an antigen by an antibody. The antibody specificity of the antibodies or fragments described herein can be determined based on affinity and / or binding affinity. Specification 29 / 49 pages 31 CN 121449754 A Affinity, expressed as the dissociation equilibrium constant (KD) between the antigen and antibody, measures the binding strength between the antigenic determinant (epitope) and the antibody binding site. Affinity is a measure of the strength of binding between an antibody and its antigen. Antibodies typically bind with a KD of 10⁻⁵ M to 10⁻¹¹ M. Any KD greater than 10⁻⁴ M is generally considered to indicate nonspecific binding. The smaller the KD value, the stronger the binding of the antigenic determinant...The stronger the binding strength between the cluster and the antibody binding site, the better. In some respects, the antibodies described herein have a KD of less than 10⁻⁴ 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 M.

[0375] Nanocages are also described herein, 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. Furthermore, fusion protein pairs are described herein, wherein the fusion protein pairs self-assemble to form nanocage monomers, and wherein the first and second nanocage monomer subunits are fused to different biologically active moieties.

[0376] It should be understood that the nanocage can be self-assembled from: multiple identical fusion proteins, multiple different fusion proteins (and thus the nanocage is multivalent and / or multispecific), combinations of fusion proteins and wild-type proteins, and any combination thereof. For example, the nanocage can be internally and / or externally modified by combining at least one fusion protein as described herein with at least one anticancer antibody for immunotherapy. In some typical aspects, about 20% to about 80% of the nanocage monomer comprises the fusion protein as described herein. Given the modular solution described herein, the nanocage can theoretically include up to twice the number of bioactive moieties in the monomers within the nanocage, since each nanocage monomer can be divided into two subunits, each of which can independently bind a different bioactive moiety. It should be understood that this modularity can be used to achieve any desired ratio of bioactive moieties as described herein in specific examples, where, in some specific examples, the four different bioactive moieties are in a 4:2:1:1 ratio. For example, the nanocage described herein can include at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different bioactive moieties. Thus, the nanocage can be multivalent and / or multispecific, and the extent to which this can be controlled relatively easily.

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

[0378] In a typical aspect, the nanocages described herein comprise a first fusion protein, a second fusion protein, and a third fusion protein, and at least one complete nanocage monomer optionally fused to a bioactive portion, wherein the bioactive portion of the first fusion protein, the second fusion protein, and the third fusion protein is different from the bioactive portion of the complete nanocage monomer.

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

[0380] In some aspects, the antibody or fragment of the second fusion protein is N49P7 or iMab A12P; and the antibody or fragment of the third fusion protein is 10E8v4. In one typical aspect, the nanocages described herein comprise the following four fusion proteins in an optional 4:2:1:1 ratio: Specification 30 / 49 pages 32 CN 121449754 A

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

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

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

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

[0385] In some aspects, the nanocages described herein comprise, or consist of, sequences having at least 70% (such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with one or more of the following sequences, wherein ferritin subunits are shown in bold, linkers are underlined, light chains are shown in italics, and heavy chains are shown in lowercase letters:

[0386] Specification 31 / 49 pages 33 CN 121449754 A

[0387] Specification 32 / 49 pages 34 CN 121449754 A

[0388]

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

[0390] In some aspects, the cargo molecule is contained within to provide T-cell epitopes, but optionally does not provide B-cell epitopes. Alternatively, the cargo molecule is fused to the fusion protein and contained within to provide T-cell epitopes, but optionally does not provide B-cell epitopes.

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

[0392] Furthermore, the nanocage may also include an antigen on its surface, which may be expressed as a fusion protein having nanocage monomers.

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

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

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

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

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

[0398] Any suitable expression vector may be used. For example, prokaryotic cloning vectors include plasmids of *E. coli*, such as colE1, pCR1, pBR322, pMB9, pUC, pKSM, and RP4. Prokaryotic vectors also include derivatives of phage DNA (such as M13 and other filamentous single-stranded DNA phages). An example of a vector for yeast is the 2μ plasmid. Suitable vectors for expression in mammalian cells include known derivatives of SV-40, adenoviruses, DNA sequences derived from retroviruses, and shuttle vectors derived from combinations of functional mammalian vectors such as those described above, as well as functional plasmids and phage DNA.

[0399] Other eukaryotic expression vectors are known in the art (e.g., PJ Southern and P. Berg, J. Mol. Appl. Genet, 1:327-341, 1982; Subramani et al., Mol. Cell. Biol, 1:854-864, 1981; Kaufhiann and Sharp, "Amplification And Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene", J. Mol. Biol, 159:601-621, 1982; Kaufhiann and Sharp, Mol. Cell. Biol, 159:601-664, 1982; Scahill et al., "Expression And Characterization Of The Product Of A Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells", Proc. Nat'l Acad. Sci. USA, 80:4654-4659, 1983; Urlaub and Chasin, Proc. Nat'l Acad. Sci USA, 77:4216-4220, 1980 (all of the above references are incorporated herein by reference).

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

[0401] Recombinant host cells comprising the expression vectors described above are also described herein. The fusion proteins described herein can be expressed in cell lines rather than in hybridomas. Nucleic acids including sequences encoding polypeptides according to the invention can be used to transform suitable mammalian host cells.

[0402] Particularly preferred cell lines are selected based on high levels of expression of the target protein, constitutive expression, and minimal contamination from the host protein. Mammalian cell lines that can be used as hosts for expression are well known in the art and include many unproliferating cell lines, such as, but not limited to, Chinese hamster ovary (CHO) cells, young hamster kidney (BHK) cells, and many other cell lines. Other suitable eukaryotic cells include yeast and other fungi. Useful prokaryotic hosts include, for example, *Escherichia coli* (such as *E. coli* SG-936, *E. coli* HB 101, *E. coli* W3110, *E. coli* X1776, *E. coli* X2282, *E. coli* DHI, and *E. coli* MRC1), *Pseudomonas*, *Bacillus* (such as *Bacillus subtilis*), and *Streptomyces*.

[0403] These recombinant host cells can be used to produce fusion proteins by culturing cells under conditions that allow peptide expression and purifying the peptide from the host cells or the culture medium surrounding the host cells. The targeted secretion of expressed peptides in recombinant host cells can be promoted by inserting a sequence encoding a signal peptide or secretory precursor peptide at the 5' end of the target gene encoding the antibody (see Shokri et al., 2003, Appl Microbiol Biotechnol., 60(6):654-664; Nielsen et al., Prot. Eng., 10:1-6, 1997; von Heinje et al., Nucl. Acids Res., 14:4683-4690, 1986, all of which are incorporated herein by reference). These secretory precursor peptide elements can be derived from prokaryotic or eukaryotic sequences.Therefore, appropriately, a secretory leader peptide is used, which is an amino acid attached to the N-terminus of a polypeptide to guide the polypeptide out of the host cell cytosol and secreted into the culture medium.

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

[0405] It should be understood that Fab nanocages can be generated by co-transfection of HC ferritin and LC. Alternatively, a single-chain Fab-ferritin nanocage, as shown in Figure 1, can be used for transfection of a plasmid, as described in CN 121449754 A, which requires only pages 34 / 49 of the specification. This can be achieved by a linker of different lengths (e.g., 60 or 70 amino acids) between LC and HC. When using single-chain Fab, it can be ensured that the heavy and light chains are paired. Tags (e.g., Flag, HA, myc, His6x, Strep, etc.) can also be added to the N-terminus of the construct or within the linker to facilitate purification as described above. Furthermore, when co-transfecting different Fab-nanoparticle plasmids, a tagging system can be used to ensure the presence of many different Fabs on the same nanoparticle via tandem / additive affinity chromatography steps. This provides multispecificity to the nanoparticles. If desired, protease sites (e.g., TEV, 3C, etc.) can be inserted to cleave the adapter and tag after expression and / or purification. An example of such constructs is scFab 10E8 for broad-spectrum anti-HIV neutralization:

[0406] YELTQETGVSVALGRTVTITCRGDSLRSHYASWYQKKPGQAPILLFYGKNNRPSGVPDRFSGSASGNRA SLTISGAQAEDDAEYYCSSRDKSGSRLSVFGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECGGSSGSGSGS TGENLYFQGSAGTTGTSASTSGYPYDVPDYAGGGGSAGGTATLEVLFQGPSSGSSSSGGTGEVQLVESGGGLVKPGG SLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAAPVEGRFTISRLNSINFLYLEMNNLRMEDS GLYFCARTGKYYDFWSGYPPGEEYFQDWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCSRGGGGGSGGSGG SGGSMSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQR GGRALFQDIKKPAEDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLT NLHRLGGPEAGLGEYLFERLTLRHD

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

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

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

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

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

[0412] The foregoing disclosure describes the invention in its entirety. A more complete understanding can be obtained by referring to the following specific embodiments. Unless otherwise stated, these embodiments are provided for illustrative purposes only and are not intended to be limiting. Therefore, the invention should in no way be construed as limited to the following embodiments, but should be understood to cover any and all variations that become apparent from the teachings provided herein.

[0413] The following examples do not include a detailed description of conventional methods, such as methods for constructing vectors and plasmids, inserting genes encoding polypeptides into such vectors and plasmids, or introducing plasmids into host cells. These methods are well known to those skilled in the art and are described in numerous publications including Sambrook, J., Fritsch, EF, and Maniatis, specification 35 / 49 pages 37 CN 121449754 AT., 1989, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, which are incorporated herein by reference.

[0414] Without further description, it is believed that those skilled in the art can use the foregoing description and the following illustrative examples to prepare and utilize the compounds of the present invention and to practice the claimed methods. Therefore, the following working examples specifically point out typical aspects of the invention and should not be construed as limiting the remainder of this disclosure in any way.

[0415] Examples

[0416] Example 1

[0417] Introduction

[0418] Despite 30 years of effort, there is still no effective vaccine or treatment for human immunodeficiency virus type I (HIV-1). However, an exciting fact in this exploration is that a small percentage of HIV-1 infected individuals have produced antibodies with excellent neutralizing potency against circulating HIV-1 isolates. Since the discovery of the first-generation broad-spectrum neutralizing antibodies (bNAb) 2F51, 4E102, 3, 2G124 and b125, 6, the catalog of bNAb has increased significantly due to new technologies such as Env-specific single B cell sorting 7–9, antibody cloning and high-throughput neutralization assays 10–13, and the recent implementation of proteomics deconvolution technology 14. Dozens of HIV bNAbs have now been described, targeting six conserved sites on the trimer HIV envelope (Env), including the V1 / V2 loop at the trimer apex, the V3 ring glycosyl group, the CD4 binding site (CD4bs), the gp120-g41 interface, the fusion peptide, and the juxtamembrane external region (MPER) 7,9,19,10,12–18.

[0419] Attention to bNAbs as therapeutic molecules in combating HIV-1 stems from some potent antiviral activities observed in challenge experiments in rhesus monkeys 20–24 and humanized mice 25–28, and the reduction of viremia in infected humans when bNAbs are therapeutically infused 29–33. Furthermore, antibodies have key advantages over oral antiretroviral therapy (ART): they have a longer circulating half-life and can form immune complexes that enhance the host's immunity to the virus. These observationsThe findings led to clinical evaluation of antibody therapy by passively administering bNAb as an alternative to or supplement to conventional immunology for HIV-1 protection, and to efforts to control and / or eliminate HIV-1 in infected individuals.

[0420] One of the major limitations of the clinical application of bNAb is the rapid selection of resistant neutralizing viral populations 30–32, 34, 35. RNA viruses such as HIV exhibit unusual genetic diversity 36, allowing them to develop resistant mutations to evade mAb recognition. However, eliminating mutations that bind to certain bNAb has a significant adverse effect on viral fitness 37–39. Similar to the combination of different drugs in HIV-1 treatment regimens, these observations suggest that successful anti-HIV-1 antibody therapy should include bNAb-specific combinations. Therefore, there have been recent studies developing different forms of antibody-like molecules with bispecificity 40–42 or trispecificity 43–45 against Env. Another consideration is the amount of antibody required for efficacy in vivo. In fact, much work has been done to improve the potency of bNAbs by using structure-guided design or bioinformatics methods (such as VRC0146, 10E847,48 and NIH45-4649), but so far no complete success has been achieved. For bispecific and trispecific antibodies targeting multiple epitopes in Env, potency is often limited by the potency of their parent mAb. Thus, to date, while neutralization width has been significantly improved, antiviral potency remains relatively weak.40,43,44,45

[0421] The ability of an antibody to interact with more than one epitope on the same virus has a great influence on its potency.50–52 This effect is often referred to as affinity (epistemic affinity enhancement), a property of IgM antibodies in nature that compensates for their typically low affinity. Therefore, the addition of the μ tail of IgM to the constant region of IgG has been investigated to produce dodecyl IgM-like molecules with improved biological activity.53,54 Engineering has produced a variety of non-natural antibody forms at a speed far exceeding that of evolution, overcoming the limitation of IgG bivalent antibodies. Some of these designs include linear head-to-tail Fab tandem fusions 55, tandem biantibody combinations (Tamdabs) 56, or biantibodies fused with the CH3 layer of IgG 57, supplemented with IgG 58–60, and the use of multi-branched scaffolds (e.g., p5361, leucine zipper helix 62, streptavidin 63, Bacillus RNase-Bacillus RNase inhibitor module 64, and Escherichia coli Vero cytotoxin B subunits that self-assemble into pentamer forms 65 and can be further engineered into decavalent 66). These antibody architectures have contributed to their successful development as therapeutic agents.Facing different challenges. Multimeric antibody forms that depend on antibody variable fragments (Fv) are generally associated with low stability and thus a high tendency to aggregate.67 Furthermore, the dissociation of dimerizing modules, determined by the affinity constant of the complex, can limit the long-term stability of the molecule in vivo. In addition, the titers achieved with most of the antibody forms mentioned above are usually at most 3-5 valences, thus excluding combinations of high affinity and multispecificity.

[0422] This paper describes a multispecific multi-affinity antibody (multabody) platform that, in some respects, uses deferroferritin as a module subunit for the multipolymerization of up to 32 antibody fragments (antigen-binding fragments [Fab] and crystallizable fragments [Fc]) in a single molecule. Using this method, we efficiently combined four different specificities into a single molecule, including the Fab portion of three of the best anti-HIV-1bNAb and the crystallizable fragment (Fc) from IgG1, to confer multispecificity, high affinity, effector function, and extended serum half-life to the molecule. Compared to combinations of their individual parental antibodies or IgGs, the resulting multispecific multi-affinity antibodies exhibited a panviral neutralizing width and significantly higher neutralizing potency. Notably, the median IC50 of the multispecific multi-affinity antibodies against 14 pseudoviruses was 1 and 2 orders of magnitude lower, respectively, in terms of mass and molar concentration, compared to anti-HIV trispecific N6 / PGDM1400x10E8 antibodies or mixtures made from the best-known bNAb. The multispecific multi-affinity antibody design described herein represents a robust and powerful plug-and-play platform for polymerizing antibodies to enhance their therapeutic properties in inhibiting HIV-1 infection.

[0423] Materials and Methods

[0424] Expression and purification of multispecific multi-affinity antibodies based on Fab-only deferroferrin. Genes encoding the human deferroferrin light chain and scFa b-human deferroferrin fusion protein were synthesized and cloned into the pHLsec expression vector via GenArt (Life Technologies). 200 mL of HEK293F cells (Thermo Fisher Scientifics) were seeded in Freestyle expression medium at a density of 0.8 × 10⁶ cells / mL and incubated in a Multitron Pro shaker (Infors HT) at 37°C, 8% CO₂, and 70% humidity with shaking at 125 rpm. Within 24 hours post-seeding, cells were transiently transfected with 50 μg of filtered DNA pre-incubated at room temperature (RT) for 10 minutes with FectoPRO (Polyplus Transfections) at a 1:1 ratio. Plasmids encoding scFab-human deferroferritin and human deferroferritin were then used...Nanoparticles with 20%, 50%, 80%, and 100% scFab titers were obtained by mixing at ratios of 1:4, 1:1, 4:1, and 1:0, respectively. After 6–7 days, the cell suspension was harvested by centrifugation at 5000×g for 15 minutes, and the supernatant was filtered through a 0.22 μm Steritop filter (EMD Millipore). The nanoparticles were purified by affinity chromatography of Fab followed by washing and elution. Fractions containing proteins were combined, concentrated, and loaded onto a Superose 6 10 / 300GL size exclusion column (GE Heathcare) in 20 mM sodium phosphate pH 8.0 and 150 mM NaCl.

[0425] Design, expression, and purification of 32-N and 32-I multispecific multi-affinity antibodies. Genes encoding scFab and scFc fragments linked to half-ferritin were generated using the KOD-plus mutagenesis kit (Toyobo, Osaka, Japan) by deleting residues 1 to 95 (C-ferritin) and 95 to 175 (N-ferritin) of the human deferroferritin light chain. Furthermore, using the same mutagenesis kit, the binding specificity of protein L to iMab-C ferritin was disrupted by site-directed mutagenesis of alanine 12 to proline residues in the antibody light chain. The 32-N multispecific and multi-affinity antibody was transiently transfected into HEK 293F cells by mixing 66 μg of plasmid PGDM1400 with a 4:2:1:1 mixture of scFab-human deferroferritin:Fc-human deferroferritin:N49P7scFab-C ferritin:10E8scFab-C ferritin. For the 32-I multispecific multi-affinity antibody, iMab scFab-C ferritin was used instead of N49P7 scFab-C ferritin particles. Before adding cell culture, the DNA mixture was filtered and incubated at room temperature with 60 μL FectoPRO. The multispecific multi-affinity antibody was first purified by affinity chromatography using a HiTrap Protien A HP column (GE Healthcare, instructions page 37 / 49, CN 121449754 A) with 20 mM Tris pH 8.0, 3 M MgCl2, and 10% glycerol elution buffer. After buffer exchange using a PD-10 desalting column (GE Healthcare), the multispecific multi-affinity antibody was further purified by a second affinity chromatography using a HiTrap Protien L column (GE Healthcare). The protein-containing fraction was concentrated and further purified by gel filtration on a Superose 610 / 300GL column (GE Healthcare).

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

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

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

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

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

[0431] Pharmacokinetic and immunogenicity studies. In vivo studies were conducted using 20g C57BL / 6 male mice. Alternative multispecific and multi-affinity antibodies consisting of scFab and scFc fragments of mouse HD37 IgG2a fused to the N-terminus of the mouse aferroferrin light chain were used. HD37 scFab-mFerritin, Fc-mFerritin, and mFerritin were transfected and purified at a ratio of 2:1:1 as described above. L35A, L234A, and P329G mutations were introduced into the mouse IgG2a Fc construct to silence the effector function 74 of the multispecific and multi-affinity antibody. Subcutaneous single injection of 5 mg / kg multispecific and multi-affinity200 μL of PBS (pH 7.5) solution was used for antibody or control samples (HD37 IgG1, HD37 IgG2a, and hpFerritin-PfCSP malaria peptide). Blood samples were collected at multiple time points, and circulating antibody and ADA levels in serum samples were evaluated by ELISA. Briefly, 96-well Pierce nickel-coated plates (Thermo Fisher) were coated with 50 μL of 0.5 μg / mL His6x-labeled antigen hCD19 to determine the specific concentration of circulating HD37 using a reagent-specific standard curve with IgG and multispecific multi-affinity antibodies. In anti-drug antibody assays, Nunc MaxiSorp plates (Biolegend) were coated with 12-mer HD37 scFab multispecific multi-affinity antibody or hpFerritin-PfCSP malaria peptide. HRP-Protein A (Invitrogen) was used as a second molecule, and the chemiluminescent signal was quantified using a Synergy Neo2 multimodal analyzer (Biotek Instruments).

[0432] Results

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

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

[0435] Deferroferrin modification resulted in efficient heterooligomerization of the 32-mer multispecific multi-affinity antibody. Secondly, we attempted to improve the width of the exceptionally efficient 24-mer PGDM1400 multispecific and multi-affinity antibody by endowing the molecule with multispecificity. To this end, in addition to the Fc fragment of the human IgG1 isotype, we combined the PGDM1400 Fab with the near-pan-neutralizing antibody 10E8v4 (a modified 10E871 with improved solubility) and the Fab of N49P7. To achieve this level of four-component (three Fabs and one Fc) heterooligomerization, we split the human deferroferrin structure into two subunits (N-ferritin and C-ferritin) and attached the Fab to the N-terminus of each hemisphere (Figure 3a). The complementarity of the split deferroferrin resulted in self-association of the two halves, thus leading to a very efficient heterodimerization process of the fusion protein. Importantly, no significant differences were observed in the biophysical and functional properties of the multispecific and multi-affinity antibodies assembled with the split and full-length deferroferrin (Figure 4). This design allows for a simple two-step purification procedure to select for multispecific and multi-affinity antibodies with four different specificities (Figure 5) and exhibits high batch-to-batch homogeneity (Figure 6). Furthermore, splitting the deferroferritin base in half allows for the inclusion of additional Fab / Fc fragments, up to 32 components per molecule, compared to the standard deferroferritin base material (Figure 3b). The eight additional sites (increasing from the conventional 24 to 32 in our engineered platform) play a significant role in providing multispecificity without sacrificing most of the potency increase observed in the 24-mer PGDM1400 multispecific and multi-affinity antibody. Therefore, the multispecific and multi-affinity antibody 32-N was designed to yield 16 copies of PGDM1400, 8 copies of Fc, 4 copies of 10E8v4, and 4 copies of N49P7 in a 4:2:1:1 ratio through co-transfection of plasmids encoding scFab and scFc (Figure 3a). Multispecific and multi-affinityAntibody 32-N forms highly modified and homogeneous particles (Fig. 3b) and exhibits unfolding and aggregation transition temperatures similar to those of the corresponding IgG molecules, and also 72 as previously reported for IgG (Figs. 3c and 7). Binding kinetics experiments were used to demonstrate that each component of the multispecific, multi-affinity antibody (PDGM1400, N49P7, 10E8v4, and the Fc fragment) can bind its epitopes via binding to epitope-specific molecules (BG505 SOSIP D368R, 93TH057 gp120, MPER peptide, and human FcRn, respectively) (Fig. 3d). Individual IgG molecules do not bind to all antigens (Fig. 8). The ability of 32-N to bind to multiple antigens suggests that the geometry conferred by the oligomeric form of the multispecific, multi-affinity antibody does not negatively impact antigen binding, presumably because the binding interface is outward-facing.

[0436] To investigate whether a multispecific, multi-affinity antibody could be designed that cross-targets HIV Env and the T-cell receptor CD4, we replaced N49P7 with iMab (a proven CD4-directed post-attachment inhibitor that effectively eradicates HIV 68, 69). The multispecific, multi-affinity antibody containing PDGM1400, iMab, 10E8v4, and Fc fragments (referred to as 32-I) exhibited similar homogeneity, thermostability, and multispecificity to 32-N (Figures 3, 7, and 8), highlighting the robust plug-and-play nature of multispecific, multi-affinity antibody platforms that allow for easy exchange of antibody sequences to alter specificity.

[0437] The HIV-1 multispecific, multi-affinity antibody exhibited exceptionally broad-spectrum neutralizing activity and potency. The neutralizing potency and breadth of the multispecific, multi-affinity antibodies 32-N and 32-I against 14 pseudoviruses were evaluated in a standardized in vitro TZM-bl neutralization assay 73. Groups of 14 PsVs were designed to include low-sensitivity PsVs, with each evaluated bNAb containing at least one resistant PsV. The IC50 values ​​and widths of the multispecific multi-affinity antibodies were compared to: (i) individual IgGs, (ii) IgG mixtures containing the same relative amounts of each IgG present in the multispecific multi-affinity antibody, and (iii) the N6 / PGDM1400x10E8 trispecific antibody 43. Both the 32-N and 32-I multispecific multi-affinity antibodies showed 100% width for their respective groups, 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 IgG mixtures and trispecific antibodies. However, the potency of the mixtures or trispecific antibody combinations against all tested PsVs was similar to that of the optimal mAbs when tested individually (Table 1). Thus, with IgG mixtures…Compared with the IC50 values ​​of trispecific antibodies, the median IC50 values ​​of multispecific multi-affinity antibodies were reduced by more than 90% and 99% in μg / mL and nM, respectively (Figures 9 and 10, Tables 1 and 2).

[0438] Table 1. Specification 40 / 49 pages 42 CN 121449754 A

[0439]

[0440] Table 2. Specification 41 / 49 pages 43 CN 121449754 A

[0441]

[0442] The in vivo pharmacokinetics and antidrug antibody profiles of the multispecific multi-affinity antibodies were similar to those of the corresponding IgGs. We then examined the in vivo toxicity, immunogenicity, and bioavailability of the multispecific multi-affinity antibodies after subcutaneous administration of 5 mg / kg to mice. Specification 42 / 49 pages 44 CN 121449754 A To evaluate our novel platform technology, we used a species-matched alternative multispecific multi-affinity antibody composed of mouse Fab and mouse Fc (IgG2a isotype) fused to mouse deferroferrin subunits, the opposite of the fully human component used in HIV-1 multispecific multi-affinity antibodies for human use. The Fab specificity selected for this alternative multispecific multi-affinity antibody is the Fab specificity that does not bind endogenous mouse proteins, similar to HIV-1 human mAbs that do not bind endogenous human proteins. Administration of the multispecific multi-affinity antibody was well-tolerated, with no observed weight loss or signs of toxicity. The alternative multispecific multi-affinity antibody did not induce a significant immunogenic response in mice; the level of anti-drug antibody (ADA) detected after 14 days was negligible for both the alternative multispecific multi-affinity antibody and its sequence-matched IgG2a (Figure 11b). This contrasts with highly immunogenic particles displaying malaria circospore protein (CSP) on the surface of Helicobacter pylori ferritin, which were used as a positive control for immunogenicity. Furthermore, alternative multispecific multi-affinity antibodies showed in vivo exposure days in close range with parental IgG1 and IgG2a molecules (Fig. 11b). The serum half-life was prolonged by introducing the LALAP mutation 74, which has been reported to silence Fc effector function. In summary, these data demonstrate the feasibility of tuning the bioavailability properties of our multispecific multi-affinity antibody platform and provide an exciting set of initial in vivo validation data for development.

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[0518] 75. Graille, M. et al., Complex between Peptostreptococcus magnus protein L and a human antibody reveals structural convergence in the interaction modesSpecification page 48 / 49 50 CN 121449754 A of Fab binding proteins. Structure, 2001, doi:10.1016 / S0969-2126(01)00630-X Specification page 49 / 49 51 CN 121449754 A Figure 1 Specification Figure 1 / 11 page 52 CN 121449754 A Figure 2 Specification Figure 2 / 11 page 53 CN 121449754 A Figure 3a Specification Figure 3 / 11 page 54 CN 121449754 A Figure 3b-Figure 3d Specification Figure 4 / 11 page 55 CN 121449754 A Figure 4 Specification Figure 5 / 11 page 56 CN 121449754 A Figure 5 Specification Figure 6 / 11 page 57 CN 121449754 A Figure 6 Figure 7 of the instruction manual, page 58 of 11, CN 121449754 A; Figure 8 of the instruction manual, page 59 of 11, CN 121449754 A; Figure 9 of the instruction manual, page 60 of 11, CN 121449754 A; Figure 10 of the instruction manual, page 61 of 10 of 11, CN 121449754 A; Figure 11 of the instruction manual, page 62 of 11 of 11, CN 121449754 A. Abstract: A fusion protein comprises a first nanocage monomer subunit of a nanocage monomer; and a bioactive 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.

Claims

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

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

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

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

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

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

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

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

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

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

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