Antibody cage nanostructure

Abstract

Provided are component polypeptides for Antibody Cage (AbC) nanostructures. The nanostructures may have 04, D2, T4, T8, or 15 architecture. Further provided are Fc-binding polypeptides that include a Z domain with defined amino acid substitutions relative to a reference Z domain. The amino acid substitution or substitutions may be D6E, S9N, N16H, M17L, N21T, A23E, G27A, T40A, or A50N, or a combination thereof, or other disclosed amino acid substitutions and combinations. The component polypeptides may be modified to enhance Fc affinity, reduce internal disulfide formation, and / or enable bioconjugation of the nanostructure. Further provided are AbC nanostructures, polynucleotides encoding the component polypeptides or nanostructures, delivery vehicles, pharmaceutical compositions, host cells, methods of making, methods of use, and other embodiments.

Description

[0001] Atorney Docket No. 24-1880-WO

[0002] ANTIBODY CAGE NANOSTRUCTURE

[0003] FUNDING STATEMENT

[0004] This invention was made with government support under Grant No. 1R21AI160030-01A1, awarded by the National Institutes of Health and Grant No. WR-20-034, awarded by the WE- REACH. The government has certain rights in the invention.

[0005] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0006] The instant application contains an electronic Sequence Listing that has been submited electronically and is hereby incorporated by reference in its entirety, The Sequence Listing was created on December 11, 2025, is named “24-1880-WO_Sequence_Listing_ST26_12-l l- 2025.xml” and is 569,273 bytes in size.

[0007] TECHNICAL FIELD

[0008] The invention relates to protein nanostructures, computational methods used to design protein nanostructures, and uses thereof in, for example, biotherapeutics.

[0009] BACKGROUND

[0010] Designed Antibody Cage (AbC™) nanostructures formed from two polypeptide components have been described in Divine et al. 2021 Science 372(6537):eabd9994 (PMID: 33795432). Such nanostructures incorporate as a first component being fragment crystallizable (Fc) domain dimer fused or bioconjugated to two effector domains, which may be a binding agent such as antigen-binding fragment of an antibody or a receptor ligand; and incorporate a second component that is multimerized through an oligomerization domain, with the two components spontaneously assembled to form a nanostructure with point group symmetry, such as an 04 nanostructure having octahedral symmetry. Advantages of AbC™ nanostructures may include lower effective concentrations due to polyvalent display of the effector domains.

[0011] However, there remains a need in the art for AbC™ nanostructures having improved pharmacokinetics and pharmacodynamics (PK / PD). The following disclosure provides nanostructures that may address that need and others. Atorney Docket No. 24-1880-WO

[0012] SUMMARY

[0013] In a first aspect, the disclosure provides an Fc-binding polypeptide, comprising a Z domain, wherein the Z domain specifically binds fragment crystallizable (Fc) domains, and wherein the Z domain comprises a polypeptide sequence disclosed in Table 1A, or a variant at least 85%, at least 90%, or at least 95% identical thereto; or wherein the Z domain comprises, relative to SEQ ID NO:498, an amino acid substitution D6E, S9N, N16H, M17L, N21T, A23E, G27A, T40A, A50N, or a combination thereof.

[0014] MGFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNLLAEAKKLADAQAPK (SEQ ID NO: 498)

[0015] In a second aspect, the disclosure provides an 04 component polypeptide modified to include a Z domain, comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 35-415 of SEQ ID NO: 1

[0016] (MG) FNKDQQSAFYEILNMPNLNEALRNGFIQLLKDDPSKSTVILTAAKVAAELSEK IRTLKESGSSYEQIAETVAKAVAKLVEKLKRNGVSEDEIALAVALI I SAVIQTLKES GSSYEVIAEIVARIVAEIVEALKRSGTSEDEIAEIVARVI SEVIRTLKESGSSYEVI AEI VARI VAEIVEALKRSGTSEDEIAKIVARVIAEVLRTLKESGSSEEVIKEI VARI ITEIKEALKRSGTSEDEIELITLMIEAALEIAKLKSSGSEYEEICEDVARRIAELVE KLKRDGTSAVEIAKIVAAI I SAVIAMLKASGSSYEVI CECVARIVAEIVEALKRSGT SAAI IALIVALVISEVIRTLKESGSSFEVILECVIRIVLEI IEALKRSGTSEQDVML IVMAVLLVVLATLQLSGS, wherein residues in parentheses are optional and may be present or may be deleted; wherein the polypeptide comprises an oligomerization domain and a Z domain that specifically binds fragment crystallizable (Fc) domains, and wherein the Z domain comprises a polypeptide sequence according to FNKEQQNAFYEILHLPNLNEEQRNAFIQSLKD (SEQ ID NO: 2) or FNKDQQSAFYEILNMPNLNEALRNGFIQLLKD (SEQ ID NO: 3), or a variant at least 85%, at least 90%, or at least 95% identical thereto; or a polypeptide sequence disclosed in Table 1A, or a variant at least 85%, at least 90%, or at least 95% identical thereto; or wherein the Z domain comprises, relative to SEQ ID NO:498, an amino acid substitution D6E, S9N, N16H, M17L, N21T, A23E, G27A, T40A, A50N, or a combination thereof.

[0017] In a third aspect, the disclosure provides an 04 component polypeptide modified to decrease internal disulfide bond formation, comprising a polypeptide sequence at least 50%, at least Atorney Docket No. 24-1880-WO

[0018] 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 35-415 of SEQ ID NO: 4

[0019] (MG) FNKDQQSAFYEILNMPNLNEALRNGFIQLLKDDPSKSTVILTAAKVAAELSEK IRTLKESGSSYEQIAETVAKAVAKLVEKLKRNGVSEDEIALAVALI ISAVIQTLKES GSSYEVIAEIVARIVAEIVEALKRSGTSEDEIAEIVARVISEVIRTLKESGSSYEVI AEI VARI VAEIVEALKRSGTSEDEIAKIVARVIAEVLRTLKESGSSEEVIKEI VARI ITEIKEALKRSGTSEDEIELITLMIEAALEIAKLKSSGSEYEEICEDVARRIAELVE KLKRDGTSAVEIAKIVAAI ISAVIAMLKASGSSYEVICECVARIVAEIVEALKRSGT SAAIIALIVALVISEVIRTLKESGSSFEVILECVIRIVLEI IEALKRSGTSEQDVML IVMAVLLVVLATLQLSGS, wherein residues in parentheses are optional and may be present or may be deleted; wherein the polypeptide comprises an oligomerization domain and a fragment crystallizable (Fc)- binding domain that specifically binds fragment crystallizable (Fc) domains, wherein the polypeptide sequence comprises an amino acid substitution relative to the SEQ ID NO:4 at position C271, C321, C323, or C373, or a combination thereof; or wherein the polypeptide sequence comprises amino acid substitutions relative to the SEQ ID NO:4 at positions C271, C321, C323, and C373.

[0020] In embodiments, the polypeptide sequence comprises, relative to the SEQ ID NO:4, an amino acid substitution C271A, C321A, C323I, or C373I, or a combination thereof; or wherein the polypeptide sequence comprises, relative to the SEQ ID NO:4, amino acid substitutions C271A, C321A, C323I, and C373I.

[0021] In embodiments, the Fc-binding domain is a Z domain, and wherein the Z domain comprises a polypeptide sequence according to FNKEQQNAFYEILHLPNLNEEQRNAFIQSLKD (SEQ ID NO: 2) or FNKDQQSAFYEILNMPNLNEALRNGFIQLLKD (SEQ ID NO: 3), or a variant at least 85%, at least 90%, or at least 95% identical thereto; or a polypeptide sequence disclosed in Table 1A, or a variant at least 85%, at least 90%, or at least 95% identical thereto.

[0022] In a fourth aspect, the disclosure provides an 04 component polypeptide modified for bioconjugation, comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 35- 415 of SEQ ID NO: 1

[0023] (MG) FNKDQQSAFYEILNMPNLNEALRNGFIQLLKDDPSKSTVILTAAKVAAELSEK IRTLKESGSSYEQIAETVAKAVAKLVEKLKRNGVSEDEIALAVALI ISAVIQTLKES Atorney Docket No. 24-1880-WO

[0024] AEI VARI VAEIVEALKRSGTSEDEIAKIVARVIAEVLRTLKESGSSEEVIKEI VARI ITEIKEALKRSGTSEDEIELITLMIEAALEIAKLKSSGSEYEEICEDVARRIAELVE KLKRDGTSAVEIAKIVAAI I SAVIAMLKASGSSYEVI CECVARIVAEIVEALKRSGT SAAI IALIVALVISEVIRTLKESGSSFEVILECVIRIVLEI IEALKRSGTSEQDVML IVMAVLLVVLATLQLSGS, wherein residues in parentheses are optional and may be present or may be deleted; wherein the polypeptide the polypeptide comprises an oligomerization domain and a fragment crystallizable (Fc)-binding domain that specifically binds fragment crystallizable (Fc) domains, wherein the polypeptide sequence comprises an amino acid substitution relative to the SEQ ID NO: 1 at position E51, T58, E61, E67, Q68, K79, K83, R86, El 11, El 17, E167, K220, T228, E232, R236, E241, S265, E269, E280, K284, S114, A224, R276, or R337, or a combination thereof; and wherein optionally the polypeptide is bioconjugated to a steric exclusion molecule, optionally a polyethylene glycol (PEG), preferably PEG-5K.

[0025] In embodiments, the polypeptide sequence comprises, relative to the SEQ ID NO: 1, an amino acid substitution E51C, T58C, E61C, E67C, Q68C, K79C, K83C, R86C, E111C, E117C, E167C, K220C, T228C, E232C, R236C, E241C, S265C, E269C, E280C, K284C, S114C, A224C, R276C, or R337C; or a combination thereof.

[0026] In embodiments, the polypeptide sequence comprises, relative to the SEQ ID NO: 1, an amino acid substitution K79C.

[0027] In embodiments, the polypeptide sequence comprises an amino acid substitution relative to the SEQ ID NO: 1 at position C271, C321, C323, or C373, or a combination thereof; or wherein the polypeptide sequence comprises amino acid substitutions relative to the SEQ ID NO: 1 at positions C271, C321, C323, and C373.

[0028] In embodiments, the polypeptide sequence comprises, relative to the SEQ ID NO: 1, an amino acid substitution C271A, C321A, C323I, or C373I, or a combination thereof; or wherein the polypeptide sequence comprises, relative to the SEQ ID NO: 1, amino acid substitutions C271A, C321A, C323I, and C373I.

[0029] In embodiments, the Fc-binding domain is a Z domain, and wherein the Z domain comprises a polypeptide sequence according to FNKEQQNAFYEILHLPNLNEEQRNAFIQSLKD (SEQ ID NO: 2) or FNKDQQSAFYEILNMPNLNEALRNGFIQLLKD (SEQ ID NO: 3), or a variant at least 85%, at least 90%, or at least 95% identical thereto; or a polypeptide sequence disclosed in Table 1A, or a variant at least 85%, at least 90%, or at least 95% identical thereto. Atorney Docket No. 24-1880-WO

[0030] In a fifth aspect, the disclosure provides an 04 component polypeptide, wherein the polypeptide comprises a polypeptide sequence comprising a first segment disclosed in Table 1A or Table 6, a second segment disclosed in Table 7, and a third segment disclosed in Table 8; or for each segment, a variant at least 85%, at least 90%, or at least 95% identical thereto.

[0031] In embodiments, the polypeptide comprises a polypeptide sequence according to any one of SEQ ID NOs: 470-479; or a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical thereto.

[0032] In embodiments, the polypeptide comprises a polypeptide sequence according to SEQ ID NO: 470; or a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical thereto.

[0033] In a sixth aspect, the disclosure provides a D2 component polypeptide modified to include a Z domain, comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 56-255 of SEQ ID NO: 480:

[0034] (M) SDEEERNELIQRIKEAAQRAREAAERTGDPRVRELARELQKIAQNAFYEVLHDP SSSEVNEALKAVVKAIELAVRALEEAEKTGDPEVRELAREVVRLAVEVAQATQAGEN DTLRKVAERALRLAKEAAKRGDAKAAKQAAKIAKLAAANAGDEDVLKKVELVRLAIE LVEIVVENAKRKGDDDKEAAEAALAAFRIVLAAAQLAGIASLEVLELALRLIKEVVE NAQREGYDIAVAAIAAAVAFAVVAVAAAAADITSSEVLELAIRLIKEVVENAQREGY VI LLAALAAAAAFVVVAAAAKRAGI TSSETLKRAI EEI RKRVEEAQREGNDI SEAAR QAAEEFRKKAEELK (GSWLEHHHHHH) , wherein residues in parentheses are optional, and may be present or may be deleted; wherein the polypeptide comprises an oligomerization domain and a Z domain that specifically binds fragment crystallizable (Fc) domains, wherein the Z domain comprises a polypeptide sequence according to (M)SDEEERNELIKRIREAAQRAREAAERTGDPRVRELARELARIAQIAFYLVLHDP (SEQ ID NO: 618); or (M)SDEEERNELIKRIREAAQRAREAAERTGDPRVRELARELAKLAQIAFYLVLHDP (SEQ ID NO: 619), or a variant at least 85%, at least 90%, or at least 95% identical thereto, or wherein the Z domain comprises one or more amino acid substitutions K12Q, R15K, A41Q, R42K, I46N, or L50E, or a combination thereof, relative to SEQ ID NO: 618. Atorney Docket No. 24-1880-WO

[0035] In a seventh aspect, the disclosure provides a T4 component polypeptide comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 36-305 of SEQ ID NO: 481:

[0036] (MG) FNKEQQNAFYLILHLPNLTEEQRNAFIQSLKDDPSKSAVVAGEAAIENARNAL KKGSPETAREAVRLALELVQQAERQARKTGSTERLIAAAKLAIEVARVALKVGSPET AREAVRTALELVQELIRQARKTGSKEVLEEAAKLALEVAKVAAEVGSPETAARAVAT AVEALKEAGASEDEIAEIVARVISEVIRILKESGSEYKVIARAVARIVAEIVEALKR SGTSEDEIAEIVARVISEVIRTLKESGSDYLI IAVIVAI IVAEIVEALKRSGTSEDE IAEIVARVISEVIRTLKESGSSYEVIKEIVQI IVLAI ILALMKSGTEVEEILLILLR VKTEVRRTLKES (GSWLEHHHHHH) , wherein residues in parentheses are optional, and may be present or may be deleted; wherein the polypeptide comprises an oligomerization domain and a Z domain that specifically binds fragment crystallizable (Fc) domains, and wherein the Z domain comprises an amino acid sequence according to FNKSQQSAFYLILNMPNLNEAQRNGFIQSLKD (SEQ ID NO:

[0037] 616), or a variant at least 85%, at least 90%, or at least 95% identical thereto, or wherein the Z domain comprises an amino acid substitution D6E, S9N, N16H, M17L, N21T, A23E, G27A, T40A, or A50N, or a combination thereof, relative to SEQ ID NO: 498.

[0038] In an eight aspect, the disclosure provides a T8 component polypeptide comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 36-266 of SEQ ID NO: 482:

[0039] (MG) FNKEQQNAFYEVLHLPNLTEEQRNAFIQSLKDDPSQSLKILIKAAAGGDSELE EVAKRI IKELAEQGRSEKEAAKEAAELIQRITRAAGGNSDLIELAVRIVKILEEQGR SPSEAAKEAVEAIEAIVRAAGGDSEAIKVAAEIAKTI ITQKESGSEYKEICRTVARI VAEIVEKLKRNGASEDEIAEIVAAI IAAVILTLKLSGSDYLI I CVCVAI I VAEI VEA LKRSGTSEDEIAEIVARVISAVIRVLKESGSSYEVIKECVQI IVLAI ILALMKSGTE VEEILLILLRVKTEVRRTLKES (GSWLEHHHHHH) ; wherein residues in parentheses are optional, and may be present or may be deleted; wherein the polypeptide comprises an oligomerization domain and a Z domain that specifically binds fragment crystallizable (Fc) domains, and wherein the Z domain comprises an amino acid sequence according to FNKDQQSAFYEVLNMPNLNEAQRNGFIQSLKD (SEQ ID NO:

[0040] 617) or a variant at least 85%, at least 90%, or at least 95% identical thereto, or wherein the Z domain comprises an amino acid substitution D6E, S9N, N16H, M17L, N21T, A23E, G27A, T40L, or A50G, or a combination thereof, relative to SEQ ID NO: 498. Atorney Docket No. 24-1880-WO

[0041] In a nineth aspect, the disclosure provides an 15 component polypeptide comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 56-227 of SEQ ID NO: 483;

[0042] (M) SDEEERNELIQRIKEAAQRAREAAERTGDPRVRELARELQKLAQNAFYEVLHDP

[0043] S S SDVNEALKL I VEAI EAAVRALEAAERAGDPELREDAREAVRLAVEAAQEVQRNPS SSTANLLLKAIVALAEALAAAANGDKEKFKKAAESALEIAKRVVEVASKEGDPEAVL EAAKVALRVAELAAKNGDKEVFKKAAESALEVAKRLVEVASKEGDPELVLEAAKVAL RVAELAAKNGDKEVFQKAAASAVEVALRLTEVASKEGDSELETEAAKVITRVRELAS KQGDAAVAILAETAEVKLEIEESKKRPQSESAKNLILIMQLLINQIRLLVLQIRMLD EQRQE (GSWLEHHHHHH) wherein residues in parentheses are optional, and may be present or may be deleted; wherein the polypeptide comprises an oligomerization domain and a Z domain that specifically binds fragment crystallizable (Fc) domains, and wherein the Z domain comprises an amino acid sequence according to

[0044] (M)SDEEERNELIKRIREAAQRAREAAERTGDPRVRELARELARLAQRAFYLVLHDP (SEQ ID NO: 620), or a variant at least 85%, at least 90%, or at least 95% identical thereto, or wherein the Z domain comprises one or more amino acid substitutions K12Q, R15K, A41Q, R42K, R46N, or L50E, or a combination thereof, relative to SEQ ID NO: 620.

[0045] In a tenth aspect, the disclosure provides an 15 component polypeptide comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 56-227 of SEQ ID NO: 484

[0046] (M) SDEEERNELIQRIKEAAQRAREAAERTGDPRVRELARELQKLAQNAFYEVLHNP ESEGTLRALDHI IRAIDAAVEAIEAALESGDEEARERAREAVRQAVEAAQRVLENPE SEAVLKLLEAVVALAEALAAAMRGDKEEFKKAAKKAIKIAREVVKLAKKQGNAELVL KAAEIALEVARLAAEKGDKEIFKEAAKAAIEIAREWKLAKKQGNAELVLKAAEIAL EVARLAAEKGDEEIFREAAKAAIEIANEVKKLAKKQGNEELVKKALEI IKEVGKLAK EKGDRSSKTLAESELIKFEIEELEKKPHSLETKALILISKLLYASIVLLSEI IDELY KI LKK (GSWLEHHHHHH) ; wherein residues in parentheses are optional, and may be present or may be deleted; wherein the polypeptide comprises an oligomerization domain and a Z domain that specifically binds fragment crystallizable (Fc) domains, and wherein the Z domain comprises an amino acid sequence according to

[0047] (M)SDEEERNELIKRIREAAQRAREAAERTGDPRVRELARELARLAQRAFYLVLHDP (SEQ ID NO: 620), or wherein the Z domain comprises one or more amino acid substitutions Atorney Docket No. 24-1880-WO

[0048] K12Q, R15K, A41Q, R42K, R46N, or L50E, or a combination thereof, relative to SEQ ID NO: 620.

[0049] In an eleventh aspect, the disclosure provides an AnglF polypeptide, comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 1-225 of SEQ ID NO: 6

[0050] KAELASEKPFRDCADVYQAGFNKSGIYTIYINNMPEPKKVFCNMDVNGGGWTVIQHR EDGSLDFQRGWKEYKMGFGNPSGEYWLGNEFI FAITSQRQYMLRIELMDWEGNRAYS QYDRFHIGNEKQNYRLYLKGHTGTAGKQSSLILHGADFSTKDADNDNCMCKCALMLT GGWWFDACGPSNLNGMFYTAGQNHGKLNGIKWHYFKGPSYSLRSTTMMIRPLDFGGS GGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDLSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK, wherein the polypeptide sequence comprises an amino acid substitution relative to the SEQ ID NO:6 at position K23, M105, H148Q, F188, R214, or S215, or a combination thereof.

[0051] In embodiments, the polypeptide sequence comprises an amino acid substitution relative to the SEQ ID NO:6 at positions K23, M105, H148, F188, R214, and S215.

[0052] In embodiments, the polypeptide sequence comprises, relative to the SEQ ID NO:6, an amino acid substitution K23T, M105E, H148Q, F188Y, R214K, or S215M, or a combination thereof.

[0053] In embodiments, the polypeptide sequence comprises, relative to the SEQ ID NO:6. amino acid substitutions K23T, M105E, H148Q, F188Y, R214K, and S215M.

[0054] In embodiments, the polypeptide comprises a polypeptide linker and a fragment crystallizable (Fc) polypeptide, wherein optionally the polypeptide sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 1-463 of SEQ ID NO: 6.

[0055] In a further aspect, the disclosure provides an antibody cage (AbC) nanostructure, comprising a plurality of first polypeptides, each first polypeptide comprising a fragment crystallizable (Fc) polypeptide that forms an Fc dimer; and a plurality of second polypeptides, wherein each second polypeptide is a polypeptide as described herein. Atorney Docket No. 24-1880-WO

[0056] In embodiments, the plurality of first polypeptides multimerize to form a plurality of first components and the plurality of second polypeptides multimerize to form a plurality of second components; and wherein the first components and the second components assemble by direct interaction of Fc dimers of the first polypeptides with Fc-binding domains of the second polypeptides.

[0057] In embodiments, the first polypeptides comprise antibody or an antigen-binding domain thereof, a polypeptide linker, and the Fc polypeptide.

[0058] In embodiments, the antibody is selected from Herceptin, which is a humanized anti-HER2 monoclonal antibody, Rituxan, which is a chimeric anti-CD20 monoclonal antibody, Panorex or (edrecolomab, which is a murine IgG2a antibody recognizing EpCAM, BEC2, which is murine IgG antibody that recognizes GD3 ganglioside, IMC-C225, which is a chimeric IgG antibody against EGFR, Vitaxin, which is a humanized antibody targeting vitronectin receptor, Smart Ml 95 which is a humanized anti-CD33 IgG antibody, LymphoCide which is a humanized IgG antibody targeting lymphocytes, Smart ID 10 which is a humanized antibody that binds to an HLA-DR determinant, Oncolym which is a murine antibody targeting HLA-DR10, Allomune which is a humanized anti-CD2 mAb, an anti-VEGF antibody, CEAcide which is a humanized anti-CEA antibody, IMC-1C11 which is an anti- KDR chimeric antibody, Cetuximab, which is an anti-EGFR chimeric antibody, Lob 7 / 6, Lucatumumab, Dacetuzumab, Selicrelumab, Bleselumab, Urelumab, Utomilumab, Drozitumab, scTRAIL-Fc, KMTR2, 16E2, Aplitabart, which is an multivalent IgM DR5 agonist antibody, optionally conatumumab, which is a human agonist antibody to death receptor 5 (DR5).

[0059] In embodiments, the first polypeptides comprise a binding agent. In embodiments, the binding agent is an Angl polypeptide, wherein optionally the first polypeptide is any AnglF polypeptide described herein.

[0060] In embodiments, the binding agent is Ang2, Ang3, Ang4 polypeptides or engineered molecules Bow-Angl, Comp-Angl, ABTAA antibody and vasculotide that binds to Tie2 receptor.

[0061] In embodiments, the binding agent is transferrin, EGF, bombesin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, interleukins such as IL-2 and IL-6, TGF-a, TGF-P, vaccinia growth factor (VGF), vascular endothelial growth factor (VEGF), insulin and Atorney Docket No. 24-1880-WO insulin-like growth factors I and II, somatostatin, lectins and apoprotein from low density lipoprotein.

[0062] In a further aspect, the disclosure provides a polynucleotide encoding any polypeptide or nanostructure described herein.

[0063] In a further aspect, the disclosure provides a delivery vehicle, comprising any polynucleotide described herein, optionally a viral vector or lipid nanoparticle.

[0064] In a further aspect, the disclosure provides a pharmaceutical composition, comprising any polypeptide, nanostructure, polynucleotide, or delivery vehicle disclosed herein, and a pharmaceutically acceptable carrier.

[0065] In a further aspect, the disclosure provides a host cell suitable for expression of any polypeptide or nanostructure described herein; and / or comprising any polynucleotide described herein.

[0066] In a further aspect, the disclosure provides a method of making a polypeptide or nanostructure, comprising culturing any host cell described herein under conditions suitable for expression of the polypeptide or nanostructure.

[0067] In a further aspect, the disclosure provides a method of treating a disease or disorder in a subject suffering from or at risk thereof, the method comprising administering to the subject any polypeptide, nanostructure, polynucleotide, delivery vehicle, or pharmaceutical composition described herein.

[0068] In embodiments, the disease or disorder is a viral infection, optionally influenza.

[0069] In embodiments, the disease or disorder is a cancer, wherein optionally the nanostructure comprises an antigen-binding fragment of conatumumab, and a cancer is a cancer responsive to conatumumab.

[0070] In yet further aspect, the disclosure provides a composition or method as described herein.

[0071] Any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.

[0072] BRIEF DESCRIPTIONS OF THE DRAWINGS

[0073] FIG. 1 and 2 shows schematic representation of O4_Fc AbC.

[0074] FIG. 3 is schematic representation of the high-throughput computational design process. Atorney Docket No. 24-1880-WO

[0075] FIG. 4 show in vitro serum stability following 24h incubation in serum at 37°C (FIG. 4A) and in vivo half-life improvement (FIG. 4B) of original, redesigned and PEGylated AbCs.

[0076] FIG. 5A shows the sites for PEG mutations. FIG. 5B shows a heatmap and FIG. 5C shows the graphical representation of % reduction in antibody binding to 04 surface in presence of PEG mutants crosslinked with different molecular weights of PEG.

[0077] FIG. 6A and FIG. 6B show % viability of Colo205 cells when exposed to InM (FIG. 6A) or 10 nM (FIG. 6B) AbCs.

[0078] FIG. 7A shows % viability of Colo205 cells when exposed to 10 nM or 50 nM AbC with O4 AI R3 PEG variant crosslinked with different molecular weights of PEG.

[0079] FIG. 7B shows % viability of SW403 cells when exposed to 10 nM or 50 nM AbC with O4 AI R3 PEG variant crosslinked with different molecular weights of PEG.

[0080] FIG. 8 shows schematic representation of 04 Z domain redesign.

[0081] FIG. 9 shows in vitro serum stability of AbCs with 04 Z domain design following 24h incubation in human serum at 37°C.

[0082] FIG. 10A shows a heatmap of 100 pM sort on yeast. FIG. 10B shows the domain structure showing SSM hotspots.

[0083] FIG. 11 shows cryo-electron microscopy (cryo-EM) and results from small-angle light scatering (SAXS) experiments of design and cryo relaxed model.

[0084] FIG. 12A shows combined SEC traces for redesigned 04 component variants FIG. 12B shows in vitro serum stability.

[0085] FIG. 13A and FIG. 13B shows in vitro serum stability and biological activity (% viability) of redesigned 04 components after 24h and 72h, respectively.

[0086] FIG. 14A show in vitro serum stability following 24h incubation in serum at 37°C (FIG. 14 A) and in vivo half-life improvement (FIG. 14B) of original or redesigned AbCs.

[0087] FIG. 15 shows representative IVIS images of 7-week-old Balb / C female mice retro-orbitally (RO) with 2 mg / kg of AbC. Images were obtained 4h after RO injections.

[0088] FIG. 16A and FIG. 16B shows in vivo stability and biodistribution of redesigned AbC compared to original. Atorney Docket No. 24-1880-WO

[0089] FIG. 17A shows representative IVIS images of mice injected with targeted vs untargeted AbCs. FIG. 17B shows in vivo stability and biodistribution.

[0090] FIG. 18 shows cell viability of SW403 when exposed to AbCs after incubation for 1 week in PBS at 37°C.

[0091] FIG. 19 and 20 show viability data from 17 cancer cell lines with TRAIL, anti-DR5 antibody, or optimized DR5-AbC variants.

[0092] FIG. 21 and FIG. 22 show in vivo efficacy of 04 variants against the buffer or dulanermin- only controls upon intratumoral (FIG. 21) or intravenous (FIG. 22) injections in a Colo205 xenograft model. Tumors were allowed to form for 14 days before beginning drug injections.

[0093] FIG. 23A shows in vivo efficacy of 04 variants against the buffer or dulanermin-only controls upon intratumoral injections in an A375 xenograft model. Tumors were allowed to form for 30 days before beginning drug injections. FIG. 23B shows % change in tumor volume of the model upon drug injections.

[0094] FIG. 24 shows caspase 3 / 7 activity of RCC4 cells when treated with PBS, TRAIL, anti-DR5 antibody or DR5-AbCs at different concentrations.

[0095] FIG. 25 shows representative Western blot and normalized signal of cleaved PARP (c-PARP) from RCC4 cells when treated with PBS, TRAIL, anti-DR5 antibody or DR5-AbCs.

[0096] FIG. 26 shows % cell viability of Colo205 cells when treated with control or 10 nM AbC and FIG. 27 shows % cell viability of SW403 cells when treated with control or 50 nM AbC in presence of pan-caspase inhibitor (Z-VAD FMK), caspase-3 inhibitor (Z-DEVD-FMK) or C- Flip inhibitor (Rocagalmide).

[0097] FIG. 28A and 28B show DR5 expression in various cancer cell lines. FIG. 28C and 28D show DR5 expression and AbC killing activity in monocyte-derived dendritic cells (moDCs).

[0098] FIG. 29 and 30 shows % cell viability of A375 (FIG. 29) and Colo205 (FIG. 30) when treated with increasing concentrations of control or redesigned AbCs.

[0099] FIG 31 shows representative flow cytometry histograms showing % translocated calreticulin in A375 (top) and Colo205 (botom) cells when treated with of control or redesigned AbCs.

[0100] FIG. 32 shows phagocyte activity of engulfed cancer cells as determined by using pHrodo- labeled cell engulfing human macrophages in A375 (top) and Colo205 (botom) cells when treated with of control or redesigned AbCs. Atorney Docket No. 24-1880-WO

[0101] FIG. 33A and 33B show qPCR analysis for different cytokines from A375 cells when treated with control or AbCs.

[0102] FIG. 34 shows T-cell proliferation when treated with control, anti-DR5 antibody, INBRX- 109, or an optimized DR5-AbC.

[0103] FIG. 35 and 36 show experimental evaluation of CD4+T cell activity when treated with a mixture of conditioned media from treating A375 (FIG. 35) and Colo205 (FIG. 36) cells with AbCs for 24h and fresh media in a 1 : 1 ratio.

[0104] FIG. 37A shows representative Western blot and FIG. 37B shows normalized signal for pAKT and pERKl / 2 in endothelial HUVEC when treated with control or AlF-AbC.

[0105] FIG. 38A shows representative images and FIG. 38B shows vascular stability of endothelial HUVEC cells when treated with control or AlF-AbC.

[0106] FIG. 39 shows activity in endothelial HUVEC cells when treated with control or optimized and PEGylated 04 bearing the AnglF6 Tie2-binding domain.

[0107] FIG. 40. 7-week-old mice were infected with 0.5LD50 dose of the H1N1 California 2009 strain of influenza (H1N 1 Ca / 09) and dosed with 2 mg / kg post-infection for 5 days with control or optimized and PEGylated 04 bearing the AnglF6 Tie2-binding domain. Weight was tracked for 12 days.

[0108] DETAILED DESCRIPTION

[0109] DEFINITIONS

[0110] All references cited are herein incorporated by reference in their entirety. Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al.,

[0111] 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185. edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), “Guide to Protein Purification” in Methods in Enzymology (M. P. Deutsheer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al.

[0112] 1990. Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, Tex.). Atorney Docket No. 24-1880-WO

[0113] As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (He; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser, S), threonine (Thr, T). tryptophan (Trp; W), tyrosine (Tyr, Y), and valine (Vai; V).

[0114] In all embodiments of polypeptides disclosed herein, any N-terminal methionine (M) residues or methionine-glycine (MG) residues are optional (i.e.: the N-terminal methionine residue or methionine-glycine residues may be present or may be absent).

[0115] Words using the singular or plural number also may include the plural and singular number, respectively. The singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0116] As used herein, the term “about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. For example, about means within a standard deviation using measurements generally acceptable in the art. For example, about means a range extending to + / - 10%, + / - 5%, + / - 3%, or + / - 1% of the specified value.

[0117] The term “at least” followed by a number is used herein to denote the start of a range beginning with that number (which may be a range having an upper limit or no upper limit, depending on the variable being defined). For example, “at least 1” means 1 or more than 1.

[0118] The term “at most” followed by a number is used herein to denote the end of a range ending with that number (which may be a range having 1 or 0 as its lower limit, or a range having no lower limit, depending upon the variable being defined). For example, “at most 4” means 4 or less than 4, and “at most 40%” means 40% or less than 40%. When, in this specification, a range is given as “(a first number) to (a second number)” or “(a first number)-(a second number)” this means a range whose lower limit is the first number and whose upper limit is the second number. For example, 25 to 100 mm means a range whose lower limit is 25 mm, and whose upper limit is 100 mm.

[0119] The term “identical” or percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence. Methods of alignment of sequences for comparison are well known in the art. Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present Atorney Docket No. 24-1880-WO in both sequences. The percent sequence identity is determined by dividing the number of matches in the alignment by the length of the reference sequence, followed by multiplying the resulting value by 100. For example, a peptide sequence that has 1166 matches when aligned with a reference sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166=1554 * 100=75.0). As the terms are used herein, gaps in the alignment do not decrease the percent sequence identity. Unless otherwise specified, optimal alignment of sequences for comparison is conducted by the global alignment algorithm of Needleman and Wunsch, Mol. Biol. 48:443 (1970) as implemented by EMBOSS Needle (on the World Wide Web at ebi.ac.uk / Tools / psa / emboss_needle / ) (Madeira et al. Nucleic Acids Res. 50(Wl):W276-W279 (2022)). Other alignment methods may be used, including without limitation those described in Devereux, et al, Nucleic Acids Res. 12:387-95 (1984); Atschul et al. J. Mo. Biol. 215:403- 10 (1990) (BLAST); Carrillo and Lipman Siam J. Appl. Math. 48(5) (1988); Computational Molecular Biology (Lesk, AM, ed., 1989); Biocomputing Informatics and Genome Projects, (Smith, DW, ed., 1993); Computer Analysis of Sequence Data, Part I, (Griffin and Griffin, eds., 1994); Sequence Analysis in Molecular Biology (von Heinje, 2012); Sequence Analysis Primer (Gribskov and Devereux, J., eds. 1993). Sequence identity is calculated using the implementation of the Needleman-Wunsch algorithm provided by the National Library of Medicine (on the World Wide Web at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=GlobalAln).

[0120] For example, sequence identity can be determined by standard methods that are commonly used to compare the similarity of two polypeptide or two polynucleotide sequences. Using a computer program such as EMBOSS Needle or BLAST, two polypeptide or two polynucleotide sequences are aligned for optimal matching of their respective residues (either along the full length of one or both sequences, or along a pre-determined portion of one or both sequences). The programs provide a default opening penalty and a default gap penalty, and a scoring matrix such as PAM 250 (a standard scoring matrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978)) that can be used in conjunction with the computer program.

[0121] The term “substantially similar” refers to two polypeptides, proteins, assemblies, nanostructures, or other physical embodiments of the present disclosure that may differ in architecture, sequence, configuration, associations, and the like yet provide about the same or similar properties, structure, activity, and / or function. In other words, some embodiments of the present disclosure may be exchanged, yet achieve a desired outcome, e.g., in properties, Atorney Docket No. 24-1880-WO structures, activities, and / or functions.

[0122] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising,” as well as “has” or “having” and “includes” or “including,” will be understood to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps; that is to say, in the sense of “including, but not limited to”.

[0123] “Consisting essentially of’ or “consists essentially” indicates exclusion of elements or steps that materially affect the basic and novel characteristics of the claimed invention.

[0124] Any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein. The words “herein,” “above,” and “below” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application. Section headings are for organization and disclosure in one section may be combined with disclosure in other sections.

[0125] “Treating” or “treatment” as used herein (and as well-understood in the art) also broadly includes any approach for obtaining beneficial or desired results in a subject’s condition, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (i.e. , not worsening) the state of disease, prevention of a disease’s transmission or spread, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission, whether partial or total and whether detectable or undetectable. In other words, “treatment” as used herein includes any cure, amelioration, or prevention of a disease. Treatment may prevent the disease from occurring; inhibit the disease’s spread; relieve the disease’s symptoms, fully or partially remove the disease’s underlying cause, shorten a disease’s duration, or do a combination of these things.

[0126] The terms “disease” or “condition” refer to a state of being or health status of a patient or subject capable of being treated with the compounds or methods provided herein. The disease may be a cancer. The disease may be an autoimmune disease. The disease may be an inflammatory disease. The disease may be an infectious disease. In some further instances, “cancer” refers to human cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, etc., including solid and lymphoid cancers, kidney, breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, glioma, Atorney Docket No. 24-1880-WO esophagus, and liver cancer, including hepatocarcinoma, lymphoma, including B-acute lymphoblastic lymphoma, non-Hodgkin’s lymphomas (e.g., Burkit’s, Small Cell, and Large Cell lymphomas), Hodgkin’s lymphoma, leukemia (including AML, ALL, and CML), or multiple myeloma.

[0127] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. A “fusion protein” refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.

[0128] In all embodiments, any N-terminal methionine residue is optional, and may be present or may be deleted.

[0129] The term “EC50” or “half maximal effective concentration” as used herein refers to the concentration of a molecule (e.g., antibody, chimeric antigen receptor or bispecific antibody) capable of inducing a response which is halfway between the baseline response and the maximum response after a specified exposure time. In embodiments, the EC50 is the concentration of a molecule (e.g., antibody, chimeric antigen receptor or bispecific antibody) that produces 50% of the maximal possible effect of that molecule.

[0130] Nanostructure: As used herein, the term refers to a protein complex made up of a plurality of a single component or pluralities of two or more components, where each component is a multimeric protein complex having at least one symmetry axis, and where component or components assemble with point group symmetry.

[0131] Antibody Cage (AbC™) nanostructure: As used herein, the term refers to any nanostructure where the first component includes an immunoglobulin domain, such as a fragment crystallizable domain. The term includes embodiments having a multimeric complex of a fusion protein such as an Fc fusion protein. That is, the term AbC nanostructure may include nanostructures that have a monoclonal antibody as the first component but other nanostructures in which the monoclonal antibody is replaced with an engineered Fc fusion protein. Illustrative AbC nanostructures are described as “antibody-bound nanoparticle structures” in US20230330033A1 (“Designed antibody-bound nanoparticles”) and US20230233707A1 (“Antibody-bound nanoparticles”), the entire contents of which are incorporated herein in their entireties. Atorney Docket No. 24-1880-WO

[0132] Fc-binding domain: As used herein, the term refers to any polypeptide or polypeptide fragment that specifically binds an Fc domain or Fc domain variant, including but not limited to Protein A and fragments thereof. Illustrative Fc-binding domains include a “D domain” and is a “Z domain,” examples of which are well known in the art — for example, see Nilsson et al., Protein Engng. 1 : 107-113 (1987) describing the Z domain as a domain that spans a portion of the IgG-binding B domain of staphylococcal protein A, and has the amino acid sequence: AVDNKFNKEQQNAFYE ILHLPNLNEEQRNAFI QSLKDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO 622). Further illustrative examples include the truncated sequence Z domain having sequence:

[0133] FNKEQQNAFYE ILHLPNLNEEQRNAFIQSLKD (SEQ ID NO: 2) or

[0134] FNKEQQNAFYE ILHLPNLTEEQRNAFIQSLKD (SEQ ID NO: 489)

[0135] Functional variants of a Z domain include a variant having hydrophobic substitutions to improve packing against adjacent helical domains, such as a variant having two leucine (L) substitutions resulting in the sequence:

[0136] FNKDQQSAFYE ILNMPNLNEALRNGFI QLLKD (SEQ ID NO: 3) or

[0137] FNKEQQNAFYE ILHLPNLTEELRNAFI QLLKD (SEQ ID NO: 490)

[0138] Further illustrative Fc-binding domains include “aflfibodies”. See WO 95 / 19374; Nord et al. (1995) Protein Engineering, Vol. 8 (6), 601-608; and Nord et al. (1997) Nature Biotechnology, Vol. 15, Tll-TTI.

[0139] Component polypeptide: As used herein, the term refers to any polypeptide that is capable of multimerizing to form a component of a nanostructure . Illustrative component polypeptides are described in US20230330033A1 (“Designed antibody-bound nanoparticles”) and US20230233707A1 (“Antibody-bound nanoparticles”), the entire contents of which are incorporated herein in their entireties.

[0140] Bioconjugation: As used herein the term “bioconjugation” refers to a process of making a protein into a bioconjugate. An illustrative example of a “bioconjugate” is a protein modified to include one or more PEG molecules bioconjugated to one or more cysteine (C) residues substituted into the polypeptide sequence at selected site(s). Another example is bioconjugation to one or more lysine (L) residues substituted into the polypeptide sequence at selected site(s). As used herein, the terms “bioconjugate” and “bioconjugate linker” refers to the resulting association between atoms or molecules of “bioconjugate reactive groups” or “bioconjugate reactive moieties”. The association can be direct or indirect. For example, a Atorney Docket No. 24-1880-WO conjugate between a first bioconjugate reactive group (e.g., -NH2, -C(O)OH, -N- hydroxysuccinimide, or -maleimide) and a second bioconjugate reactive group (e.g., sulfhydryl, sulfur-containing amino acid, amine, amine sidechain containing amino acid, or carboxylate) provided herein can be direct, e.g., by covalent bond or linker (e.g. a first linker of second linker), or indirect, e.g., by non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like). In embodiments, bioconjugates or bioconjugate linkers are formed using bioconjugate chemistry (i.e. the association of two bioconjugate reactive groups) including, but are not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition). These and other useful reactions are discussed in, for example, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, D.C., 1982. In embodiments, the first bioconjugate reactive group (e.g., maleimide moiety) is covalently attached to the second bioconjugate reactive group (e.g. a sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., haloacetyl moiety) is covalently atached to the second bioconjugate reactive group (e.g. a sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., pyridyl moiety) is covalently atached to the second bioconjugate reactive group (e.g. a sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., -N- hydroxysuccinimide moiety) is covalently atached to the second bioconjugate reactive group (e.g. an amine). In embodiments, the first bioconjugate reactive group (e.g., maleimide moiety) is covalently atached to the second bioconjugate reactive group (e.g. a sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., -sulfo-N-hydroxy succinimide moiety) is covalently atached to the second bioconjugate reactive group (e.g. an amine).

[0141] Useful bioconjugate reactive moieties used for bioconjugate chemistries herein include, for example:

[0142] (a) carboxyl groups and various derivatives thereof including, but not limited to, N-hydroxy succinimide esters, N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters; Atorney Docket No. 24-1880-WO

[0143] (b) hydroxyl groups which can be converted to esters, ethers, aldehydes, etc.

[0144] (c) haloalkyl groups wherein the halide can be later displaced with a nucleophilic group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the site of the halogen atom;

[0145] (d) dienophile groups which are capable of participating in Diels-Alder reactions such as, for example, maleimido or maleimide groups;

[0146] (e) aldehyde or ketone groups such that subsequent derivatization is possible via formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or via such mechanisms as Grignard addition or alkyllithium addition;

[0147] (f) sulfonyl halide groups for subsequent reaction with amines, for example, to form sulfonamides;

[0148] (g) thiol groups, which can be converted to disulfides, reacted with acyl halides, or bonded to metals such as gold, or react with maleimides;

[0149] (h) amine or sulfhydryl groups (e.g., present in cysteine), which can be, for example, acylated, alkylated or oxidized;

[0150] (i) alkenes, which can undergo, for example, cycloadditions, acylation, Michael addition, etc;

[0151] (j) epoxides, which can react with, for example, amines and hydroxyl compounds;

[0152] (k) phosphoramidites and other standard functional groups useful in nucleic acid synthesis;

[0153] (l) metal silicon oxide bonding;

[0154] (m) metal bonding to reactive phosphorus groups (e.g. phosphines) to form, for example, phosphate diester bonds;

[0155] (n) azides coupled to alkynes using copper catalyzed cycloaddition click chemistry; and Atorney Docket No. 24-1880-WO

[0156] (o) biotin conjugate can react with avidin or strepavidin to form a avidin-biotin complex or streptavidin-biotin complex.

[0157] The bioconjugate reactive groups can be chosen such that they do not participate in, or interfere with, the chemical stability of the conjugate described herein. Alternatively, a reactive functional group can be protected from participating in the crosslinking reaction by the presence of a protecting group. In embodiments, the bioconjugate comprises a molecular entity derived from the reaction of an unsaturated bond, such as a maleimide, and a sulfhydryl group.

[0158] DESCRIPTION OF EMBODIMENTS

[0159] The disclosure relates generally to designed nanostructures, including but not limited to AbC nanostructures, such as 04 nanostructures having an 04 architecture, such as T4 nanostructures having an T4 architecture, such as T8 nanostructures having an T8 architecture, such as 15 nanostructures having an 15 architecture. The nanostructures described herein include as a component a multimerized component polypeptides.

[0160] In some embodiments, the polypeptide comprises a variant further comprising an N-terminal tag, wherein the N-terminal tag is M or MG.

[0161] An illustrative 04 polypeptide may have the polypeptide sequence below (SEQ ID NO: 202 or 501), or a variant thereof, where the N-terminal MG and the C-terminal hexahistidine tags in parentheses are optional:

[0162] 1 (MG) FNKDQQSA FYEILNMPNL NEALRNGFIQ LLKDDPSKST

[0163] 41 VILTAAKVAA ELSEKIRTLK ESGSSYEQIA ETVAKAVAKL

[0164] 81 VEKLKRNGVS EDEIALAVAL I I SAVIQTLK ESGSSYEVIA

[0165] 121 EIVARIVAEI VEALKRSGTS EDEIAEIVAR VI SEVIRTLK

[0166] 161 ESGSSYEVIA EIVARIVAEI VEALKRSGTS EDEIAKIVAR

[0167] 201 VIAEVLRTLK ESGSSEEVIK EIVARI ITEI KEALKRSGTS

[0168] 241 EDEIELITLM IEAALEIAKL KSSGSEYEEI CEDVARRIAE

[0169] 281 LVEKLKRDGT SAVEIAKIVA AI I SAVIAML KASGSSYEVI

[0170] 321 CECVARIVAE IVEALKRSGT SAAI IALIVA LVI SEVIRTL

[0171] 361 KESGSSFEVI LECVIRIVLE I IEALKRSGT SEQDVMLIVM

[0172] 401 AVLLVVLATL QLS (GSLEHHH HHH) ( SEQ ID NO : 202 )

[0173] 1 (MG) FNKDQQSA FYEILNMPNL NEALRNGFIQ LLKDDPSKST

[0174] 41 VILTAAKVAA ELSEKIRTLK ESGSSYEQIA ETVAKAVAKL Atorney Docket No. 24-1880-WO

[0175] 81 VEKLKRNGVS EDEIALAVAL I ISAVIQTLK ESGSSYEVIA

[0176] 121 EIVARIVAEI VEALKRSGTS EDEIAEIVAR VI SEVIRTLK

[0177] 161 ESGSSYEVIA EIVARIVAEI VEALKRSGTS EDEIAKIVAR

[0178] 201 VIAEVLRTLK ESGSSEEVIK EIVARI ITEI KEALKRSGTS

[0179] 241 EDEIELITLM IEAALEIAKL KSSGSEYEEI AEDVARRIAE

[0180] 281 LVEKLKRDGT SAVEIAKIVA AI I SAVIAML KASGSSYEVI

[0181] 321 AEIVARIVAE IVEALKRSGT SAAI IALIVA LVI SEVIRTL

[0182] 361 KESGSSFEVI LEIVIRIVLE I IEALKRSGT SEQDVMLIVM

[0183] 401 AVLLVVLATL QLS (GSWLEHH HHHH) ( SEQ ID NO : 501 )

[0184] The disclosure provides improvement to 04 polypeptides including but not limited to modifications to the Fc-binding domain (e.g., Z domain); removal of cysteine residues; introduction of artificial sites for bioconjugations (e.g., PEG at cysteine residues); and computational re-modelling of polypeptide segments validated experimentally, as well as combinations thereof. The disclosure provides improvement to D2, T4, T8, and 15 polypeptides including but not limited to modifications to the Fc-binding domain (e.g., Z domain).

[0185] The disclosed Z domains may also be used in any polypeptides where an Fc-binding domain is desired to perform intended function, including but not limited to component polypeptides of AbC nanostructures.

[0186] Z domains

[0187] In a first aspect, the disclosure provides an Fc-binding polypeptide, comprising a Z domain, wherein the Z domain specifically binds fragment crystallizable (Fc) domains, and wherein the Z domain comprises a polypeptide sequence disclosed in Table 1A or Table IB, or a variant at least 85%, at least 90%, or at least 95% identical thereto.

[0188] Table 1A Atorney Docket No. 24-1880-WO Atorney Docket No. 24-1880-WO Atorney Docket No. 24-1880-WO Atorney Docket No. 24-1880-WO Atorney Docket No. 24-1880-WO Atorney Docket No. 24-1880-WO

[0189] Table IB

[0190] Component Polypeptides

[0191] 04 component polypeptides modified to include a Z domain In a second aspect, the disclosure provides an 04 component polypeptide modified to include a Z domain, comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 35-415 of SEQ ID NO: 1

[0192] (MG) FNKDQQSAFYEILNMPNLNEALRNGFIQLLKDDPSKSTVILTAAKVAAELSEK Atorney Docket No. 24-1880-WO

[0193] IRTLKESGSSYEQIAETVAKAVAKLVEKLKRNGVSEDEIALAVALI I SAVIQTLKES GSSYEVIAEIVARIVAEIVEALKRSGTSEDEIAEIVARVI SEVIRTLKESGSSYEVI AEI VARI VAEIVEALKRSGTSEDEIAKIVARVIAEVLRTLKESGSSEEVIKEI VARI ITEIKEALKRSGTSEDEIELITLMIEAALEIAKLKSSGSEYEEICEDVARRIAELVE KLKRDGTSAVEIAKIVAAI I SAVIAMLKASGSSYEVI CECVARIVAEIVEALKRSGT SAAI IALIVALVISEVIRTLKESGSSFEVILECVIRIVLEI IEALKRSGTSEQDVML IVMAVLLVVLATLQLSGS, wherein residues in parentheses are optional and may be present or may be deleted; wherein the polypeptide comprises an oligomerization domain and a Z domain that specifically binds fragment crystallizable (Fc) domains, and wherein the Z domain comprises a polypeptide sequence according to FNKEQQNAFYEILHLPNLNEEQRNAFIQSLKD (SEQ ID NO: 2), FNKDQQSAFYEILNMPNLNEALRNGFIQLLKD (SEQ ID NO: 3), FNKEQQNAFYEILHLPNLTEEQRNAFIQSLKD (SEQ ID NO: 489), or FNKEQQNAFYEILHLPNLTEELRNAFIQLLKD (SEQ ID NO: 490), or a variant at least 85%, at least 90%, or at least 95% identical thereto; or a polypeptide sequence a polypeptide sequence disclosed in Table 1A, or a variant at least 85%, at least 90%, or at least 95% identical thereto.

[0194] In embodiments, the polypeptide comprises an oligomerization domain and a fragment crystallizable (Fc)-binding domain that specifically binds fragment crystallizable (Fc) domains, wherein the polypeptide sequence comprises an amino acid substitution relative to the SEQ ID NO: 1 at position D6, S9, N16, Ml 7, N21, A23, G27, T40, orA50 or a combination thereof; or wherein the polypeptide sequence comprises amino acid substitutions relative to the SEQ ID NO: 1 at positions D6, S9, N16, M17, N21, A23, G27, T40, or A50.

[0195] In embodiments, the polypeptide sequence comprises, relative to the SEQ ID NO: 1, an amino acid substitution D6E, S9N, N16H, M17L, N21T, A23E, G27A, T40A, or A50N or a combination thereof; or wherein the polypeptide sequence comprises, relative to the SEQ ID NO: 1, amino acid substitutions D6E, S9N, N16H, M17L, N21T, A23E, G27A, T40A, A50N.

[0196] 04 component polypeptides modified to decrease internal disulfide bond formation

[0197] In a third aspect, the disclosure provides an 04 component polypeptide modified to decrease internal disulfide bond formation, comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 35-415 of SEQ ID NO: 202, in which selected cysteine (C) sites selected for possible removal are emphasized by underline and bold. Atorney Docket No. 24-1880-WO

[0198] 1 (MG) FNKDQQSA FYEILNMPNL NEALRNGFIQ LLKDDPSKST

[0199] 41 VILTAAKVAA ELSEKIRTLK ESGSSYEQIA ETVAKAVAKL

[0200] 81 VEKLKRNGVS EDEIALAVAL I I SAVIQTLK ESGSSYEVIA

[0201] 121 EIVARIVAEI VEALKRSGTS EDEIAEIVAR VI SEVIRTLK

[0202] 161 ESGSSYEVIA EIVARIVAEI VEALKRSGTS EDEIAKIVAR

[0203] 201 VIAEVLRTLK ESGSSEEVIK EIVARI ITEI KEALKRSGTS

[0204] 241 EDEIELITLM IEAALEIAKL KSSGSEYEEI CEDVARRIAE

[0205] 281 LVEKLKRDGT SAVEIAKIVA AI I SAVIAML KASGSSYEVI

[0206] 321 CECVARIVAE IVEALKRSGT SAAI IALIVA LVI SEVIRTL

[0207] 361 KESGSSFEVI LECVIRIVLE I IEALKRSGT SEQDVMLIVM

[0208] 401 AVLLVVLATL QLS (GSLEHHH HHH) wherein residues in parentheses are optional and may be present or may be deleted; wherein the polypeptide comprises an oligomerization domain and a fragment crystallizable (Fc)- binding domain that specifically binds fragment crystallizable (Fc) domains, wherein the polypeptide sequence comprises an amino acid substitution relative to SEQ ID NO:202 or 501 at position C271, C321, C323, or C373, or a combination thereof; or wherein the polypeptide sequence comprises amino acid substitutions relative to SEQ ID NO:202 or 501 at positions C271, C321, C323, and C373.

[0209] In embodiments, the polypeptide sequence comprises an amino acid substitution C271A, C321A, C323I, or C373I, or a combination thereof; or wherein the polypeptide sequence comprises, relative to SEQ ID NO:202 or 501, amino acid substitutions C271A, C321A, C323I, and C373I.

[0210] In embodiments, the Fc-binding domain is a Z domain, and wherein the Z domain comprises a polypeptide sequence according to FNKEQQNAFYEILHLPNLNEEQRNAFIQSLKD (SEQ ID NO: 2), FNKDQQSAFYEILNMPNLNEALRNGFIQLLKD (SEQ ID NO: 3), FNKEQQNAFYEILHLPNLTEEQRNAFIQSLKD (SEQ ID NO: 489), or FNKEQQNAFYEILHLPNLTEELRNAFIQLLKD (SEQ ID NO: 490), or a variant at least 85%, at least 90%, or at least 95% identical thereto; or a polypeptide sequence disclosed in Table 1A, or a variant at least 85%, at least 90%, or at least 95% identical thereto.

[0211] 04 component polypeptide modified for bioconjugation

[0212] In a fourth aspect, the disclosure provides an 04 component polypeptide modified for bioconjugation, comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 35- 415 of SEQ ID NO: 1 Atorney Docket No. 24-1880-WO

[0213] (MG) FNKDQQSAFYEILNMPNLNEALRNGFIQLLKDDPSKSTVILTAAKVAAELSEK

[0214] IRTLKESGSSYEQIAETVAKAVAKLVEKLKRNGVSEDEIALAVALI I SAVIQTLKES

[0215] GSSYEVIAEIVARIVAEIVEALKRSGTSEDEIAEIVARVI SEVIRTLKESGSSYEVI

[0216] AEI VARI VAEIVEALKRSGTSEDEIAKIVARVIAEVLRTLKESGSSEEVIKEI VARI ITEIKEALKRSGTSEDEIELITLMIEAALEIAKLKSSGSEYEEICEDVARRIAELVE KLKRDGTSAVEIAKIVAAI I SAVIAMLKASGSSYEVI CECVARIVAEIVEALKRSGT SAAI IALIVALVISEVIRTLKESGSSFEVILECVIRIVLEI IEALKRSGTSEQDVML IVMAVLLVVLATLQLSGS, wherein residues in parentheses are optional and may be present or may be deleted; wherein the polypeptide the polypeptide comprises an oligomerization domain and a fragment crystallizable (Fc)-binding domain that specifically binds fragment crystallizable (Fc) domains, wherein the polypeptide sequence comprises an amino acid substitution relative to SEQ ID NO: 1 at position E51, T58, E61, E67, Q68, K79, K83, R86, El 11, El 17, E167, K220, T228, E232, R236, E241, S265, E269, E280, K284, S 114, A224, R276, R337, or R387, or a combination thereof; and wherein optionally the polypeptide is bioconjugated to a steric exclusion molecule, optionally a polyethylene glycol (PEG), preferably PEG-5K.

[0217] In embodiments, the polypeptide sequence is bioconjugated with polyethylene glycol (PEG) of various sizes including but not limited to IK, 2K, 5K, 10K, or 20K.

[0218] In embodiments, the polypeptide sequence comprises, relative to SEQ ID NO: 1, an amino acid substitution E51C, T58C, E61C, E67C, Q68C, K79C, K83C, R86C, E111C, E117C, E167C, K220C, T228C, E232C, R236C, E241C, S265C, E269C, E280C, K284C, S114C, A224C, R276C, R337C, or R387C, or a combination thereof.

[0219] In embodiments, the polypeptide sequence comprises, relative to SEQ ID NO: 1, an amino acid substitution K79C.

[0220] In embodiments, the polypeptide sequence comprises an amino acid substitution relative to SEQ ID NO: 1 at position C271, C321, C323, or C373, or a combination thereof; or wherein the polypeptide sequence comprises amino acid substitutions relative to SEQ ID NO: lat positions C271, C321, C323, and C373.

[0221] In embodiments, the polypeptide sequence comprises, relative to SEQ ID NO: 1, an amino acid substitution C271A, C321A, C323I, or C373I, or a combination thereof; or wherein the polypeptide sequence comprises, relative to SEQ ID NO: 1, amino acid substitutions C271A, C321A, C323I, and C373I. Atorney Docket No. 24-1880-WO

[0222] In embodiments, the Fc-binding domain is a Z domain, and wherein the Z domain comprises a polypeptide sequence according to FNKEQQNAFYEILHLPNLNEEQRNAFIQSLKD (SEQ ID NO: 2), FNKDQQSAFYEILNMPNLNEALRNGFIQLLKD (SEQ ID NO: 3), FNKEQQNAFYEILHLPNLTEEQRNAFIQSLKD (SEQ ID NO: 489), or FNKEQQNAFYEILHLPNLTEELRNAFIQLLKD (SEQ ID NO: 490), or a variant at least 85%, at least 90%, or at least 95% identical thereto; or a polypeptide sequence disclosed in Table 1A, or a variant at least 85%, at least 90%, or at least 95% identical thereto.

[0223] 04 component polypeptides combining designed polypeptide segments

[0224] In a fifth aspect, the disclosure provides an 04 component polypeptide, wherein the polypeptide comprises a polypeptide sequence comprising a first segment disclosed in Table 1A or Table 6, a second segment disclosed in Table 7, and a third segment disclosed in Table 8; or for each segment, a variant at least 85%, at least 90%, or at least 95% identical thereto.

[0225] In embodiments, the polypeptide comprises a polypeptide sequence according to any one of SEQ ID NOs: 470-479; or a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical thereto.

[0226] In embodiments, the polypeptide comprises a polypeptide sequence according to SEQ ID NO: 470; or a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical thereto.

[0227] In a sixth aspect, the disclosure provides an AnglF polypeptide, comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 1-225 of SEQ ID NO: 6

[0228] KAELASEKPFRDCADVYQAGFNKSGIYTIYINNMPEPKKVFCNMDVNGGGWTVIQHR EDGSLDFQRGWKEYKMGFGNPSGEYWLGNEFI FAITSQRQYMLRIELMDWEGNRAYS QYDRFHIGNEKQNYRLYLKGHTGTAGKQSSLILHGADFSTKDADNDNCMCKCALMLT GGWWFDACGPSNLNGMFYTAGQNHGKLNGIKWHYFKGPSYSLRSTTMMIRPLDFGGS GGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDLSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK, wherein the polypeptide sequence comprises an amino acid substitution relative to SEQ ID NO:6 at position K23, M105, H148Q, F188, R214, or S215, or a combination thereof. Attorney Docket No. 24-1880-WO

[0229] In embodiments, the polypeptide sequence comprises an amino acid substitution relative to SEQ ID NO:6 at positions K23, M105, H148, F188, R214, and S215.

[0230] In embodiments, the polypeptide sequence comprises, relative to SEQ ID NO:6, an amino acid substitution K23T, M105E, H148Q, F188Y, R214K, or S215M, or a combination thereof.

[0231] In embodiments, the polypeptide sequence comprises, relative to SEQ ID NO:6, amino acid substitutions K23T, M105E, H148Q, F188Y, R214K, and S215M.

[0232] In embodiments, the polypeptide comprises a polypeptide linker and a fragment crystallizable (Fc) polypeptide, wherein optionally the polypeptide sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 1-463 of SEQ ID NO: 6.An illustrative modified AnglF polypeptide may have the polypeptide sequence below (SEQ ID NO: 203, with six amino acid substitutions emphasized:

[0233] 1 KAELASEKPF RDCADVYQAG FNKSGIYTIY INNMPEPKKV 41 FCNMDVNGGG WTVIQHREDG SLDFQRGWKE YKMGFGNPSG

[0234] 81 EYWLGNEFI F AITSQRQYML RIELMDWEGN RAYSQYDRFH

[0235] 121 IGNEKQNYRL YLKGHTGTAG KQSSLILHGA DFSTKDADND 161 NCMCKCALML TGGWWFDACG PSNLNGMFYT AGQNHGKLNG 201 IKWHYFKGPS YSLKMTTMMI RPLDF

[0236] Table 2A: Sequence and building blocks used for AbCs of other geometries (residues in parentheses are optional, and may be present or may be deleted) Atorney Docket No. 24-1880-WO Attorney Docket No. 24-1880-WO

[0237] Table 2B: Sequences of AbCs of different geometries (residues in parentheses are optional, and may be present or may be deleted) Atorney Docket No. 24-1880-WO

[0238] D2 component polypeptides

[0239] The disclosure provides non-limiting examples of nanostructure comprising various D2 component polypeptides. In embodiments, the second polypeptide comprises an amino acid sequence disclosed in Table 2B or a variant thereof. The variant may be at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical thereto.

[0240] The disclosure provides a D2 component polypeptide modified to include a Z domain, comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 56-255 of SEQ ID NO: 480:

[0241] (M) SDEEERNELIQRIKEAAQRAREAAERTGDPRVRELARELQKIAQNAFYEVLHDP SSSEVNEALKAVVKAIELAVRALEEAEKTGDPEVRELAREVVRLAVEVAQATQAGEN DTLRKVAERALRLAKEAAKRGDAKAAKQAAKIAKLAAANAGDEDVLKKVELVRLAIE LVEIVVENAKRKGDDDKEAAEAALAAFRIVLAAAQLAGIASLEVLELALRLIKEVVE NAQREGYDIAVAAIAAAVAFAVVAVAAAAADITSSEVLELAIRLIKEVVENAQREGY VI LLAALAAAAAFVVVAAAAKRAGI TSSETLKRAI EEI RKRVEEAQREGNDI SEAAR QAAEEFRKKAEELK (GSWLEHHHHHH) , wherein residues in parentheses are optional, and may be present or may be deleted and wherein the polypeptide comprises an oligomerization domain and a Z domain that specifically binds fragment crystallizable (Fc) domains.

[0242] In embodiments, the D2 component polypeptide comprising the Z domain comprises a polypeptide sequence according to (M)SDEEERNELIKRIREAAQRAREAAERTGDPRVRELARELARIAQIAFYLVLHDP (SEQ ID NO: 618); or Atorney Docket No. 24-1880-WO

[0243] (M)SDEEERNELIKRIREAAQRAREAAERTGDPRVRELARELAKLAQIAFYLVLHDP (SEQ ID NO: 619); or a variant at least 85%, at least 90%, or at least 95% identical thereto.

[0244] In embodiments, the D2 component polypeptide comprising the Z domain comprises one or more amino acid substitutions at positions K12, R15, A41, R42, 146, or L50, or a combination thereof, relative to SEQ ID NO: 618.

[0245] In embodiments, the D2 component polypeptide comprising the Z domain comprises one or more amino acid substitutions K12Q, R15K, A41Q, R42K, I46N, or L50E, or a combination thereof, relative to SEQ ID NO: 618.

[0246] T4 component polypeptides

[0247] The disclosure provides non-limiting examples of nanostructure comprising various T4 component polypeptides. In embodiments, the second polypeptide comprises an amino acid sequence disclosed in Table 2B or a variant thereof. The variant may be at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical thereto.

[0248] Thr disclosure provides a T4 component polypeptide comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 36-305 of SEQ ID NO: 481:

[0249] (MG) FNKEQQNAFYLILHLPNLTEEQRNAFIQSLKDDPSKSAVVAGEAAIENARNAL KKGSPETAREAVRLALELVQQAERQARKTGSTERLIAAAKLAIEVARVALKVGSPET AREAVRTALELVQELIRQARKTGSKEVLEEAAKLALEVAKVAAEVGSPETAARAVAT AVEALKEAGASEDEIAEIVARVISEVIRILKESGSEYKVIARAVARIVAEIVEALKR SGTSEDEIAEIVARVISEVIRTLKESGSDYLI IAVIVAI IVAEIVEALKRSGTSEDE IAEIVARVISEVIRTLKESGSSYEVIKEIVQI IVLAI ILALMKSGTEVEEILLILLR VKTEVRRTLKES (GSWLEHHHHHH) , wherein residues in parentheses are optional, and may be present or may be deleted; wherein the polypeptide comprises an oligomerization domain and a Z domain that specifically binds fragment crystallizable (Fc) domains.

[0250] In embodiments, the T4 component polypeptide comprising the Z domain comprises an amino acid sequence according to FNKSQQSAFYLILNMPNLNEAQRNGFIQSLKD (SEQ ID NO: 616), or a variant at least 85%, at least 90%, or at least 95% identical thereto. Atorney Docket No. 24-1880-WO

[0251] In embodiments, the T4 component polypeptide comprising the Z domain comprises one or more amino acid substitutions D6, S9, N16, Ml 7, N21, A23, G27, T40, or A50, or a combination thereof, relative to SEQ ID NO: 498.

[0252] In embodiments, the T4 component polypeptide comprising the Z domain comprises an amino acid substitution D6E, S9N, N16H, M17L, N21T, A23E, G27A, T40A, A50N, or a combination thereof, relative to SEQ ID NO: 498.

[0253] T8 Component Polypeptides

[0254] The disclosure provides non-limiting examples of nanostructure comprising various T8 component polypeptides. In embodiments, the second polypeptide comprises an amino acid sequence disclosed in Table 2B or a variant thereof. The variant may be at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical thereto.

[0255] The disclosure provides a T8 component polypeptide comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 36-266 of SEQ ID NO: 482:

[0256] (MG) FNKEQQNAFYEVLHLPNLTEEQRNAFIQSLKDDPSQSLKILIKAAAGGDSELE EVAKRI IKELAEQGRSEKEAAKEAAELIQRITRAAGGNSDLIELAVRIVKILEEQGR SPSEAAKEAVEAIEAIVRAAGGDSEAIKVAAEIAKTI ITQKESGSEYKEICRTVARI VAEIVEKLKRNGASEDEIAEIVAAI IAAVILTLKLSGSDYLI I CVCVAI I VAEI VEA LKRSGTSEDEIAEIVARVISAVIRVLKESGSSYEVIKECVQI IVLAI ILALMKSGTE VEEILLILLRVKTEVRRTLKES (GSWLEHHHHHH) ; wherein residues in parentheses are optional, and may be present or may be deleted; wherein the polypeptide comprises an oligomerization domain and a Z domain that specifically binds fragment crystallizable (Fc) domains.

[0257] In embodiments, the T8 component polypeptide comprising the Z domain comprises an amino acid sequence according to FNKDQQSAFYEVLNMPNLNEAQRNGFIQSLKD (SEQ ID NO: 617) or a variant at least 85%, at least 90%, or at least 95% identical thereto.

[0258] In embodiments, the T8 component polypeptide comprising the Z domain comprises one or more amino acid substitutions D6, S9, N16, Ml 7, N21, A23, G27, T40, or A50, or a combination thereof, relative to SEQ ID NO: 498. Atorney Docket No. 24-1880-WO

[0259] In embodiments, the T8 component polypeptide comprising the Z domain comprises an amino acid substitution D6E, S9N, N16H, M17L, N21T, A23E, G27A, T40L, or A50G, or a combination thereof, relative to SEQ ID NO: 498.

[0260] 15 Component Polypeptides

[0261] The disclosure provides non-limiting examples of nanostructure comprising various 15 component polypeptides. In embodiments, the second polypeptide comprises an amino acid sequence disclosed in Table 2B or a variant thereof. The variant may be at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical thereto.

[0262] The disclosure provides an 15 component polypeptide comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 56-227 of SEQ ID NO: 483:

[0263] (M) SDEEERNELIQRIKEAAQRAREAAERTGDPRVRELARELQKLAQNAFYEVLHDP

[0264] S S SDVNEALKL I VEAI EAAVRALEAAERAGDPELREDAREAVRLAVEAAQEVQRNPS SSTANLLLKAIVALAEALAAAANGDKEKFKKAAESALEIAKRVVEVASKEGDPEAVL EAAKVALRVAELAAKNGDKEVFKKAAESALEVAKRLVEVASKEGDPELVLEAAKVAL RVAELAAKNGDKEVFQKAAASAVEVALRLTEVASKEGDSELETEAAKVITRVRELAS KQGDAAVAILAETAEVKLEIEESKKRPQSESAKNLILIMQLLINQIRLLVLQIRMLD EQRQE (GSWLEHHHHHH) wherein residues in parentheses are optional, and may be present or may be deleted; wherein the polypeptide comprises an oligomerization domain and a Z domain that specifically binds fragment crystallizable (Fc) domains.

[0265] The disclosure provides an 15 component polypeptide comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 56-227 of SEQ ID NO: 484:

[0266] (M) SDEEERNELIQRIKEAAQRAREAAERTGDPRVRELARELQKLAQNAFYEVLHNP ESEGTLRALDHI IRAIDAAVEAIEAALESGDEEARERAREAVRQAVEAAQRVLENPE SEAVLKLLEAVVALAEALAAAMRGDKEEFKKAAKKAIKIAREVVKLAKKQGNAELVL KAAEIALEVARLAAEKGDKEIFKEAAKAAIEIAREWKLAKKQGNAELVLKAAEIAL EVARLAAEKGDEEIFREAAKAAIEIANEVKKLAKKQGNEELVKKALEI IKEVGKLAK EKGDRSSKTLAESELIKFEIEELEKKPHSLETKALILISKLLYASIVLLSEI IDELY KI LKK (GSWLEHHHHHH) ; Atorney Docket No. 24-1880-WO wherein residues in parentheses are optional, and may be present or may be deleted; wherein the polypeptide comprises an oligomerization domain and a Z domain that specifically binds fragment crystallizable (Fc) domains.

[0267] In embodiments, the 15 component polypeptide comprising the Z domain further comprises an amino acid sequence according to (M)SDEEERNELIKRIREAAQRAREAAERTGDPRVRELARELARLAQRAFYLVLHDP (SEQ ID NO: 620), or a variant at least 85%, at least 90%, or at least 95% identical thereto.

[0268] In embodiments, the 15 component polypeptide comprising the Z domain further comprises one or more amino acid substitutions at positions K12, R15, A41, R42, R46, or L50, or a combination thereof, relative to SEQ ID NO: 620.

[0269] In embodiments, the 15 component polypeptide comprising the Z domain further comprises one or more amino acid substitutions K12Q, R15K, A41Q, R42K, R46N, or L50E, or a combination thereof, relative to SEQ ID NO: 620.

[0270] Antibody Cage (AbC) Nanostructures

[0271] In an eighth aspect, the disclosure provides an antibody cage (AbC) nanostructure, comprising a plurality of first polypeptides, each first polypeptide comprising a fragment crystallizable (Fc) polypeptide that forms an Fc dimer; and a plurality of second polypeptides, wherein each second polypeptide is a polypeptide as described herein.

[0272] In embodiments, the plurality of first polypeptides multimerize to form a plurality of first components and the plurality of second polypeptides multimerize to form a plurality of second components; and wherein the first components and the second components assemble by direct interaction of Fc dimers of the first polypeptides with Fc-binding domains of the second polypeptides.

[0273] In embodiments, the first polypeptides comprise an antibody or an antigen-binding domain thereof, a polypeptide linker, and the Fc polypeptide.

[0274] In embodiments, the AbC nanostructures can comprise an d2, o4, t32, or i52 component polypeptide.

[0275] In embodiments, the antibody is selected from Herceptin, which is a humanized anti-HER2 monoclonal antibody, Rituxan, which is a chimeric anti-CD20 monoclonal antibody, Panorex or (edrecolomab, which is a murine IgG2a antibody recognizing EpCAM, BEC2, which is Atorney Docket No. 24-1880-WO murine IgG antibody that recognizes GD3 ganglioside, IMC-C225, which is a chimeric IgG antibody against EGFR, Vitaxin, which is a humanized antibody targeting vitronectin receptor, Smart Ml 95 which is a humanized anti-CD33 IgG antibody, LymphoCide which is a humanized IgG antibody targeting lymphocytes, Smart ID 10 which is a humanized antibody that binds to an HLA-DR determinant, Oncolym which is a murine antibody targeting HLA-DR10, Allomune which is a humanized anti-CD2 mAb, an anti-VEGF antibody, CEAcide which is a humanized anti-CEA antibody, IMC-1C11 which is an anti- KDR chimeric antibody, Cetuximab, which is an anti-EGFR chimeric antibody, Lob 7 / 6, Lucatumumab, Dacetuzumab, Selicrelumab, Bleselumab, Urelumab, Utomilumab, Drozitumab, scTRAIL-Fc, KMTR2, 16E2, Aplitabart, which is an multivalent IgM DR5 agonist antibody, optionally conatumumab, which is a human agonist antibody to death receptor 5 (DR5).

[0276] In embodiments, the first polypeptides comprise a binding agent.

[0277] In embodiments, the binding agent is an AnglF polypeptide, wherein optionally the first polypeptide is any AnglF polypeptide described herein.

[0278] In embodiments, the binding agent is Ang2, Ang3, Ang4 polypeptides or engineered molecules Bow-Angl, Comp-Angl, ABTAA antibody and vasculotide polypeptide that binds to Tie2 receptor.

[0279] In embodiments, the binding agent is transferrin, EGF, bombesin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, TGF-a, TGF-P, vaccinia growth factor (VGF), insulin and insulin-like growth factors I and II, somatostatin, lectins and apoprotein from low density lipoprotein.

[0280] Polynucleotides and Delivery Vehicles

[0281] In a further aspect, the disclosure provides a polynucleotide encoding any polypeptide or nanostructure described herein. The polynucleotide may comprise single stranded or double stranded RNA or DNA in genomic or cDNA form, or DNA-RNA hybrids, each of which may include chemically or biochemically modified, non-natural, or derivatized nucleotide bases. Such polynucleotides may comprise additional residues useful for promoting expression and / or purification of the encoded polypeptide or nanoparticle, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization Atorney Docket No. 24-1880-WO signals. It will be apparent to those of skill in the art, based on the teachings herein, what polynucleotide sequences will encode the polypeptides or nanoparticles of the disclosure. The polynucleotide may comprise an expression vector comprising the polynucleotide encoding the polypeptide or nanoparticle, operatively linked to a suitable control sequence. “Expression vector” includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product, such as a promoter. “Control sequences” operably linked to the nucleic acid sequences of the disclosure are nucleic acid sequences capable of effecting the expression of the polynucleotides. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the polynucleotide and the promoter sequence can still be considered “operably linked” to the polynucleotide. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type, including but not limited plasmid and viral-based expression vectors. The control sequence used to drive expression of the disclosed polynucleotides may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive). The expression vector must be replicable in the host organisms either as an episome or by integration into host chromosomal DNA.

[0282] In a further aspect, the disclosure provides a delivery vehicle, comprising any polynucleotide described herein, optionally a viral vector or lipid nanoparticle.

[0283] Pharmaceutical Compositions

[0284] The disclosure also provides pharmaceutical compositions. Such pharmaceutical compositions can be used for generating an immune response against an infectious disease in a subject. The pharmaceutical compositions of the disclosure may include a pharmaceutically acceptable carrier. A thorough discussion of such carriers is available in Chapter 30 of Remington: The Science and Practice of Pharmacy (23rd ed., 2021). Atorney Docket No. 24-1880-WO

[0285] In some embodiments, the pharmaceutical composition can also include excipients and / or additives. Examples of these are surfactants, stabilizers, complexing agents, antioxidants, or preservatives which prolong the duration of use of the finished pharmaceutical formulation, flavorings, vitamins, or other additives known in the art. Complexing agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA) or a salt thereof, such as the disodium salt, citric acid, nitrilotriacetic acid and the salts thereof. In some embodiments, preservatives include, but are not limited to, those that protect the solution from contamination with pathogenic particles, including benzalkonium chloride or benzoic acid, or benzoates such as sodium benzoate. Antioxidants include, but are not limited to, vitamins, provitamins, ascorbic acid, vitamin E, salts or esters thereof.

[0286] Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, faty acid esters, hydroxymethyl cellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, weting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present disclosure.

[0287] In some embodiments, one or more tonicity agents may be added to provide the desired ionic strength. Tonicity agents for use herein include those which display no or only negligible pharmacological activity after administration. Both inorganic and organic tonicity adjusting agents may be used.

[0288] In a further aspect, the disclosure provides a pharmaceutical composition, comprising any polypeptide, nanostructure, polynucleotide, or delivery vehicle disclosed herein, and a pharmaceutically acceptable carrier.

[0289] Host Cells and Methods of Making

[0290] In a further aspect, the disclosure provides a host cell suitable for expression of any polynucleotide, polypeptide or nanostructure described herein; and / or comprising any polynucleotide described herein. The host cells can be either prokaryotic or eukaryotic. The Atorney Docket No. 24-1880-WO cells can be transiently or stably engineered to incorporate, for example, an expression vector of the disclosure, using techniques including but not limited to bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection.

[0291] In a further aspect, the disclosure provides a method of making a polypeptide or nanostructure, comprising culturing any host cell described herein under conditions suitable for expression of the polypeptide or nanostructure

[0292] Methods of Treating

[0293] In a further aspect, the disclosure provides a method of treating a disease or disorder in a subject suffering from or at risk thereof, the method comprising administering to the subject any polypeptide, nanostructure, polynucleotide, delivery vehicle, or pharmaceutical composition described herein.

[0294] In embodiments, the disease or disorder is a viral infection, optionally influenza.

[0295] In embodiments, the disease or disorder is a cancer, wherein optionally the nanostructure comprises an antigen-binding fragment of conatumumab or another DR5 -binding protein, and a cancer is a cancer responsive to the conatumumab or other DR5 -binding protein nanostructure.

[0296] In embodiments, the disease or disorder is condition or dysfunction associated with endothelial function, wherein optionally the nanostructure comprises an antigen-binding fragment of Ang 1 or another Tie2-binding protein, and endothelial dysfunction is responsive to the Angl or other Tie2-binding protein nanostructure.

[0297] EXAMPLES

[0298] EXAMPLE 1: PEGYLATION SITE SELECTION AND TESTING

[0299] This example explores selection of an optimum PEGylation site and evaluates its biological stability and biological activity.

[0300] A series of site-specific mutations (amino acid substitutions) were characterized for half-life extension through linear methoxy-PEG conjugation (at sites identified in Table 2) and / or internal native disulfide removal and / or engineering by amino acid substitutions at C271A, C321A, C323I, C373I relative to SEQ ID NO: 1. A series of cysteines on different Attorney Docket No. 24-1880-WO surface-exposed sites of the 04 component as described in SEQ ID NO: 204 were engineered based on structural analysis and 25 different sites and conjugated PEG molecules of 6 different chain lengths using maleimide chemistry to single cysteine point mutants at each site were identified (Table 3). These substitutions can be combined so that the protein includes one or more of the mutations (amino acid substitutions) listed in Table 3. The residue K79C in the JI region as provided the optimal site for conjugation of a 5 kDa PEG molecule to increase stability in serum without compromising in vitro activity (Fig. 5A, 6A, and 6B).

[0301] The original cage component benefited from PEGylation, but its overall activity was almost always impacted whereas the optimized and stabilized variants could be conjugated with high molecular weight PEG without an impact on activity (Figs. 4 and 5). After production and characterization, several mutants led to better yield, solubility, monodispersity, serum stability, and were also amenable to cross-linking to the Fc. Crosslinking has initially been evaluated using a homobifiinctional bis-maleimide PEG crosslinker (bMP) with different sized PEG (3.4kDa, 5kDa, and lOkDa). In summary, all sites evaluated for linear PEG conjugation were also evaluated for bMP -based crosslinking. After thorough characterization, 5Meq of DTT at site R86 using a 5 and 10 kDa bMP showed monodisperse AbC that cross-linked to native hinge-region cysteines and retained signaling activity. Consequently, these reinforced crosslinked AbCs exhibited over 800-fold improved blood half-life (Fig. 7).

[0302] Table 3: Polyethylene glycol (PEG) site modifications. Atorney Docket No. 24-1880-WO

[0303] EXAMPLE 2: D TO Z DOMAIN OF PROTEIN A TO INCREASE FC BINDING AND / OR REDUCE OFF- TARGET FAB BINDING

[0304] This example explores modulation of Fc binding capacity upon a switch from domain D to Z of protein A of AbCs (Table 4). The original AbCs were generated using WORMS to connect a series of designed symmetric oligomers to an antibody binding interface derived from protein A (PDB IDEE). Upon closer inspection, IDEE was derived from a protein A fragment (domain D) that also binds to Fab regions of antibodies with substantial binding affinity, this may contribute to instability because multiple competing interactions could occur at the intended interface with the Fc region. Consequently, new sequences that had higher specificity for only one site on the antibody were pursued. A number of variants by inserting amino acids from the interface region of our designed protein with mutations from a protein A fragment that held greater specificity for the Fc region (domain Z, PDB 5U4Y, SEQ ID NO: 2) were generated (Fig. 8). Simultaneously, the native disulfide bond cysteine residues in the original o42. 1 (SEQ ID NO: 1) were engineered to be replaced with the hydrophobic residues alanine (“A”) and isoleucine (“I”) predicted to provide favorable core packing (SEQ ID NO: 204). This was done in order to prime the entire polypeptide for chemical conjugation / PEGylation through insertion of the single site cysteine mutations described in Example 1, and limit risk for off-target conjugation to native disulfide cysteines. The Z mutations are applied to O4_AI_C6His to generate O4_AI_ZN23T_C6His are D6E, S9N, N16H, M17L, N21T, A23E, G27A, T40A, and A50N.

[0305] The sequence for O4_AI_C6His (SEQ ID NO: 230) is as follows: (residues in parentheses are optional, and may be present or may be deleted)

[0306] (MG)FNKDQQSAFYEILNMPNLNEALRNGFIQLLKDDPSKSTVILTAAKVAAEL SEKIRTLKESGSSYEQIAETVAKAVAKLVEKLKRNGVSEDEIALAVALIISAVIQTLKES GSSYEVIAEIVARIVAEIVEALKRSGTSEDEIAEIVARVISEVIRTLKESGSSYEVIAEIVA RIVAEIVEALKRSGTSEDEIAKIVARVIAEVLRTLKESGSSEEVIKEIVARIITEIKEALKR SGTSEDEIELITLMIEAALEIAKLKSSGSEYEEIAEDVARRIAELVEKLKRDGTSAVEIA KIVAAIISAVIAMLKASGSSYEVIAEIVARIVAEIVEALKRSGTSAAIIALIVALVISEVIRT Atorney Docket No. 24-1880-WO

[0307] LKESGSSFEVILEIVIRIVLEIIEALKRSGTSEQDVMLIVMAVLLWLATLQL(SGSWLEH HHHHH).

[0308] Table 4: Sequences for Domains of Protein A

[0309] A serum stability assay was developed in order to understand the relationship between redesign and serum stability. Briefly, fluorescently-labeled AbCs with full antibody were incubated in both 100% human serum and also human serum depleted of Ig antibodies for 24h at 25°C and 37°C. The intact AbC were separated from free fluorescent Fc by sizeexclusion chromatography in order to determine how much the AbC disassembles over time. The swap from D- to Z-domain mutations at the interface reduced serum disassembly by about 60% (Fig. 9). To further improve serum stability of the interface, a combo sitesaturation mutagenesis (SSM) was performed at select sites on the designed protein interface and displayed these on the surface of yeast using the Z domain as a scaffold. The yeast were then mixed with fluorescent antibodies, washed, and incubated in 100% human serum for 72 hours at 37°C. After sorting and sequencing the retained binding population, a series of mutations that improved serum stability over the three days were identified (Figs. 10A, 10B), and a combo library of these mutations was ordered, which comprised 192 different variants where in the J 1 region, AA position at 2 can be N or Q, AA at position 3 can be K or R, AA at position 5 can be Q or M, AA at position 11 can be E or N, AA at position 12 can be I or V, Attorney Docket No. 24-1880-WO

[0310] AA at position 19 can be N or S, and AA at position 25 can be A, N, or F (Tables 1 and 5). All super Z sequences were assembled with 2J0 and 10 for a full length 04 to assess each combined clone’s stability. These were subsequently evaluated to assess which mutations produced solubly, and top stabilized candidates were selected using a serum stability assay.

[0311] Table 5: Super Z Sequences Atorney Docket No. 24-1880-WO Atorney Docket No. 24-1880-WO Atorney Docket No. 24-1880-WO Atorney Docket No. 24-1880-WO Atorney Docket No. 24-1880-WO Atorney Docket No. 24-1880-WO Atorney Docket No. 24-1880-WO Atorney Docket No. 24-1880-WO Atorney Docket No. 24-1880-WO Atorney Docket No. 24-1880-WO Atorney Docket No. 24-1880-WO Atorney Docket No. 24-1880-WO Atorney Docket No. 24-1880-WO

[0312] EXAMPLE 3: COMPUTATIONAL REDESIGN AND SCREENING OF STRUCTURAL DOMAINS OF 04

[0313] This example describes computational redesign, screening, and product optimization of 04 AbCs. Investigations into the possible factors contributing to the serum instability observed in the original AbCs led to the discovery that multiple structural regions likely underwent significant conformational changes in order to assemble and adopt the AbC architecture in its designed state. Specifically, cryo-electron microscopy (cryo-EM) and small-angle light scatering (SAXS) experiments revealed multiple alpha helices were severely displaced in order for the tetrahedral designed component (04) to assemble with Fc into its corresponding octahedral AbC (Fig. 11). By redesigning the 04 component in its unbound state towards the designed configuration in its assembled octahedral AbC state, improved thermodynamics of AbC assembly and consequently its serum stability was expected. This was performed by addressing three distinct designed regions of the 04 component, which are termed junction 1 (JI), junction 2 (J2), and interface (I). Input models for the interface region were informed by multiple cryo-EM structures of the tetrameric building block, whereas junction regions 1 and 2 were informed by the single cryo-EM structure of the 04 component assembled in the AbC state. Each region was subjected to two unique RosettaScripts protocols (PlaceMotifs and FastDesign) with multiple options enabled. Favorable Roseta metrics were scored on the total pool of output prior to down-selection to a final set of redesigned variants for experimental characterization. Gene fragments encoding redesigned sequences for each of the distinct structural regions of the 04 component (JI, J2, and I) were synthesized and cloned through a Golden Gate protocol (Figs. 1-3, 12A, Tables 6-9). Full genes were assembled combinatorially across the JI, J2, and I regions (in the format JI -J2-I) through robotic liquid handling in order to sample a sufficiently large number of output sequences, while also increasing overall Attorney Docket No. 24-1880-WO sequence diversity by multiple orders of magnitude. Each variant was evaluated based on yield, monodispersity, assembly competency, and serum stability. Over 300 components successfully formed intact AbCs with fluorescently-labeled Fc. The best behaved variants that spanned a range of improved serum stabilities were identified (TableslOA and 10B). Ultimately, a series of components that exhibited nearly 100% intact AbC after 24h, and >80% after 72h were identified (Figs. 12B, 13 and 14, Table 10).

[0314] Table 6: Sequences for Junction 1 (JI) Atorney Docket No. 24-1880-WO

[0315] Table 7: Sequences for Junction 2 (J2) Atorney Docket No. 24-1880-WO

[0316] Table 8: Sequences for Interface (I) Attorney Docket No. 24-1880-WO

[0317] Table 9A: Sequences for Al (Alanine & Isoleucine pairwise mutations made to the cysteine residues) & Z Domain Mutations (residues in parentheses are optional, and may be present or may be deleted) Atorney Docket No. 24-1880-WO Attorney Docket No. 24-1880-WO

[0318] Table 9B: Sequences for Al (Alanine & Isoleucine pairwise mutations made to the cysteine residues) & Z Domain Mutations (residues in parentheses are optional, and may be present or may be deleted) Attorney Docket No. 24-1880-WO

[0319] Table 10A: Stable designs in order of J1-J2-I assessed by the serum exchange assay. Atorney Docket No. 24-1880-WO Atorney Docket No. 24-1880-WO

[0320] Table 1OB: List and sequences of exemplary designs based on serum exchange assay. Attorney Docket No. 24-1880-WO

[0321] Example 4: SERUM-STABLE ABCS EXHIBIT SIGNIFICANTLY IMPROVED BIODISTRIBUTION

[0322] AND PHARMACOKINETICS IN VIVO

[0323] This example explores and evaluates biodistribution (BioD) and in vivo pharmacokinetics (PK) of serum-stable AbCs.

[0324] To assess biodistribution of the original 04 compared to one of the highest yield and serum stable redesigned variant (Z49-6-11AL-14AL) formed as AbCs with fluorescently labeled Fc, 7-week-old Balb / C female mice retro-orbitally injected (RO) with 2 mg / kg of AbC. After 4 hours, the mice were sacrificed and imaged with an in vivo imaging system (IVIS). The original 04 exhibited poor distribution as a large percentage of the injected dose remained at the site of injection (FIGs. 15 and 16). However, the optimized variant could freely distribute post-RO administration and traffic to the liver for clearance (FIGs. 15 and 16). Atorney Docket No. 24-1880-WO

[0325] Comparison of the blood half-life of the 5k PEG conjugated K79C variant against the original 04 component revealed that the original exhibited a half-life of <1 minute while the optimized exhibited that of approximately 5.5 hours (over a 1500X increase in blood half-life). Injecting the optimized variant formed as an AbC with the AnglF6-Fc protein (as described in Example 7) targeting the endothelial receptor Tie2, showed that the particle localized most heavily to the lung and was retained out to at least 24 hours (FIG. 17) . However, it was cleared from blood circulation almost immediately, indicating a marked difference and completely new PK / BioD profde of AbC compared to Fc component alone. This data shows that improving the stability of the 04 AbC was critical in enabling the AbC to become bioavailable and efficiently localize to the tissue / region of highest target expression (FIG. 17). This capability opens up a wide range of new therapeutic opportunities by targeting AbCs to the lung but also to other tissues simply by virtue of swapping out different antibodies.

[0326] Example 5: GENERATION OF OTHER ABC GEOMETRIES INCORPORATING Z DO AIN MUTATIONS

[0327] This example describes generation of AbCs with different geometries incorporating Z domain mutations and assess their stability. To extend the stability improvements and Fc- binding specificity made to the original o42.1 through the Z domain mutations that yielded O4_AI_Z and related sequences, structural alignment of the N-terminal regions of the following AbC components from Divine et al. 2021, Science, 373(6537): 10.1126 / science.abd9994 (proA domain was performed. Next, Z mutations used to convert O4_AI (SEQ ID NO: 204) to O4_AI_ZN23T (SEQ ID NO: 463) were inserted as described in Example 2 in the structurally homologous positions. This yielded the following Z variants of the following designs d2.4 to D2_ZN23T (SEQ ID NO: 480), t3.4 to T4AI_rl_ZN23T (SEQ ID NO: 481), t3.8 to T8_ZN23T (SEQ ID NO: 482), and i52.3 to I5_ZN23T (SEQ ID NO: 483). T4AI_rl_N23T also had its internal disulfides converted to alanine ("A") and isoleucine ("I") similarly as what was done with o42.1 to O4_AI. I5_mpnn2_ZN23T (SEQ ID NO: 484) was the result of one additional round of ProteinMPNN sequence design performed over the rest of the non-proA backbone in order to improve stability, yield, and / or activity. Dauparas et al. 2022 Science, 378(6615): 10.1126 / science.add2187. Atorney Docket No. 24-1880-WO

[0328] Example 6: REDESIGNED 04 ABC INCORPORATING CONATUMUMAB

[0329] This example explores biological activity measured as the ability to induce cancer cell death in vitro using redesigned 04 AbC incorporated with the anti-Death Receptor 5 (DR5) targeting antibody, Conatumumab (also known as and abbreviated “AMG655”).

[0330] An AbC incorporated with the DR5 targeting antibody is a strong agonist capable of inducing potent cancer cell death in vitro and the AbCs with higher valencies that were able to bind a higher number of receptors tended to increase potency of cell death. This was further evaluated by measured cell death in 13 other cancer cell lines in vitro. The study showed that activity was largely conserved across cancer types, although degree of sensitivity correlated with documented sensitivities of the cell lines to the DR5 and caspase 3-mediated apoptotic pathway. In all cell lines, both original and optimized O4-AMG655 AbCs outperformed TRAIL and AMG655 in killing activity (Figs. 19 and 20).

[0331] Next, 7-week-old CD1 nude mice were dosed with 5 mg / kg of O4-original, 04- optimized, a combination treatment of AMG655 and the former clinical candidate (recombinant TRAIL) and found that both AbCs outperformed the antibody-ligand combo and elicited sustained tumor remission even after the study had ended. Comparison of the original and serum-stable optimized 04 in both a cell killing and xenograft tumor study showed that both original and serum -optimized molecules performed effectively (Figs. 21, 22, and 23) and this could be a result of the nude mice possessing the human DR5 target only at the target tumor site (only non-cross-reactive murine DR5 present in other healthy tissue), combined with the absence of an adaptive immune system and high levels endogenous IgG in the nude mice to impact circulating AbC stability. The in vitro and in vivo data corroborated that AMG655-AbCs induce caspase-mediated cell death (Figs. 24-27), and overall magnitude of cell killing was found to be similar between both original and optimized O4-AMG655 AbCs.

[0332] To understand the Mechanism of Action (Mo A) of AbCs derived from AMG655 on cancer cells, cancer cells were treated with several redesigned AbCs and cell viability was measured. The decrease in viable cell count may result from inducing cell death (cytotoxic effect). AMG655, a DR5 -targeting antibody from Amgen, did not yield significant benefits in clinical studies. The expression profile of DR5 was confirmed across multiple cell lines using flow cytometry (Fig. 28). To confirm the effectiveness of the redesigned O4AI-AMG655, G4AI-AMG000 was used as a negative control. Treating with Dulanermin / TRAIL and AMG655 together was a positive control. Among several redesigned O4AI-AMG655 variants, Atorney Docket No. 24-1880-WO namely O4AI_Z_N23T (SEQ ID NO: 463), T8_W_Z_N23T (SEQ ID NO: 482), I5_mpnn2- 2_Z_N23T (SEQ ID NO: 484), and 6_K79-5K-ll-0 (SEQ ID NO: X), we investigated their effects on cancer cells and immune cells. These were determined based on the number of composed antibodies. O4AI_Z_N23T consists of 12, T8_W_Z_N23T has 4, I5_mpnn2- 2_Z_N23T has 30, and 6_K79-5K-ll-0 is composed of 12 antibodies. As seen in Figs. 29 and 30, they are highly effective in reducing cell viability compared to Dulanermin / TRAIL + AMG655 (by comparing IC50 values). The findings revealed apoptotic cell death triggered by Dulanermin / TRAIL + AMG655 (Figs. 25). Since the CellTiter-Glo™ Reagent was used to measure cellular ATP levels and this reagent contains inhibitors that block the activity of ATPases thus preventing the degradation of ATP after treatment with AbC, a rapid shutdown of ATP upon AbC treatment indicated a decrease in metabolism due to mitochondrial dysfunction within the cell. Changes in cellular metabolism can influence pathways leading to cell death, but metabolic shutdown alone does not always induce apoptosis. Recently, the impact of metabolic changes on novel strategies to enhance cancer immunotherapy has become apparent. This is because metabolic reprogramming has the potential to directly affect tumor cells and regulate immune cell differentiation and activation. O4AI-AMG655 showed significant differences in cell viability among diverse mutant cancer cell lines (Fig. 19). However, the challenge in cancer treatment lies in the unpredictable mutations that can lead to drug resistance and recurrence, which remain significant hurdles to overcome. The redesigned AbCs used here were able to effectively reduce cell survival across various mutant conditions.

[0333] Calreticulin (CRT), which is normally located in the endoplasmic reticulum (ER) lumen, becomes exposed during the process where it is displayed on the surface of stressed and dying cancer cells. This exposure facilitates the uptake of these cells by macrophages or dendritic cells and allows for the presentation of tumor-associated antigens to T lymphocytes, triggering an anticancer immune response. Once translocated, CALR stimulates macrophages to engulf stressed or dying cell portions through interaction with the CD91 cell surface receptor, significantly influencing the immunogenicity of cancer cell death. Chemokines, particularly human interleukin-8 (IL8), play a role in the immunogenic translocation of CRT to the outer leaflet of the plasma membrane. AbCs have been found to stimulate the production of IL8 by human cancer cells in vitro, further enhancing the immunogenicity of these cancer cells and regulating immune cell differentiation and activation. Treatment with AbCs leads to the translocation of CALR to the outer surface of the plasma membrane, correlating with an increase in immunogenic cell death induced by AbC treatment (Fig. 31). Atorney Docket No. 24-1880-WO

[0334] To investigate the potential facilitation of phagocytosis by AbCs, CD14+cells were isolated from human blood and induced their differentiation into macrophages. pHrodo Red dye, which acts as a specific sensor of phagocytic events based on its pH-sensitive properties, was utilized to stain engulfed cancer cells for visualization. The red fluorescence of pHrodo occurs due to phagosome acidification following phagocytosis. Phagocyte activity was measured by utilizing a flow cytometer for fluorescence intensity measurement (Fig. 32). The results show that the level of CRT translocation to the extracellular region correlates with the extent of cancer cell engulfinent by macrophages.

[0335] To investigate potential changes in cytokines that might impact immune cells during cancer cell death induced by AbCs, qPCR analysis was performed. IL8, known to induce CRT translocation, was included as a positive control. qPCRwas performed using A375 cells treated with 100 pM for 24 hrs, revealing a significant increase in the expression of cytokines that influence immune cell activity in cells treated with AbCs (Figs. 33A and 33B). While the cytokine response varied depending on the diversity of AbCs over the same duration, this observation was not investigated in cell viability results, suggesting that diversity through AbCs redesign can be applied with varying efficacy in inducing ICD.

[0336] To assess the impact of cytokine expression induced by 1 OnM AbCs-treated cancer cells on immune cells, CD4+T cells isolated from human blood were utilized. Additionally, a mixture of conditioned media from treating A375 and Colo205 cells with AbCs for 24 hrs and fresh media in a 1 : 1 ratio was prepared for the experimental evaluation of CD4+T cell activity (Figs. 35 and 36). Similar to CD4+T cells, the activity of CD8+T cells was enhanced when cultured in conditioned media where AbCs were treated in cancer cells.

[0337] Example 7: ENGINEERED ANGIF DOMAIN TESTED IN VIVO WITH INFLUENZA

[0338] This example evaluates on-target biological activity of redesigned and PEGylated AbC that exhibited improved bioavailability. AbCs have previously been shown to be able to potently agonize the receptor tyrosine kinase angiopoietin-1 (Tie2) receptor in vitro. Activation of this pathway induces phosphorylation of both AKT and extracellular signal -regulated kinase 1 and 2 (ERK1 / 2) (Figs. 37 and 38). Biologies that activate these effectors have been investigated as potential therapeutic candidates for the treatment and prevention of endothelial cell death during cases of severe inflammation from conditions such as sepsis and acute respiratory distress syndrome (ARDS). The fibrinogen-like binding domain, of Angiopoietin 1 (AngIF) (SEQ ID NO: 484) was optimized using the PROSS server to introduce 6 mutations Attorney Docket No. 24-1880-WO

[0339] K23T, M105E, H148Q, F188Y, R214K, and S215M to improve stability ofthe AnglF domain (AnglF6) (SEQ ID NO: 486) and its activity was validated in human endothelial umbilical vein cells (HUVECs) (Fig. 39). This optimization significantly improved activity of the PEGylated and stabilized variant compared to previous AbC iterations (Fig. 39). Next, to assess in vivo activity for the serum stabilized 04 component combined with the AnglF6 domain, 7- week-old mice were treated following infection with a 0.5LD50 dose of the H1N1 California 2009 strain of influenza (H1N1 Ca / 09). This strain was selected because it induces severe weight loss over the course of the infection in mice but does not result in death. H1N1 Ca / 09 is a widely used strain that provides a therapeutically relevant model for infection-mediated vascular dysfunction and consequent weight loss in mice . The mice were mice at 2 mg / kg postinfection for 5 days and their weight loss was tracked for a total of twelve days. This study showed that mice treated with our optimized and PEGylated 04 bearing the AnglF6 Tie2- binding domain exhibited no weight change throughout the course of infection, indicating a robust therapeutic & efficacious response to treatment (Fig. 40). Table 12: Sequences for AnglF and AnglF6 Attorney Docket No. 24-1880-WO

[0340] INCORPORATION BY REFERENCE

[0341] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavor to which this specification relates. EQUIVALENTS

[0342] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. The scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

Atorney Docket No. 24-1880-WOCLAIMS1. A Fc-binding polypeptide, comprising a Z domain, wherein the Z domain specifically binds fragment crystallizable (Fc) domains, and wherein the Z domain comprises a polypeptide sequence disclosed in Table 1A or Table IB, or a variant at least 85%, at least 90%, or at least 95% identical thereto; or wherein the Z domain comprises an amino acid substitution D6E, S9N, N16H, M17L, N21T, A23E, G27A, T40A, or A50N, or a combination thereof, relative to SEQ ID NO:498.MGFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNLLAEAKKLA DAQAPK (SEQ ID NO: 498).

2. An 04 component polypeptide modified to include a Z domain, comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 35-415 of SEQ ID NO: 1(MG) FNKDQQSAFYEILNMPNLNEALRNGFIQLLKDDPSKSTVILTAAKVAAELSEKIRTLKESGSSYEQIAETVAKAVAKLVEKLKRNGVSEDEIALAVALI I SAVIQTLKES GSSYEVIAEIVARIVAEIVEALKRSGTSEDEIAEIVARVI SEVIRTLKESGSSYEVI AEI VARI VAEIVEALKRSGTSEDEIAKIVARVIAEVLRTLKESGSSEEVIKEI VARI ITEIKEALKRSGTSEDEIELITLMIEAALEIAKLKSSGSEYEEICEDVARRIAELVE KLKRDGTSAVEIAKIVAAI I SAVIAMLKASGSSYEVI CECVARIVAEIVEALKRSGT SAAI IALIVALVISEVIRTLKESGSSFEVILECVIRIVLEI IEALKRSGTSEQDVMLIVMAVLLVVLATLQLSGS, wherein residues in parentheses are optional, and may be present or may be deleted; wherein the polypeptide comprises an oligomerization domain and a Z domain that specifically binds fragment crystallizable (Fc) domains, and wherein the Z domain comprises a polypeptide sequence according toFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKD (SEQ ID NO: 2) orFNKDQQSAFYEILNMPNLNEALRNGFIQLLKD (SEQ ID NO: 3), or a variant at least 85%, at least 90%, or at least 95% identical thereto; or a polypeptide sequence disclosed in Table 1A, or a variant at least 85%, at least 90%, or at least 95% identical thereto; or wherein the Z domain comprises an amino acid substitution D6E, S9N, N16H,Atorney Docket No. 24-1880-WOM17L, N21T, A23E, G27A, T40A, or A50N, or a combination thereof, relative to SEQ ID NO:498.

3. An 04 component polypeptide modified to decrease internal disulfide bond formation, comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 35-415 of SEQ ID NO: 4(MG) FNKDQQSAFYEILNMPNLNEALRNGFIQLLKDDPSKSTVILTAAKVAAELSEK IRTLKESGSSYEQIAETVAKAVAKLVEKLKRNGVSEDEIALAVALI I SAVIQTLKES GSSYEVIAEIVARIVAEIVEALKRSGTSEDEIAEIVARVI SEVIRTLKESGSSYEVI AEI VARI VAEIVEALKRSGTSEDEIAKIVARVIAEVLRTLKESGSSEEVIKEI VARI ITEIKEALKRSGTSEDEIELITLMIEAALEIAKLKSSGSEYEEICEDVARRIAELVE KLKRDGTSAVEIAKIVAAI I SAVIAMLKASGSSYEVI CECVARIVAEIVEALKRSGT SAAI IALIVALVISEVIRTLKESGSSFEVILECVIRIVLEI IEALKRSGTSEQDVML IVMAVLLVVLATLQLSGS, wherein residues in parentheses are optional and may be present or may be deleted; wherein the polypeptide comprises an oligomerization domain and a fragment crystallizable (Fc)-binding domain that specifically binds fragment crystallizable (Fc) domains, wherein the polypeptide sequence comprises an amino acid substitution, relative to SEQ ID NO:4, at position C271, C321, C323, or C373, or a combination thereof; or wherein the polypeptide sequence comprises amino acid substitutions relative to SEQ ID NO:4 at positions C271, C321, C323, and C373.

4. The polypeptide of claim 3, wherein the polypeptide sequence comprises, relative to SEQ ID NO:4, an amino acid substitution C271A, C321A, C323I, or C373I, or a combination thereof; or wherein the polypeptide sequence comprises, relative to SEQ ID NO:4, amino acid substitutions C271A, C321A, C323I, and C373I.

5. The polypeptide of any one of claims 3-4, wherein the Fc-binding domain is a Z domain, and wherein the Z domain comprises a polypeptide sequence according toFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKD (SEQ ID NO: 2) orFNKDQQSAFYEILNMPNLNEALRNGFIQLLKD (SEQ ID NO: 3), or a variant at least 85%, at least 90%, or at least 95% identical thereto; orAtorney Docket No. 24-1880-WO a polypeptide sequence disclosed in Table 1A, or a variant at least 85%, at least 90%, or at least 95% identical thereto.

6. An 04 component polypeptide modified for bioconjugation, comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 35-415 of SEQ ID NO: 1(MG) FNKDQQSAFYEILNMPNLNEALRNGFIQLLKDDPSKSTVILTAAKVAAELSEK IRTLKESGSSYEQIAETVAKAVAKLVEKLKRNGVSEDEIALAVALI I SAVIQTLKES GSSYEVIAEIVARIVAEIVEALKRSGTSEDEIAEIVARVI SEVIRTLKESGSSYEVI AEI VARI VAEIVEALKRSGTSEDEIAKIVARVIAEVLRTLKESGSSEEVIKEI VARI ITEIKEALKRSGTSEDEIELITLMIEAALEIAKLKSSGSEYEEICEDVARRIAELVE KLKRDGTSAVEIAKIVAAI I SAVIAMLKASGSSYEVI CECVARIVAEIVEALKRSGT SAAI IALIVALVISEVIRTLKESGSSFEVILECVIRIVLEI IEALKRSGTSEQDVML IVMAVLLVVLATLQLSGS, wherein residues in parentheses are optional and may be present or may be deleted; wherein the polypeptide comprises an oligomerization domain and a fragment crystallizable (Fc)-binding domain that specifically binds fragment crystallizable (Fc) domains, wherein the polypeptide sequence comprises an amino acid substitution, relative to SEQ ID NO: 1, at position E51, T58, E61, E67, Q68, K79, K83, R86, El 11, El 17, E167, K220, T228, E232, R236, E241, S265, E269, E280, K284, S 114, A224, R276, or R337, or a combination thereof; and wherein optionally the polypeptide is bioconjugated to a steric exclusion molecule, optionally a polyethylene glycol (PEG), preferably PEG-5K.

7. The polypeptide of claim 6, wherein the polypeptide sequence comprises, relative to SEQ ID NO: 1, an amino acid substitution E51C, T58C, E61C, E67C, Q68C, K79C, K83C, R86C, E111C, E117C, E167C, K220C, T228C, E232C, R236C, E241C, S265C, E269C, E280C, K284C, S114C, A224C, R276C, or R337C; or a combination thereof.

8. The polypeptide of claim 7, wherein the polypeptide sequence comprises, relative to SEQ ID NO: 1, an amino acid substitution K79C.

9. The polypeptide of any one of claims 6-8, wherein the polypeptide sequenceAtorney Docket No. 24-1880-WO comprises, relative to SEQ ID NO: 1, an amino acid substitution at position C271, C321, C323, or C373, or a combination thereof; or wherein the polypeptide sequence comprises, relative to SEQ ID NO: 1, amino acid substitutions at positions C271, C321, C323, and C373.

10. The polypeptide of claim 9, wherein the polypeptide sequence comprises, relative to SEQ ID NO: 1, an amino acid substitution C271A, C321A, C323I, or C373I, or a combination thereof; or wherein the polypeptide sequence comprises, relative to SEQ ID NO: 1, amino acid substitutions C271A, C321A, C323I, and C373I.

11. The polypeptide of any one of claims 6-10, wherein the Fc-binding domain is a Z domain, and wherein the Z domain comprises a polypeptide sequence according toFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKD (SEQ ID NO: 2) or FNKDQQSAFYEILNMPNLNEALRNGFIQLLKD (SEQ ID NO: 3), or a variant at least 85%, at least 90%, or at least 95% identical thereto; or a polypeptide sequence disclosed in Table 1A, or a variant at least 85%, at least 90%, or at least 95% identical thereto.

12. An 04 component polypeptide, wherein the polypeptide comprises a polypeptide sequence comprising a first segment disclosed in Table 1A or Table 6, a second segment disclosed in Table 7, and a third segment disclosed in Table 8; or for each segment, a variant at least 85%, at least 90%, or at least 95% identical thereto.

13. The polypeptide of any one of claims 6-12, wherein the polypeptide comprises a polypeptide sequence according to any one of SEQ ID NOs: 470-479; or a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical thereto.

14. The polypeptide of claim 6-13, wherein the polypeptide comprises a polypeptide sequence according to SEQ ID NO: 470; or a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical thereto.

15. An D2 component polypeptide modified to include a Z domain, comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at leastAtorney Docket No. 24-1880-WO85%, at least 90%, at least 95%, at least 98% identical to residues 56-255 of SEQ IDNO: 480:(M) SDEEERNELIQRIKEAAQRAREAAERTGDPRVRELARELQKIAQNAFYEVLHDP SSSEVNEALKAVVKAIELAVRALEEAEKTGDPEVRELAREVVRLAVEVAQATQAGEN DTLRKVAERALRLAKEAAKRGDAKAAKQAAKIAKLAAANAGDEDVLKKVELVRLAIE LVEIVVENAKRKGDDDKEAAEAALAAFRIVLAAAQLAGIASLEVLELALRLIKEVVE NAQREGYDIAVAAIAAAVAFAVVAVAAAAADITSSEVLELAIRLIKEVVENAQREGY VI LLAALAAAAAFVVVAAAAKRAGI TSSETLKRAI EEI RKRVEEAQREGNDI SEAAR QAAEEFRKKAEELK (GSWLEHHHHHH) , wherein residues in parentheses are optional, and may be present or may be deleted; wherein the polypeptide comprises an oligomerization domain and a Z domain that specifically binds fragment crystallizable (Fc) domains, and wherein the Z domain comprises a polypeptide sequence according to (M)SDEEERNELIKRIREAAQRAREAAERTGDPRVRELARELARIAQIAFYLVLH DP (SEQ ID NO: 618); or a variant at least 85%, at least 90%, or at least 95% identical thereto; or (M)SDEEERNELIKRIREAAQRAREAAERTGDPRVRELARELAKLAQIAFYLVL HDP (SEQ ID NO: 619); or a variant at least 85%, at least 90%, or at least 95% identical thereto; or wherein the Z domain comprises one or more amino acid substitutions K12Q, R15K, A41Q, R42K, I46N, or L50E, or a combination thereof, relative to SEQ ID NO: 618.

16. A T4 component polypeptide comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 36-305 of SEQ ID NO: 481(MG) FNKEQQNAFYLILHLPNLTEEQRNAFIQSLKDDPSKSAVVAGEAAIENARNAL KKGSPETAREAVRLALELVQQAERQARKTGSTERLIAAAKLAIEVARVALKVGSPET AREAVRTALELVQELIRQARKTGSKEVLEEAAKLALEVAKVAAEVGSPETAARAVAT AVEALKEAGASEDEIAEIVARVISEVIRILKESGSEYKVIARAVARIVAEIVEALKR SGTSEDEIAEIVARVISEVIRTLKESGSDYLI IAVIVAI IVAEIVEALKRSGTSEDE IAEIVARVISEVIRTLKESGSSYEVIKEIVQI IVLAI ILALMKSGTEVEEILLILLR VKTEVRRTLKES (GSWLEHHHHHH) , wherein residues in parentheses are optional, and may be present or may be deleted;Atorney Docket No. 24-1880-WO wherein the polypeptide comprises an oligomerization domain and a Z domain that specifically binds fragment crystallizable (Fc) domains, and wherein the Z domain comprises an amino acid sequence according to FNKSQQSAFYLILNMPNLNEAQRNGFIQSLKD (SEQ ID NO: 616), or a variant at least 85%, at least 90%, or at least 95% identical thereto; or wherein the Z domain comprises an amino acid substitution D6E, S9N, N16H, M17L, N21T, A23E, G27A, T40A, or A50N, or a combination thereof, relative to SEQ ID NO: 498.

17. A T8 component polypeptide comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 36-266 of SEQ ID NO: 482:(MG) FNKEQQNAFYEVLHLPNLTEEQRNAFIQSLKDDPSQSLKILIKAAAGGDSELEEVAKRI IKELAEQGRSEKEAAKEAAELIQRITRAAGGNSDLIELAVRIVKILEEQGR SPSEAAKEAVEAIEAIVRAAGGDSEAIKVAAEIAKTI ITQKESGSEYKEICRTVARI VAEIVEKLKRNGASEDEIAEIVAAI IAAVILTLKLSGSDYLI I CVCVAI I VAEI VEA LKRSGTSEDEIAEIVARVISAVIRVLKESGSSYEVIKECVQI IVLAI ILALMKSGTE VEEILLILLRVKTEVRRTLKES (GSWLEHHHHHH) ; wherein residues in parentheses are optional, and may be present or may be deleted; wherein the polypeptide comprises an oligomerization domain and a Z domain that specifically binds fragment crystallizable (Fc) domains, and wherein the Z domain comprises an amino acid sequence according toFNKDQQSAFYEVLNMPNLNEAQRNGFIQSLKD (SEQ ID NO: 617) or a variant at least 85%, at least 90%, or at least 95% identical thereto; or wherein the Z domain comprises an amino acid substitution D6E, S9N, N16H, M17L, N21T, A23E, G27A, T40L, or A50G, or a combination thereof, relative to SEQ ID NO: 498.

18. A 15 component polypeptide comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 56-227 of SEQ ID NO: 483;(M) SDEEERNELIQRIKEAAQRAREAAERTGDPRVRELARELQKLAQNAFYEVLHDPS S SDVNEALKL I VEAI EAAVRALEAAERAGDPELREDAREAVRLAVEAAQEVQRNPS SSTANLLLKAIVALAEALAAAANGDKEKFKKAAESALEIAKRVVEVASKEGDPEAVLAtorney Docket No. 24-1880-WOEAAKVALRVAELAAKNGDKEVFKKAAESALEVAKRLVEVASKEGDPELVLEAAKVAL RVAELAAKNGDKEVFQKAAASAVEVALRLTEVASKEGDSELETEAAKVITRVRELAS KQGDAAVAILAETAEVKLEIEESKKRPQSESAKNLILIMQLLINQIRLLVLQIRMLD EQRQE (GSWLEHHHHHH) wherein residues in parentheses are optional, and may be present or may be deleted; wherein the polypeptide comprises an oligomerization domain and a Z domain that specifically binds fragment crystallizable (Fc) domains, and wherein the Z domain comprises an amino acid sequence according to (M)SDEEERNELIKRIREAAQRAREAAERTGDPRVRELARELARLAQRAFYLVL HDP (SEQ ID NO: 620), or a variant at least 85%, at least 90%, or at least 95% identical thereto, or wherein the Z domain comprises one or more amino acid substitutions K12Q, R15K, A41Q, R42K, R46N, or L50E, or a combination thereof, relative to SEQ ID NO: 620.

19. A 15 component polypeptide comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 56-227 of SEQ ID NO: 484(M) SDEEERNELIQRIKEAAQRAREAAERTGDPRVRELARELQKLAQNAFYEVLHNP ESEGTLRALDHI IRAIDAAVEAIEAALESGDEEARERAREAVRQAVEAAQRVLENPE SEAVLKLLEAVVALAEALAAAMRGDKEEFKKAAKKAIKIAREVVKLAKKQGNAELVL KAAEIALEVARLAAEKGDKEIFKEAAKAAIEIAREWKLAKKQGNAELVLKAAEIAL EVARLAAEKGDEEIFREAAKAAIEIANEVKKLAKKQGNEELVKKALEI IKEVGKLAK EKGDRSSKTLAESELIKFEIEELEKKPHSLETKALILISKLLYASIVLLSEI IDELY KI LKK ( GSWLEHHHHHH ) ; wherein residues in parentheses are optional, and may be present or may be deleted; wherein the polypeptide comprises an oligomerization domain and a Z domain that specifically binds fragment crystallizable (Fc) domains, and wherein the Z domain comprises an amino acid sequence according to(M)SDEEERNELIKRIREAAQRAREAAERTGDPRVRELARELARLAQRAFYLVL HDP (SEQ ID NO: 620), or a variant at least 85%, at least 90%, or at least 95% identical thereto, or wherein the Z domain comprises one or more amino acid substitutions K12Q,Atorney Docket No. 24-1880-WOR15K, A41Q, R42K, R46N, or L50E, or a combination thereof, relative to SEQ ID NO: 620.

20. An AnglF polypeptide, comprising a polypeptide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 1-225 of SEQ ID NO: 6KAELASEKPFRDCADVYQAGFNKSGIYTIYINNMPEPKKVFCNMDVNGGGWTVIQHR EDGSLDFQRGWKEYKMGFGNPSGEYWLGNEFI FAITSQRQYMLRIELMDWEGNRAYS QYDRFHIGNEKQNYRLYLKGHTGTAGKQSSLILHGADFSTKDADNDNCMCKCALMLT GGWWFDACGPSNLNGMFYTAGQNHGKLNGIKWHYFKGPSYSLRSTTMMIRPLDFGGS GGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDLSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK, wherein the polypeptide sequence comprises an amino acid substitution, relative to SEQ ID NO:6, at position K23, M105, H148Q, F188, R214, or S215, or a combination thereof.

21. The polypeptide of claim 20, wherein the polypeptide sequence comprises an amino acid substitution, relative to SEQ ID NO:6, at positions K23, M105, H148, F188, R214, and S215.

22. The polypeptide of claim 20, wherein the polypeptide sequence comprises, relative to SEQ ID NO:6, an amino acid substitution K23T, M105E, H148Q, F188Y, R214K, or S215M, or a combination thereof.

23. The polypeptide of claim 20, wherein the polypeptide sequence comprises, relative to SEQ ID NO:6, amino acid substitutions K23T, M105E, H148Q, F188Y, R214K, and S215M.

24. The polypeptide sequence of any one of claims 20-23, wherein the polypeptide comprises a polypeptide linker and a fragment crystallizable (Fc) polypeptide, wherein optionally the polypeptide sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to residues 1-463 of SEQ ID NO:6.Atorney Docket No. 24-1880-WO25. An antibody cage (AbC) nanostructure, comprising: a plurality of first polypeptides, each first polypeptide comprising a fragment crystallizable (Fc) polypeptide that forms an Fc dimer; and a plurality of second polypeptides, wherein each second polypeptide is a polypeptide according to any one of claims 1-19.

26. The nanostructure of claim 25, wherein the plurality of first polypeptides multimerize to form a plurality of first components and the plurality of second polypeptides multimerize to form a plurality of second components; and wherein the first components and the second components assemble by direct interaction of Fc dimers of the first polypeptides with Fc-binding domains of the second polypeptides.

27. The nanostructure of any one claims 25-26, wherein the first polypeptides comprise an antibody or an antigen-binding domain thereof, a polypeptide linker, and the Fc polypeptide.

28. The nanostructure of claim 27, wherein the antibody is selected from Herceptin, Rituxan, Panorex, BEC2, IMC-C225,Vitaxin, Smart M195, LymphoCide, Smart ID 10, Oncolym, Allomune, an anti-VEGF antibody, CEAcide, IMC-1C11, Cetuximab, Lob 7 / 6, Lucatumumab, Dacetuzumab, Selicrelumab, Bleselumab, Urelumab, Utomilumab, Drozitumab, scTRAIL-Fc, KMTR2, 16E2, Aplitabart, optionally conatumumab.

29. The nanostructure of any one claims 25-26, wherein the first polypeptides comprise a binding agent.

30. The nanostructure of claim 29, wherein the binding agent is an AnglF polypeptide, wherein optionally the first polypeptide is an AnglF polypeptide according to any one of claims 20-24.

31. The nanostructure of claim 29, wherein the binding agent is Ang2, Ang3, Ang4 polypeptides or engineered molecules Bow-Angl, Comp-Angl, ABTAA antibody and vasculotide, transferrin, EGF, bombesin, gastrin, gastrin-releasing peptide, platelet- derived growth factor, IL-2, IL-6, TGF-a, TGF-P, vaccinia growth factor (VGF),Atorney Docket No. 24-1880-WO insulin and insulin-like growth factors I and II, somatostatin, lectins and apoprotein from low density lipoprotein.

32. A polynucleotide encoding the polypeptide of any of claims 1-24 or the nanostructure of any one of claims 25-31.

33. A delivery vehicle, comprising the polynucleotide of claims 32, and optionally a viral vector or lipid nanoparticle.

34. A pharmaceutical composition, comprising the polypeptide of any of claims 1-24, the nanostructure of any one of claims 25-31, the polynucleotide of claim 32, or the delivery vehicle of claim 33, and a pharmaceutically acceptable carrier.

35. A host cell suitable for expression of the polypeptide of any of claims 1-24 or the nanostructure of any one of claims 25-31; and / or comprising the polynucleotide of claim 32.

36. A method of making a polypeptide or nanostructure, comprising culturing the host cell of claim 35 under conditions suitable for expression of the polypeptide or nanostructure.

37. A method of treating a disease or disorder in a subject suffering from or at risk thereof, the method comprising administering to the subject the polypeptide of any of claims 1-24, the nanostructure of any one of claims 25-31, the polynucleotide of claim 32, or the delivery vehicle of claim 33, or the pharmaceutical composition of claims 34.

38. The method of claim 37, wherein the disease or disorder is a viral infection, optionally influenza.

39. The method of claim 37, wherein the disease or disorder is a cancer, wherein optionally the nanostructure comprises an antigen-binding fragment of conatumumab, and a cancer is a cancer responsive to conatumumab.

40. A composition or method as described herein.

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