DR5-Targeted Multabody for the Treatment of Cancer

Abstract

A self-assembled polypeptide complex comprising: 1) a fusion polypeptide comprising an Fc polypeptide linked to a nanocage monomer or a subunit thereof, and 2) a fusion polypeptide comprising an antigen-binding antibody fragment capable of binding to DR5.The present invention addresses this need by providing a self-assembled polypeptide complex comprising: (a) a fusion polypeptide comprising (1) an Fc polypeptide and (2) a nanocage monomer or a subunit thereof, and (b) a fusion polypeptide comprising (1) an antibody fragment capable of binding to DR5 and (2) a nanocage monomer or a subunit thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 243,372, filed September 13, 2021, the entire contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML file was created on Sep. 13, 2022, is named Sequence Listing Sep-2022 3206-5069_R, and is 57.6 kilobytes in size. [Background technology]

[0003] Death receptor 5 (DR5) is a member of the TNF receptor superfamily. DR5 is activated by receptor trimerization upon ligand binding. Once activated, DR5 delivers intracellular apoptotic signals to cells. DR5 is upregulated in various types of cancer cells, presenting an attractive target for cancer therapy. However, the efficacy of candidate therapeutics based on targeting DR5 may be limited by factors such as insufficient ability to crosslink receptors in the cell membrane.

[0004] There is a need for improved compositions and methods for targeting DR5. Summary of the Invention [Means for solving the problem]

[0005] The present invention addresses this need by providing a self-assembled polypeptide complex comprising (a) (1) an Fc polypeptide and (2) a fusion polypeptide comprising a nanocage monomer or a subunit thereof, and (b) (1) an antibody fragment capable of binding to DR5 and (2) a nanocage monomer or a subunit thereof. In certain embodiments, the Fc polypeptide comprises certain amino acid residues at specific positions, and the self-assembled polypeptide complex.

[0006] In one aspect, (a) a plurality of first fusion polypeptides, each first fusion polypeptide comprising (1) an Fc polypeptide and (2) a nanocage monomer or a subunit thereof; (b) a plurality of second fusion polypeptides, each second fusion polypeptide comprising: (1) an antibody fragment capable of binding to DR5; and (2) a nanocage monomer or a subunit thereof; The Fc polypeptide comprises an IgG1 Fc chain, which comprises (1) an amino acid residue other than glycine at position 237 and (2) a proline residue at position 329, according to the EU numbering system.

[0007] In some embodiments, the nanocage monomer or subunit thereof in each first fusion polypeptide and each second fusion polypeptide is a ferritin monomer or subunit thereof.

[0008] In some embodiments, the ferritin monomer or subunit thereof is a ferritin light chain or subunit thereof.

[0009] In some embodiments, the ferritin monomer or subunit thereof is human ferritin or a subunit thereof.

[0010] In some embodiments, the self-assembled polypeptide complex does not include any ferritin heavy chain or subunits of a ferritin heavy chain.

[0011] In some embodiments, the ferritin monomer or subunit thereof is a ferritin monomer subunit.

[0012] In some embodiments, a. each first fusion polypeptide comprises a C-half ferritin and each second fusion polypeptide comprises an N-half ferritin; or b. Each first fusion polypeptide contains an N-half ferritin and each second fusion polypeptide contains a C-half ferritin.

[0013] In some embodiments, each first fusion polypeptide comprises an Fc polypeptide linked to the N-terminus of a C-half ferritin via an amino acid linker. In some embodiments, the amino acid linker is n S) m In some embodiments, the linker (G n S) m The linker is (GGGGS) m (SEQ ID NO:54) is a linker.

[0014] In some embodiments, the Fc polypeptide comprises a single chain Fc (scFc) comprising two Fc chains, the two Fc chains being linked by an amino acid linker, e.g., (G n S) m Linkers, e.g., (GGGGS) m (SEQ ID NO:54) are linked via an amino acid linker.

[0015] In some embodiments, the Fc polypeptide comprises an IgG1 Fc chain.

[0016] In some embodiments, the IgG1 Fc chain comprises an alanine at position 237, according to EU numbering. In some embodiments, the IgG1 Fc chain comprises an alanine at position 234, an alanine at position 235, an arginine at position 236, and a leucine at position 330, according to EU numbering.

[0017] In some embodiments, within each second fusion polypeptide, the antigen-binding antibody fragment is linked to the N-terminus of a nanocage monomer or a subunit thereof.

[0018] In some embodiments, the antigen-binding antibody fragment of each second fusion polypeptide is a Fab fragment. In some embodiments, each second fusion polypeptide does not include any antibody CH2 or CH3 domains.

[0019] In some embodiments, the self-assembled polypeptide complex further comprises a plurality of third fusion polypeptides, each third fusion polypeptide comprising (1) an antigen-binding antibody fragment and (2) a nanocage monomer or a subunit thereof, and the third fusion polypeptide is different from the second fusion polypeptide.

[0020] In some embodiments, the antigen-binding antibody fragment of each third fusion polypeptide is a Fab fragment.

[0021] In some embodiments, each third fusion polypeptide does not contain any antibody CH2 or CH3 domains.

[0022] In some embodiments, the nanocage monomer or subunit thereof of each first fusion polypeptide and each second fusion polypeptide is a ferritin monomer or subunit thereof; a. each first fusion polypeptide comprises a C-half ferritin and each second fusion polypeptide comprises an N-half ferritin; or b. Each first fusion polypeptide contains an N-half ferritin and each second fusion polypeptide contains a C-half ferritin.

[0023] In some embodiments, the self-assembled polypeptide complex is characterized by a 1:1 ratio of the first fusion polypeptide to the second fusion polypeptide.

[0024] In some embodiments, the self-assembled polypeptide complex comprises a total of 24 to 48 fusion polypeptides.

[0025] In some embodiments, the self-assembled polypeptide complex comprises a total of at least 24 fusion polypeptides.

[0026] In some embodiments, the self-assembled polypeptide complex comprises a total of at least 32 fusion polypeptides.

[0027] In some embodiments, the self-assembled polypeptide complex has a total of about 32 fusion polypeptides.

[0028] In some embodiments, after administration of a composition comprising the self-assembling polypeptide complex, the concentration of the self-assembling polypeptide complex is substantially similar to the concentration of a reference IgG molecule administered by the same route of administration and in a similar composition for the first 7 days after administration to a subject in need thereof.

[0029] In some embodiments, the self-assembled polypeptide complex does not exhibit binding to at least one human Fcγ receptor as determined in an in vitro assay.

[0030] In some embodiments, the self-assembled polypeptide complex does not exhibit binding to one or more human Fcγ receptors selected from the group consisting of hFcγRI, hFcγRIIa, hFcγRIIb, hFcγRIIIa, hFcγRIIIb, and combinations thereof, as determined by in vitro assays.

[0031] In some embodiments, the self-assembled polypeptide complex does not exhibit binding to hFcγRI as determined by in vitro assay. In some embodiments, the self-assembled polypeptide complex does not exhibit binding to hFcγRIIa as determined by in vitro assay. In some embodiments, the self-assembled polypeptide complex does not exhibit binding to hFcγRIIIa as determined by in vitro assay. In some embodiments, the self-assembled polypeptide complex does not exhibit binding to hFcγRIIb as determined by in vitro assay. In some embodiments, the self-assembled polypeptide complex does not exhibit binding to hFcγRIIIb as determined by in vitro assay.

[0032] In certain embodiments, (a) a plurality of first fusion polypeptides, each of the first fusion polypeptides comprising (1) an scFc and (2) a ferritin monomer or subunit thereof; (b) a plurality of second fusion polypeptides, each second fusion polypeptide comprising (1) an antibody fragment capable of binding to DR5 and (2) a ferritin monomer or a subunit thereof, wherein the scFc comprises two IgG1 Fc chains, each IgG1 Fc chain comprising an alanine at position 234, an alanine at position 235, an arginine at position 236, an alanine at position 237, a proline at position 329, and a leucine at position 330 according to EU numbering.

[0033] In some embodiments, a. each first fusion polypeptide comprises a C-half ferritin and each second fusion polypeptide comprises an N-half ferritin; or b. Each first fusion polypeptide contains an N-half ferritin and each second fusion polypeptide contains a C-half ferritin.

[0034] In some embodiments, each first fusion polypeptide comprises an scFc linked to the N-terminus of a C-half ferritin, and each second fusion polypeptide comprises a Fab linked to the N-terminus of an N-half ferritin.

[0035] In some embodiments, (1) in each first fusion polypeptide, the scFc is linked to the N-terminus of the C-half ferritin via an amino acid linker, and / or (2) in each second fusion polypeptide, the Fab is linked to the N-terminus of the N-half ferritin via an amino acid linker.

[0036] In some embodiments, the self-assembled polypeptide complex is characterized by a 1:1 ratio of the first fusion polypeptide to the second fusion polypeptide.

[0037] In some embodiments, the self-assembled polypeptide complex further comprises a plurality of third fusion polypeptides, each third fusion polypeptide comprising (1) an antigen-binding antibody fragment and (2) a nanocage monomer or a subunit thereof, and the third fusion polypeptide is different from the second fusion polypeptide.

[0038] In some embodiments, the self-assembled polypeptide complex comprises a total of 24 to 48 fusion polypeptides.

[0039] In some embodiments, the self-assembled polypeptide complex comprises a total of at least 24 fusion polypeptides.

[0040] In some embodiments, the self-assembled polypeptide complex comprises a total of at least 32 fusion polypeptides.

[0041] In some embodiments, the self-assembled polypeptide complex has a total of about 32 fusion polypeptides.

[0042] In some embodiments, the antibody fragment comprises: (1) a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3; and (2) a light chain comprising CDR-L1, CDR-L2, and CDR-L3; (a)(i) CDR-H1 has the sequence of SEQ ID NO: 27, or a sequence which differs therefrom by one or two amino acids; (ii) CDR-H2 has the sequence of SEQ ID NO: 28 or a sequence which differs therefrom by one or two amino acids; (iii) CDR-H3 has the sequence of SEQ ID NO: 29 or a sequence which differs therefrom by one or two amino acids; (iv) CDR-L1 has the sequence of SEQ ID NO: 24 or a sequence which differs therefrom by one or two amino acids; (v) CDR-L2 has the sequence of SEQ ID NO: 25 or a sequence which differs therefrom by one or two amino acids; (vi) CDR-L3 has the sequence of SEQ ID NO: 26 or a sequence which differs therefrom by one or two amino acids; (b)(i) CDR-H1 has the sequence of SEQ ID NO: 35, or a sequence which differs therefrom by one or two amino acids; (ii) CDR-H2 has the sequence of SEQ ID NO: 36 or a sequence which differs therefrom by one or two amino acids; (iii) CDR-H3 has the sequence of SEQ ID NO: 37, or a sequence which differs therefrom by one or two amino acids; (iv) CDR-L1 has the sequence of SEQ ID NO: 32 or a sequence which differs therefrom by one or two amino acids; (v) CDR-L2 has the sequence of SEQ ID NO: 33 or a sequence which differs therefrom by one or two amino acids; (vi) CDR-L3 has the sequence of SEQ ID NO: 34 or a sequence which differs therefrom by one or two amino acids; (c)(i) CDR-H1 has the sequence of SEQ ID NO: 43, or a sequence which differs therefrom by one or two amino acids; (ii) CDR-H2 has the sequence of SEQ ID NO: 44 or a sequence which differs therefrom by one or two amino acids; (iii) CDR-H3 has the sequence of SEQ ID NO: 45 or a sequence which differs therefrom by one or two amino acids; (iv) CDR-L1 has the sequence of SEQ ID NO: 40 or a sequence which differs therefrom by one or two amino acids; (v) CDR-L2 has the sequence of SEQ ID NO: 41 or a sequence which differs therefrom by one or two amino acids; (vi) CDR-L3 has the sequence of SEQ ID NO: 42 or a sequence which differs therefrom by one or two amino acids; or (d)(i) CDR-H1 has the sequence of SEQ ID NO: 51, or a sequence which differs therefrom by one or two amino acids; (ii) CDR-H2 has the sequence of SEQ ID NO: 52 or a sequence which differs therefrom by one or two amino acids; (iii) CDR-H3 has a sequence of SEQ ID NO: 53 or a sequence that differs therefrom by one or two amino acids; (iv) CDR-L1 has the sequence of SEQ ID NO: 48 or a sequence which differs therefrom by one or two amino acids; (v) CDR-L2 has the sequence of SEQ ID NO: 49 or a sequence which differs therefrom by one or two amino acids; (vi) CDR-L3 has the sequence of SEQ ID NO: 50, or a sequence which differs therefrom by one or two amino acids.

[0043] In some embodiments, the antibody fragment comprises: (1) a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3; and (2) a light chain comprising CDR-L1, CDR-L2, and CDR-L3; (a)(i) CDR-H1 has the sequence of SEQ ID NO: 27; (ii) CDR-H2 has the sequence of SEQ ID NO: 28, (iii) CDR-H3 has the sequence of SEQ ID NO: 29, (iv) CDR-L1 has the sequence of SEQ ID NO: 24, (v) CDR-L2 has the sequence of SEQ ID NO: 25; (vi) CDR-L3 has the sequence of SEQ ID NO: 26; (b)(i) CDR-H1 has the sequence of SEQ ID NO: 35; (ii) CDR-H2 has the sequence of SEQ ID NO: 36, (iii) CDR-H3 has the sequence of SEQ ID NO: 37, (iv) CDR-L1 has the sequence of SEQ ID NO: 32, (v) CDR-L2 has the sequence of SEQ ID NO: 33, (vi) CDR-L3 has the sequence of SEQ ID NO: 34; (c)(i) CDR-H1 has the sequence of SEQ ID NO: 43; (ii) CDR-H2 has the sequence of SEQ ID NO: 44; (iii) CDR-H3 has the sequence of SEQ ID NO: 45; (iv) CDR-L1 has the sequence of SEQ ID NO: 40, (v) CDR-L2 has the sequence of SEQ ID NO: 41; (vi) CDR-L3 has the sequence of SEQ ID NO: 42; or (d)(i) CDR-H1 has the sequence of SEQ ID NO: 51; (ii) CDR-H2 has the sequence of SEQ ID NO: 52, (iii) CDR-H3 has the sequence of SEQ ID NO: 53; (iv) CDR-L1 has the sequence of SEQ ID NO: 48, (v) CDR-L2 has the sequence of SEQ ID NO: 49, (vi) CDR-L3 has the sequence of SEQ ID NO:50.

[0044] In some embodiments, the antibody fragment comprises: (1) a heavy chain variable region that is at least 85% identical to a reference VH sequence; (2) a light chain variable region that is at least 85% identical to a reference VL sequence; (a) the reference VH sequence has the sequence of SEQ ID NO: 23 and the reference VL sequence has the sequence of SEQ ID NO: 22; or (b) the reference VH sequence has the sequence of SEQ ID NO: 31 and the reference VL sequence has the sequence of SEQ ID NO: 30; or (c) the reference VH sequence has the sequence of SEQ ID NO: 39 and the reference VL sequence has the sequence of SEQ ID NO: 38; or (d) the reference VH sequence has the sequence of SEQ ID NO: 47 and the reference VL sequence has the sequence of SEQ ID NO: 46.

[0045] In one aspect, a method is provided that includes administering to a mammalian subject a composition that includes a self-assembled polypeptide complex disclosed herein.

[0046] In some embodiments, the subject is a human.

[0047] In some embodiments, the subject is diagnosed with or at risk of developing a tumor at the time of administration.

[0048] In some embodiments, the administering step results in the slowing or inhibition of tumor progression.

[0049] In some embodiments, the administering step results in tumor regression.

[0050] In some embodiments, the administering step results in complete regression of the tumor.

[0051] In some embodiments, the methods include administration by a systemic route, including, for example, subcutaneous, intravenous, or intramuscular injection, inhalation, or intranasal administration.

[0052] In one aspect, there is provided a use of a composition comprising a self-assembled polypeptide complex as described herein for administration to a mammalian subject.

[0053] In some embodiments, the subject is a human.

[0054] In some embodiments, the subject is diagnosed with or at risk of developing a tumor at the time of administration.

[0055] In some embodiments, the use is to slow or inhibit tumor progression.

[0056] In some embodiments the use is for causing tumor regression.

[0057] In some embodiments, the use is to cause complete regression of a tumor.

[0058] In some embodiments, the use is for systemic administration.

[0059] In some embodiments, systemic administration includes subcutaneous, intravenous, or intramuscular injection, inhalation, or intranasal administration.

[0060] In one aspect, there is provided a composition comprising a self-assembled polypeptide complex as described herein for use in administration to a mammalian subject.

[0061] In some embodiments, the subject is a human.

[0062] In some embodiments, the subject is diagnosed with or at risk of developing a tumor at the time of administration.

[0063] In some embodiments, the composition is for slowing or inhibiting tumor progression.

[0064] In some embodiments, the composition is for causing tumor regression.

[0065] In some embodiments, the composition is for causing complete regression of the tumor.

[0066] In some embodiments, the composition is for systemic administration.

[0067] In some embodiments, systemic administration includes subcutaneous, intravenous, or intramuscular injection, inhalation, or intranasal administration. [Brief description of the drawings]

[0068] [Figure 1A] FIG. 1 is a diagrammatic representation of human ferritin light chain (hFTL) and exemplary N-half ferritin (N-hFTL) and C-half ferritin (C-hFTL) molecules.

[0069] [Figure 1B] 1 is a diagrammatic representation of the fusion polypeptides that together form an exemplary Multabody of the present disclosure.

[0070] [Diagram 2] 1 shows Biolayer Interferometry (BLI) time response curves for binding of a DR5-targeting Multabody (Cona MB IgG1 LLRAL) to human DR5, human DR4, human osteoprotegerin (OPG), human decoy receptor 1 (DcR1), and human DcR2, respectively.

[0071] [Diagram 3] 1 shows exemplary BLI time response curves for binding of DR5-targeted Multabodies containing various Fc chains to human, cynomolgus, and mouse FcRn measured at pH 6.0 for association and pH 7.4 for dissociation.

[0072] [Figure 4] 1 shows dose-dependent killing of cancer cells by Cona MB IgG1 wt, Cona MB IgG1 LLRAL, and conatumumab (Cona) in different human tumor cell lines, quantified and expressed as the percentage of viable cells following multibody or antibody treatment relative to vehicle-treated tumor cells.

[0073] [Figure 5A] FIG. 1 is a schematic showing the design of the pharmacokinetic study described in Example 6.

[0074] [Figure 5B] 1 shows plots showing plasma levels of Cona MB IgG1 wt, Cona MB IgG1 LLRAL, or conatumumab (Cona) after a single intraperitoneal (ip) or intravenous (iv) dose administered to severe combined immunodeficiency (SCID) mice.

[0075] [Figure 5C] 1 shows plasma levels of Cona MB IgG1 LLRAL or conatumumab in SCID mice administered two intraperitoneal doses, 96 hours apart.

[0076] [Figure 6A] FIG. 1 is a schematic diagram showing the design of the in vivo efficacy experiment described in Example 7 and performed in the COLO205 xenograft mouse model.

[0077] [Figure 6B] Tumor volumes at various time points (FIG. 6B) and at day 88 (FIG. 6C) in mice bearing COLO205 xenograft tumors and treated with vehicle, conatumumab, or Cona MB IgG1 LLRAL are shown. [Figure 6C] Tumor volumes at various time points (FIG. 6B) and at day 88 (FIG. 6C) in mice bearing COLO205 xenograft tumors and treated with vehicle, conatumumab, or Cona MB IgG1 LLRAL are shown.

[0078] [Figure 6D] Tumor growth curves of individual COLO205 xenograft tumor-bearing mice treated with vehicle (FIG. 6D), conatumumab (FIG. 6E), or Cona MB IgG1 LLRAL (FIGS. 6F and 6G) ​​are shown. [Figure 6E]Tumor growth curves of individual COLO205 xenograft tumor-bearing mice treated with vehicle (FIG. 6D), conatumumab (FIG. 6E), or Cona MB IgG1 LLRAL (FIGS. 6F and 6G) ​​are shown. [Figure 6F] Tumor growth curves of individual COLO205 xenograft tumor-bearing mice treated with vehicle (FIG. 6D), conatumumab (FIG. 6E), or Cona MB IgG1 LLRAL (FIGS. 6F and 6G) ​​are shown. [Figure 6G] Tumor growth curves of individual COLO205 xenograft tumor-bearing mice treated with vehicle (FIG. 6D), conatumumab (FIG. 6E), or Cona MB IgG1 LLRAL (FIGS. 6F and 6G) ​​are shown.

[0079] [Figure 6H] 1 is a time plot showing plasma levels of Cona MB IgG1 LLRAL or conatumumab following intraperitoneal administration to COLO205 xenograft tumor-bearing mice.

[0080] [Figure 7A] 1 is a plot showing tumor volumes in mice in various groups over time since the first dose. "MB" indicates the Cona MB IgG1 LLRAL group. Triangles on the x-axis indicate treatment time points. [Figure 7B] Tumor volumes of individual mice within the vehicle (Figure 7B) and 5 mg / kg, 1 mg / kg, 0.25 mg / kg, and 0.1 mg / kg Cona MB IgG1 LLRAL-treated groups (Figures 7C, 7D, 7E, and 7F, respectively) are shown. In Figures 7B-7F, "CR" indicates complete regression. [Figure 7C] Tumor volumes of individual mice within the vehicle (Figure 7B) and 5 mg / kg, 1 mg / kg, 0.25 mg / kg, and 0.1 mg / kg Cona MB IgG1 LLRAL-treated groups (Figures 7C, 7D, 7E, and 7F, respectively) are shown. In Figures 7B-7F, "CR" indicates complete regression. [Figure 7D]Tumor volumes of individual mice within the vehicle (Figure 7B) and 5 mg / kg, 1 mg / kg, 0.25 mg / kg, and 0.1 mg / kg Cona MB IgG1 LLRAL-treated groups (Figures 7C, 7D, 7E, and 7F, respectively) are shown. In Figures 7B-7F, "CR" indicates complete regression. [Figure 7E] Tumor volumes of individual mice within the vehicle (Figure 7B) and 5 mg / kg, 1 mg / kg, 0.25 mg / kg, and 0.1 mg / kg Cona MB IgG1 LLRAL-treated groups (Figures 7C, 7D, 7E, and 7F, respectively) are shown. In Figures 7B-7F, "CR" indicates complete regression. [Figure 7F] Tumor volumes of individual mice within the vehicle (Figure 7B) and 5 mg / kg, 1 mg / kg, 0.25 mg / kg, and 0.1 mg / kg Cona MB IgG1 LLRAL-treated groups (Figures 7C, 7D, 7E, and 7F, respectively) are shown. In Figures 7B-7F, "CR" indicates complete regression.

[0081] [Figure 7G] 1 is a plot showing the amount of Cona MB IgG1 LLRAL detectable in blood samples collected at various time points after the first dose was administered. Cona MB IgG1 LLRAL was detectable at all time points tested in the 0.1 mg / kg, 0.25 mg / kg, 1 mg / kg, and 5 mg / kg Cona MB IgG1 LLRAL treatment groups, and pharmacokinetics appeared linear in all dose groups.

[0082] [Figure 8] 1 is a schematic diagram showing the design of the study of large tumor penetration and apoptosis induction by DR5-targeted Multabody in COLO205 xenograft mouse model. The experiment is described in Example 9.

[0083] [Figure 9]Representative tumor sections from untreated (FIGS. 9A and 9C) or DR5-targeted Multabody ("MB")-treated (FIGS. 9B and 9D) mice are shown. Sections were stained with an antibody against cleaved caspase-3, a marker of apoptosis. Experiments are described in Example 9. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0084] definition The terms "about" and "approximately", when used herein in reference to a value, are used interchangeably and refer to a value similar to the referenced value. Generally, a person of ordinary skill in the art familiar with the context will understand the relevant degree of variation encompassed by "about" or "approximately" in that context. For example, in some embodiments, the terms "about" and "approximately" can encompass values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referenced value.

[0085] As used herein, the terms "modify," "modified," "reduce," "reduced," "increase," "increased," or "reduction," "reduced" (e.g., with respect to a particular outcome or effect) have a meaning relative to a reference level. In some embodiments, in the context of considering a mutation in an Fc chain or Fc polypeptide, the reference level is a level known or determined in an IgG that does not contain the referenced mutation(s) in the Fc region.

[0086] As used herein, the term "binding" refers to a non-covalent association between or among two or more entities, unless otherwise specified. "Direct" binding involves physical contact between the entities or moieties, and indirect binding involves physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts, including when the interacting entities or moieties are studied alone or in the context of a more complex system (e.g., while covalently or otherwise associated with a carrier entity, and / or within a biological system or cell). As used herein, "non-binding" or "no binding" between two entities, or similar phrases, refers to 1) a lack of detectable binding, or 2) binding below a set threshold, corresponding to no binding in an appropriate assay, e.g., an in vitro binding assay, such as biolayer interferometry. For example, in some embodiments, in an in vitro biolayer interferometry assay, when a test article is present at a concentration of 20 nM, a maximum association binding response of less than 0.1 nm after 180 seconds to a biosensor loaded with 0.8 nm target is classified as "unbound."

[0087] The terms "ferritin" and "apoferritin" are used interchangeably herein and generally refer to a polypeptide (e.g., a ferritin chain) that can be assembled into a ferritin complex, typically comprising 24 protein subunits. In some embodiments, the ferritin is a human ferritin, e.g., a human ferritin light chain, e.g., a human ferritin light chain having at least 85% sequence identity with SEQ ID NO: 1 or UniProt P02792. In some embodiments, the ferritin is a wild-type ferritin. For example, the ferritin may be a wild-type human ferritin.

[0088] The term "ferritin monomer" is used herein to refer to a single chain of ferritin that can self-assemble, in the presence of other ferritin chains, into a polypeptide complex containing multiple ferritin chains, for example, 24 or more ferritin chains.

[0089] As used herein, the term "linker" is used to refer to an entity that connects two or more elements to form a multi-element agent. For example, one of skill in the art will understand that polypeptides having structures that include two or more functional or organizational domains (e.g., fusion polypeptides) often include a stretch of amino acids between such domains that link them together. In some embodiments, polypeptides that include linker elements have an overall structure of the general form S1-L-S2, where S1 and S2, which may be the same or different, represent two domains that are associated with each other by a linker (L). In some embodiments, the linker comprises an "amino acid linker" i.e., amino acid residues, e.g., the amino acid linker can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acid residues. In some embodiments, the linker is characterized in that it does not tend to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide.

[0090] The term "multispecificity" as used herein refers to the characteristic of having at least two binding sites to which at least two different binding partners, e.g., antigens or receptors (e.g., Fc receptors), can bind. For example, a polypeptide complex comprising at least two Fab fragments, each of which can bind a different antigen, is "multispecific". As an additional example, a polypeptide complex comprising an Fc fragment (capable of binding to an Fc receptor) and a Fab fragment (capable of binding to an antigen) is "multispecific".

[0091] As used herein, the term "multivalent" refers to the characteristic of having at least two binding sites to which a binding partner, e.g., an antigen or a receptor (e.g., an Fc receptor), can bind. The binding partners capable of binding to the at least two binding sites can be the same or different.

[0092] The term "nanocage monomer" as used herein refers to a single chain of a polypeptide that can self-assemble with other nanocage monomers to form a self-assembled polypeptide complex comprising a plurality of nanocage monomers. In some embodiments, the nanocage monomer is selected from monomers of ferritin, apoferritin, encapsulin, sulfur oxygenase reductase (SOR), lumazine synthase, pyruvate dehydrogenase, carboxysome, vault protein, GroEL, heat shock proteins, E2P coat protein, MS2 coat protein, fragments thereof, and mutants thereof.

[0093] The term "polypeptide" as used herein generally has its art-recognized meaning of a polymer of at least three amino acids, e.g., a polymer linked together by peptide bonds. Those of skill in the art will appreciate that the term "polypeptide" is intended to be general enough to encompass not only polypeptides having the complete sequences listed herein, but also polypeptides that represent functional fragments of such complete polypeptides (i.e., fragments that retain at least one activity). Moreover, those of skill in the art will appreciate that protein sequences generally tolerate some substitutions without destroying activity. Thus, any polypeptide that retains activity and shares an overall sequence identity with another polypeptide of the same class that is at least about 30-40%, often more than about 50%, about 60%, about 70%, or about 80%, and more usually more than 90% or even more than 95%, 96%, 97%, 98%, or 99%, usually encompassing at least 3-4, and often up to 20 or more amino acids, is encompassed by the related term "polypeptide" as used herein. Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, glycosylation, etc. In some embodiments, proteins may contain natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof.

[0094] The term "self-assembly," when used in reference to a macromolecular complex (e.g., a polypeptide complex), refers to the spontaneous formation of the complex when sufficient components of the complex to be formed (e.g., a fusion polypeptide) are present. In some embodiments, the complex self-assembles at physiological conditions or in a buffer (e.g., a solution) that corresponds to physiological conditions.

[0095] As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human). In some embodiments, the subject is afflicted with or susceptible to the relevant disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a person having one or more characteristics characteristic of susceptibility or risk for a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is a subject to whom and / or to whom diagnosis and / or treatment is being administered.

[0096] As used herein, the term "treatment" (also "treat" or "treating") refers to the administration of any therapy that partially or completely alleviates, alleviates, relieves, inhibits, delays the onset, reduces the severity, and / or reduces the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be of subjects who do not show signs of the associated disease, disorder, and / or condition, and / or who show only early signs of the disease, disorder, and / or condition. Alternatively, or additionally, such treatment may be of subjects who show one or more established signs of the associated disease, disorder, and / or condition. In some embodiments, the treatment may be of subjects who have been diagnosed as suffering from the associated disease, disorder, and / or condition. In some embodiments, the treatment may be of subjects who are known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the associated disease, disorder, and / or condition.

[0097] A. Fusion Polypeptides In many embodiments, a fusion polypeptide compatible with the compositions and methods disclosed herein generally comprises a nanocage monomer or subunit thereof linked to either an Fc polypeptide or an antigen-binding antibody fragment. Within the fusion polypeptide, the Fc polypeptide or antigen-binding antibody fragment may be linked to the nanocage monomer or subunit thereof at a particular terminus, e.g., the N-terminus or C-terminus, of the nanocage monomer or subunit thereof. In some embodiments, the Fc polypeptide or antigen-binding antibody fragment is linked via an amino acid linker, such as the linkers described herein.

[0098] 1. Nanocage monomer and its subunits In some embodiments, the nanocage monomer is a ferritin monomer.

[0099] The term "ferritin monomer" is used herein to refer to a single chain of ferritin that can self-assemble into a polypeptide complex containing multiple ferritin chains, e.g., 24 or more ferritin chains, in the presence of other ferritin chains. In some embodiments, the ferritin monomer is a ferritin light chain. In some embodiments, the ferritin monomer does not contain a ferritin heavy chain or other ferritin components that can bind iron or are capable of ferroxidase activity.

[0100] In some embodiments, each fusion polypeptide in the self-assembled polypeptide complex comprises a ferritin light chain or a subunit of a ferritin light chain. In these embodiments, the self-assembled polypeptide complex does not comprise any ferritin heavy chain or a subunit of a ferritin heavy chain.

[0101] In some embodiments, the ferritin monomer is a human ferritin chain, e.g., a human ferritin light chain, e.g., a human ferritin light chain having a sequence of at least residues 2-175 of SEQ ID NO:1.

[0102] A "subunit" of a ferritin monomer refers to a portion of a ferritin monomer that can spontaneously associate with another, separate subunit of a ferritin monomer such that the subunits together form a ferritin monomer, which can then self-assemble with other ferritin monomers to form a polypeptide complex.

[0103] In some embodiments, the ferritin monomer subunit comprises about half of a ferritin monomer. As used herein, the term "N-half ferritin" refers to about half of a ferritin chain that includes the N-terminus of the ferritin chain. As used herein, the term "C-half ferritin" refers to about half of a ferritin chain that includes the C-terminus of the ferritin chain. The exact point at which a ferritin chain can be split to form N-half ferritin and C-half ferritin can vary depending on the embodiment. For example, in the context of a ferritin monomer subunit based on the human ferritin light chain, the half can be split at a point corresponding to positions from about 75 to about 100 (or a substantial portion thereof) of SEQ ID NO:1. For example, in some embodiments, an N-half-ferritin based on the human ferritin light chain has an amino acid sequence corresponding to residues 1-95 of SEQ ID NO:1 (or a substantial portion thereof, e.g., residues 2-95 of SEQ ID NO:1), and a C-half-ferritin based on the human ferritin light chain has an amino acid sequence corresponding to residues 96-175 of SEQ ID NO:1 (or a substantial portion thereof).

[0104] In some embodiments, the halves are split at a point corresponding to positions from about 85 to about 92 of SEQ ID NO: 1. For example, in some embodiments, an N-half ferritin based on the human ferritin light chain has an amino acid sequence corresponding to residues 1-90 of SEQ ID NO: 1 (or a substantial portion thereof, e.g., residues 2-90 of SEQ ID NO: 1), and a C-half ferritin based on the human ferritin light chain has an amino acid sequence corresponding to residues 91-175 of SEQ ID NO: 1 (or a substantial portion thereof).

[0105] 2. Fc Polypeptides In certain embodiments, the fragment crystallizable (Fc) polypeptides each comprise an Fc chain having one or more mutations relative to a reference Fc chain of the same Ig class, which may be, for example, of the IgG1 class, as further described below.

[0106] Unless otherwise stated, numbering of residues in antibody fragments, e.g., the Fc chain, throughout this disclosure is according to EU numbering.

[0107] In some embodiments, the Fc polypeptide comprises one or more human IgG1 Fc chains, i.e., except for the mutations described herein, the Fc polypeptide comprises an Fc chain that is substantially similar to that of the Fc chain in wild-type human IgG1.

[0108] In some embodiments, the Fc polypeptide comprises one or more IgG1 Fc chains (e.g., human IgG1 Fc chains or human Fc chains), i.e., the Fc polypeptide comprises an Fc chain that has an amino acid sequence substantially similar to that of a chain in a wild-type IgG1 Fc, except for having a particular residue(s) at certain positions described herein (which may differ from the residue(s) in the corresponding wild-type Fc chain). In some embodiments, the wild-type IgG1 Fc is a human IgG1 Fc, and each Fc chain has the amino acid sequence of SEQ ID NO:4. For example, an Fc polypeptide may comprise an Fc chain having an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of the Fc chain in wild-type IgG1 Fc. In some embodiments, an Fc polypeptide comprises an Fc chain having an amino acid sequence that includes a particular residue(s) at certain position(s) specifically described for that Fc chain, but is otherwise 100% identical to the corresponding Fc chain in a wild-type Fc chain, e.g., a wild-type IgG1 Fc chain. In some embodiments, an Fc polypeptide comprises an Fc chain having an amino acid sequence that differs from the sequence of SEQ ID NO:4 by at least one, at least two, at least three, or at least four amino acid residues. In some embodiments, the Fc polypeptide comprises an Fc chain having an amino acid sequence that differs by no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, or no more than 4 amino acid residues from the sequence of SEQ ID NO:4.

[0109] In some embodiments, the Fc polypeptide is a single chain Fc (scFc) that comprises two Fc chains linked together by a covalent linker, e.g., via an amino acid linker.

[0110] In certain embodiments, the Fc chain comprises (1) an amino acid residue other than glycine at position 237, and (2) a proline residue at position 329. In some embodiments, the Fc chain comprises an alanine at position 237.

[0111] In some embodiments, the Fc chain is an IgG1 Fc chain and further comprises a mutation or set of mutations at one or more positions selected from 234, 235, 236, 330, and combinations thereof.

[0112] For example, in some embodiments, the Fc chain is an IgG1 Fc chain comprising an alanine at position 234, an alanine at position 235, an arginine at position 236, an alanine at position 237, a proline at position 329, and a leucine at position 330.

[0113] In some embodiments, the Fc chain further comprises a mutation at a position associated with glycosylation, eg, position 297 (eg, by including a glutamine at position 297).

[0114] In some embodiments, the Fc chain comprises a mutation or set of mutations (relative to the corresponding wild-type Fc chain) associated with an altered characteristic as further described herein. By "associated with" it is meant that the mutation or set of mutations has been previously characterized as conferring an altered characteristic (e.g., altered binding to FcRn, altered effector function, etc.) in the context of an antibody, such as an IgG antibody. By "altered" it is meant that the property (e.g., binding to an Fc receptor (e.g., Fcγ receptor or FcRn)) is different from that observed without the mutation or set of mutations.

[0115] For example, in some embodiments, the altered characteristics include altered binding to an Fc receptor.

[0116] In some embodiments, the altered characteristics include altered binding to an Fcγ receptor, e.g., a human FcγR, in some embodiments, the FcγR is a human FcγR selected from the group consisting of hFcγRI, hFcγRIIa, hFcγRIIb, hFcγRIIIa, hFcγRIIIb, and combinations thereof.

[0117] In some embodiments, altered binding comprises no binding or significantly reduced binding in an assay, e.g., an in vitro assay, compared to a corresponding control (e.g., the level of binding typically observed under similar circumstances with the corresponding wild-type chain).

[0118] 3. Antibody fragments capable of binding to DR5 The antibody fragment is typically capable of binding to an epitope within DR5, exemplary sequences of which are shown in SEQ ID NO:18 to SEQ ID NO:21.

[0119] In some embodiments, the antibody fragment is a Fab. In some embodiments, the antibody fragment is a single chain Fab (scFab), e.g., a fusion polypeptide is used that includes both the heavy and light chains of a Fab, optionally linked by a linker (e.g., an amino acid linker disclosed herein).

[0120] In certain embodiments, the antibody fragment comprises a heavy chain variable region (e.g., V H In certain embodiments, the antibody fragment comprises a heavy chain variable domain (e.g., V H ) and a light chain variable domain (e.g., V L or V K In certain embodiments, the antibody fragment comprises a heavy chain variable domain (e.g., V H ) and a light chain variable domain (e.g., V L or V K ) containing Fab.

[0121] In certain embodiments, the antibody fragment does not include any domain from the Fc region, e.g., does not include any CH2 or CH3 domain. In certain embodiments, the antibody fragment that can bind to DR5 is an antibody fragment or derived from any of various DR5 antibodies, including, for example, fully human, humanized, or chimeric DR5 antibodies. The DR5 antibody from which the antibody fragment is obtained or derived can be any of various antibody classes, including, for example, IgG1, IgG2, IgG4 antibodies. In some embodiments, the antibody fragment is obtained or derived from an agonist DR5 antibody, e.g., an agonist humanized DR5 antibody.

[0122] Non-limiting examples of DR5 antibodies include, for example, conatumumab, tigatuzumab, lexatumumab, and dorozitumab.

[0123] In some embodiments, an antibody fragment capable of binding to DR5 comprises heavy and light chain CDRs having sequences similar to those of the heavy and light chain CDRs of the DR5 antibody (e.g., each CDR is identical or has one or two amino acid substitutions).

[0124] In some embodiments, an antibody fragment capable of binding to DR5 comprises: (1) a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3; and (2) a light chain comprising CDR-L1, CDR-L2, and CDR-L3; (a)(i) CDR-H1 has the sequence of SEQ ID NO: 27, or a sequence which differs therefrom by one or two amino acid residues; (ii) CDR-H2 has the sequence of SEQ ID NO: 28 or a sequence which differs therefrom by one or two amino acid residues; (iii) CDR-H3 has the sequence of SEQ ID NO: 29 or a sequence that differs therefrom by one or two amino acid residues; (iv) CDR-L1 has the sequence of SEQ ID NO: 24 or a sequence which differs therefrom by one or two amino acid residues; (v) CDR-L2 has the sequence of SEQ ID NO: 25 or a sequence which differs therefrom by one or two amino acid residues; (vi) CDR-L3 has the sequence of SEQ ID NO: 26 or a sequence which differs therefrom by one or two amino acid residues; (b)(i) CDR-H1 has the sequence of SEQ ID NO: 35, or a sequence which differs therefrom by one or two amino acid residues; (ii) CDR-H2 has the sequence of SEQ ID NO: 36 or a sequence which differs therefrom by one or two amino acid residues; (iii) CDR-H3 has a sequence of SEQ ID NO: 37 or a sequence that differs therefrom by one or two amino acid residues; (iv) CDR-L1 has the sequence of SEQ ID NO: 32 or a sequence which differs therefrom by one or two amino acid residues; (v) CDR-L2 has the sequence of SEQ ID NO: 33 or a sequence which differs therefrom by one or two amino acid residues; (vi) CDR-L3 has the sequence of SEQ ID NO: 34 or a sequence that differs therefrom by one or two amino acid residues; or (c)(i) CDR-H1 has the sequence of SEQ ID NO: 43, or a sequence which differs therefrom by one or two amino acid residues; (ii) CDR-H2 has the sequence of SEQ ID NO: 44 or a sequence which differs therefrom by one or two amino acid residues; (iii) CDR-H3 has the sequence of SEQ ID NO: 45, or a sequence which differs therefrom by one or two amino acid residues; (iv) CDR-L1 has the sequence of SEQ ID NO: 40 or a sequence which differs therefrom by one or two amino acid residues; (v) CDR-L2 has the sequence of SEQ ID NO: 41 or a sequence which differs therefrom by one or two amino acid residues; (vi) CDR-L3 has the sequence of SEQ ID NO: 42 or a sequence which differs therefrom by one or two amino acid residues; or (d)(i) CDR-H1 has the sequence of SEQ ID NO: 51, or a sequence which differs therefrom by one or two amino acid residues; (ii) CDR-H2 has the sequence of SEQ ID NO: 52 or a sequence which differs therefrom by one or two amino acid residues; (iii) CDR-H3 has a sequence of SEQ ID NO: 53 or a sequence that differs therefrom by one or two amino acid residues; (iv) CDR-L1 has the sequence of SEQ ID NO: 48 or a sequence which differs therefrom by one or two amino acid residues; (v) CDR-L2 has the sequence of SEQ ID NO: 49 or a sequence which differs therefrom by one or two amino acid residues; (vi) CDR-L3 has a sequence of SEQ ID NO: 50, or a sequence which differs therefrom by one or two amino acid residues.

[0125] In some embodiments, an antibody fragment capable of binding to DR5 comprises heavy and light chain CDRs that have sequences that are identical to those of the heavy and light chain CDRs of the DR5 antibody, except for a total of one or two amino acid substitutions across all six CDRs.

[0126] In some embodiments, an antibody fragment capable of binding to DR5 comprises heavy and light chain complementarity determining regions (CDRs) having the same sequences as the CDRs of a DR5 antibody.

[0127] In some embodiments, an antibody fragment capable of binding to DR5 comprises: (1) a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3; and (2) a light chain comprising CDR-L1, CDR-L2, and CDR-L3; (a)(i) CDR-H1 has the sequence of SEQ ID NO: 27; (ii) CDR-H2 has the sequence of SEQ ID NO: 28, (iii) CDR-H3 has the sequence of SEQ ID NO: 29, (iv) CDR-L1 has the sequence of SEQ ID NO: 24, (v) CDR-L2 has the sequence of SEQ ID NO: 25; (vi) CDR-L3 has the sequence of SEQ ID NO: 26; (b)(i) CDR-H1 has the sequence of SEQ ID NO: 35; (ii) CDR-H2 has the sequence of SEQ ID NO: 36, (iii) CDR-H3 has the sequence of SEQ ID NO: 37, (iv) CDR-L1 has the sequence of SEQ ID NO: 32, (v) CDR-L2 has the sequence of SEQ ID NO: 33, (vi) CDR-L3 has the sequence of SEQ ID NO: 34; (c)(i) CDR-H1 has the sequence of SEQ ID NO: 43; (ii) CDR-H2 has the sequence of SEQ ID NO: 44; (iii) CDR-H3 has the sequence of SEQ ID NO: 45; (iv) CDR-L1 has the sequence of SEQ ID NO: 40, (v) CDR-L2 has the sequence of SEQ ID NO: 41; (vi) CDR-L3 has the sequence of SEQ ID NO: 42; or (d)(i) CDR-H1 has the sequence of SEQ ID NO: 51; (ii) CDR-H2 has the sequence of SEQ ID NO: 52, (iii) CDR-H3 has the sequence of SEQ ID NO: 53; (iv) CDR-L1 has the sequence of SEQ ID NO: 48, (v) CDR-L2 has the sequence of SEQ ID NO: 49, (vi) CDR-L3 has the sequence of SEQ ID NO:50.

[0128] In some embodiments, the antibody fragment capable of binding to DR5 comprises (1) a heavy chain variable region that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a heavy chain variable region from a DR5 antibody; (2) a light chain variable region that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a light chain variable region from a DR5 antibody.

[0129] In some embodiments, the antibody fragment capable of binding to DR5 comprises (1) Reference V H a heavy chain variable region that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence (2) Reference VL and a light chain variable region that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of (a) Reference V H The sequence has the sequence of SEQ ID NO: 23 and the reference V L The sequence has the sequence of SEQ ID NO: 22 or (b) Reference V H The sequence has the sequence of SEQ ID NO: 31 and the reference V L The sequence has the sequence of SEQ ID NO: 30; or (c) Reference V H The sequence has the sequence of SEQ ID NO: 39 and the reference V L The sequence has the sequence of SEQ ID NO: 38, or (d) Reference V H The sequence has the sequence of SEQ ID NO: 47 and the reference V L The sequence has the sequence of SEQ ID NO:46.

[0130] In embodiments in which more than one type of fusion polypeptide with an antibody fragment is used, the antibody fragments in the various types of fusion polypeptides may be capable of binding to the same epitope on DR5, may be capable of binding to distinct and non-overlapping epitopes on DR5, or may be capable of binding to distinct but overlapping epitopes on DR5.

[0131] 4. Linker In certain embodiments, linkers are used in fusion polypeptides and / or single chain molecules such as scFc. In some embodiments, the linker is an amino acid linker. For example, a linker as used herein can contain about 1 to about 100 amino acid residues, e.g., about 1 to about 70, about 2 to about 70, about 1 to about 30, or about 2 to about 30 amino acid residues. In some embodiments, the linker contains at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid residues.

[0132] In certain embodiments, the linker comprises a glycine serine sequence, e.g., (G n S) m The sequence includes, for example, GGS, GGGS (SEQ ID NO:55), or GGGGS (SEQ ID NO:54).

[0133] B. Self-assembled Polypeptide Complexes In one embodiment, a self-assembled polypeptide complex is provided that includes a plurality of fusion polypeptides disclosed herein. In general, the self-assembled polypeptide complex provided includes: (a) a plurality of first fusion polypeptides, each of which includes (1) an Fc polypeptide and (2) an Fc polypeptide linked to a nanocage monomer or a subunit thereof, and the Fc polypeptide includes an Fc chain having one or more mutations relative to a reference Fc chain of the same Ig class; and (b) a plurality of second fusion polypeptides, each of which includes (1) an antigen-binding antibody fragment and (2) an antigen-binding antibody fragment linked to a nanocage monomer or a subunit thereof.

[0134] In some embodiments, the nanocage monomer is a ferritin monomer and each fusion polypeptide in the self-assembled polypeptide complex comprises a ferritin light chain or a subunit of a ferritin light chain. In these embodiments, the self-assembled polypeptide complex does not comprise any ferritin heavy chain, subunit of a ferritin heavy chain, or other ferritin components capable of binding iron or capable of ferroxidase activity.

[0135] In some embodiments, the nanocage monomer or subunit thereof is a ferritin monomer subunit, and (a) each first fusion polypeptide comprises a ferritin monomer subunit that is a C-half ferritin and each second fusion polypeptide comprises a ferritin monomer subunit that is an N-half ferritin, or (b) each first fusion polypeptide comprises a ferritin monomer subunit that is an N-half ferritin and each second fusion polypeptide comprises a ferritin monomer subunit that is a C-half ferritin.

[0136] In some embodiments, the self-assembling polypeptide complex comprises a total of 24 to 48 fusion polypeptides. In some embodiments, the self-assembling polypeptide complex comprises a total of 24 fusion polypeptides. In some embodiments, the self-assembling polypeptide complex comprises a total of more than 24 fusion polypeptides, for example, at least 26, at least 28, at least 30, at least 32 fusion polypeptides, at least 34 fusion polypeptides, at least 36 fusion polypeptides, at least 38 fusion polypeptides, at least 40 fusion polypeptides, at least 42 fusion polypeptides, at least 44 fusion polypeptides, at least 46 fusion polypeptides, or at least 48 fusion polypeptides. In some embodiments, the self-assembling polypeptide complex comprises about 32 fusion polypeptides.

[0137] In some embodiments, the self-assembled polypeptide complex comprises at least 4, at least 5, at least 6, at least 7, or at least 8 first fusion polypeptides.

[0138] In some embodiments, the self-assembled polypeptide complex comprises at least 4, at least 5, at least 6, at least 7, or at least 8 second fusion polypeptides.

[0139] In some embodiments, the self-assembled polypeptide complex further comprises at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 third fusion polypeptides.

[0140] In some embodiments, the self-assembled polypeptide complex comprises a ratio of about 1:1, 11:13, 3:5, 1:2, 7:17, 1:3, 2:7, 5:19, 1:4, 1:5, 1:6, 1:7, 1:8, 1:12, 1:24 of a first fusion polypeptide to all other fusion polypeptides.

[0141] Pharmacokinetic characteristics In certain embodiments, when administered to a subject in need of a self-assembling polypeptide complex, the provided self-assembling polypeptide complex has one or more pharmacokinetic characteristics similar to that of a reference IgG molecule (e.g., an IgG molecule that matches the class of the Fc chain in the Fc polypeptide of the first fusion polypeptide in the self-assembling polypeptide complex). In some embodiments, the pharmacokinetic characteristics discussed herein (e.g., half-life, AUC, and / or C max ) are achieved when the self-assembling polypeptide complex is administered to a human subject. In some embodiments, the range of pharmacokinetic characteristics discussed herein are achieved when the self-assembling polypeptide complex is administered via a systemic route, e.g., intravenous or subcutaneous administration.

[0142] In some embodiments, the self-assembled polypeptide complexes disclosed herein have a half-life similar to that of a reference IgG molecule. The reference IgG molecule may be, for example, an antibody from which an antigen-binding antibody fragment in a second and / or third fusion polypeptide in a self-assembled polypeptide complex is derived. For example, if an antigen-binding fragment in a second and / or third fusion polypeptide comprises a variable region from "antibody A", then in some embodiments the reference IgG molecule may be "antibody A".

[0143] In some embodiments, after administration to a subject in need of the self-assembling polypeptide complex, the self-assembling polypeptide complex remains viable for about 3 to 35 days, about 3 to about 28 days, about 3 to about 21 days, about 3 to about 14 days, about 3 to about 10 days, about 3 to about 7 days, about 3 to about 5 days, about 5 to about 35 days, about 5 to about 28 days, about 5 to about 21 days, about 5 to about 14 days, about 5 to about 3 ...35 days, about 5 to about 28 days, about 5 to about 21 days, about 5 to about 14 days, about 5 to about about 10 days, about 5 to about 7 days, about 7 to about 35 days, about 7 to about 28 days, about 7 to about 21 days, about 7 to about 14 days, about 7 to about 10 days, about 10 to about 35 days, about 10 to about 28 days, about 10 to about 21 days, about 10 to about 14 days, about 14 to about 35 days, about 14 to about 28 days, about 14 to about 21 days, about 21 to about 35 days, or about 21 to about 28 days. In some embodiments, after administration to a subject in need of the self-assembling polypeptide complex, the self-assembling polypeptide complex has a half-life of at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, or at least 28 days. In some embodiments, after administration to a subject in need thereof, the self-assembled polypeptide complex is detectable in serum at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, or at least 28 days.

[0144] In some embodiments, the self-assembling polypeptide complexes disclosed herein have a bioavailability similar to that of a reference IgG molecule, e.g., an antibody from which a Fab fragment included in the self-assembling polypeptide complex is derived. For example, in some embodiments, after administration to a subject in need of the self-assembling polypeptide complex, the self-assembling polypeptide complex has a bioavailability of about 10 to about 8000 days·μg / mL, about 10 to about 7000 days·μg / mL, about 10 to about 6000 days·μg / mL, about 10 to about 5000 days·μg / mL, about 10 to about 4000 days·μg / mL, about 10 to about 3000 days·μg / mL, about 10 to about 2500 days·μg / mL, about 10 to about 1000 days·μg / mL, about 10 to about 1500 days·μg / mL, about 10 to about 100 0 days μg / mL, about 10 to about 750 days μg / mL, about 10 to about 500 days μg / mL, about 10 to about 400 days μg / mL, about 10 to about 300 days μg / mL, about 10 to about 200 days μg / mL, about 10 to about 100 days μg / mL, about 10 to about 5 0 days μg / mL, about 10 to about 25 days μg / mL, about 25 to about 8000 days μg / mL, about 25 to about 7000 days μg / mL, about 25 to about 6000 days μg / mL, about 25 to about 5000 days μg / mL, about 25 to about 4000 days μg / mL, about 2 5 to approximately 3000 days μg / mL, approximately 25 to approximately 2500 days μg / mL, approximately 25 to approximately 1000 days μg / mL, approximately 25 to approximately 1500 days μg / mL, approximately 25 to approximately 1000 days μg / mL, approximately 25 to approximately 750 days μg / mL, approximately 25 to approximately 500 days μg / mL, about 25 to about 400 days μg / mL, about 25 to about 300 days μg / mL, about 25 to about 200 days μg / mL, about 25 to about 100 days μg / mL, about 25 to about 50 days μg / mL, about 50 to about 8000 days μg / mL, about 50 to about 7000 days μg / mL, about 50 to about 6000 days, μg / mL, about 50 to about 5000 days, μg / mL, about 50 to about 4000 days, μg / mL, about 50 to about 3000 days, μg / mL, about 50 to about 2500 days, μg / mL, about 50 to about 2000 days, μg / mL, about 50 ~1500 days μg / mL, approximately 50 to approximately 1000 days μg / mL, approximately 50 to approximately 750 days μg / mL, approximately 50 to approximately 500 days μg / mL, approximately 50 to approximately 400 days μg / mL, approximately 50 to approximately 300 days μg / mL, approximately 50 to approximately 200 days μg / mL,About 50 to about 100 day·μg / mL, about 100 to about 8000 day·μg / mL, about 100 to about 7000 day·μg / mL, about 100 to about 6000 day·μg / mL, about 100 to about 5000 day·μg / mL, about 100 to about 4000 day·μg / mL, about 100 to about 3000 day·μg / mL, about 100 to about 2500 day·μg / mL, about 100 to about 1000 day·μg / mL, about 100 to about 1500 day·μg / mL, about 100 to about 1000 day·μg / mL, about 100 to about 750 day·μg / mL, about 100 to about 500 day·μg / mL, about 100 to about 400 day·μg / mL, about 100 to about 300 day·μg / mL, about 100 to about 200 day·μg / mL, about 200 to about 8000 day·μg / mL, about 200 to about 7000 day·μg / mL, about 200 to about 6000 day·μg / mL, about 200 to about 5000 day·μg / mL, about 200 to about 4000 day·μg / mL, about 200 to about 3000 day·μg / mL, about 200 to about 2000 day·μg / mL, about 200 to about 1000 day·μg / mL, about 200 to about 1500 day·μg / mL, about 200 to about 1000 day·μg / mL, about 200 to about 750 day·μg / mL, about 200 to about 500 day·μg / mL, about 200 to about 400 day·μg / mL, about 200 to about 300 day·μg / mL, about 300 to about 8000 day·μg / mL, about 300 to about 7000 day·μg / mL, about 300 to about 6000 day·μg / mL, about 300 to about 5000 day·μg / mL, about 300 to about 4000 day·μg / mL, about 300 to about 3000 day·μg / mL, about 300 to about 2500 day·μg / mL, about 300 to about 2000 day·μg / mL, about 300 to about 1500 day·μg / mL, about 300 to about 1000 day·μg / mL, about 300 to about 750 day·μg / mL, about 300 to about 500 day·μg / mL, about 300 to about 400 day·μg / mL, about 400 to about 8000 day·μg / mL, about 400 to about 7000 day·μg / mL, about 400 to about 6000 day·μg / mL, about 400 to about 5000 day·μg / mL, about 400 to about 4000 day·μg / mL, about 400 to about 3000 day·μg / mL, about 400 to about 2500 day·μg / mL, about 400 to about 2000 day·μg / mL, about 400 to about 1500 day·μg / mL, about 400 to about 1000 day·μg / mL, about 400 to about 750 day·μg / mL, about 400 to about 500 day·μg / mL, about 500 to about 8000 day·μg / mL,about 500 to about 7000 day·μg / mL, about 500 to about 6000 day·μg / mL, about 500 to about 5000 day·μg / mL, about 500 to about 4000 day·μg / mL, about 500 to about 3000 day·μg / mL, about 500 to about 2500 day·μg / mL, about 500 to about 2000 day·μg / mL, about 500 to about 1500 day·μg / mL, about 500 to about 1000 day·μg / mL, about 500 to about 750 day·μg / mL, about 750 to about 8000 day·μg / mL, about 750 to about 7000 day·μg / mL, about 750 to about 6000 day·μg / mL, about 750 to about 5000 day·μg / mL, about 750 to about 4000 day·μg / mL, about 750 to about 3000 day·μg / mL, about 750 to about 2500 day·μg / mL, about 750 to about 2000 day·μg / mL, about 750 to about 1500 day·μg / mL, about 750 to about 1000 day·μg / mL, about 1000 to about 8000 day·μg / mL, about 1000 to about 7000 day·μg / mL, about 1000 to about 6000 day·μg / mL, about 1000 to about 5000 day·μg / mL, about 1000 to about 4000 day·μg / mL, about 1000 to about 3000 day·μg / mL, about 1000 to about 2500 day·μg / mL, about 1000 to about 2000 day·μg / mL, about 1000 to about 1500 day·μg / mL, about 1500 to about 8000 day·μg / mL, about 1500 to about 7000 day·μg / mL, about 1500 to about 6000 day·μg / mL, about 1500 to about 5000 day·μg / mL, about 1500 to about 4000 day·μg / mL, about 1500 to about 3000 day·μg / mL, about 1500 to about 2500 day·μg / mL, about 1500 to about 2000 day·μg / mL, about 2000 to about 8000 day·μg / mL, about 2000 to about 7000 day·μg / mL, about 2000 to about 6000 day·μg / mL, about 2000 to about 5000 day·μg / mL, about 2000 to about 4000 day·μg / mL, about 2000 to about 3000 day·μg / mL, about 2000 to about 2500 day·μg / mL, about 2500 to about 8000 day·μg / mL, about 2500 to about 7000 day·μg / mL, about 2500 to about 6000 day·μg / mL, about 2500 to about 5000 day·μg / mL, about 2500 to about 4000 day·μg / mL, about 2500 to about 3000 day·μg / mL, about 3000 to about 8000 day·μg / mL, about 3000 to about 7000 day·μg / mL, about 3000 to about 6000 day·μg / mL,About 3000 to about 5000 days μg / mL, about 3000 to about 4000 days μg / mL, about 4000 to about 8000 days μg / mL, about 400 0 to approximately 7000 days μg / mL, approximately 4000 to approximately 6000 days μg / mL, approximately 4000 to approximately 5000 days μg / mL, approximately 5000 to approximately 80 It has an area under the curve (AUC) of 00 days·μg / mL, about 5000 to about 7000 days·μg / mL, about 5000 to about 6000 days·μg / mL, about 6000 to about 8000 days·μg / mL, about 6000 to about 7000 days·μg / mL, or about 7000 to about 8000 days·μg / mL. In some embodiments, after administration to a subject in need thereof, the self-assembling polypeptide complex has an AUC of at least 10 days·μg / mL, at least 25 days·μg / mL, at least 50 days·μg / mL, at least 100 days·μg / mL, at least 200 days·μg / mL, at least 300 days·μg / mL, at least 400 days·μg / mL, at least 500 days·μg / mL, at least 750 days·μg / mL, at least 1000 days·μg / mL, at least 1500 days·μg / mL, at least 2000 days·μg / mL, at least 2500 days·μg / mL, at least 3000 days·μg / mL, at least 4000 days·μg / mL, at least 5000 days·μg / mL, at least 6000 days·μg / mL, at least 7000 days·μg / mL, or at least 8000 days·μg / mL.

[0145] In some embodiments, the self-assembling polypeptide complexes disclosed herein have a bioavailability similar to that of a reference IgG molecule. For example, in some embodiments, after administration to a subject in need thereof, the self-assembling polypeptide complex has a bioavailability of about 10 μg / mL to about 750 mg / mL, about 25 μg / mL to about 750 mg / mL, about 50 μg / mL to about 750 mg / mL, about 75 μg / mL to about 750 mg / mL, about 100 μg / mL to about 750 mg / mL, about 250 μg / mL to about 750 mg / mL, about 500 μg / mL to about 750 mg / mL, about 750 μg / mL to about 750 mg / mL, about 1 mg / mL to about 750 mg / mL, or about 2 mg / mL to about 750 mg / mL. mL, about 10 mg / mL to about 750 mg / mL, about 25 mg / mL to about 750 mg / mL, about 50 mg / mL to about 750 mg / mL, about 75 mg / mL to about 750 mg / mL, about 100 mg / mL to about 750 mg / mL, about 250 mg / mL to about 750 mg / mL mL, about 500mg / mL to about 750mg / mL, about 10μg / mL to about 500mg / mL, about 25μg / mL to about 500mg / mL, about 50μg / mL to about 500mg / mL, about 75μg / mL to about 500mg / mL, about 100μg / mL to about 500mg / m L, about 250 μg / mL to about 500 mg / mL, about 500 μg / mL to about 500 mg / mL, about 750 μg / mL to about 500 mg / mL, about 1 mg / mL to about 500 mg / mL, about 10 mg / mL to about 500 mg / mL, about 25 mg / mL to about 500 mg / m L, about 50 mg / mL to about 500 mg / mL, about 75 mg / mL to about 500 mg / mL, about 100 mg / mL to about 500 mg / mL, about 250 mg / mL to about 500 mg / mL, about 10 μg / mL to about 250 mg / mL, about 25 μg / mL to about 250 mg / mL , about 50μg / mL to about 250mg / mL, about 75μg / mL to about 250mg / mL, about 100μg / mL to about 250mg / mL, about 250μg / mL to about 250mg / mL, about 500μg / mL to about 250mg / mL, about 750μg / mL to about 250mg / mL mL, about 1 mg / mL to about 250 mg / mL, about 10 mg / mL to about 250 mg / mL, about 25 mg / mL to about 250 mg / mL, about 50 mg / mL to about 250 mg / mL, about 75 mg / mL to about 250 mg / mL, about 100 mg / mL to about 250 mg / mL,from about 10 μg / mL to about 100 mg / mL, from about 25 μg / mL to about 100 mg / mL, from about 50 μg / mL to about 100 mg / mL, from about 75 μg / mL to about 100 mg / mL, from about 100 μg / mL to about 100 mg / mL, from about 250 μg / mL to about 100 mg / mL, from about 500 μg / mL to about 100 mg / mL, from about 750 μg / mL to about 100 mg / mL, from about 1 mg / mL to about 100 mg / mL, from about 10 mg / mL to about 100 mg / mL, from about 25 mg / mL to about 100 mg / mL, from about 50 mg / mL to about 100 mg / mL, from about 75 mg / mL to about 100 mg / mL, from about 10 μg / mL to about 75 mg / mL, from about 25 μg / mL to about 75 mg / mL, from about 50 μg / mL to about 75 mg / mL, from about 75 μg / mL to about 75 mg / mL, from about 100 μg / mL to about 75 mg / mL, from about 250 μg / mL to about 75 mg / mL, from about 500 μg / mL to about 75 mg / mL, from about 750 μg / mL to about 75 mg / mL, from about 1 mg / mL to about 75 mg / mL, from about 10 mg / mL to about 75 mg / mL, from about 25 mg / mL to about 75 mg / mL, from about 50 mg / mL to about 75 mg / mL, from about 10 μg / mL to about 50 mg / mL, from about 25 μg / mL to about 50 mg / mL, from about 50 μg / mL to about 50 mg / mL, from about 75 μg / mL to about 50 mg / mL, from about 100 μg / mL to about 50 mg / mL, from about 250 μg / mL to about 50 mg / mL, from about 500 μg / mL to about 50 mg / mL, from about 750 μg / mL to about 50 mg / mL, from about 1 mg / mL to about 50 mg / mL, from about 10 mg / mL to about 50 mg / mL, from about 25 mg / mL to about 50 mg / mL, from about 10 μg / mL to about 25 mg / mL, from about 25 μg / mL to about 25 mg / mL, from about 50 μg / mL to about 25 mg / mL, from about 75 μg / mL to about 25 mg / mL, from about 100 μg / mL to about 25 mg / mL, from about 250 μg / mL to about 25 mg / mL, from about 500 μg / mL to about 25 mg / mL, from about 750 μg / mL to about 25 mg / mL, from about 1 mg / mL to about 25 mg / mL, from about 10 mg / mL to about 25 mg / mL, from about 10 μg / mL to about 10 mg / mL, from about 25 μg / mL to about 10 mg / mL, from about 50 μg / mL to about 10 mg / mL, from about 75 μg / mL to about 10 mg / mL, from about 100 μg / mL to about 10 mg / mL, from about 250 μg / mL to about 10 mg / mL, from about 500 μg / mL to about 10 mg / mL, from about 750 μg / mL to about 10 mg / mL,Approximately 1 mg / mL to approximately 10 mg / mL, approximately 10 μg / mL to approximately 1 mg / mL, approximately 25 μg / mL to approximately 1 mg / mL, approximately 50 μg / mL to approximately 1 mg / mL, approximately 75 μg / mL to approximately 1 mg / mL, approximately 1 00μg / mL ~ approx. 1mg / mL, approx. 250μg / mL ~ approx. 1mg / mL, approx. 500μg / mL ~ approx. 1mg / mL, approx. 750μg / mL ~ approx. 1mg / mL, approx. 10μg / mL ~ approx. 750μg / m L, about 25μg / mL to about 750μg / mL, about 50μg / mL to about 750μg / mL, about 75μg / mL to about 750μg / mL, about 100μg / mL to about 750μg / mL, about 250μg / m L ~ approx. 750 μg / mL, approx. 500 μg / mL ~ approx. 750 μg / mL, approx. 10 μg / mL ~ approx. 500 μg / mL, approx. 25 μg / mL ~ approx. 500 μg / mL, approx. 50 μg / mL ~ approx. 500 μg / mL , about 75μg / mL to about 500μg / mL, about 100μg / mL to about 500μg / mL, about 250μg / mL to about 500μg / mL, about 10μg / mL to about 250μg / mL, about 25μg / mL ~about 250μg / mL, about 50μg / mL to about 250μg / mL, about 75μg / mL to about 250μg / mL, about 100μg / mL to about 250μg / mL, about 10μg / mL to about 100μg / mL, A maximum concentration (C,) of about 25 μg / mL to about 100 μg / mL, about 50 μg / mL to about 100 μg / mL, about 75 μg / mL to about 100 μg / mL, about 10 μg / mL to about 75 μg / mL, about 25 μg / mL to about 75 μg / mL, about 50 μg / mL to about 75 μg / mL, about 10 μg / mL to about 50 μg / mL, about 25 μg / mL to about 50 μg / mL, or about 10 μg / mL to about 25 μg / mL max In some embodiments, after administration to a subject in need thereof, the self-assembling polypeptide complex has a maximum concentration (C) of at least 10 μg / mL, at least 25 μg / mL, at least 50 μg / mL, at least 100 μg / mL, at least 250 μg / mL, at least 500 μg / mL, at least 750 μg / mL, at least 1 mg / mL, at least 10 mg / mL, at least 25 mg / mL, at least 50 mg / mL, at least 75 mg / mL, at least 100 mg / mL, at least 250 mg / mL, at least 500 mg / mL, or at least 750 mg / mL.max ).

[0146] effect In certain embodiments, the provided self-assembled polypeptide complexes can induce multimerization (eg, trimerization) of DR5 receptors on target cells (eg, cancer cells).

[0147] In some embodiments, administration of the self-assembling polypeptide complex disclosed herein to a subject results in an improvement in a clinical outcome or metric in the subject. For example, in a subject with a tumor, administration of the self-assembling polypeptide complex can cause tumor inhibition or delay in progression, such as tumor regression. In some embodiments, administration of the self-assembling polypeptide complex results in complete tumor regression.

[0148] C. Treatment method In one aspect, methods are provided that may be useful for treating, alleviating, or preventing a disease or condition (e.g., an infectious disease, cancer, or an autoimmune disease) generally comprising administering to a subject a composition comprising a self-assembling polypeptide complex of the present disclosure.

[0149] In some embodiments, the subject is a mammal, such as a human.

[0150] Compositions for administration to a subject generally comprise the self-assembling polypeptide complexes disclosed herein. In some embodiments, such compositions further comprise a pharma- ceutically acceptable excipient.

[0151] The compositions can be formulated for administration by any of a variety of routes, including systemic routes (eg, oral, inhalation, intranasal, intravenous, intraperitoneal, subcutaneous, or intramuscular administration).

[0152] In some embodiments, the administering step results in an improvement in one or more clinical outcomes or metrics in the subject.

[0153] In some embodiments, the administering step results in a slowing or inhibition of tumor progression, e.g., regression of the tumor. In some embodiments, the administering step results in complete regression of the tumor. EXAMPLES

[0154] Example 1. Construction and Expression of Representative DR5-Targeted Multabodies This example illustrates a method for producing a ferritin light chain comprising: (1) a human ferritin light chain or a subunit thereof; and (2) a glycerol-binding domain (Gly) as described herein. n -Ser) m The generation of DR5-targeting Multabodies (MBs) comprising combinations of fusion proteins comprising a single-chain Fab (scFab) or a single-chain Fc-dimer (scFc) fused via a linker, such as an amino acid linker, is described.

[0155] Genetically encoded fusion proteins, (1) scFab of anti-DR5 antibody (in this case, conatumumab) fused to the N-terminus of hFTL (aDR5-hFTL, SEQ ID NO: 9), (2) scFab of anti-DR5 antibody fused to the N-terminus of N-half ferritin (aDR5-N_hFTL, SEQ ID NO: 11), and (3) scFc fused to the N-terminus of C-half ferritin (various scFc-C_hFTL constructs described further below), were prepared, mixed in a 2:1:1 molar ratio, and transiently transfected into ExpiCHO-S cells for the production and formation of DR5-targeted MBs. See FIG. 1B.

[0156] The scFc-ferritin fusion proteins contained wild-type (WT) or engineered IgG1 Fc chains. The engineered IgG1 Fc chains contained various combinations of L234A, L235A, G236R, G237A, P329G, and A330L mutations, as shown in Table 1 below. The numbering in Table 1 follows the EU numbering scheme.

[0157] Table 1. Residues at certain positions within the IgG1 Fc chain used in the multibody and corresponding sequence numbers of the scFc-C_hFTL construct In the rows corresponding to a set of engineered Fc chains, residues are shown only at positions that differ from the wild-type residue. [Table 1]

[0158] Example 2. Target Binding of Multabody Determined by Biolayer Interferometry The binding kinetics and affinity of exemplary MBs generated as described in Example 1 to recombinant human DR5 (hDR5), cynomolgus monkey DR5 (cDR5), mouse DR5 (mDR5), and rat DR5 (rDR5) were determined by Biolayer Interferometry (BLI) using an Octet RED96 instrument. Binding characteristics were determined for (1) conatumumab, a fully human monoclonal IgG1 antibody that binds to DR5, and (2) Cona MB IgG1 LLRAL, a multibody that comprises a fusion polypeptide containing an scFc with the "LLRAL" mutations listed in Table 1, and also comprises a fusion polypeptide containing an scFab derived from conatumumab.

[0159] Briefly, Ni-NTA biosensors were coated with hDR5-His, cDR5-His, mDR5-His, or rDR5-His (extracellular domains of hDR5, cDR5, mDR5, or rDR5 with a C-terminal polyhistidine tag) to reach a signal response of 0.8 nm. The coated biosensors were immersed in wells containing serial dilutions of test MBs (20-10-5-2.5-1.25-0.63 nM) in PBS-0.02%T-0.01%BSA (PBS supplemented with 0.02% (v / v) Tween® 20 and 0.01% (w / v) BSA) for 180 s (association phase), and then in PBS-0.02%T-0.01%BSA for 180 s (dissociation phase). All measurements were performed at 30°C in PBS-0.02%T-0.01%BSA, pH 7.4, with a shaking speed of 1000 rpm and monitored in real time. During the experiment, 10 mM glycine, pH 1.7, was applied four times for 5 seconds, followed by 10 mM NiSO. 4The biosensor was regenerated by refilling with for 1 min.

[0160] The maximum association binding response at the end of the association phase, the dissociation rate (k off ), and / or the equilibrium dissociation constant (K D ) Target binding was assessed based on the maximum association binding response below 0.1 nm at 20 nM test MB was classified as "non-binding."

[0161] k on , k off , and the resulting K D The values ​​determined for are summarized in Table 2. Cona MB IgG1 LLRAL, which bound to human and cynomolgus DR5 with approximately sub-picomolar affinity, showed no binding to mouse or rat DR5. (The instrument has a detection limit of 1 picomolar.) These observations are consistent with the binding profile of conatumumab ("Cona"). [Table 2]

[0162] Example 3. Binding specificity of Multabody determined by biolayer interferometry BLI was used to assess non-specific binding of exemplary DR-targeted MBs to relevant tumor necrosis factor receptor superfamily (TNFRSF) members.

[0163] The experiments were performed similarly as described in Example 2, except that His-tagged extracellular domains of human DR4, human osteoprotegerin (OPG), human decoy receptor 1 (DcR1), or human DcR2-hDR4-His, hOPG-His, hDcR1-His, or hDcR2-His were coated onto Ni-NTA biosensors and titrated with test MBs at various concentrations.

[0164] Figures 2A, 2B, 2C, 2D, and 2E show representative examples of relevant segments of the resulting sensorgrams. Cona MB IgG1 LLRAL bound to DR5 with high specificity and showed no binding to other tested TNFRSF members.

[0165] Example 4. Binding of Multabody to Fc Receptors Determined by Biolayer Interferometry The binding kinetics and affinity of different DR5-targeted MBs (each containing a different set of Fc mutations and generated as described in Example 1) to different Fc receptors was determined by BLI.

[0166] All MBs tested in this example contained a polypeptide containing an scFab derived from conatumumab that binds to DR5. These DR5-targeted MBs ("Cona MBs") also contain polypeptides with scFc with either a wild-type Fc chain (IgG1 wt) or an Fc chain with a certain combination of mutations (IgG1 LLRAL). (See Table 1)

[0167] Binding to the following human, cynomolgus monkey, and mouse Fc receptors was determined: human Fc gamma receptor type I (hFcγRI), hFcγRIIa, hFcγRIIb, hFcγRIIIa, hFcγRIIIb, human neonatal Fc receptor (hFcRn), cynomolgus monkey FcγRI (cFcγRI), cFcγRIIa, cFcγRIIb, cFcγRIII, cFcRn, mouse FcγRI (mFcγRI), mFcγRIIb, mFcγRIII, mFcγRIV, and mFcRn.

[0168] The experiment was performed similarly as described in Example 2, except that His-tagged Fc receptors were coated onto Ni-NTA biosensors and titrated with test MBs at various concentrations. To assess the potential for MBs to undergo endosomal recycling, binding to FcRn was measured at pH 6.0 for association and pH 7.4 for dissociation.

[0169] Table 3 shows the k determined for MBs binding to various FcγRs. on , k off , and K D The values ​​are summarized: Cona MB with wild type IgG1 Fc (IgG1 WT) bound to human, cynomolgus, and mouse FcγR. Cona MB with IgG1 LLRAL showed reduced binding to human, cynomolgus, and mouse FcγR.

[0170] Figure 3 shows representative examples of relevant segments of sensorgrams obtained from FcRn binding studies. All tested MBs bound to human, cynomolgus, and mouse FcRn at pH 6.0 and dissociated from the receptor at pH 7.4. Cona MB IgG1 LLRAL dissociated from human or cynomolgus FcRn at pH 7.4 at a rate comparable to Cona MB IgG1 WT. [Table 3-1] [Table 3-2]

[0171] Example 5. Evaluation of in vitro tumor cytotoxicity The cytotoxicity of exemplary DR5-targeted MBs (generated as described in Example 1) was evaluated using different tumor cell lines. Cona MB IgG1 wt and Cona MB IgG1 LLRAL were tested against COLO205 (human colon cancer), HCT-15 (human colon cancer), NCI-H2122 (human lung cancer), SNU-5 (human gastric cancer), Capan-1 (human pancreatic cancer), MDA-MB-231 (invasive ductal carcinoma), BxPC-3 (human pancreatic cancer), and NCI-H2228 (human lung cancer) cell lines.

[0172] Tumor cell lines were seeded at 5000 cells per well in 96-well plates and incubated overnight to facilitate binding. The next day, cells were treated with serial dilutions of DR5-targeted MB or anti-DR5 (conatumumab) and incubated at 37°C for 24 hours. Cell viability was measured using the CellTiter-Glo Luminescence Cell Viability Assay (Promega) according to the manufacturer's instructions. Briefly, assay reagent (100 μL) was added to 100 μL of cells at room temperature, mixed at 500 rpm for 2 minutes using a plate shaker, and incubated at room temperature for 10 minutes. Luminescence signals were read using a Synergy Neo2 Multi-Mode Assay Microplate Reader (BioTek Instruments).

[0173] Figure 4 shows the results from these experiments. In Figure 4, the percentage of viable cells (compared to vehicle-treated tumor cells) is plotted on the y-axis against the test molecule concentration on the x-axis. Data were fitted using non-linear regression (log inhibitor vs. response, variable slope, 4 parameters) using Prism 9.1.2 software (GraphPad). Table 4 shows the resulting IC 50 Values ​​are provided for the DR5-targeted MBs tested (Cona MB IgG1 wt and Cona MB IgG1 LLRAL) were both able to induce cytotoxicity in human cancer cell lines, whereas the IgG1 antibody conatumumab did not or only very poorly induced tumor cytotoxicity. [Table 4]

[0174] Example 6. Pharmacokinetics of Multabody in Mice The pharmacokinetics (PK) of exemplary DR5-targeted MBs (Cona MB IgG1 LLRAL and conatumumab) generated as described in Example 1 were analyzed in 8-week-old female BALB / c SCID mice (Jackson Labs).

[0175] For single-dose PK studies, mice received a single bolus injection of 200 μL of test molecule at 5 mg / kg intraperitoneally (ip) or intravenously (iv) (N=8 mice per group, N=4 mice per time point). Blood samples of 50-100 μL were collected at 3 hours, 24 hours, 48 ​​hours, 72 hours, 5 days, 7 days, 15 days, and 21 days.

[0176] For multiple dose PK studies, mice were injected with 200 μL of test molecule at 5 mg / kg on day 9, followed by a second injection of 200 μL of test molecule at 5 mg / kg 96 hours later. Blood samples of 50-100 μL were collected 3, 24, 48, 72, and 96 hours after the first dose, and 3, 24, 48, 72 hours, 5, 7, and 14 days after the second dose.

[0177] Blood samples were collected from the saphenous vein into heparin-coated tubes and finger vortexed to ensure mixing, after which samples were centrifuged at 1500×g for 15 min at 4° C. Plasma samples were collected and immediately stored at −80° C. until use in the ELISA assay.

[0178] To measure plasma drug levels by ELISA, recombinant DR5 at 2 μg / mL in PBS was coated onto Maxisorp plates (Fisher Scientific) overnight at 4° C. After washing twice with PBS-0.05%T (PBS supplemented with 0.05% (v / v) Tween®-20), plates were blocked with 3% (w / v) BSA in PBS for 1 h at room temperature and then washed twice with PBS-0.05%T. Plasma samples were diluted in PBS-0.05%T-0.5%BSA (PBS supplemented with 0.05% (v / v) Tween®-20 and 0.5% (w / v) BSA), added to wells and incubated for 1 h at room temperature and 500 rpm, followed by another washing step with PBS-0.05%T. Bound molecules were detected by incubation with goat polyclonal anti-human Fc-HRP secondary antibody (Jackson Immunoresearch) diluted 1:10000. After an additional washing step with PBS-0.05%T, the OptEIA™ TMB substrate reagent set (BD Biosciences) was used for detection according to the manufacturer's instructions, and the absorbance was read at 450 nm using a Synergy Neo2 Multi-Mode Assay Microplate Reader (BioTek Instruments). A calibration curve was prepared using dilutions of the test molecules in PBS-0.05%T-0.5%BSA.

[0179] FIG. 5A is a schematic showing the design of these pharmacokinetic studies.

[0180] FIG. 5B shows plots of plasma concentrations over time in a single-dose PK study of Cona MB IgG1 wt, Cona MB IgG1 LLRAL, and conatumumab ("Cona"). Table 5 presents a summary of the calculated half-lives. Cona MB IgG1 LLRAL exhibited significantly enhanced PK properties compared to Cona MB IgG1 wt. Over the first 7 days, plasma concentrations of Cona MB IgG1 LLRAL remained comparable to those of conatumumab. Cona MB IgG1 LLRAL plasma concentrations on day 15 were significantly higher than the IC200 determined in an in vitro cytotoxicity assay (see Example 5). 50 IC value (approximately 0.04 μg / mL) and on day 21 50 It was 40 times the value.

[0181] FIG. 5C shows plots of plasma concentrations over time in a multiple dose PK study for Cona MB IgG1 LLRAL and conatumumab. [Table 5]

[0182] Example 7. Therapeutic Efficacy of DR5-Targeted Multabody in Xenograft Mouse Model The therapeutic efficacy of the exemplary Multabody was evaluated in a colon cancer xenograft model. Figure 6A shows a schematic diagram illustrating the study design of the in vivo efficacy study.

[0183] 5×10 6 COLO205 cells were injected subcutaneously into the flank of BALB / c SCID mice (n=12 per group). Tumors were grown to 200 mm 3Tumors were allowed to grow to an average size of 100 μg / kg. Mice were divided into treatment or control groups so that the average tumor volume was equal between groups (Figure 6B). Mice received treatment (5 mg / kg per dose) or control via intraperitoneal (ip) injection once or twice weekly for 2 weeks (Figure 6A). Tumor volumes were measured twice weekly using calipers. Blood samples were collected 24 hours, 7 days, 14 days, and 21 days after the first dose, and drug levels in plasma were measured as described in Example 6.

[0184] Figures 6B-6G are plots showing the tumor volumes of mice in the various groups. The plots in Figures 6B and 6D-6G show the tumor volumes over time, and the plot in Figure 6C shows the tumor volumes at 88 days after treatment initiation. Treatment with Cona MB IgG1 LLRAL once or twice weekly significantly inhibited the growth of large established tumors, suggesting that Cona MB IgG1 LLRAL was able to invade tumors even at their large size. Cona MB IgG1 LLRAL inhibited tumor growth more strongly than conatumumab, resulting in 9 / 12 and 11 / 12 complete regressions in the once weekly and twice weekly treatment groups, respectively. This compares to only 1 / 12 complete remission in the conatumumab treatment group.

[0185] Figure 6H is a plot showing the concentration of the test molecule in plasma (y-axis) as a function of time after the first dose (x-axis). The pharmacokinetic profile of Cona MB IgG1 LLRAL observed in the COLO205 colon cancer mouse model was similar to that observed in the multiple dose PK study described in Example 6. (See Figure 5B). These results suggest that the presence of tumors does not affect the clearance of the Multabody.

[0186] These experiments were repeated in a second study with the same study design using vehicle, conatumumab, or Cona MB IgG1 LLRAL at 5 mg / kg per dose once per week for 2 weeks. The results of this second study were similar to those of the first study, with no tumor regression observed in the vehicle group, none of the 10 mice in the conatumumab group showing complete tumor regression, and all 10 mice in the Cona MB IgG1 LLRAL group showing complete tumor regression (data not shown). The results of this second study indicate that the therapeutic effect of Cona MB IgG1 LLRAL is highly reproducible with weekly dosing.

[0187] Example 8. Dose-ranging efficacy of DR5-targeted multibodies in xenograft mouse models The dose ranging efficacy of Cona IgG1 MB LLRAL was also investigated in the same colon cancer xenograft model used in Example 6.

[0188] 5×10 6 COLO205 cells were injected subcutaneously into the flank of Balb / c SCID mice. Tumors were grown to 200 mm 3 Tumors were allowed to grow to an average size of 100 x 100 cm. Mice were sorted into one of five groups (vehicle or Cona MB IgG1 LLRAL at doses of 0.1 mg / kg, 0.25 mg / kg, 1 mg / kg, or 5 mg / kg) so that the average tumor volume was equal across each group. Mice received treatment or vehicle via intraperitoneal (ip) injection once a week for three weeks. Tumor volumes were measured twice a week using calipers. Blood samples were taken throughout the experiment.

[0189] FIG. 7A is a plot showing the tumor volumes of mice in various groups over time after the first dose. "MB" indicates the Cona MB IgG1 LLRAL group. FIGS. 7B-7F show the tumor volumes of individual mice within the vehicle (FIG. 7B) and 5 mg / kg, 1 mg / kg, 0.25 mg / kg, and 0.1 mg / kg Cona MB IgG1 LLRAL treatment groups (FIGS. 7C, 7D, 7E, and 7F, respectively). As shown in FIG. 7A, mice treated with Cona MB IgG1 LLRAL at doses of 0.25 mg / kg, 1 mg / kg, and 5 mg / kg showed significant improvement over the vehicle group. Cona MB IgG1 LLRAL initially appeared to have similar efficacy at doses of 1 mg / kg and 5 mg / kg. However, 1 mg / kg showed less complete responses and more regrowth than the 5 mg / kg treatment group, which saw complete regression in 12 of 12 mice.

[0190] Figure 7G is a plot showing the amount of Cona MB IgG1 LLRAL detectable in blood samples collected at various time points after the first dose was administered. Cona MB IgG1 LLRAL was detectable in plasma at all time points tested in the 0.1 mg / kg, 0.25 mg / kg, 1 mg / kg, and 5 mg / kg Cona MB IgG1 LLRAL treatment groups, and pharmacokinetics appeared linear in all dose groups.

[0191] These results demonstrate that Cona MB IgG1 LLRAL demonstrated therapeutic efficacy at multiple doses tested, including comparable efficacy at dose levels 5-fold lower than those tested in Example 6. However, while complete tumor regression was observed at the 5 mg / kg dose level, some tumor regrowth was observed when the dose level was reduced to 1 mg / kg.

[0192] Example 9. Induction of invasion and apoptosis in large tumors by DR5-targeted multibody Exemplary Multabodies generated as described in Example 1 were evaluated for their ability to invade large tumors and induce apoptosis in a colon xenograft model.

[0193] 5×10 6 COLO205 cells were injected subcutaneously into the flank of Balb / c SCID mice. Tumors were grown to 500 mm 3 Tumor volumes were measured twice weekly using calipers. Mice were cultured until the mean tumor size was identical across the two groups, approximately 500 mm in each group. 3 Mice were then randomly assigned to vehicle or treatment groups such that the dose was 0.01 mg / kg. Mice were then administered a single dose of vehicle or 5 mg / kg of Cona MB IgG1 LLRAL by intraperitoneal (ip) injection. Blood samples were collected 24 hours after injection of vehicle or Cona MB IgG1 LLRAL, and tumors were harvested for subsequent histological analysis. Figure 8 shows a schematic and timeline of these experiments.

[0194] Tumors were formalin fixed, embedded in paraffin, and sectioned. Tumor sections were immunohistochemically stained for cleaved caspase-3, a marker of apoptosis. Representative images of stained tissue sections are shown in Figures 9A and 9C (vehicle-treated mice) and Figures 9B and 9D (Cona MB IgG1 LLRAL-treated mice). As shown in these figures, few apoptotic cells were detected in tumor sections from vehicle-treated mice. In contrast, tissue sections from Cona MB IgG1 LLRAL-treated mice contained the majority of apoptotic cells throughout the tumor, including deep within the core of the tumor away from the tumor margins (see stained cells in Figures 9B and 9D).

[0195] These results confirm that Cona MB IgG1 LLRAL induced apoptosis of tumor cells throughout the tumor. Furthermore, these results confirm that the multibody is able to penetrate the core of large established tumors. Sequence Listing The underlines in the fusion sequences indicate linker sequences. The bold text within the fusion sequence indicates ferritin or ferritin subunit sequences. Within the variable region sequences, the underlining and bolding together indicate the sequences of the complementarity determining regions. The boxed and bolded residues indicate residues that were mutated relative to the reference molecule, e.g., IgG1 Fc. Sequence number 1 hFTL MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD Sequence number 2 N_hFTL MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEW Sequence number 3 C_hFTL GKTPDAMKAAMALEKKLNQALLLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD SEQ ID NO:4 IgG1 Fc DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:5 IgG1 scFc [ka] SEQ ID NO:6 Cona LC EIVLTQSPGTLSLSPGERATLSCRASQGISRSYLAWYQQKPGQAPSLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFGSSPWTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:7 Cona HC QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYFWSWIRQLPGKGLEWIGHIHNSGTTYYNPSLKSRVTISVDTSKKQFSLRLSSVTAADTAVYYCARDRGGDYYYGMDVWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:8 Cona scFab [ka] SEQ ID NO:9 aDR5-hFTL [ka] SEQ ID NO: 10 aDR5-hFTL, alternative sequence [ka] SEQ ID NO:11 aDR5-N_hFTL [ka] SEQ ID NO: 12 aDR5-N_hFTL, alternative sequence [ka] SEQ ID NO: 13 scFc-C_hFTL IgG1 WT [ka] SEQ ID NO: 14 scFc-C_hFTL IgG1 LALA [ka] SEQ ID NO: 15 scFc-C_hFTL IgG1 LALAP [ka] SEQ ID NO: 16 scFc-C_hFTL IgG1 LLRAL [ka] SEQ ID NO: 17 scFc-C_hFTL IgG1 LLGRAL [ka] SEQ ID NO: 18 Human DR5 (GenBank Accession No. AAB67109.1) [ka] SEQ ID NO: 19 Human DR5 (GenBank Accession No. AAB71412.1) [ka] SEQ ID NO: 20 Human DR5 (GenBank Accession No. AAC01565.1) [ka] SEQ ID NO: 21 Human DR5 (GenBank Accession No. AAB67103.1) [ka] SEQ ID NO: 22 V from conatumumab L [ka] SEQ ID NO: 23 V from conatumumab H [ka] SEQ ID NO: 24 CDR-L1 from conatumumab RASQGISRSYLA SEQ ID NO: 25 CDR-L2 from conatumumab GASSRAT SEQ ID NO: 26 CDR-L3 from conatumumab QQFGSSPWT SEQ ID NO: 27 CDR-H1 from conatumumab GGSISSGDYFWS SEQ ID NO: 28 CDR-H2 from conatumumab HIHNSGTTYYNPSLKS SEQ ID NO: 29 CDR-H3 from conatumumab DRGGDYYYGMDV SEQ ID NO: 30 V from tigatuzumab L [ka] SEQ ID NO: 31 V from tigatuzumab H [ka] SEQ ID NO: 32 CDR-L1 from tigatuzumab KASQDVGTAVA SEQ ID NO: 33 CDR-L2 from tigatuzumab WASTRHT SEQ ID NO: 34 CDR-L3 from tigatuzumab QQYSSYRT SEQ ID NO: 35 CDR-H1 from tigatuzumab GFTFSSYVMS SEQ ID NO: 36 CDR-H2 from tigatuzumab TISSGGSYTYYPDSVKG SEQ ID NO: 37 CDR-H3 from tigatuzumab RGDSMITTDY SEQ ID NO: 38 V from Dorozitumab L [ka] SEQ ID NO: 39 V from Dorozizumab H [ka] SEQ ID NO: 40 CDR-L1 from Drozizumab SGDSLRSYYAS SEQ ID NO: 41 CDR-L2 from dorozizumab GANNRPS SEQ ID NO: 42 CDR-L3 from dorozizumab NSADSSGNHVV SEQ ID NO: 43 CDR-H1 from Drozitumab GFTFDDYAMS SEQ ID NO: 44 CDR-H2 from Drozitumab INWQGGSTGYADSVKG SEQ ID NO: 45 CDR-H3 from Drozitumab ILGAGRGWYFDY SEQ ID NO: 46 V from lexatumumab L [ka] SEQ ID NO: 47 V from lexatumumab H [ka] SEQ ID NO: 48 CDR-L1 from lexatumumab QGDSLRSYYAS SEQ ID NO: 49 CDR-L2 from lexatumumab GKNNRPS SEQ ID NO: 50 CDR-L3 from lexatumumab NSRDSSGNHVV SEQ ID NO: 51 CDR-H1 from lexatumumab GFTFDDYGMS SEQ ID NO: 52 CDR-H2 from lexatumumab INWNGGSTGYADSVKG SEQ ID NO: 53 CDR-H3 from lexatumumab ILGAGRGWYFDL SEQ ID NO:54 GS linker sequence GGGGS SEQ ID NO: 55 GS linker sequence GGGS

[0196] Equivalents / Alternative Embodiments While the invention has been described in relation to specific embodiments thereof, it will be understood that further modifications are possible, and this application is intended to cover generally any variations, uses, or adaptations of the invention in accordance with the principles of the invention, including such departures from the present disclosure that are within known or customary practice in the art to which this invention pertains, and may be applied to the essential features of the invention as described hereinabove.

Claims

1. 1. A self-assembling polypeptide complex comprising: (a) a plurality of first fusion polypeptides, each first fusion polypeptide comprising: (1) an Fc polypeptide, and (2) the plurality of first fusion polypeptides comprising nanocage monomers or subunits thereof; (b) a plurality of second fusion polypeptides, each second fusion polypeptide comprising: (1) an antibody fragment capable of binding to DR5, and (2) the plurality of second fusion polypeptides comprising nanocage monomers or subunits thereof; the Fc polypeptide comprises an IgG1 Fc chain, the IgG1 chain being, according to EU numbering: (1) an amino acid residue other than glycine at position 237, and (2) The self-assembled polypeptide complex, which contains a proline residue at position 329.

2. The self-assembled polypeptide complex of claim 1, wherein the nanocage monomer or subunit thereof in each first fusion polypeptide and each second fusion polypeptide is a ferritin monomer or subunit thereof.

3. The self-assembled polypeptide complex of claim 2 , wherein the ferritin monomer or subunit thereof is a ferritin light chain or subunit thereof.

4. The self-assembled polypeptide complex of claim 2, which does not contain any ferritin heavy chain or ferritin heavy chain subunit.

5. The self-assembled polypeptide complex of claim 2 , wherein the ferritin monomer or subunit thereof is a ferritin monomer subunit. (a) each first fusion polypeptide comprises a C-half ferritin and each second fusion polypeptide comprises an N-half ferritin; or (b) each first fusion polypeptide comprises N-half ferritin and each second fusion polypeptide comprises C-half ferritin.

7. The self-assembled polypeptide complex of claim 6, wherein each first fusion polypeptide comprises an Fc polypeptide linked to the N-terminus of the C-half ferritin via an amino acid linker.

8. 2. The self-assembled polypeptide complex of claim 1, wherein the Fc polypeptide comprises a single-chain Fc (scFc) comprising two Fc chains, the two Fc chains being linked via an amino acid linker.

9. The self-assembling polypeptide complex of claim 1 , wherein the Fc polypeptide comprises an IgG1 Fc chain.

10. The self-assembling polypeptide complex of claim 9 , wherein the IgG1 Fc chain comprises an alanine at position 237 according to EU numbering.

11. 10. The self-assembled polypeptide complex of claim 9, wherein the IgGl Fc chain comprises alanine at position 234, alanine at position 235, arginine at position 236, and leucine at position 330 according to EU numbering.

12. The self-assembled polypeptide complex of claim 1, wherein within each second fusion polypeptide, the antigen-binding antibody fragment is linked to the N-terminus of the nanocage monomer or a subunit thereof.

13. The self-assembling polypeptide complex of claim 1 , wherein the antigen-binding antibody fragment of each second fusion polypeptide is a Fab fragment.

14. and further comprising a plurality of third fusion polypeptides, each third fusion polypeptide comprising: (1) an antigen-binding antibody fragment, and (2) The self-assembled polypeptide complex of claim 1, comprising a nanocage monomer or a subunit thereof, wherein the third fusion polypeptide is different from the second fusion polypeptide.

15. The self-assembling polypeptide complex of claim 14, wherein the antigen-binding antibody fragment of each third fusion polypeptide is a Fab fragment.

16. the nanocage monomer or subunit thereof of each first fusion polypeptide and each second fusion polypeptide is a ferritin monomer subunit; (a) each first fusion polypeptide comprises a C-half ferritin and each second fusion polypeptide comprises an N-half ferritin; or 2. The self-assembled polypeptide complex of claim 1, wherein (b) each first fusion polypeptide comprises an N-half ferritin and each second fusion polypeptide comprises a C-half ferritin.

17. The self-assembling polypeptide complex of claim 1, characterized in that after administration of a composition comprising the self-assembling polypeptide complex, the concentration of the self-assembling polypeptide complex is substantially similar to the concentration of a reference IgG molecule administered by the same route of administration and in a similar composition for the first seven days after administration to a subject in need thereof.

18. The self-assembling polypeptide complex of claim 1, which does not exhibit binding to at least one human Fcγ receptor as determined in an in vitro assay.

19. 1. A self-assembling polypeptide complex comprising: (a) a plurality of first fusion polypeptides, each first fusion polypeptide comprising: (1) scFc, and (2) the plurality of first fusion polypeptides comprising a ferritin monomer or a subunit thereof; (b) a plurality of second fusion polypeptides, each second fusion polypeptide comprising: (1) an antibody fragment capable of binding to DR5, and (2) the plurality of second fusion polypeptides comprising a ferritin monomer or a subunit thereof, wherein the scFc comprises two IgG1 Fc chains, each of which comprises alanine at position 234, alanine at position 235, arginine at position 236, alanine at position 237, proline at position 329, and leucine at position 330, according to EU numbering. (a) each first fusion polypeptide comprises a C-half ferritin and each second fusion polypeptide comprises an N-half ferritin; or 20. The self-assembled polypeptide complex of claim 19, wherein (b) each first fusion polypeptide comprises an N-half ferritin and each second fusion polypeptide comprises a C-half ferritin.

21. The self-assembling polypeptide complex of claim 20, wherein each first fusion polypeptide comprises an scFc linked to the N-terminus of the C-half ferritin, and each second fusion polypeptide comprises an Fab linked to the N-terminus of the N-half ferritin.

22. (1) in each first fusion polypeptide, the scFc is linked to the N-terminus of the C-half ferritin via an amino acid linker; and / or (2) The self-assembled polypeptide complex of claim 21, wherein in each second fusion polypeptide, the Fab is linked to the N-terminus of the N-half ferritin via an amino acid linker.

23. and further comprising a plurality of third fusion polypeptides, each third fusion polypeptide comprising: (1) an antigen-binding antibody fragment, and (2) The self-assembled polypeptide complex of claim 19, comprising a nanocage monomer or a subunit thereof, wherein the third fusion polypeptide is different from the second fusion polypeptide.

24. the antibody fragment (1) a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3; (2) a light chain comprising CDR-L1, CDR-L2, and CDR-L3; (a)(i) the CDR-H1 has the sequence of SEQ ID NO: 27, or a sequence that differs therefrom by one or two amino acids; (ii) the CDR-H2 has the sequence of SEQ ID NO: 28 or a sequence that differs therefrom by one or two amino acids; (iii) the CDR-H3 has the sequence of SEQ ID NO: 29, or a sequence that differs therefrom by one or two amino acids; (iv) the CDR-L1 has the sequence of SEQ ID NO: 24 or a sequence that differs therefrom by one or two amino acids; (v) the CDR-L2 has the sequence of SEQ ID NO: 25, or a sequence that differs therefrom by one or two amino acids; (vi) the CDR-L3 has the sequence of SEQ ID NO: 26 or a sequence that differs therefrom by one or two amino acids; (b)(i) the CDR-H1 has the sequence of SEQ ID NO: 35, or a sequence that differs therefrom by one or two amino acids; (ii) the CDR-H2 has the sequence of SEQ ID NO: 36, or a sequence that differs therefrom by one or two amino acids; (iii) the CDR-H3 has the sequence of SEQ ID NO: 37, or a sequence that differs therefrom by one or two amino acids; (iv) the CDR-L1 has the sequence of SEQ ID NO: 32, or a sequence that differs therefrom by one or two amino acids; (v) the CDR-L2 has the sequence of SEQ ID NO: 33, or a sequence that differs therefrom by one or two amino acids; (vi) the CDR-L3 has the sequence of SEQ ID NO: 34 or a sequence that differs therefrom by one or two amino acids; (c)(i) the CDR-H1 has the sequence of SEQ ID NO: 43, or a sequence that differs therefrom by one or two amino acids; (ii) the CDR-H2 has the sequence of SEQ ID NO: 44, or a sequence that differs therefrom by one or two amino acids; (iii) the CDR-H3 has the sequence of SEQ ID NO: 45, or a sequence that differs therefrom by one or two amino acids; (iv) the CDR-L1 has the sequence of SEQ ID NO: 40, or a sequence that differs therefrom by one or two amino acids; (v) the CDR-L2 has the sequence of SEQ ID NO: 41, or a sequence that differs therefrom by one or two amino acids; (vi) the CDR-L3 has the sequence of SEQ ID NO: 42 or a sequence that differs therefrom by one or two amino acids; or (d)(i) the CDR-H1 has the sequence of SEQ ID NO: 51, or a sequence that differs therefrom by one or two amino acids; (ii) the CDR-H2 has the sequence of SEQ ID NO: 52, or a sequence that differs therefrom by one or two amino acids; (iii) the CDR-H3 has the sequence of SEQ ID NO: 53, or a sequence that differs therefrom by one or two amino acids; (iv) the CDR-L1 has the sequence of SEQ ID NO: 48, or a sequence that differs therefrom by one or two amino acids; (v) the CDR-L2 has the sequence of SEQ ID NO: 49, or a sequence that differs therefrom by one or two amino acids; (vi) The self-assembled polypeptide complex of claim 1, wherein the CDR-L3 has the sequence of SEQ ID NO: 50 or a sequence that differs therefrom by one or two amino acids.

25. the antibody fragment (1) a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3; (2) a light chain comprising CDR-L1, CDR-L2, and CDR-L3; (a)(i) the CDR-H1 has the sequence of SEQ ID NO: 27; (ii) the CDR-H2 has the sequence of SEQ ID NO: 28; (iii) the CDR-H3 has the sequence of SEQ ID NO: 29; (iv) the CDR-L1 has the sequence of SEQ ID NO: 24; (v) the CDR-L2 has the sequence of SEQ ID NO: 25; (vi) the CDR-L3 has the sequence of SEQ ID NO: 26; (b)(i) the CDR-H1 has the sequence of SEQ ID NO: 35; (ii) the CDR-H2 has the sequence of SEQ ID NO: 36; (iii) the CDR-H3 has the sequence of SEQ ID NO: 37; (iv) the CDR-L1 has the sequence of SEQ ID NO: 32; (v) the CDR-L2 has the sequence of SEQ ID NO: 33; (vi) the CDR-L3 has the sequence of SEQ ID NO: 34; (c)(i) the CDR-H1 has the sequence of SEQ ID NO: 43; (ii) the CDR-H2 has the sequence of SEQ ID NO: 44; (iii) the CDR-H3 has the sequence of SEQ ID NO: 45; (iv) the CDR-L1 has the sequence of SEQ ID NO: 40; (v) the CDR-L2 has the sequence of SEQ ID NO: 41; (vi) the CDR-L3 has the sequence of SEQ ID NO: 42; or (d)(i) the CDR-H1 has the sequence of SEQ ID NO: 51; (ii) the CDR-H2 has the sequence of SEQ ID NO: 52; (iii) the CDR-H3 has the sequence of SEQ ID NO: 53; (iv) the CDR-L1 has the sequence of SEQ ID NO: 48; (v) the CDR-L2 has the sequence of SEQ ID NO: 49; (vi) The self-assembled polypeptide complex of claim 24, wherein the CDR-L3 has the sequence of SEQ ID NO:

50.

26. the antibody fragment (1) Reference V Ｈ a heavy chain variable region having at least 85% sequence identity with the sequence (2) Reference V Ｌ a light chain variable region having at least 85% sequence identity to the sequence (a) the reference V Ｈ The sequence has the sequence of SEQ ID NO: 23, and the reference V Ｌ the sequence has the sequence of SEQ ID NO: 22; (b) Reference V above. Ｈ The sequence has the sequence of SEQ ID NO: 31, and the reference V Ｌ the sequence has the sequence of SEQ ID NO: 30; (c) Reference V above. Ｈ The sequence has the sequence of SEQ ID NO: 39, and the reference V Ｌ the sequence has the sequence of SEQ ID NO: 38, or (d) Reference V above. Ｈ The sequence has the sequence of SEQ ID NO: 47, and the reference V Ｌ The self-assembling polypeptide complex of claim 1 , wherein the sequence has the sequence of SEQ ID NO:

46.

27. A composition comprising the self-assembled polypeptide complex of any one of claims 1 to 26 for use in a mammalian subject.

28. 28. The composition for use according to claim 27, wherein the subject is a human.

29. 28. The composition of claim 27, wherein the subject is diagnosed with or at risk of developing a tumor.

30. 30. The composition of claim 29 for delaying or inhibiting the progression of said tumor.

31. 31. The composition of claim 30 for causing regression of the tumor.

32. 32. The composition of claim 31 for causing complete regression of the tumor.

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