Treatment of chemotherapy-resistant tumors using Maltabodies

Abstract

A method of treating a subject having or at risk of having a chemotherapy-resistant tumor, comprising administering a pharmaceutical composition comprising a self-assembling polypeptide complex, thereby resulting in slowing, inhibition of tumor progression, or tumor regression. The present invention provides, for example, the use of a pharmaceutical composition comprising a self-assembling polypeptide complex to treat a subject having or at risk of having a chemotherapy-resistant tumor, wherein the self-assembling polypeptide complex comprises a plurality of first fusion polypeptides, each comprising (1) an antibody or antibody fragment capable of binding to a tumor-associated antigen expressed on the chemotherapy-resistant tumor, and (2) a nanocage monomer or subunit thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 375,242, filed September 11, 2022, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] Sequence Listing This specification refers to a Sequence Listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML file, created on September 11, 2023, is named RBT_055WO_ST26.xml and is 52kb in size. [Background technology]

[0003] background The development of anti-cancer drugs, such as chemotherapeutic agents, has greatly improved the treatment options for cancer patients.However, in many cases, even if patients respond favorably to chemotherapeutic drugs, they may end up experiencing relapse and recurrence due to the development of chemotherapy resistance by tumors.In some cases, as with intrinsically resistant tumors, no favorable response is observed even at the early stage.

[0004] There is a need for improved compositions and methods for treating chemotherapy-resistant tumors. Summary of the Invention [Means for solving the problem]

[0005] overview The present invention addresses this need by providing methods and related uses for treating chemotherapy-resistant tumors.

[0006] In one aspect, a method of treating a subject having or at risk of having a chemotherapy-resistant tumor is provided, comprising administering to the subject a pharmaceutical composition comprising a self-assembling polypeptide complex, wherein the self-assembling polypeptide complex comprises a plurality of first fusion polypeptides, each of which comprises (1) an antibody or antibody fragment capable of binding to a tumor-associated antigen expressed on the chemotherapy-resistant tumor, and (2) a nanocage monomer or a subunit thereof. Also provided is a use of a pharmaceutical composition comprising a self-assembling polypeptide complex for treating a chemotherapy-resistant tumor in a subject having or at risk of having a chemotherapy-resistant tumor, wherein the self-assembling polypeptide complex comprises a plurality of first fusion polypeptides, each of which comprises (1) an antibody or antibody fragment capable of binding to a tumor-associated antigen expressed on the chemotherapy-resistant tumor, and (2) a nanocage monomer or a subunit thereof.

[0007] In some embodiments, the complex further comprises a plurality of second fusion polypeptides, each of which comprises (1) an Fc polypeptide and (2) a nanocage monomer or a subunit thereof.

[0008] In some embodiments, each second fusion polypeptide comprises an Fc polypeptide linked to a nanocage monomer or subunit thereof via an amino acid linker.

[0009] In some embodiments, the amino acid linker is n S) m Linkers, e.g., (GGGGS) m (SEQ ID NO: 50) including the linker.

[0010] In some embodiments, the Fc polypeptide comprises a single chain Fc (scFc) comprising two Fc chains, wherein the two Fc chains are separated by an amino acid linker, e.g., (G n S) m Linkers, e.g., (GGGGS) m (SEQ ID NO: 50) linked via a linker.

[0011] In some embodiments, the Fc polypeptide comprises an IgG1 Fc chain. In some embodiments, the IgG1 Fc chain comprises (1) an amino acid residue other than glycine at position 237, and (2) a proline residue at position 329, according to EU numbering. In some embodiments, the IgG1 Fc chain comprises an alanine at position 237, according to EU numbering.

[0012] 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.

[0013] In some embodiments, the nanocage monomer or subunit thereof is a ferritin monomer or subunit thereof, for example, a ferritin light chain or subunit thereof and / or human ferritin or subunit thereof.

[0014] In some embodiments, the self-assembling polypeptide complex does not comprise a ferritin heavy chain or a subunit of a ferritin heavy chain.

[0015] In some embodiments, at least a portion of the first plurality of fusion polypeptides and / or a portion of the second plurality of fusion polypeptides are fusion polypeptides each comprising a ferritin monomer subunit.

[0016] 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 comprises an N half-ferritin and each second fusion polypeptide comprises a C half-ferritin.

[0017] In some embodiments, the self-assembling polypeptide complex is characterized by a 1:1 ratio of the first fusion polypeptide to the second fusion polypeptide.

[0018] In some embodiments, the total number of fusion polypeptides in each self-assembling polypeptide complex is at least 24 or at least 32.

[0019] In some embodiments, the total number of fusion polypeptides in each self-assembling polypeptide complex is about 24 or about 32.

[0020] In some embodiments, the total number of fusion polypeptides in each self-assembling polypeptide complex is between 24 and 48.

[0021] In some embodiments, the antibody or antibody fragment in each of the first fusion polypeptides binds to the N-terminus of a nanocage monomer or subunit thereof.

[0022] In some embodiments, each first fusion polypeptide comprises (1) a Fab fragment and (2) a nanocage monomer or subunit thereof. In some embodiments, each first fusion polypeptide does not comprise an antibody CH2 domain or an antibody CH3 domain.

[0023] In some embodiments, the tumor-associated antigen is a member of the TNF receptor superfamily, eg, a death receptor, eg, DR5 (also known as TRAIL receptor 2 (TRAILR2)).

[0024] In some embodiments, the antibody or antibody fragment is conatumumab, tigatuzumab, lexatumumab, drozitumab, or a DR5-binding fragment of any of the foregoing.

[0025] In some embodiments, the antibody or antibody fragment is conatumumab or a DR5-binding fragment thereof.

[0026] In some embodiments, the antibody or 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, wherein

[0027] (a) (i) CDR-H1 has the sequence of SEQ ID NO: 23 or a sequence that differs therefrom by one or two amino acids; (ii) CDR-H2 has the sequence of SEQ ID NO: 24 or a sequence that differs therefrom by one or two amino acids; (iii) CDR-H3 has the sequence of SEQ ID NO: 25 or a sequence that differs therefrom by one or two amino acids; (iv) CDR-L1 has the sequence of SEQ ID NO: 20 or a sequence that differs therefrom by one or two amino acids; (v) CDR-L2 has the sequence of SEQ ID NO: 21 or a sequence that differs therefrom by one or two amino acids; (vi) CDR-L3 has the sequence of SEQ ID NO: 22 or a sequence that differs therefrom by one or two amino acids;

[0028] (b) (i) CDR-H1 has the sequence of SEQ ID NO: 31 or a sequence that differs therefrom by one or two amino acids; (ii) CDR-H2 has the sequence of SEQ ID NO: 32 or a sequence that differs therefrom by one or two amino acids; (iii) CDR-H3 has the sequence of SEQ ID NO: 33 or a sequence that differs therefrom by one or two amino acids; (iv) CDR-L1 has the sequence of SEQ ID NO: 28 or a sequence that differs therefrom by one or two amino acids; (v) CDR-L2 has the sequence of SEQ ID NO: 29 or a sequence that differs therefrom by one or two amino acids; (vi) CDR-L3 has the sequence of SEQ ID NO: 30 or a sequence that differs therefrom by one or two amino acids;

[0029] (c) (i) CDR-H1 has the sequence of SEQ ID NO: 39 or a sequence that differs therefrom by one or two amino acids; (ii) CDR-H2 has the sequence of SEQ ID NO: 40 or a sequence that differs therefrom by one or two amino acids; (iii) CDR-H3 has the sequence of SEQ ID NO: 41 or a sequence that differs therefrom by one or two amino acids; (iv) CDR-L1 has the sequence of SEQ ID NO: 36 or a sequence that differs therefrom by one or two amino acids; (v) CDR-L2 has the sequence of SEQ ID NO: 37 or a sequence that differs therefrom by one or two amino acids; (vi) CDR-L3 has the sequence of SEQ ID NO: 38 or a sequence that differs therefrom by one or two amino acids; or

[0030] (d) (i) CDR-H1 has the sequence of SEQ ID NO: 47 or a sequence that differs therefrom by one or two amino acids; (ii) CDR-H2 has the sequence of SEQ ID NO: 48 or a sequence that differs therefrom by one or two amino acids; (iii) CDR-H3 has the sequence of SEQ ID NO: 49 or a sequence that differs therefrom by one or two amino acids; (iv) CDR-L1 has the sequence of SEQ ID NO: 44 or a sequence that differs therefrom by one or two amino acids; (v) CDR-L2 has the sequence of SEQ ID NO: 45 or a sequence that differs therefrom by one or two amino acids; (vi) CDR-L3 has the sequence of SEQ ID NO: 46 or a sequence which differs therefrom by one or two amino acids.

[0031] 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, wherein

[0032] (a)(i) CDR-H1 has the sequence of SEQ ID NO: 23; (ii) CDR-H2 has the sequence of SEQ ID NO: 24; (iii) CDR-H3 has the sequence of SEQ ID NO: 25; (iv) CDR-L1 has the sequence of SEQ ID NO: 20; (v) CDR-L2 has the sequence of SEQ ID NO: 21; (vi) CDR-L3 has the sequence of SEQ ID NO: 22;

[0033] (b)(i) CDR-H1 has the sequence of SEQ ID NO: 31; (ii) CDR-H2 has the sequence of SEQ ID NO: 32; (iii) CDR-H3 has the sequence of SEQ ID NO: 33; (iv) CDR-L1 has the sequence of SEQ ID NO: 28; (v) CDR-L2 has the sequence of SEQ ID NO: 29; (vi) CDR-L3 has the sequence of SEQ ID NO: 30;

[0034] (c)(i) CDR-H1 has the sequence of SEQ ID NO: 39; (ii) CDR-H2 has the sequence of SEQ ID NO: 40; (iii) CDR-H3 has the sequence of SEQ ID NO: 41; (iv) CDR-L1 has the sequence of SEQ ID NO: 36; (v) CDR-L2 has the sequence of SEQ ID NO: 37; (vi) CDR-L3 has the sequence of SEQ ID NO: 38; or

[0035] (d)(i) CDR-H1 has the sequence of SEQ ID NO: 47; (ii) CDR-H2 has the sequence of SEQ ID NO: 48; (iii) CDR-H3 has the sequence of SEQ ID NO: 49; (iv) CDR-L1 has the sequence of SEQ ID NO: 44; (v) CDR-L2 has the sequence of SEQ ID NO: 45; (vi) CDR-L3 has the sequence of SEQ ID NO: 46.

[0036] In some embodiments, the antibody fragment comprises: (1) a heavy chain variable region having at least 85% identity to a reference VH sequence; and (2) a light chain variable region having at least 85% identity to a reference VL sequence, wherein (a) the reference VH sequence has the sequence of SEQ ID NO: 19 and the reference VL sequence has the sequence of SEQ ID NO: 18; or (b) the reference VH sequence has the sequence of SEQ ID NO: 27 and the reference VL sequence has the sequence of SEQ ID NO: 26; or (c) the reference VH sequence has the sequence of SEQ ID NO: 35 and the reference VL sequence has the sequence of SEQ ID NO: 34; or (d) the reference VH sequence has the sequence of SEQ ID NO: 43 and the reference VL sequence has the sequence of SEQ ID NO: 42;

[0037] In some embodiments, the subject is a mammal, for example, a human.

[0038] In some embodiments, the tumor is a colon tumor.

[0039] In some embodiments, the subject has chemotherapy-resistant tumor, for example, the tumor that is resistant to taxane, vinca alkaloid, anthracycline, platinum-containing compound or any combination thereof.For example, the tumor can be resistant to taxane, for example, paclitaxel, taxane or both; vinca alkaloid, for example, vincristine; anthracycline, for example, doxorubicin; and / or platinum-containing compound, for example, satraplatin and oxaliplatin.

[0040] In some embodiments, the administering step comprises administration by a systemic route, for example, subcutaneous, intravenous or intramuscular, inhalation, or intranasal administration.

[0041] In some embodiments, the administering step comprises administering more than one dose of the pharmaceutical composition (i.e., administering more than one dose of the pharmaceutical composition to the subject). For example, the pharmaceutical composition may be administered once a week for at least two weeks, at least three weeks, or longer.

[0042] In some embodiments, the administering step or use results in slowing, inhibition of tumor progression, or tumor regression.

[0043] In some embodiments, methods are provided for treating a subject having or at risk of having a chemotherapy-resistant tumor, comprising the step of administering to the subject a pharmaceutical composition comprising a self-assembling polypeptide complex, wherein the self-assembling polypeptide complex comprises: (a) a plurality of first fusion polypeptides, each comprising (1) an antibody fragment capable of binding to DR5 and (2) a ferritin monomer or a subunit thereof; and (b) a plurality of second fusion polypeptides, each comprising (1) an scFc and (2) a ferritin monomer or a subunit thereof, wherein the scFc comprises two IgG1 Fc chains, each 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.

[0044] In some embodiments, there is provided a use of a pharmaceutical composition comprising a self-assembling polypeptide complex for treating a subject having or at risk of having a chemotherapy-resistant tumor, wherein the self-assembling polypeptide complex comprises (a) a plurality of first fusion polypeptides, each comprising (1) an antibody fragment capable of binding to DR5 and (2) a ferritin monomer or a subunit thereof, and (b) a plurality of second fusion polypeptides, each comprising (1) an scFc and (2) a ferritin monomer or a subunit thereof, wherein the scFc comprises two IgG1 Fc chains, each 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.

[0045] In some embodiments, the administering step or use results in slowing, inhibition of tumor progression, or tumor regression.

[0046] In one aspect, there is provided a use of a self-assembling polypeptide complex in the manufacture of a medicament for treating a chemotherapy-resistant tumor, wherein the self-assembling polypeptide complex comprises a plurality of first fusion polypeptides, each comprising (1) an antibody or antibody fragment capable of binding to a tumor-associated antigen expressed on a chemotherapy-resistant tumor, and (2) a nanocage monomer or a subunit thereof. [Brief explanation of the drawings]

[0047] [Figure 1A] FIG. 1A is a diagrammatic representation of human ferritin light chain (hFTL) and exemplary N-half ferritin (N-hFTL) and C-half ferritin (C-hFTL) molecules.

[0048] [Figure 1B] FIG. 1B is a diagrammatic representation of fusion polypeptides that together form an exemplary maltabody of the present disclosure.

[0049] [Figure 2A] Figure 2A depicts tumor growth curves showing tumor volume plotted against time after tumor cell injection in mice injected with vehicle (black curve), conatumumab ("parent IgG", green curve) or DR5-targeted maltabody ("MB", orange curve). Red arrows indicate treatment time points. The experiment is described in Example 2.

[0050] [Figure 2B] Figure 2B shows survival curves of HCT-15 xenograft mice for mice injected with vehicle (black curve), conatumumab ("parent IgG", green curve), or DR5-targeted maltabody ("MB", orange curve). The experiment is described in Example 2.

[0051] [Figure 3] Figure 3 is a schematic diagram depicting the design of a dose-ranging efficacy study of DR5-targeted maltabodies in the HCT-15 xenograft mouse model. The experiment is described in Example 3.

[0052] [Figure 4A] Figure 4A depicts tumor growth curves showing tumor volume plotted against time after treatment for HCT-15 xenografted mice administered vehicle or 0.1 mg / kg, 0.25 mg / kg, 1 mg / kg, or 5 mg / kg of DR5-targeted maltabody ("MB"). See Example 3.

[0053] [Figure 4B] FIG. 4B depicts the amount of maltabody detected in the blood of mice over time from the first dose in the dose-ranging efficacy study described in Example 3.

[0054] [Figure 5] 5 is a schematic diagram depicting the design of a study of large tumor penetration and apoptosis induction by DR5-targeted maltabodies in the HCT-15 xenograft mouse model. The experiment is described in Example 4.

[0055] [Figure 6] Figures 6A-6B show representative tumor sections from mice untreated (Figures 6A and 6B) or treated with DR5-targeted maltabody ("MB") (Figures 6C and 6D). Sections were stained with an antibody against cleaved caspase 3, a marker of apoptosis. The experiment is described in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0056] Detailed Description The present disclosure provides methods of treating a subject having or at risk of having a chemotherapy-resistant tumor by administering to the subject a pharmaceutical composition comprising a self-assembling polypeptide complex ("Multabody") as further described herein. The methods disclosed herein have demonstrated therapeutic efficacy, e.g., the ability to cause tumor suppression and / or regression, at least over a period of time, in highly aggressive, chemotherapy-resistant xenograft tumor models.

[0057] definition The terms "about" and "approximately" are used interchangeably herein when referring to values, and refer to values ​​that are close to a reference value. Generally, a person skilled in the art who is familiar with the context will understand the appropriate degree of variation encompassed by "about" or "approximately" in that context. For example, in some embodiments, the terms "about" and "approximately" can encompass a range of 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 reference value.

[0058] As used herein, the terms "alter," "altered," "decrease," "decreased," "increase," "increased," or "reduction," "reduced" have their meaning (e.g., with respect to a particular result or effect) relative to a reference level. In some embodiments, in the context of discussing mutations in an Fc chain or Fc polypeptide, the reference level is a known level or a level determined with an IgG that does not contain the reference mutation(s) in its Fc region.

[0059] As used herein, the term "binding" refers to a non-covalent association between two or more entities, unless otherwise specified. "Direct" binding includes physical contact between entities or moieties, and indirect binding includes physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in a variety of contexts, including when the interacting entities or moieties are tested in isolation, or in the context of more complex systems (e.g., covalently or otherwise associated with a carrier entity and / or in a biological system or cell). As used herein, the phrase "unbound" or "unbound" between two entities or similar phrases refers to 1) the absence of detectable binding, or 2) binding below a set threshold corresponding to no binding in an appropriate assay, such as an in vitro binding assay, e.g., biolayer interferometry. For example, in some embodiments, in an in vitro biolayer interferometry assay, a maximum association binding response of less than 0.1 nm after 180 seconds for a biosensor loaded with 0.8 nm target when the test article is present at a concentration of 20 nM is classified as "unbound."

[0060] As used herein, the term "chemo-resistance" refers to the tumor's characteristic of poorly responding to chemotherapeutic drugs when used to describe tumors. The term "chemo-resistance" encompasses both intrinsic resistance (i.e., resistance without prior exposure) to one or more chemotherapeutic drugs and acquired resistance (i.e., resistance that occurs after exposure) to one or more chemotherapeutic drugs. Chemo-resistant tumors can be resistant to one or more chemotherapeutic drugs of the same class and / or one or more classes of chemotherapeutic drugs.

[0061] The terms "ferritin" and "apoferritin" are used interchangeably herein and generally refer to a polypeptide (e.g., a ferritin chain) that can assemble into a ferritin complex, typically comprising 24 protein subunits. In some embodiments, the ferritin is human ferritin, e.g., a human ferritin light chain, e.g., a human ferritin light chain having at least 85% sequence identity to SEQ ID NO: 1 or UniProt P02792. In some embodiments, the ferritin is wild-type ferritin. For example, the ferritin can be wild-type human ferritin.

[0062] 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.

[0063] 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, those skilled in the art understand that polypeptides whose structures include two or more functional or structural domains (e.g., fusion polypeptides) often include a stretch of amino acids between such domains that connects them to one another. 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 one another by the linker (L). In some embodiments, the linker is an "amino acid linker." That is, the linker includes amino acid residues. For example, an 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, linkers are characterized in that they do not tend to adopt rigid three-dimensional structures, but rather provide flexibility to the polypeptide.

[0064] The term "multispecific," as used herein, refers to the property of having at least two binding sites capable of binding at least two different binding partners, e.g., antigens or receptors (e.g., Fc receptors). For example, a polypeptide complex comprising at least two Fab fragments, each capable of binding to a different antigen, is "multispecific." As a further 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."

[0065] The term "multivalent," as used herein, refers to the property of having at least two binding sites to which a binding partner, e.g., an antigen or 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.

[0066] The term "nanocage monomer," as used herein, refers to a single-chain polypeptide that can self-assemble with other nanocage monomers to form a self-assembled polypeptide complex comprising multiple 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 variants thereof.

[0067] The term "polypeptide," as used herein, generally has its art-recognized meaning of a polymer of at least three amino acids joined together, e.g., by peptide bonds. Those of skill in the art will understand that the term "polypeptide" is intended to be general enough to encompass not only polypeptides having the complete sequences recited herein, but also polypeptides representing functional fragments of such complete polypeptides (i.e., fragments that retain at least one activity). Moreover, those of skill in the art will understand that protein sequences generally tolerate some substitutions without destroying activity. Thus, the related term "polypeptide" as used herein encompasses any polypeptide that retains activity, shares at least about 30-40%, often greater than about 50%, 60%, 70%, or 80% overall sequence identity, usually further comprises at least one region with one or more highly conserved regions, often greater than 90% or even much higher, such as 95%, 96%, 97%, 98%, or 99%, identity, and typically encompasses at least 3-4, and often up to 20 or more, amino acids with another polypeptide of the same species. 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.

[0068] The term "self-assembled," when used with respect to a macromolecular complex (e.g., a polypeptide complex), refers to the spontaneous formation of a complex (a fusion polypeptide) when sufficient components are present for the complex to form. In some embodiments, the complex self-assembles under physiological conditions or in a buffer (e.g., a solution) that corresponds to physiological conditions.

[0069] As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human). In some embodiments, the subject is suffering from or susceptible to the relevant disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject is one who possesses one or more characteristics that characterize a susceptibility to or risk for the disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is one for whom diagnosis and / or therapy is performed and / or has been performed.

[0070] As used herein, the term "treatment" (also "treat" or "treating") refers to any administration of a therapy that partially or completely alleviates, ameliorate, relieves, inhibits, delays the onset of, reduces the severity of, 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 treatment of subjects who do not exhibit signs of the associated disease, disorder, and / or condition and / or who exhibit only early signs of the disease, disorder, and / or condition. Alternatively, or in addition, such treatment may be treatment of subjects who exhibit one or more established signs of the associated disease, disorder, and / or condition. In some embodiments, treatment may be treatment of subjects who have been diagnosed as suffering from the associated disease, disorder, and / or condition. In some embodiments, treatment may be treatment of subjects who are known to have one or more susceptibility factors that statistically correlate with an elevated risk of developing the associated disease, disorder, and / or condition.

[0071] A. Fusion Polypeptides In many embodiments, fusion polypeptides compatible with the compositions and methods disclosed herein generally comprise a nanocage monomer or subunit thereof that binds to an antibody fragment capable of binding to a tumor-associated antigen expressed on a chemotherapy-resistant tumor or any of the "Fc polypeptides" described herein. Within the fusion polypeptide, the antibody fragment or Fc polypeptide may be linked to the nanocage monomer or subunit thereof at a specific terminus, e.g., the N-terminus or C-terminus, of the nanocage monomer or subunit. In some embodiments, the antibody fragment or Fc fragment is linked via an amino acid linker, e.g., a linker described herein.

[0072] 1. Nanocage monomer and its subunits In some embodiments, the nanocage monomer is a ferritin monomer.

[0073] The term "ferritin monomer" is used herein to refer to a single-chain 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 capable of binding iron or ferroxidase activity.

[0074] In some embodiments, each fusion polypeptide in the self-assembling polypeptide complex comprises a ferritin light chain or a subunit of a ferritin light chain. In these embodiments, the self-assembling polypeptide complex does not comprise a ferritin heavy chain or a subunit of a ferritin heavy chain.

[0075] In some embodiments, the ferritin monomer is a human ferritin chain, eg, a human ferritin light chain, eg, a human ferritin light chain having the sequence of at least residues 2-175 of SEQ ID NO:1.

[0076] A "subunit" of a ferritin monomer refers to a portion of a ferritin monomer that can spontaneously associate with another, different subunit of a ferritin monomer such that the subunits together form a ferritin monomer that can then self-assemble with other ferritin monomers to form a polypeptide complex.

[0077] In some embodiments, the ferritin monomer subunit comprises approximately half of a ferritin monomer. As used herein, the term "N-half ferritin" refers to approximately half of a ferritin chain, including the N-terminus of the ferritin chain. As used herein, the term "C-half ferritin" refers to approximately half of a ferritin chain, including 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 75 to 100 (or a substantial portion thereof) of SEQ ID NO: 1. For example, in some embodiments, an N-half ferritin based on a 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 a human ferritin light chain has an amino acid sequence corresponding to residues 96-175 of SEQ ID NO:1 (or a substantial portion thereof).

[0078] 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).

[0079] 2. Antibody fragments capable of binding to tumor-associated antigens expressed on chemotherapy-resistant tumors An antibody fragment may comprise a heavy chain variable region (e.g., V H In certain embodiments, the antibody fragment typically comprises a heavy chain variable domain (e.g., a 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.

[0080] In certain embodiments, the fusion polypeptide (e.g., the first fusion polypeptide) does not comprise a full-length antibody, but rather comprises an antibody fragment lacking one or more domains from a full-length antibody. For example, in some embodiments, the antibody fragment does not comprise any domains from the Fc region, e.g., does not comprise a CH2 domain or a CH3 domain.

[0081] In some embodiments, the antibody fragment is a Fab. In some embodiments, the antibody fragment is a single-chain Fab (scFab), e.g., using a fusion polypeptide comprising both the heavy and light chains of a Fab, optionally joined by a linker (e.g., an amino acid linker disclosed herein).

[0082] In some embodiments, the tumor-associated antigen expressed on chemotherapy-resistant tumors is a member of the tumor necrosis factor (TNF) receptor superfamily. Cytokine receptors of the TNF receptor superfamily can bind to TNF via their extracellular domains and, upon activation, typically form a trimeric complex in the cell membrane. In some embodiments, the tumor-associated antigen is a death receptor (e.g., TNFR1, Fas receptor, DR4 (also known as TRAIL receptor 1), or DR5 (also known as TRAIL receptor 2)), a class of receptors within the TNF receptor superfamily that contain a "death domain." Death receptors play a role in apoptosis.

[0083] The suitability of antibody fragments for use in the methods disclosed herein can be evaluated using any of a variety of methods known in the art.For example, the binding of an antibody or antibody fragment to a desired target, for example, an antigen expressed on a chemotherapy-resistant tumor, can be evaluated using surface plasmon resonance or biolayer interferometry.Alternatively, or in addition, if the tumor-associated antigen is a member of the TNF receptor superfamily, the antibody fragment can be evaluated for its ability to induce trimerization of TNF receptors.Alternatively, or in addition, if the tumor-associated antigen is a death receptor, the ability of the antibody fragment to induce apoptosis can be evaluated, for example, in a cell-based apoptosis assay.

[0084] In some embodiments, the tumor-associated antigen is DR5. For example, the antibody fragment may be capable of binding to an epitope within DR5, exemplary sequences of which are set forth in SEQ ID NOs: 14 to 17. In certain embodiments, the antibody fragment capable of binding to DR5 is or is derived from any of a variety of DR5 antibodies, including, for example, fully human, humanized, or chimeric DR5 antibodies. The DR5 antibody from which the antibody fragment is derived can be any of a variety of antibody classes, including, for example, IgG1, IgG2, or IgG4 antibodies. In some embodiments, the antibody fragment is derived from or is derived from a DR5 agonist antibody, for example, a humanized DR5 agonist antibody.

[0085] Non-limiting examples of DR5 antibodies include, for example, conatumumab, tigatuzumab, lexatumumab, and drozitumab.

[0086] In some embodiments, the antibody fragment capable of binding to DR5 comprises heavy and light chain CDRs with 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).

[0087] In some embodiments, the 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, wherein (a)(i) CDR-H1 has the sequence of SEQ ID NO: 23 or a sequence that differs therefrom by one or two amino acid residues; (ii) CDR-H2 has the sequence of SEQ ID NO: 24 or a sequence that differs therefrom by one or two amino acid residues; (iii) CDR-H3 has the sequence of SEQ ID NO: 25 or a sequence that differs therefrom by one or two amino acid residues; (iv) CDR-L1 has the sequence of SEQ ID NO: 20 or a sequence that differs therefrom by one or two amino acid residues; (v) CDR-L2 has the sequence of SEQ ID NO: 21 or a sequence that differs therefrom by one or two amino acid residues; (vi) CDR-L3 has the sequence of SEQ ID NO: 22 or a sequence that differs therefrom by one or two amino acid residues; (b)(i) CDR-H1 has the sequence of SEQ ID NO: 31 or a sequence that differs therefrom by one or two amino acid residues; (ii) CDR-H2 has the sequence of SEQ ID NO: 32 or a sequence that differs therefrom by one or two amino acid residues; (iii) CDR-H3 has the sequence of SEQ ID NO: 33 or a sequence that differs therefrom by one or two amino acid residues; (iv) CDR-L1 has the sequence of SEQ ID NO: 28 or a sequence that differs therefrom by one or two amino acid residues; (v) CDR-L2 has the sequence of SEQ ID NO: 29 or a sequence that differs therefrom by one or two amino acid residues; (vi) CDR-L3 has the sequence of SEQ ID NO: 30 or a sequence that differs therefrom by one or two amino acid residues; (c)(i) CDR-H1 has the sequence of SEQ ID NO: 39 or a sequence that differs therefrom by one or two amino acid residues; (ii) CDR-H2 has the sequence of SEQ ID NO: 40 or a sequence that differs therefrom by one or two amino acid residues; (iii) CDR-H3 has the sequence of SEQ ID NO: 41 or a sequence that differs therefrom by one or two amino acid residues; (iv) CDR-L1 has the sequence of SEQ ID NO: 36 or a sequence that differs therefrom by one or two amino acid residues; (v) CDR-L2 has the sequence of SEQ ID NO: 37 or a sequence that differs therefrom by one or two amino acid residues; (vi) CDR-L3 has the sequence of SEQ ID NO: 38 or a sequence that differs therefrom by one or two amino acid residues; or (d)(i) CDR-H1 has the sequence of SEQ ID NO: 47 or a sequence that differs therefrom by one or two amino acid residues; (ii) CDR-H2 has the sequence of SEQ ID NO: 48 or a sequence that differs therefrom by one or two amino acid residues; (iii) CDR-H3 has the sequence of SEQ ID NO: 49 or a sequence that differs therefrom by one or two amino acid residues; (iv) CDR-L1 has the sequence of SEQ ID NO: 44 or a sequence that differs therefrom by one or two amino acid residues; (v) CDR-L2 has the sequence of SEQ ID NO: 45 or a sequence that differs therefrom by one or two amino acid residues; (vi) CDR-L3 has the sequence of SEQ ID NO: 46 or a sequence which differs therefrom by one or two amino acid residues.

[0088] In some embodiments, the antibody fragment capable of binding to DR5 comprises heavy and light chain CDRs that have sequences identical to those of the DR5 antibody, except for a total of one or two amino acid substitutions across all six CDRs.

[0089] In some embodiments, the antibody fragment capable of binding to DR5 comprises a heavy chain and a light chain complementarity determining region (CDR) having sequences identical to the CDRs of the DR5 antibody.

[0090] In some embodiments, the 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, wherein (a)(i) CDR-H1 has the sequence of SEQ ID NO: 23; (ii) CDR-H2 has the sequence of SEQ ID NO: 24; (iii) CDR-H3 has the sequence of SEQ ID NO: 25; (iv) CDR-L1 has the sequence of SEQ ID NO: 20; (v) CDR-L2 has the sequence of SEQ ID NO: 21; (vi) CDR-L3 has the sequence of SEQ ID NO: 22; (b)(i) CDR-H1 has the sequence of SEQ ID NO: 31; (ii) CDR-H2 has the sequence of SEQ ID NO: 32; (iii) CDR-H3 has the sequence of SEQ ID NO: 33; (iv) CDR-L1 has the sequence of SEQ ID NO: 28; (v) CDR-L2 has the sequence of SEQ ID NO: 29; (vi) CDR-L3 has the sequence of SEQ ID NO: 30; (c)(i) CDR-H1 has the sequence of SEQ ID NO: 39; (ii) CDR-H2 has the sequence of SEQ ID NO: 40; (iii) CDR-H3 has the sequence of SEQ ID NO: 41; (iv) CDR-L1 has the sequence of SEQ ID NO: 36; (v) CDR-L2 has the sequence of SEQ ID NO: 37; (vi) CDR-L3 has the sequence of SEQ ID NO: 38; or (d)(i) CDR-H1 has the sequence of SEQ ID NO: 47; (ii) CDR-H2 has the sequence of SEQ ID NO: 48; (iii) CDR-H3 has the sequence of SEQ ID NO: 49; (iv) CDR-L1 has the sequence of SEQ ID NO: 44; (v) CDR-L2 has the sequence of SEQ ID NO: 45; (vi) CDR-L3 has the sequence of SEQ ID NO: 46.

[0091] In some embodiments, the antibody fragment capable of binding to DR5 comprises: (1) a heavy chain variable region having 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% identity to the heavy chain variable region derived from the DR5 antibody; (2) a light chain variable region having 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% identity to the light chain variable region derived from the DR5 antibody; Includes.

[0092] In some embodiments, the antibody fragment capable of binding to DR5 comprises: (1) Reference V H a heavy chain variable region having 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% identity to the sequence; (2) Reference V L a light chain variable region having 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% identity to the sequence where (a) Reference V H The sequence has the sequence of SEQ ID NO: 19 and the reference V L the sequence has the sequence of SEQ ID NO: 18; (b) Reference VH The sequence has the sequence of SEQ ID NO: 27 and the reference V L the sequence has the sequence of SEQ ID NO: 26; (c) Reference V H The sequence has the sequence of SEQ ID NO: 35 and the reference V L the sequence has the sequence of SEQ ID NO: 34, or (d) Reference V H The sequence has the sequence of SEQ ID NO: 43 and the reference V L The sequence has the sequence of SEQ ID NO:42.

[0093] In embodiments using multiple types of fusion polypeptides with antibody fragments, 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 different non-overlapping epitopes on DR5, or may be capable of binding to different, but overlapping epitopes on DR5.

[0094] 3. Fc polypeptide In certain embodiments, the fragment crystallizable (Fc) polypeptides comprise Fc chains, each having one or more mutations compared to a reference Fc chain of the same Ig class. As further described herein below, the reference Fc chain may be, for example, of the IgG1 class. Unless otherwise stated, the numbering of residues within an antibody fragment, e.g., an Fc chain, follows EU numbering throughout this disclosure.

[0095] 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 substantially similar to an Fc chain in wild-type human IgG1.

[0096] In some embodiments, the Fc polypeptide comprises one or more IgG1 Fc chains (e.g., human IgG1 Fc chains or human Fc chains). That is, the Fc polypeptide comprises Fc chains having an amino acid sequence substantially similar to the sequence of a chain in wild-type IgG1 Fc, except for having 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, each Fc chain having the amino acid sequence of SEQ ID NO: 4. For example, the Fc polypeptide may comprise Fc chains having an amino acid sequence 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 that includes particular residue(s) at certain position(s) described in detail for that Fc chain, but otherwise has an amino acid sequence that is 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 that has an amino acid sequence that differs by at least 1, at least 2, at least 3, or at least 4 amino acid residues from the sequence of SEQ ID NO: 4. In some embodiments, an Fc polypeptide comprises an Fc chain that has 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.

[0097] In some embodiments, the Fc polypeptide is a single chain Fc (scFc) comprising two Fc chains linked together by a covalent linker, eg, via an amino acid linker.

[0098] 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. 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.

[0099] 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.

[0100] 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).

[0101] In some embodiments, the Fc chain comprises a mutation or set of mutations (compared to the corresponding wild-type Fc chain) that is associated with an altered property, as further described herein. By "associated with" is meant that the mutation or set of mutations has been previously characterized as conferring an altered property (e.g., altered binding to FcRn, altered effector function, etc.) in the context of an antibody, e.g., an IgG antibody. By "altered" is meant that the property (e.g., binding to an Fc receptor (e.g., an Fcγ receptor or FcRn)) is different from the property observed without the mutation or set of mutations.

[0102] For example, in some embodiments, the altered property comprises altered binding to an Fc receptor.

[0103] In some embodiments, the altered property comprises 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.

[0104] In some embodiments, altered binding includes 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 with the corresponding wild-type strand under similar circumstances).

[0105] 4. Linker In certain embodiments, linkers are used within fusion polypeptides and / or single-chain molecules, e.g., scFcs. 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.

[0106] In certain embodiments, the linker comprises a glycine-serine sequence, e.g., (G n S) m The sequence includes, for example, the sequence GGS, GGGS, or GGGGS (SEQ ID NO: 50).

[0107] B. Self-Assembled Polypeptide Complexes In one embodiment, a self-assembling polypeptide complex is provided comprising a plurality of fusion polypeptides disclosed herein. Generally, the self-assembling polypeptide complex comprises: (a) a plurality of first fusion polypeptides, each comprising (1) an Fc polypeptide that binds to (2) nanocage monomers or subunits thereof, wherein the Fc polypeptides comprise an Fc chain having one or more mutations compared to a reference Fc chain of the same Ig class; and (b) a plurality of second fusion polypeptides, each comprising (1) an antigen-binding antibody fragment that binds to (2) nanocage monomers or subunits thereof.

[0108] 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 a ferritin heavy chain, a subunit of a ferritin heavy chain, or other ferritin components capable of binding iron or ferroxidase activity.

[0109] In some embodiments, the nanocage monomers or subunits thereof are ferritin monomer subunits, and (a) each of the first fusion polypeptides comprises a ferritin monomer subunit that is a C-half ferritin and each of the second fusion polypeptides comprises a ferritin monomer subunit that is an N-half ferritin, or (b) each of the first fusion polypeptides comprises a ferritin monomer subunit that is an N-half ferritin and each of the second fusion polypeptides comprises a ferritin monomer subunit that is a C-half ferritin.

[0110] 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, e.g., at least 26 fusion polypeptides, at least 28 fusion polypeptides, at least 30 fusion polypeptides, 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.

[0111] In some embodiments, the self-assembling polypeptide complex comprises at least 4, at least 5, at least 6, at least 7, or at least 8 first fusion polypeptides.

[0112] In some embodiments, the self-assembling polypeptide complex comprises at least 4, at least 5, at least 6, at least 7, or at least 8 second fusion polypeptides.

[0113] In some embodiments, the self-assembling 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.

[0114] In some embodiments, the self-assembling polypeptide complex comprises a first fusion polypeptide to all other fusion polypeptides in a ratio of approximately 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.

[0115] effect In certain embodiments, the provided self-assembling polypeptide complexes are capable of inducing multimerization (e.g., trimerization) of a TNF receptor superfamily receptor (e.g., a death receptor, e.g., a DR5 receptor) on a target cell (e.g., a cancer cell).

[0116] In some embodiments, administration of the self-assembling polypeptide complexes disclosed herein to a subject results in an improvement in clinical outcomes or metrics in the subject. For example, in a subject with a tumor, administration of the self-assembling polypeptide complexes or compositions thereof can inhibit or slow tumor progression, e.g., cause tumor regression. In some embodiments, administration of the self-assembling polypeptide complexes results in complete tumor regression.

[0117] C. Treatment Method In one aspect, provided are methods that may be useful for treating, ameliorating, or preventing a disease or condition (e.g., cancer, e.g., chemotherapy-resistant cancer) generally comprising administering to a subject a composition comprising a self-assembling polypeptide complex described herein.

[0118] In some embodiments, the subject is a mammal, for example, a human.

[0119] In many embodiments, the subject has or is at risk of having a chemotherapy-resistant tumor.

[0120] In some embodiments, the subject has a chemotherapy-resistant tumor at the time of administration of the pharmaceutical composition.

[0121] In some embodiments, the subject has not been previously treated with a chemotherapeutic agent, for example, the subject may have or be at risk of having a tumor that is intrinsically chemoresistant or has characteristics that suggest that the tumor has the potential to acquire chemotherapy resistance.

[0122] In some embodiments, the subject and / or the subject's tumor sample possesses a genetic mutation or genetic variation associated with intrinsic chemotherapy resistance or the likelihood of acquiring chemotherapy resistance. In some embodiments, the subject and / or the subject's tumor sample possesses an epigenetic change associated with intrinsic chemotherapy resistance or the likelihood of acquiring chemotherapy resistance. In some embodiments, the subject has or is at risk of having a genetically heterogeneous tumor. In some embodiments, the subject has or is at risk of having a tumor containing cancer stem cells. In some embodiments, the subject has one or more clinical markers associated with having or being at risk of having a chemotherapy-resistant tumor.

[0123] In some embodiments, the subject has not been previously treated with one or more chemotherapeutic agents. In some such embodiments, the subject exhibits one or more clinical signs of tumor recurrence or relapse.

[0124] In some embodiments, the subject has a tumor of a histology known to be prone to chemotherapy resistance.

[0125] In some embodiments, the tumor is a solid tumor, for example, a colon tumor.

[0126] Compositions for administration to a subject generally comprise a self-assembling polypeptide complex as disclosed herein. In some embodiments, such compositions further comprise a pharmaceutically acceptable excipient.

[0127] The compositions may be formulated for administration for any of a variety of routes of administration, including systemic routes (eg, oral, inhalation, intranasal, intravenous, intraperitoneal, subcutaneous, or intramuscular administration).

[0128] In some embodiments, the administering step results in an improvement in one or more clinical outcomes or metrics in the subject.

[0129] In some embodiments, the administering step results in a slowing or inhibition of tumor progression, e.g., tumor regression, hi some embodiments, the administering step results in complete tumor regression. [Example]

[0130] Example 1 Construction and expression of DR5-targeted Maltabodies In this example, a linker, such as a (Gly) n -Ser) m We describe the generation of a DR5-targeting maltabody (MB) comprising a combination of fusion proteins comprising (1) human ferritin light chain or a subunit thereof, and (2) a single-chain Fab (scFab) or a single-chain Fc dimer (scFc), fused via an amino acid linker.

[0131] Genes encoding fusion proteins (1) an anti-DR5 antibody scFab (in this case, conatumumab) fused to the N-terminus of hFTL (aDR5-hFTL, SEQ ID NO: 9), (2) an anti-DR5 antibody scFab fused to the N-terminus of N-half ferritin (aDR5-N_hFTL, SEQ ID NO: 11), and (3) scFc-LLRAL (described further below) fused to the N-terminus of C-half ferritin (scFc-LLRAL-C_hFTL, SEQ ID NO: 13) were prepared, mixed in a molar ratio of 2:1:1, and transiently transfected into ExpiCHO-S cells to generate and form DR5-targeted MBs. See Figure 1B. scFc-LLRAL is a single-chain Fc dimer based on IgG1 Fc and has the following Fc mutations: L234A, L235A, G236R, G237A, and A330L. The residue at position 329 is a proline (numbering according to the EU numbering scheme).

[0132] The resulting maltabody is a DR5-targeting maltabody and is hereafter referred to as Cona MB IgG1 LLRAL.

[0133] Example 2 Therapeutic efficacy of DR5-targeted maltabodies in a chemotherapy-resistant xenograft mouse model The therapeutic efficacy of exemplary maltabodies generated as described in Example 1 was evaluated in a chemotherapy-resistant xenograft model. HCT-15 (colon adenocarcinoma) cells are known to be intrinsically resistant to chemotherapy drugs including paclitaxel, docetaxel, vincristine, doxorubicin, satraplatin, and oxaliplatin.

[0134] 5×10 6 HCT-15 cells were injected subcutaneously into the flanks of Balb / c SCID mice. Tumors were grown to 200 mm 3 The tumors were allowed to grow to an average size of 1000 mg / kg. Mice were sorted into one of three groups (vehicle, parental IgG (conatumumab), or Cona MB IgG1 LLRAL) to ensure that the average tumor volume was equal across groups. Mice received intraperitoneal (ip) injections of 5 mg / kg parental IgG, 5 mg / kg Cona MB IgG1 LLRAL, or vehicle once a week for two weeks. Tumor volumes were measured twice a week using calipers.

[0135] Figure 2A is a plot of tumor volume in various groups of mice over time from tumor injection. Red arrows indicate treatment time points. "MB" indicates the Cona MB IgG1 LLRAL group.

[0136] As shown in Figure 2A, treatment with Cona MB IgG1 LLRAL caused regression of established tumors over a period of time, after which the tumors began to grow again. Although tumors in Cona MB IgG1 LLRAL-treated mice grew again, tumor growth was still suppressed compared to tumor growth in the control (vehicle) group. Tumor growth in the parental IgG-treated group was found to be indistinguishable from that in the vehicle group.

[0137] Cona MB IgG1 LLRAL-treated mice also showed statistically significant (p<0.0001) improved survival compared to vehicle- or parental IgG-treated mice (see Figure 2B, which depicts a plot of event-free survival over time for the three groups).

[0138] These results suggest that Cona MB IgG1 LLRAL demonstrated therapeutic efficacy and improved survival in a chemotherapy-resistant tumor model that was also resistant to treatment with the parental IgG administered at the same dose.

[0139] Example 3 Dose-ranging efficacy of DR5-targeted maltabodies in a chemotherapy-resistant xenograft mouse model The dose ranging efficacy of Cona IgG1 MB LLRAL was also investigated in the same chemotherapy-resistant xenograft model used in Example 2.

[0140] 5×10 6 HCT-15 cells were injected subcutaneously into the flanks of Balb / c SCID mice. Tumors were grown to 200 mm 3 The tumors were allowed to grow to an average size of 1000 mg / kg. 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 groups. Mice received treatment or vehicle by intraperitoneal (ip) injection once a week for three weeks. Tumor volumes were measured twice a week using calipers. Blood samples were obtained throughout the experiment, as indicated by the red arrows in Figure 3, which depicts the timeline of these experiments.

[0141] Figure 4A is a plot of tumor volume over time for various groups of mice from the initial dose. "MB" refers to the Cona MB IgG1 LLRAL group. As shown in Figure 4A, treatment with 0.25 mg / kg, 1 mg / kg, and 5 mg / kg doses of Cona MB IgG1 LLRAL slowed tumor progression, with these treatment groups showing significant improvement over the vehicle group, and the 1 mg / kg treatment group exhibiting comparable treatment efficacy to the 5 mg / kg group.

[0142] Figure 4B is a plot depicting the amount of detectable Cona MB IgG1 LLRAL in blood samples taken at various time points after administration of the first dose. Cona MB IgG1 LLRAL was detectable at all time points examined in the 0.25 mg / kg, 1 mg / kg, and 5 mg / kg Cona MB IgG1 LLRAL treatment groups.

[0143] These results demonstrate that Cona MB IgG1 LLRAL exhibited therapeutic efficacy at multiple doses tested, including comparable efficacy at dose levels one-fifth the levels examined in Example 2.

[0144] Example 4 Maltabodies penetrate large chemotherapy-resistant tumors and induce apoptosis The ability of exemplary maltabodies, generated as described in Example 1, to penetrate large tumors and induce apoptosis was evaluated in a chemotherapy-resistant xenograft model.

[0145] 5×10 6 HCT-15 cells were injected subcutaneously into the flanks of Balb / c SCID mice. Tumors were allowed to grow until well established (approximately 31 days). Tumor volume was estimated using calipers. The mean tumor size was identical across the two groups, approximately 400 mm in each group. 3Mice were randomly assigned to vehicle or treatment groups so that the maximal response was 100%. Mice were then administered vehicle or 5 mg / kg of Cona MB IgG1 LLRAL via intraperitoneal injection. Blood samples were collected 24 hours after injection of vehicle or Cona MB IgG1 LLRAL, and tumors were collected for subsequent histological analysis. Figure 5 shows a schematic diagram and timeline of these experiments.

[0146] Tumors were fixed in formalin, embedded in paraffin, and sectioned. Tumor sections were immunohistochemically stained for cleaved caspase 3, an apoptosis marker. Representative images of stained tissue sections are shown in Figures 6A and 6B (vehicle-treated mice) and Figures 6C and 6D (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 many apoptotic cells throughout the tumor, away from the tumor periphery and deep within the tumor core (see stained cells in Figures 6C and 6D).

[0147] These results confirm that Cona MB IgG1 LLRAL induced tumor cell apoptosis in the tumor core. Furthermore, these results confirm that Maltabodies are able to penetrate established large tumor cores. Sequence Listing The underlined parts in the fusion sequences indicate linker sequences. Bold text within the fusion sequence indicates ferritin or ferritin subunit sequences. The underlined and bolded text within the variable region sequences together indicate the complementarity determining region sequences. Boxed and bolded residues indicate residues that were mutated compared to a reference molecule, eg, IgG1 Fc. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0148] Numbered Embodiments Embodiment 1: A method of treating a subject having or at risk of having a chemotherapy-resistant tumor, comprising administering to the subject a pharmaceutical composition comprising a self-assembling polypeptide complex, wherein the self-assembling polypeptide complex comprises a plurality of first fusion polypeptides, each of the first fusion polypeptides comprising (1) an antibody or antibody fragment capable of binding to a tumor-associated antigen expressed on the chemotherapy-resistant tumor, and (2) a nanocage monomer or subunit thereof, wherein said administering results in slowing, inhibition of tumor progression, or tumor regression.

[0149] Embodiment 2: The method of embodiment 1, wherein the complex further comprises a plurality of second fusion polypeptides, each comprising (1) an Fc polypeptide and (2) a nanocage monomer or subunit thereof.

[0150] Embodiment 3: The method of embodiment 2, wherein each second fusion polypeptide comprises an Fc polypeptide linked to a nanocage monomer or a subunit thereof via an amino acid linker.

[0151] Embodiment 4: The amino acid linker is selected from the group consisting of (G n S) m 4. The method of embodiment 3, comprising a linker.

[0152] Embodiment 5: (G n S) m The linker is (GGGGS) m 5. The method of embodiment 4, wherein the linker is (SEQ ID NO: 50).

[0153] Embodiment 6: The method of any one of embodiments 2 to 5, wherein the Fc polypeptide comprises a single-chain Fc (scFc) comprising two Fc chains, and the two Fc chains are linked via an amino acid linker.

[0154] Embodiment 7: The amino acid linker joining the two Fc chains is n S) m 7. The method of embodiment 6, comprising a linker.

[0155] Embodiment 8: (G n S) m The linker is (GGGGS) m 13. The method of embodiment 12, wherein the linker is (SEQ ID NO: 50).

[0156] Embodiment 9: The method of any one of embodiments 2 to 8, wherein the Fc polypeptide comprises an IgG1 Fc chain.

[0157] Embodiment 10: The method of embodiment 9, wherein the IgG1 Fc chain comprises (1) an amino acid residue other than glycine at position 237, and (2) a proline residue at position 329 according to EU numbering.

[0158] Embodiment 11: The method of embodiment 10, wherein the IgG1 Fc chain comprises an alanine at position 237 according to EU numbering.

[0159] Embodiment 12: The method of embodiment 10 or 11, wherein 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.

[0160] Embodiment 13: The method of any one of embodiments 1 to 12, wherein the nanocage monomer or subunit thereof is a ferritin monomer or subunit thereof.

[0161] Embodiment 14: The method of embodiment 13, wherein the ferritin monomer or subunit thereof is a ferritin light chain or subunit thereof.

[0162] Embodiment 15: The method of embodiment 13 or 14, wherein the ferritin monomer or subunit thereof is human ferritin or a subunit thereof.

[0163] Embodiment 16: The method of any one of embodiments 13 to 15, wherein the self-assembling polypeptide complex does not comprise a ferritin heavy chain or a subunit of a ferritin heavy chain.

[0164] Embodiment 17: The method of any one of embodiments 13 to 16, wherein at least a portion of the first fusion polypeptides and / or a portion of the second fusion polypeptides are fusion polypeptides each comprising a ferritin monomer subunit.

[0165] Embodiment 18: The method of embodiment 17, wherein (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 an N half-ferritin and each second fusion polypeptide comprises a C half-ferritin.

[0166] Embodiment 19: The method of embodiment 18, wherein the self-assembling polypeptide complex is characterized by a ratio of the first fusion polypeptide to the second fusion polypeptide of 1:1.

[0167] Embodiment 20: The method of any one of embodiments 1 to 19, wherein the total number of fusion polypeptides in each self-assembling polypeptide complex is at least 24.

[0168] Embodiment 21: The method of claim 20, wherein the total number of fusion polypeptides in each self-assembling polypeptide complex is at least 32.

[0169] Embodiment 22: The method of any one of embodiments 1 to 19, wherein the total number of fusion polypeptides in each self-assembling polypeptide complex is about 24.

[0170] Embodiment 23: The method of any one of embodiments 1 to 19, wherein the total number of fusion polypeptides in each self-assembling polypeptide complex is about 32.

[0171] Embodiment 24: The method of any one of embodiments 1 to 19, wherein the total number of fusion polypeptides in each self-assembling polypeptide complex is between 24 and 48.

[0172] Embodiment 25: The method of any one of embodiments 1 to 24, wherein the antibody or antibody fragment in each of the first fusion polypeptides is bound to the N-terminus of the nanocage monomer or subunit thereof.

[0173] Embodiment 26: The method of any one of embodiments 1 to 25, wherein each first fusion polypeptide comprises (1) a Fab fragment and (2) a nanocage monomer or a subunit thereof.

[0174] Embodiment 27: The method of any one of embodiments 1 to 26, wherein each of the first fusion polypeptides does not comprise an antibody CH2 domain or an antibody CH3 domain.

[0175] Embodiment 28: The method of any one of embodiments 1 to 27, wherein the tumor-associated antigen is a member of the TNF receptor superfamily.

[0176] Embodiment 29: The method of embodiment 28, wherein the member of the TNF receptor superfamily is a death receptor.

[0177] Embodiment 30: The method of embodiment 29, wherein the death receptor is DR5.

[0178] Embodiment 31: The method of embodiment 30, wherein the antibody or antibody fragment is conatumumab, tigatuzumab, lexatumumab, drozitumumab, or a DR5-binding fragment of any of the foregoing.

[0179] Embodiment 32: The method of embodiment 31, wherein the antibody or antibody fragment is conatumumab or a DR5-binding fragment thereof.

[0180] Embodiment 33: The antibody or 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: 23 or a sequence that differs therefrom by one or two amino acids; (ii) CDR-H2 has the sequence of SEQ ID NO: 24 or a sequence that differs therefrom by one or two amino acids; (iii) CDR-H3 has the sequence of SEQ ID NO: 25 or a sequence that differs therefrom by one or two amino acids; (iv) CDR-L1 has the sequence of SEQ ID NO: 20 or a sequence that differs therefrom by one or two amino acids; (v) CDR-L2 has the sequence of SEQ ID NO: 21 or a sequence that differs therefrom by one or two amino acids; (vi) CDR-L3 has the sequence of SEQ ID NO: 22 or a sequence that differs therefrom by one or two amino acids; (b) (i) CDR-H1 has the sequence of SEQ ID NO: 31 or a sequence that differs therefrom by one or two amino acids; (ii) CDR-H2 has the sequence of SEQ ID NO: 32 or a sequence that differs therefrom by one or two amino acids; (iii) CDR-H3 has the sequence of SEQ ID NO: 33 or a sequence that differs therefrom by one or two amino acids; (iv) CDR-L1 has the sequence of SEQ ID NO: 28 or a sequence that differs therefrom by one or two amino acids; (v) CDR-L2 has the sequence of SEQ ID NO: 29 or a sequence that differs therefrom by one or two amino acids; (vi) CDR-L3 has the sequence of SEQ ID NO: 30 or a sequence that differs therefrom by one or two amino acids; (c) (i) CDR-H1 has the sequence of SEQ ID NO: 39 or a sequence that differs therefrom by one or two amino acids; (ii) CDR-H2 has the sequence of SEQ ID NO: 40 or a sequence that differs therefrom by one or two amino acids; (iii) CDR-H3 has the sequence of SEQ ID NO: 41 or a sequence that differs therefrom by one or two amino acids; (iv) CDR-L1 has the sequence of SEQ ID NO: 36 or a sequence that differs therefrom by one or two amino acids; (v) CDR-L2 has the sequence of SEQ ID NO: 37 or a sequence that differs therefrom by one or two amino acids; (vi) CDR-L3 has the sequence of SEQ ID NO: 38 or a sequence that differs therefrom by one or two amino acids; or (d) (i) CDR-H1 has the sequence of SEQ ID NO: 47 or a sequence that differs therefrom by one or two amino acids; (ii) CDR-H2 has the sequence of SEQ ID NO: 48 or a sequence that differs therefrom by one or two amino acids; (iii) CDR-H3 has the sequence of SEQ ID NO: 49 or a sequence that differs therefrom by one or two amino acids; (iv) CDR-L1 has the sequence of SEQ ID NO: 44 or a sequence that differs therefrom by one or two amino acids; (v) CDR-L2 has the sequence of SEQ ID NO: 45 or a sequence that differs therefrom by one or two amino acids; (vi) The method of embodiment 31, wherein the CDR-L3 has the sequence of SEQ ID NO: 46 or a sequence that differs therefrom by one or two amino acids.

[0181] Embodiment 34: 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: 23; (ii) CDR-H2 has the sequence of SEQ ID NO: 24; (iii) CDR-H3 has the sequence of SEQ ID NO: 25; (iv) CDR-L1 has the sequence of SEQ ID NO: 20; (v) CDR-L2 has the sequence of SEQ ID NO: 21; (vi) CDR-L3 has the sequence of SEQ ID NO: 22; (b)(i) CDR-H1 has the sequence of SEQ ID NO: 31; (ii) CDR-H2 has the sequence of SEQ ID NO: 32; (iii) CDR-H3 has the sequence of SEQ ID NO: 33; (iv) CDR-L1 has the sequence of SEQ ID NO: 28; (v) CDR-L2 has the sequence of SEQ ID NO: 29; (vi) CDR-L3 has the sequence of SEQ ID NO: 30; (c)(i) CDR-H1 has the sequence of SEQ ID NO: 39; (ii) CDR-H2 has the sequence of SEQ ID NO: 40; (iii) CDR-H3 has the sequence of SEQ ID NO: 41; (iv) CDR-L1 has the sequence of SEQ ID NO: 36; (v) CDR-L2 has the sequence of SEQ ID NO: 37; (vi) CDR-L3 has the sequence of SEQ ID NO: 38; or (d)(i) CDR-H1 has the sequence of SEQ ID NO: 47; (ii) CDR-H2 has the sequence of SEQ ID NO: 48; (iii) CDR-H3 has the sequence of SEQ ID NO: 49; (iv) CDR-L1 has the sequence of SEQ ID NO: 44; (v) CDR-L2 has the sequence of SEQ ID NO: 45; (vi) The method of embodiment 33, wherein the CDR-L3 has the sequence of SEQ ID NO: 46.

[0182] Embodiment 35: The antibody fragment is (1) a heavy chain variable region having at least 85% identity to a reference VH sequence; (2) a light chain variable region having at least 85% identity to a reference VL sequence; Including, (a) the reference VH sequence has the sequence of SEQ ID NO: 19 and the reference VL sequence has the sequence of SEQ ID NO: 18; or (b) the reference VH sequence has the sequence of SEQ ID NO: 27 and the reference VL sequence has the sequence of SEQ ID NO: 26; or (c) the reference VH sequence has the sequence of SEQ ID NO: 35 and the reference VL sequence has the sequence of SEQ ID NO: 34; or (d) The method of any one of embodiments 1 to 34, wherein the reference VH sequence has the sequence of SEQ ID NO: 43 and the reference VL sequence has the sequence of SEQ ID NO: 42.

[0183] Embodiment 36: The method of any one of embodiments 1 to 35, wherein the subject is a mammal.

[0184] Embodiment 37: The method of embodiment 36, wherein the subject is a human.

[0185] Embodiment 38: The method of any one of embodiments 1 to 37, wherein the tumor is a colon tumor.

[0186] Embodiment 39: The method of any one of embodiments 1 to 38, wherein the subject has a chemotherapy-resistant tumor.

[0187] Embodiment 40: The method of any one of embodiments 1 to 39, wherein the chemotherapy-resistant tumor is resistant to a taxane, a vinca alkaloid, an anthracycline, or any combination of the foregoing.

[0188] Embodiment 41: The method of embodiment 40, wherein the chemotherapy-resistant tumor is resistant to a taxane.

[0189] Embodiment 42: The method of embodiment 41, wherein the taxane is paclitaxel, a taxane, or both.

[0190] Embodiment 43: The method of embodiment 40, 41 or 42, wherein the chemotherapy-resistant tumor is resistant to a vinca alkaloid.

[0191] Embodiment 44: The method of embodiment 43, wherein the vinca alkaloid is vincristine.

[0192] Embodiment 45: The method of any one of embodiments 41 to 44, wherein the chemotherapy-resistant tumor is resistant to anthracyclines.

[0193] Embodiment 46: The method of embodiment 45, wherein the anthracycline is doxorubicin.

[0194] Embodiment 47: The method of any one of embodiments 41 to 46, wherein the chemotherapy-resistant tumor is resistant to a platinum-containing compound.

[0195] Embodiment 48: The method of embodiment 47, wherein the platinum-containing compound is satraplatin, oxaliplatin, or both.

[0196] Embodiment 49: The method of any one of embodiments 1 to 46, wherein the administering step comprises administration by a systemic route.

[0197] Embodiment 50: The method of embodiment 47, wherein the systemic route comprises subcutaneous administration, intravenous administration, intramuscular administration, inhalation administration, or intranasal administration.

[0198] Embodiment 51: The method of any one of embodiments 1 to 48, wherein the administering step comprises administering more than one dose of the pharmaceutical composition.

[0199] Embodiment 52: The method of embodiment 49, wherein the administering step comprises administering the pharmaceutical composition once a week for at least two weeks.

[0200] Embodiment 53: The method of embodiment 50, wherein the administering step comprises administering the pharmaceutical composition once a week for at least three weeks.

[0201] Embodiment 54: A method of treating a subject having or at risk of having a chemotherapy-resistant tumor, comprising: administering to a subject a pharmaceutical composition comprising the self-assembling polypeptide complex. Including, the self-assembling polypeptide complex is (a) a plurality of first fusion polypeptides, each of which comprises (1) an antibody fragment capable of binding to DR5 and (2) a ferritin monomer or subunit thereof; (b) a plurality of second fusion polypeptides, each comprising (1) an scFc and (2) a ferritin monomer or subunit thereof, wherein the scFc comprises two IgG1 Fc chains, each 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; wherein said administering step results in slowing, inhibition of tumor progression, or tumor regression.

[0202] Equivalents / Alternative Embodiments While the invention has been described in connection with particular embodiments thereof, it will be understood that it is capable of further modifications, and this application is intended to cover any variations, uses, or adaptations of the invention including departures from the present disclosure which come within known or customary practice in the art to which this invention pertains, in accordance with the principles of the invention generally, and which may be adapted to the essential features described hereinabove.

Claims

1. 1. Use of a pharmaceutical composition comprising a self-assembling polypeptide complex for treating a subject having or at risk of having a chemotherapy-resistant tumor, comprising: the self-assembling polypeptide complex a plurality of first fusion polypeptides, each comprising (1) an antibody or antibody fragment capable of binding to a tumor-associated antigen expressed on said chemotherapy-resistant tumor, and (2) a nanocage monomer or subunit thereof; Including, use.

2. The complex is a plurality of second fusion polypeptides, each comprising (1) an Fc polypeptide and (2) a nanocage monomer or subunit thereof; The use of claim 1 further comprising:

3. The use of claim 2 , wherein each second fusion polypeptide comprises the Fc polypeptide linked to the nanocage monomer or a subunit thereof via an amino acid linker.

4. The amino acid linker is ｎ S) ｍ The use according to claim 3, comprising a linker.

5. The above (G ｎ S) ｍ The linker is (GGGGS) ｍ (SEQ ID NO: 50) is a linker.

6. The use according to any one of claims 2 to 5, 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.

7. the amino acid linker connecting the two Fc chains is ｎ S) ｍ The use according to claim 6, comprising a linker.

8. The above (G ｎ S) ｍ The linker is (GGGGS) ｍ (SEQ ID NO: 50) is a linker.

9. The use according to any one of claims 2 to 8, wherein the Fc polypeptide comprises an IgG1 Fc chain.

10. 10. The use of claim 9, wherein the IgG1 Fc chain comprises (1) an amino acid residue other than glycine at position 237 and (2) a proline residue at position 329 according to EU numbering.

11. The use of claim 10, wherein the IgG1 Fc chain comprises an alanine at position 237 according to EU numbering.

12. 12. The use of claim 10 or 11, wherein 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.

13. The use according to any one of claims 1 to 12, wherein said nanocage monomer or subunit thereof is a ferritin monomer or subunit thereof.

14. The use according to claim 13, wherein the ferritin monomer or subunit thereof is a ferritin light chain or subunit thereof.

15. The use according to claim 13 or 14, wherein the ferritin monomer or subunit thereof is human ferritin or a subunit thereof.

16. The use according to any one of claims 13 to 15, wherein the self-assembling polypeptide complex does not comprise a ferritin heavy chain or a subunit of a ferritin heavy chain.

17. The use according to any one of claims 13 to 16, wherein at least a portion of the first plurality of fusion polypeptides and / or a portion of the second plurality of fusion polypeptides are fusion polypeptides each comprising a ferritin monomer subunit.

18. (a) each first fusion polypeptide comprises a C-half ferritin and each second fusion polypeptide comprises an N-half ferritin; or (b) each of the first fusion polypeptides comprises an N-half ferritin and each of the second fusion polypeptides comprises a C-half ferritin; 18. The use according to claim 17.

19. 19. The use of claim 18, wherein the self-assembling polypeptide complex is characterized by a 1:1 ratio of first fusion polypeptide to second fusion polypeptide.

20. The use according to any one of claims 1 to 19, wherein the total number of fusion polypeptides in each self-assembling polypeptide complex is at least 24.

21. 21. The use of claim 20, wherein the total number of fusion polypeptides in each self-assembling polypeptide complex is at least 32.

22. The use according to any one of claims 1 to 19, wherein the total number of fusion polypeptides in each self-assembling polypeptide complex is about 24.

23. The use according to any one of claims 1 to 19, wherein the total number of fusion polypeptides in each self-assembling polypeptide complex is about 32.

24. The use according to any one of claims 1 to 19, wherein the total number of fusion polypeptides in each self-assembling polypeptide complex is between 24 and 48.

25. The use according to any one of claims 1 to 24, wherein the antibody or antibody fragment in each first fusion polypeptide is bound to the N-terminus of the nanocage monomer or subunit thereof.

26. The use according to any one of claims 1 to 25, wherein each first fusion polypeptide comprises (1) a Fab fragment and (2) a nanocage monomer or a subunit thereof.

27. The use according to any one of claims 1 to 26, wherein each of the first fusion polypeptides does not comprise an antibody CH2 domain or an antibody CH3 domain.

28. The use according to any one of claims 1 to 27, wherein the tumor-associated antigen is a member of the TNF receptor superfamily.

29. 29. The use of claim 28, wherein the member of the TNF receptor superfamily is a death receptor.

30. 30. The use according to claim 29, wherein the death receptor is DR5.

31. 31. The use of claim 30, wherein the antibody or antibody fragment is conatumumab, tigatuzumab, lexatumumab, drozitumab, or a DR5-binding fragment of any of the foregoing.

32. 32. The use of claim 31 , wherein the antibody or antibody fragment is conatumumab or a DR5-binding fragment thereof.

33. the antibody or 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) the CDR-H1 has the sequence of SEQ ID NO: 23 or a sequence that differs therefrom by one or two amino acids; (ii) the CDR-H2 has the sequence of SEQ ID NO: 24 or a sequence which differs therefrom by one or two amino acids; (iii) the CDR-H3 has the sequence of SEQ ID NO: 25 or a sequence that differs therefrom by one or two amino acids; (iv) the CDR-L1 has the sequence of SEQ ID NO: 20 or a sequence which differs therefrom by one or two amino acids; (v) the CDR-L2 has the sequence of SEQ ID NO: 21 or a sequence which differs therefrom by one or two amino acids; (vi) the CDR-L3 has the sequence of SEQ ID NO: 22 or a sequence which differs therefrom by one or two amino acids; (b) (i) the CDR-H1 has the sequence of SEQ ID NO: 31 or a sequence that differs therefrom by one or two amino acids; (ii) the CDR-H2 has the sequence of SEQ ID NO: 32 or a sequence which differs therefrom by one or two amino acids; (iii) the CDR-H3 has the sequence of SEQ ID NO: 33 or a sequence which differs therefrom by one or two amino acids; (iv) the CDR-L1 has the sequence of SEQ ID NO: 28 or a sequence which differs therefrom by one or two amino acids; (v) the CDR-L2 has the sequence of SEQ ID NO: 29 or a sequence that differs therefrom by one or two amino acids; (vi) the CDR-L3 has the sequence of SEQ ID NO: 30 or a sequence which differs therefrom by one or two amino acids; (c) (i) the CDR-H1 has the sequence of SEQ ID NO: 39 or a sequence which differs therefrom by one or two amino acids; (ii) the CDR-H2 has the sequence of SEQ ID NO: 40 or a sequence which differs therefrom by one or two amino acids; (iii) the CDR-H3 has the sequence of SEQ ID NO: 41 or a sequence which differs therefrom by one or two amino acids; (iv) the CDR-L1 has the sequence of SEQ ID NO: 36 or a sequence which differs therefrom by one or two amino acids; (v) the CDR-L2 has the sequence of SEQ ID NO: 37 or a sequence which differs therefrom by one or two amino acids; (vi) the CDR-L3 has the sequence of SEQ ID NO: 38 or a sequence which differs therefrom by one or two amino acids; or (d) (i) the CDR-H1 has the sequence of SEQ ID NO: 47 or a sequence which differs therefrom by one or two amino acids; (ii) the CDR-H2 has the sequence of SEQ ID NO: 48 or a sequence which differs therefrom by one or two amino acids; (iii) the CDR-H3 has the sequence of SEQ ID NO: 49 or a sequence that differs therefrom by one or two amino acids; (iv) the CDR-L1 has the sequence of SEQ ID NO: 44 or a sequence which differs therefrom by one or two amino acids; (v) the CDR-L2 has the sequence of SEQ ID NO: 45 or a sequence which differs therefrom by one or two amino acids; (vi) The use according to claim 31, wherein the CDR-L3 has the sequence of SEQ ID NO: 46 or a sequence which differs therefrom by one or two amino acids.

34. 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) the CDR-H1 has the sequence of SEQ ID NO: 23; (ii) the CDR-H2 has the sequence of SEQ ID NO: 24; (iii) the CDR-H3 has the sequence of SEQ ID NO: 25; (iv) the CDR-L1 has the sequence of SEQ ID NO: 20; (v) the CDR-L2 has the sequence of SEQ ID NO: 21; (vi) the CDR-L3 has the sequence of SEQ ID NO: 22; (b)(i) the CDR-H1 has the sequence of SEQ ID NO: 31; (ii) the CDR-H2 has the sequence of SEQ ID NO: 32; (iii) the CDR-H3 has the sequence of SEQ ID NO: 33; (iv) the CDR-L1 has the sequence of SEQ ID NO: 28; (v) the CDR-L2 has the sequence of SEQ ID NO: 29; (vi) the CDR-L3 has the sequence of SEQ ID NO: 30; (c)(i) the CDR-H1 has the sequence of SEQ ID NO: 39; (ii) the CDR-H2 has the sequence of SEQ ID NO: 40; (iii) the CDR-H3 has the sequence of SEQ ID NO: 41; (iv) the CDR-L1 has the sequence of SEQ ID NO: 36; (v) the CDR-L2 has the sequence of SEQ ID NO: 37; (vi) the CDR-L3 has the sequence of SEQ ID NO: 38; or (d)(i) the CDR-H1 has the sequence of SEQ ID NO: 47; (ii) the CDR-H2 has the sequence of SEQ ID NO: 48; (iii) the CDR-H3 has the sequence of SEQ ID NO: 49; (iv) the CDR-L1 has the sequence of SEQ ID NO: 44; (v) the CDR-L2 has the sequence of SEQ ID NO: 45; (vi) The use according to claim 33, wherein the CDR-L3 has the sequence of SEQ ID NO:

46.

35. the antibody fragment (1) a heavy chain variable region having at least 85% identity to a reference VH sequence; (2) a light chain variable region having at least 85% identity to a reference VL sequence; Including, (a) the reference VH sequence has the sequence of SEQ ID NO: 19 and the reference VL sequence has the sequence of SEQ ID NO: 18; or (b) the reference VH sequence has the sequence of SEQ ID NO: 27 and the reference VL sequence has the sequence of SEQ ID NO: 26; or (c) the reference VH sequence has the sequence of SEQ ID NO: 35 and the reference VL sequence has the sequence of SEQ ID NO: 34; or (d) the reference VH sequence has the sequence of SEQ ID NO: 43 and the reference VL sequence has the sequence of SEQ ID NO:

42.

36. The use according to any one of claims 1 to 35, wherein the subject is a mammal.

37. 37. The use of claim 36, wherein the subject is a human.

38. The use according to any one of claims 1 to 37, wherein the tumor is a colon tumor.

39. The use according to any one of claims 1 to 38, wherein the subject has a chemotherapy-resistant tumor.

40. 40. The use of any one of claims 1 to 39, wherein the chemotherapy-resistant tumor is resistant to taxanes, vinca alkaloids, anthracyclines or any combination of the foregoing.

41. 41. The use of claim 40, wherein the chemotherapy-resistant tumor is resistant to taxanes.

42. 42. The use of claim 41, wherein the taxane is paclitaxel, a taxane, or both.

43. 43. The use of claim 40, 41 or 42, wherein the chemotherapy-resistant tumor is resistant to a vinca alkaloid.

44. 44. The use of claim 43, wherein the vinca alkaloid is vincristine.

45. The use according to any one of claims 41 to 44, wherein the chemotherapy-resistant tumor is resistant to anthracyclines.

46. 46. ​​The use of claim 45, wherein the anthracycline is doxorubicin.

47. The use according to any one of claims 41 to 46, wherein the chemotherapy-resistant tumor is resistant to platinum-containing compounds.

48. 48. The use of claim 47, wherein the platinum-containing compound is satraplatin, oxaliplatin, or both.

49. 47. The use of any one of claims 1 to 46, wherein the administering step comprises administration by a systemic route.

50. 48. The use of claim 47, wherein the systemic route comprises subcutaneous administration, intravenous administration, intramuscular administration, inhalation administration or intranasal administration.

51. The use according to any one of claims 1 to 48, wherein more than one dose of the pharmaceutical composition is administered to the subject.

52. 50. The use of claim 49, wherein the pharmaceutical composition is administered to the subject once a week for at least two weeks.

53. 51. The use of claim 50, wherein the pharmaceutical composition is administered to the subject once a week for at least three weeks.

54. 54. The use of any one of claims 1 to 53, wherein said use results in a slowing or inhibition of progression of said tumor or regression of said tumor.

55. 1. Use of a pharmaceutical composition comprising a self-assembling polypeptide complex for treating a subject having or at risk of having a chemotherapy-resistant tumor, comprising: the self-assembling polypeptide complex (a) a plurality of first fusion polypeptides, each of which comprises (1) an antibody fragment capable of binding to DR5 and (2) a ferritin monomer or subunit thereof; (b) a plurality of second fusion polypeptides, each comprising (1) an scFc and (2) a ferritin monomer or subunit thereof; and wherein the scFc comprises two IgG1 Fc chains, each 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.

56. 56. The use of claim 55, wherein said use results in a slowing or inhibition of progression of said tumor or regression of said tumor.

57. 1. Use of a self-assembling polypeptide complex in the manufacture of a medicament for treating chemotherapy-resistant tumors, the self-assembling polypeptide complex comprising: a plurality of first fusion polypeptides, each comprising (1) an antibody or antibody fragment capable of binding to a tumor-associated antigen expressed on said chemotherapy-resistant tumor and (2) a nanocage monomer or subunit thereof; Including, use.

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