Multivalent and multispecific cytokine antagonists

Abstract

A fusion polypeptide comprising: (1) a cytokine binding moiety and (2) a nanocage monomer or subunit thereof; self-assembling polypeptide complexes comprising such fusion polypeptides. Self-assembling polypeptide complexes comprising an Fc fusion polypeptide and the fusion polypeptides and / or tumor-binding fusion polypeptides disclosed herein, as well as related compositions and methods.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 433,476 (filed December 18, 2022), the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

[0003] Sequence Listing

[0004] This specification refers to a sequence listing submitted electronically in XML format, which is hereby incorporated by reference in its entirety. The XML file, created on December 13, 2023, is named RBT-052WO_SL.xml and is 330 kb in size. Background Art

[0005] Cytokines are small proteins that have different effects on the growth and activity of control cells (such as immune cells and tumor cells). Anti-cytokine therapy is an emerging treatment paradigm for a variety of human diseases and conditions (including cancer and autoimmune diseases). A challenge faced by this treatment approach stems from complex disease biology, which may involve multiple pathophysiological processes driven by different cytokines. Therefore, therapeutic agents that can only target a single cytokine may lack efficacy.

[0006] There is a need for anti-cytokine therapeutics with improved efficacy. Summary of the Invention

[0007] The present invention addresses this need by providing a self-assembling polypeptide complex, the specificity and potency of which can be adjusted to enhance efficacy. In certain embodiments, the complex provided is multispecific, for example, capable of binding (e.g., specifically binding) to more than one cytokine. The self-assembling polypeptide complex provided typically displays one or more types of cytokine binding moieties and optionally displays one or more tumor binding moieties. Such self-assembling polypeptide complexes can be used to isolate cytokines, thereby interfering with cytokine signaling. Related fusion proteins, complexes, compositions and methods are also disclosed.

[0008] In one aspect, a fusion polypeptide is provided comprising: (1) a cytokine binding portion and (2) a nanocage monomer or a subunit thereof.

[0009] In some embodiments, the cytokine binding moiety comprises an antibody or an antigen-binding fragment thereof.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain variable region (V H ) and immunoglobulin light chain variable region (V L or V K ).

[0011] In some embodiments, the antibody or antigen-binding fragment thereof comprises a Fab fragment.

[0012] In some embodiments, the Fab fragment is a single-chain Fab fragment (scFab).

[0013] In some embodiments, the cytokine binding portion is capable of binding interleukin-6 (IL-6) or interleukin-11 (IL-11).

[0014] In some embodiments, the cytokine binding portion comprises an antibody fragment comprising a heavy chain variable region (V H ) and light chain variable region (V L or V K ), where V H and the V L or V K The CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of the IL-6 or IL-11 antibody have sequences that each differ from the CDRs of the IL-6 or IL-11 antibody by a maximum of two amino acids.

[0015] In some embodiments, the V H and the V L or V K The CDRs have sequences identical to those of the heavy and light chain CDRs of the IL-6 or IL-11 antibody, except for a total of one or two amino acid substitutions among all six CDRs.

[0016] In some embodiments, the V H and the V L or V K The CDRs are identical to those of the heavy and light chain CDRs of IL-6 or IL-11 antibodies.

[0017] In some embodiments, the cytokine binding portion comprises an antibody fragment comprising heavy and light chain variable regions whose sequences are at least 85% identical to those of the heavy and light chain variable regions of an IL-6 or IL-11 antibody.

[0018] In some embodiments, the cytokine binding portion is capable of binding to an epitope of interleukin-6 (IL-6) that is involved in the binding of IL-6 to the IL-6 receptor (IL-6Rα).

[0019] In some embodiments, the cytokine binding portion is capable of binding an epitope of IL-6 that is involved in the binding of IL-6 to glycoprotein 130 (gpl30).

[0020] In some embodiments, the nanocage monomer is a ferritin monomer or a subunit thereof.

[0021] In some embodiments, the ferritin monomer is a human ferritin monomer.

[0022] In some embodiments, the ferritin monomer is a ferritin light chain.

[0023] In some embodiments, the cytokine binding moiety is linked to the nanocage monomer or subunit thereof via an amino acid linker.

[0024] In some embodiments, the cytokine binding moiety is attached via the N-terminus of the nanocage monomer or a subunit thereof.

[0025] In one aspect, a self-assembling polypeptide complex is provided, comprising: (a) a plurality of first cytokine binding fusion polypeptides, each first cytokine binding fusion polypeptide being a fusion polypeptide as disclosed herein; and (b) a plurality of Fc fusion polypeptides, each Fc fusion polypeptide comprising (1) an Fc polypeptide and (2) a nanocage monomer or a subunit thereof.

[0026] In some embodiments, the self-assembling polypeptide complex further comprises (c) a plurality of second cytokine binding fusion polypeptides, each second cytokine binding fusion polypeptide being a fusion polypeptide as disclosed herein, wherein the first and second cytokine binding fusion polypeptides respectively comprise a first and a second cytokine binding portion capable of binding to different epitopes.

[0027] In some embodiments, the first and second cytokine binding moieties are capable of binding different cytokines.

[0028] In some embodiments, the first cytokine binding moiety is capable of binding IL-6 and the second cytokine binding moiety is capable of binding IL-11.

[0029] In some embodiments, the self-assembling polypeptide complex further comprises (d) a plurality of tumor-binding fusion polypeptides, each tumor-binding fusion polypeptide comprising (1) a tumor-binding portion and (2) a nanocage monomer or a subunit thereof.

[0030] In some embodiments, in each Fc fusion polypeptide, the Fc polypeptide is linked to the nanocage monomer or subunit thereof via an amino acid linker.

[0031] In some embodiments, the Fc polypeptide is linked via the N-terminus of the nanocage monomer or a subunit thereof.

[0032] In some embodiments, the Fc polypeptide is linked via the C-terminus of the nanocage monomer or a subunit thereof.

[0033] In some embodiments, within each tumor-binding fusion polypeptide, the tumor-binding moiety is linked to the nanocage monomer or subunit thereof via an amino acid linker.

[0034] In some embodiments, the tumor-binding moiety is attached via the N-terminus of the nanocage monomer or a subunit thereof.

[0035] In some embodiments, the tumor-binding moiety comprises an antibody or an antigen-binding fragment thereof.

[0036] In some embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain variable region (V H ) and immunoglobulin light chain variable region (V L or V K ).

[0037] In some embodiments, the antibody or antigen-binding fragment thereof comprises a Fab fragment. In some embodiments, the Fab fragment is a single-chain Fab fragment (scFab).

[0038] In some embodiments, the tumor binding moiety is a PD-L1 binding moiety.

[0039] In some embodiments, the tumor binding moiety comprises a heavy chain variable region (V H ) and light chain variable region (V L or V K ) of an antibody fragment, wherein the V H and the V L or V K The CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of the PD-L1 antibody have sequences that each differ from the CDRs of the PD-L1 antibody by a maximum of two amino acids.

[0040] In some embodiments, the V H and the V L or V K The CDRs of the PD-L1 antibody have sequences identical to those of the heavy and light chain CDRs of the PD-L1 antibody, except for a total of one or two amino acid substitutions among all six CDRs.

[0041] In some embodiments, the V H and the V L or V K The CDRs of are identical to those of the heavy and light chain CDRs of the PD-L1 antibody.

[0042] In some embodiments, the tumor-binding portion comprises an antibody fragment comprising heavy and light chain variable regions whose sequences are at least 85% identical to the sequences of the heavy and light chain variable regions of a PD-L1 antibody.

[0043] In some embodiments, each nanocage monomer within each Fc fusion polypeptide, each cytokine-binding fusion polypeptide, and / or each tumor-binding fusion polypeptide is a ferritin monomer or a subunit thereof.

[0044] In some embodiments, the ferritin monomer is a human ferritin monomer.

[0045] In some embodiments, the ferritin monomer is a ferritin light chain.

[0046] In some embodiments, the self-assembling polypeptide complex does not comprise any ferritin heavy chain or subunit of a ferritin heavy chain.

[0047] In some embodiments, the self-assembling polypeptide complex does not comprise any iron-binding moiety.

[0048] In one aspect, a pharmaceutical composition is provided, comprising a self-assembling polypeptide complex as disclosed herein and a pharmaceutically acceptable excipient.

[0049] In one aspect, provided is a use of a self-assembling polypeptide complex or pharmaceutical composition as disclosed herein for treating, ameliorating or preventing a disease or condition in a subject.

[0050] In one aspect, provided is a method of treating, ameliorating, or preventing a disease or condition, the method comprising administering to a subject a self-assembling polypeptide complex or pharmaceutical composition as disclosed herein.

[0051] In some embodiments, the subject is a mammal, eg, a human.

[0052] In some embodiments, the disease or condition is cancer, eg, a solid cancer or a hematological cancer.

[0053] In some embodiments, the disease or condition is an autoimmune disease or condition. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1A is a schematic representation of human ferritin light chain (hFTL) and example N-half ferritin (N-hFTL) and C-half ferritin (C-hFTL) molecules.

[0055] Figure 1B and 1C is a schematic representation of a set of fusion polypeptides that can together form exemplary multibodies of the present disclosure.

[0056] Figure 2A –2F shows a representative Octet binding sensorgram from an experiment to measure the interaction affinity and kinetics between IL-6 and one of the following: siltuximab immunoglobulin (IgG) ( Figure 2A ), clazakizumab IgG ( Figure 2B ), olokizumab IgG ( Figure 2C )、Siltuximab Multabody (MB)( Figure 2D ), Clazazumab MB ( Figure 2E ) or ologizumab MB ( Figure 2F ).

[0057] Figures 3A-3C IL-6 binding curves of ELISA experiments of anti-IL-6 Multibody and corresponding parental anti-IL-6 IgG are shown: Siltuximab ( Figure 3A ), clazakizumab ( Figure 3B ) and ologizumab ( Figure 3C ). IL-11 and bovine serum albumin (BSA) binding curves of the same anti-IL-6 Multibody and the corresponding parental anti-IL-6 IgG are also shown.

[0058] Figure 4 Western blots of extracts of HCT-15 tumor cells incubated with IL-6 alone or with anti-IL-6 Multibody were shown. The blots were stained for STAT3 to detect phosphorylation of STAT3 (pSTAT3), a marker of IL-6-induced IL-6Rα signaling activation. Actin staining was also depicted as a loading control.

[0059] Figures 5A-5C Shown are dose response curves for IL-6 signaling reporter assays performed on cells incubated with anti-IL-6 Multibody or parental anti-IL-6 IgG: Siltuximab ( Figure 5A ), clazakizumab ( Figure 5B ) and clazakizumab ( Figure 5C ).

[0060] Figure 6A and 6B Shown are the results of incubation of HCT-15 colon cancer cells ( Figure 6A ) or THP-1 leukemic mononuclear cells ( Figure 6B Dose response curve of STAT3 activation assay (MesoScale Discovery) performed on STAT3 cells. The parental antibody was olokizumab.

[0061] Figure 7A and 7BShown are dose-response curves of a cell proliferation assay (CellTiter-Glo Assay) performed on IL-6-dependent cells (INA-6 cells) incubated with anti-IL6 Multibody (MB) or with the corresponding parental anti-IL6 antibody (IgG). The parental antibody was Siltuximab ( Figure 7A ) and ologizumab ( Figure 7B ).

[0062] Figure 8 Shown are the binding profiles of anti-IL-11 MB and IgG to IL-11 or IL-6 antigens as determined by ELISA.

[0063] Figure 9 Shown are dose responses of a STAT3 activation assay (Meso Scale Discovery) performed on Panc-1 pancreatic cancer cells incubated with anti-IL-11 Multibody (MB) or with the corresponding parental anti-IL-11 antibody (IgG). The parental antibody was TPP-29536. DETAILED DESCRIPTION

[0064] Disclosed herein are fusion polypeptides each comprising (1) a nanocage monomer or a subunit thereof and (2) an antibody fragment (such as a cytokine binding agent or a tumor binding agent) or an Fc polypeptide. The nanocage monomer or a subunit thereof drives the fusion polypeptide to self-assemble into a complex that displays one or more of the aforementioned antibody fragments and Fc polypeptides.

[0065] definition

[0066] The terms "about" and "approximately", when used herein with reference to a value, are used interchangeably and refer to a value similar to the reference value. Generally, one skilled in the art familiar with the context will understand the relative degree of variation encompassed by "about" or "approximately" in this context. For example, in some embodiments, the terms "about" and "approximately" may 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.

[0067] As used herein, the terms "alter," "altered," "decrease," "decreased," "increase," "increased," or "reduction," "reduced" (e.g., with respect to some outcome or effect) have a meaning 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 level that is known or as determined using an IgG that does not contain the reference mutation in the Fc region.

[0068] As used herein, the term "antigen binding fragment" of an antibody refers to one or more fragments of an antibody that retains the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term "antigen binding fragment" of an antibody include Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, scFv fragments, dAb fragments (Ward et al., (1989) Nature 341:544-546), and separate complementary determining regions (CDRs). In some embodiments, an "antigen binding fragment" comprises a heavy chain variable region and a light chain variable region. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and fragments can be screened for effectiveness in the same manner as complete antibodies.

[0069] As used herein, unless otherwise indicated, the term "binding" refers to a non-covalent association between or among two or more entities. "Direct" binding involves physical contact between entities or moieties; indirect binding involves physical interactions by way of physical contact with one or more intermediate entities. Binding between two or more entities can generally be assessed in any of a variety of contexts—including where the interacting entities or moieties are studied in isolation or in the context of a more complex system environment (e.g., in the case of covalent or otherwise associated with a carrier entity and / or in a biological system or cell). As used herein, the phrase "non-binding" or "no binding" between two entities, or similar phrases, refers to 1) a lack of detectable binding or 2) binding below a set threshold corresponding to no binding in an appropriate assay, e.g., an in vitro binding assay such as a biolayer interferometry assay, surface plasmon resonance, a cell binding assay such as flow cytometry or an enzyme-linked immunosorbent assay (ELISA). For example, in some embodiments, in an in vitro biolayer interferometry assay, a maximum associative binding response of less than 0.1 nm after 180 seconds to a biosensor loaded with a 0.8 nm target when the test article is present at a concentration of 20 nM is classified as "no binding."

[0070] The terms "ferritin" and "apoferritin" are used interchangeably herein and generally refer to polypeptides (e.g., ferritin chains) that are capable of assembling into a ferritin complex, which typically comprises 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.

[0071] The term "ferritin monomer" is used herein to refer to a single chain of ferritin that is capable of self-assembling into a polypeptide complex comprising multiple ferritin chains (e.g., 24 or more ferritin chains) in the presence of other ferritin chains.

[0072] 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, it is understood by those of ordinary skill in the art that a polypeptide whose structure includes two or more functional domains or organizational domains (e.g., a fusion polypeptide) typically includes a stretch of amino acids between such domains that connect them to each other. In some embodiments, a polypeptide comprising a linker element has an overall structure of the general form S1-L-S2, wherein S1 and S2 can be the same or different and represent two domains associated with each other by a linker (L). In some embodiments, the linker is an "amino acid linker," i.e., it comprises amino acid residues, e.g., 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, the linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide.

[0073] As used herein, the term "multispecific" refers to a feature of having at least two binding sites at which at least two different binding partners, e.g., antigens or receptors (e.g., Fc receptors), can bind. For example, a polypeptide complex comprising at least two Fab fragments, wherein each of the two Fab fragments is capable of binding (e.g., specifically binding) to a different antigen, is "multispecific." As another example, a polypeptide complex comprising an Fc fragment (which is capable of binding (e.g., specifically binding) to an Fc receptor) and a Fab fragment (which is capable of binding (e.g., specifically binding) to an antigen) is "multispecific."

[0074] As used herein, the term "multivalent" refers to a feature of having at least two binding sites to which a binding partner, e.g., an antigen or a receptor (e.g., an Fc receptor), can bind. The binding partners that can bind to the at least two binding sites can be the same or different.

[0075] As used herein, the term "nanocage monomer" refers to a single chain of a polypeptide that is capable of self-assembling with other nanocage monomers to form a self-assembled polypeptide complex comprising a plurality of nanocage monomers. In some embodiments, the nanocage monomer is selected from monomers selected from ferritin, apoferritin, encapsulin, sulfuroxygenase reductase (SOR), lumazine synthase, pyruvate dehydrogenase, carboxysome, vault protein, GroEL, heat shock protein, E2P coat protein, MS2 coat protein, fragments thereof, and variants thereof.

[0076] As used herein, the term "polypeptide" generally has its art-recognized meaning: a polymer of at least three amino acids, for example, connected to each other by a peptide bond. It will be understood by those of ordinary skill in the art that the term "polypeptide" is intended to be sufficiently general, not only encompassing polypeptides with complete sequences as described herein, but also encompassing polypeptides representing functional fragments of such complete polypeptides (i.e., fragments retaining at least one activity). In addition, it will be understood by those of ordinary skill in the art that protein sequences generally tolerate some substitutions without destroying activity. Therefore, activity is retained and shares at least about 30-40% overall sequence identity (generally greater than about 50%, 60%, 70% or 80%) with another polypeptide of the same class, and further generally includes in one or more highly conserved regions (generally encompassing at least 3-4 amino acids, generally up to 20 or more amino acids) any polypeptide in a region much higher (generally greater than 90% or even 95%, 96%, 97%, 98% or 99%) is encompassed within the associated term "polypeptide" used herein. Polypeptide can contain L-amino acid, D-amino acid or both, and can contain any one of multiple amino acid modifications known in the art or analogs. Useful modifications include, for example, terminal acetylation, amidation, methylation, glycosylation, etc. In some embodiments, the protein can comprise natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof.

[0077] As used herein, the terms "specifically binds," "specifically binding," "binds specifically," or similar terms refer to the formation of a relatively stable complex between a binding moiety (e.g., an antibody or antigen-binding fragment thereof) and an antigen under physiological conditions. Specific binding can be measured by 1x10 -6M or smaller, 1x10 -7 M or smaller, 1x10 -8 M or smaller or 1x10 -9 M or smaller equilibrium dissociation constant (e.g., smaller K D Indicates tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, enzyme-linked immunosorbent assay, or biolayer interferometry. In some embodiments, "specific binding" and similar terms refer to a characteristic of a binding moiety wherein the binding moiety is able to bind to a target antigen but not to other antigens, such as distantly related family members of the antigen.

[0078] The term "self-assembled," when used in reference to a macromolecular complex (e.g., a polypeptide complex), refers to the spontaneous formation of the complex when sufficient components of the complex to be formed (e.g., a fusion polypeptide) are present. In some embodiments, the complex self-assembles under physiological conditions or in a buffer (e.g., a solution) corresponding to physiological conditions.

[0079] 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 a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a subject having one or more characteristics characterized by a susceptibility or risk for a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is a subject to whom diagnosis and / or therapy is being administered and / or has been administered.

[0080] As used herein, the term "treatment" (also "treat" or "treating") refers to any administration of a therapy that partially or completely alleviates, ameliorates, alleviates, suppresses, delays onset, reduces severity, and / or reduces the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be directed to subjects who do not exhibit signs of the relevant disease, disorder, and / or condition, and / or to subjects who only exhibit early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be directed to subjects who exhibit one or more established signs of the relevant disease, disorder, and / or condition. In some embodiments, treatment may be directed to subjects who have been diagnosed with the relevant disease, disorder, and / or condition. In some embodiments, treatment may be directed to subjects who are known to have one or more susceptibility factors that are statistically associated with an increased risk of developing the relevant disease, disorder, and / or condition.

[0081] A. Fusion polypeptide

[0082] In certain embodiments, fusion polypeptides compatible with the compositions and methods disclosed herein generally comprise (1) a nanocage monomer (or a subunit thereof) as described herein and (2) a cytokine binding moiety, a tumor binding moiety, or an Fc polypeptide, which may be connected via a linker, such as a linker described herein.

[0083] 1. Nanocage monomers and their subunits

[0084] In some embodiments, the nanocage monomer is a ferritin monomer.

[0085] The term "ferritin monomer" is used herein to refer to a single chain of ferritin that is capable of self-assembling into a polypeptide complex comprising 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 include a ferritin heavy chain or other ferritin components capable of binding iron or capable of ferroxidase activity.

[0086] In some embodiments, each fusion polypeptide within 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 any ferritin heavy chain or a subunit of a ferritin heavy chain. In such embodiments, the self-assembling polypeptide complex does not comprise an Fc 3+ And not with Fe 3+ Association, Fe 3+ It is an iron ion normally carried by the ferritin complex including the ferritin heavy chain.

[0087] In some embodiments, the ferritin monomer is a human ferritin chain, e.g., a human ferritin light chain, e.g., a human ferritin light chain having a sequence of at least residues 2-175 of SEQ ID NO: 1.

[0088] In some embodiments, the ferritin monomer is a full-length human ferritin light chain. As used herein, "full-length ferritin light chain" refers to a ferritin polypeptide capable of forming a four-helix bundle of two pairs of antiparallel α-helices connected by a loop and further comprising a C-terminal α-helix. In some embodiments, the full-length ferritin light chain comprises at least 85%, at least 90%, at least 95%, or 100% of the length of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the full-length ferritin light chain has an amino acid sequence that has at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the full-length ferritin light chain has the amino acid sequence of SEQ ID NO: 1. In some embodiments, the full-length ferritin light chain is a single continuous ferritin polypeptide.

[0089] A "subunit" of a ferritin monomer refers to a portion of a ferritin monomer that is capable of spontaneously associating with another, different subunit of a ferritin monomer such that these subunits together form a ferritin monomer that is, in turn, capable of self-assembling with other ferritin monomers to form a polypeptide complex.

[0090] In some embodiments, the ferritin monomer subunit comprises about half of a ferritin monomer. As used herein, the term "N-half ferritin" refers to about half of a ferritin chain, which half comprises the N-terminus of the ferritin chain. As used herein, the term "C-half ferritin" refers to about half of a ferritin chain, which half comprises the C-terminus of the ferritin chain. The exact point at which the ferritin chain can be split to form N-half ferritin and C-half ferritin can vary according to the embodiment. For example, in the context of a ferritin monomer subunit based on human ferritin light chain, these two halves can be separated at a point corresponding to a position (or substantial portion thereof) between about position 75 and about position 100 of SEQ ID NO: 1. For example, in some embodiments, the N-half ferritin based on the human ferritin light chain has an amino acid sequence corresponding to residues 1-95 of SEQ ID NO: 1 (or a substantial portion thereof, e.g., residues 2-95 of SEQ ID NO: 1), and the C-half ferritin based on the human ferritin light chain has an amino acid sequence corresponding to residues 96-175 of SEQ ID NO: 1 (or a substantial portion thereof).

[0091] In some embodiments, the two halves are separated at a point corresponding to a position between about position 85 and about position 92 of SEQ ID NO: 1. For example, in some embodiments, the 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 the 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).

[0092] 2. Combined part

[0093] The binding moiety (eg, a cytokine binding moiety or a tumor binding moiety) typically comprises an antibody fragment.

[0094] In some embodiments, the antibody fragment is a Fab. In some embodiments, the antibody fragment is a single-chain Fab (scFab); for example, a fusion polypeptide comprising both the heavy and light chains of a Fab optionally connected by a linker (e.g., an amino acid linker as disclosed herein) is used.

[0095] In certain embodiments, an antibody fragment comprises a heavy chain variable region (e.g., V H In certain embodiments, the antibody fragment comprises a heavy chain variable domain (e.g., V H ) and light chain variable domains (e.g., V L or V K In certain embodiments, the antibody fragment comprises a Fab comprising a heavy chain variable domain (e.g., V H ) and light chain variable domains (e.g., V L or V K ).

[0096] In certain embodiments, the antibody fragment does not include any domain from the Fc region, for example, does not include any constant heavy chain (CH2 or CH3) domain. In some embodiments, the antibody fragment is an antibody fragment of a fully human or humanized antibody or is derived from a fully human or humanized antibody. In some embodiments, the antibody fragment is an antibody fragment of a chimeric antibody or is derived from a chimeric antibody. The antibody from which the antibody fragment is obtained or derived can be any of a variety of antibody classes, including, for example, IgG1 antibodies, IgG2 antibodies, or IgG4 antibodies. In some embodiments, the antibody fragment is obtained from or is derived from an antagonist antibody, for example, an antagonist humanized antibody.

[0097] In embodiments using multiple types of fusion polypeptides with antibody fragments, the antibody fragments in the multiple types of fusion polypeptides may be capable of binding to the same epitope on a given antigen (e.g., an antigen on a cytokine or a tumor-associated antigen), capable of binding to different and non-overlapping epitopes on the antigen, or capable of binding to different but overlapping epitopes on the same antigen.

[0098] A. Cytokine Binding Moiety

[0099] The cytokine binding portion is typically capable of binding (eg, specifically binding) to an epitope on the cytokine of interest (eg, IL-6 and / or IL-11).

[0100] In some embodiments, the cytokine binding portion comprises a heavy chain variable region (V H ) and light chain variable region (V L or V K ) of an antibody fragment, wherein the V H and the V L or V K The CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of the antibody have sequences that are similar (e.g., differ in at most one or two amino acids each) to the sequences of the heavy and light chain CDRs of an IL-6 or IL-11 antibody (e.g., a human or humanized IL-6 or IL-11 antibody).

[0101] In some embodiments, the cytokine binding portion comprises a heavy chain variable region (V H ) and light chain variable region (V L or V K ) of an antibody fragment, wherein the V H and the V L or V K The CDRs have sequences identical to those of the heavy and light chain CDRs of an IL-6 or IL-11 antibody (e.g., a human or humanized IL-6 or IL-11 antibody), except for a total of one or two amino acid substitutions among all six CDRs.

[0102] In some embodiments, the cytokine binding portion comprises a heavy chain variable region (V H ) and light chain variable region (V L or V K ) of an antibody fragment, wherein the V H and the V L or V K The CDRs are identical to those of the heavy and light chain CDRs of an IL-6 or IL-11 antibody (eg, a human or humanized IL-6 or IL-11 antibody).

[0103] In some embodiments, the cytokine binding portion is a protein comprising heavy and light chain variable regions (e.g., V H and V L or V K ), the heavy and light chain variable regions having sequences that are 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 sequences of the heavy and light chain variable regions of an IL-6 or IL-11 antibody (e.g., a human or humanized IL-6 or IL-11 antibody). In some embodiments, the cytokine binding portion is an antibody fragment comprising heavy and light chain variable regions (e.g., V H and V L or V K ), the heavy and light chain variable regions having sequences identical to those of the heavy and light chain variable regions of an IL-6 or IL-11 antibody (e.g., a human or humanized IL-6 or IL-11 antibody).

[0104] Non-limiting examples of IL-6 antibodies include clazakizumab, EBI-029, EBI-031, elsilimomab, MEDI 5117, olokizumab, siltuximab, and sirukumab, whose heavy chain variable regions, light chain variable regions, and CDR sequences are depicted in Tables 1A-1H.

[0105] Non-limiting examples of IL-11 antibodies include 3C6 2.1, 3C6 2.2, 3C6 2.2b, TPP-29536, TPP-30003, YU100-G08, hz8C8m7, and hz18A10m19, whose heavy chain variable region, light chain variable region, and CDR sequences are depicted in Tables 2A-2H.

[0106] Table 1A: IL-6 Antibody Sequences (Clazolizumab)

[0107]

[0108] Table 1B: IL-6 antibody sequence (EBI-029)

[0109]

[0110] Table 1C: IL-6 antibody sequence (EBI-031)

[0111]

[0112] Table 1D: IL-6 antibody sequences (ecitumomab / B-E8)

[0113]

[0114] Table 1E: IL-6 antibody sequence (MEDI 5117)

[0115]

[0116] Table 1F: IL-6 antibody sequence (Ologizumab / CDP6038)

[0117]

[0118] Table 1G: IL-6 antibody sequence (siltuximab / CNTO-328)

[0119]

[0120] Table 1H: IL-6 antibody sequence (sirukumab / CNTO 136)

[0121]

[0122] Table 2A: IL-11 antibody sequence (3C6 2.1)

[0123]

[0124] Table 2B: IL-11 antibody sequence (3C6 2.2)

[0125]

[0126] Table 2C: IL-11 antibody sequence (3C6 2.2b)

[0127]

[0128] Table 2D: IL-11 antibody sequence (TPP-29536)

[0129]

[0130] Table 2E: IL-11 antibody sequence (TPP-30003)

[0131]

[0132] Table 2F: IL-11 antibody sequence (YU100-G08)

[0133]

[0134] Table 2G: IL-11 antibody sequence (hz8C8m7)

[0135]

[0136] Table 2H: IL-11 antibody sequence (hz18A10m19)

[0137]

[0138] B. Tumor-binding moiety

[0139] Tumor-binding moieties are generally capable of binding (e.g., specifically binding) to a tumor-associated antigen (e.g., a tumor-specific antigen) expressed on tumor cells or cells supporting a tumor (e.g., stromal cells). In some embodiments, the tumor-binding moiety is capable of binding (e.g., specifically binding) to PD-L1.

[0140] In some embodiments, the tumor binding moiety comprises a heavy chain variable region (V H ) and light chain variable region (V L or V K ) of an antibody fragment, wherein the V H and the V L or V K The CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of the antibody have sequences similar to (e.g., each differs by at most one or two amino acids) the CDRs of a PD-L1 antibody (e.g., a human or humanized PD-L1 antibody).

[0141] In some embodiments, the tumor binding moiety comprises a heavy chain variable region (V H ) and light chain variable region (V L or V K ) of an antibody fragment, wherein the V H and the V L or V K The CDRs of the PD-L1 antibody have sequences identical to those of the heavy and light chain CDRs of a PD-L1 antibody (e.g., a human or humanized PD-L1 antibody), except for a total of one or two amino acid substitutions among all six CDRs.

[0142] In some embodiments, the tumor binding moiety comprises a heavy chain variable region (V H ) and light chain variable region (V L or V K ) of an antibody fragment, wherein the V H and the V L or V K The CDRs of are identical to those of the heavy and light chain CDRs of a PD-L1 antibody (e.g., a human or humanized PD-L1 antibody).

[0143] In some embodiments, the tumor binding moiety comprises a heavy chain and a light chain variable region (e.g., V H and V L or V K), the heavy and light chain variable regions having sequences that are 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 sequences of the heavy and light chain variable regions of a PD-L1 antibody (e.g., a human or humanized PD-L1 antibody). In some embodiments, the tumor-binding portion is an antibody fragment comprising heavy and light chain variable regions (e.g., V H and V L or V K ), the heavy and light chain variable regions having sequences identical to those of the heavy and light chain variable regions of a PD-L1 antibody (e.g., a human or humanized PD-L1 antibody).

[0144] Non-limiting examples of PD-L1 antibodies include atezolizumab, avelumab, and durvalumab, whose heavy chain variable regions, light chain variable regions, and CDR sequences are depicted in Tables 3A-3C.

[0145] Table 3A: PD-L1 Antibody Sequences (Atezolizumab)

[0146]

[0147] Table 3B: PD-L1 Antibody Sequences (Avelumab)

[0148]

[0149] Table 3C: PD-L1 Antibody Sequences (Durvalumab)

[0150]

[0151] 3. Fc peptide

[0152] In some embodiments, the Fc polypeptide is a single-chain Fc (scFc), which comprises two Fc chains linked together by a covalent linker, eg, via an amino acid linker.

[0153] An IgG Fc chain (eg, an IgG1 Fc chain) typically contains two constant heavy chain domains (CH2 and CH3) and a hinge region connected to the CH2 domain.

[0154] In some embodiments, the Fc polypeptide comprises one or more IgG1 Fc chains (eg, human IgG1 Fc chains or human Fc chains), i.e., the Fc polypeptide comprises an Fc chain having an amino acid sequence substantially similar or identical to the amino acid sequence of a chain within wild-type IgG1 Fc.

[0155] In some embodiments, the Fc polypeptide comprises one or more human IgG1 Fc chains; that is, the Fc polypeptide comprises an Fc chain that is substantially similar or identical to an Fc chain within wild-type human IgG1. In some embodiments, the wild-type human IgG1 Fc has the amino acid sequence of SEQ ID NO: 4.

[0156] In certain embodiments, fragment crystallizable (Fc) polypeptides comprise Fc chains each having one or more mutations relative to a reference Fc chain of the same Ig class. As explained further below, the reference Fc chain can be, for example, of the IgG1 class.

[0157] Unless otherwise indicated, throughout this disclosure the numbering of residues within antibody fragments (eg, Fc chains) is according to the EU numbering.

[0158] In some embodiments, the Fc polypeptide comprises one or more human IgG1 Fc chains, ie, the Fc polypeptide comprises an Fc chain substantially similar to an Fc chain within wild-type human IgG1, except for the mutations described herein.

[0159] In some embodiments, the Fc polypeptide comprises one or more IgG1 Fc chains (e.g., human IgG1 Fc chains or human Fc chains), i.e., the Fc polypeptide comprises an Fc chain having an amino acid sequence substantially similar to the amino acid sequence of a chain within a wild-type IgG1 Fc, except for specific residues at certain positions described herein (which may differ from the residues in the corresponding wild-type Fc chain). In some embodiments, the wild-type IgG1 Fc is a human IgG1 Fc, wherein each Fc chain has the amino acid sequence of SEQ ID NO: 4. For example, the Fc polypeptide can comprise an Fc chain having an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of an Fc chain within a wild-type IgG1 Fc. In some embodiments, the Fc polypeptide comprises an Fc chain comprising the specific residues at certain positions specifically described for that Fc chain, but having an amino acid sequence that is otherwise 100% identical to a corresponding Fc chain within a wild-type Fc chain (e.g., a wild-type IgG1 Fc chain).

[0160] In some embodiments, the Fc polypeptide comprises an Fc chain having an amino acid sequence that differs from the sequence of SEQ ID NO: 4 by at least one, at least two, at least three, or at least four amino acid residues. In some embodiments, the Fc polypeptide comprises an Fc chain having an amino acid sequence that differs from the sequence of SEQ ID NO: 4 by no more than ten, no more than nine, no more than eight, no more than seven, no more than six, no more than five, or no more than four amino acid residues.

[0161] In some embodiments, the Fc chain comprises one or more IgG1, IgG2, IgG3, or IgG4 heavy chain constant regions, e.g., one or more heavy chain constant regions that are at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or identical to the heavy chain constant region of SEQ ID NO: 371, 372, 373, or 374.

[0162] In some embodiments, the Fc polypeptide comprises an "effector-silenced" Fc chain that is unable to bind, or binds only at a significantly reduced level, to one or more Fcγ receptors (e.g., human Fcγ receptors hFcγRI, hFcγRIIa, hFcγRIIb, hFcγRIIIa, hFcγRIIIb). In some embodiments, the Fc polypeptide comprises an Fc chain having a half-life extending mutation or set of mutations.

[0163] For example, in some embodiments, the Fc polypeptide comprises an IgG1 Fc having the "LLRAL" mutations: L234A, L235A, G236R, G237A, and A330L, wherein the amino acid residue at position P329 is proline (same as wild type).

[0164] 4. Connector

[0165] In certain embodiments, the linker is used in a fusion polypeptide and / or in a single-chain molecule such as scFc. In some embodiments, the linker is an amino acid linker. For example, the linker as used herein can comprise about 1 to about 100 amino acid residues, for example, 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 comprises 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.

[0166] In certain embodiments, the linker comprises a glycine-serine sequence, e.g., (G n S) msequences (e.g., GGS, GGGS (SEQ ID NO: 6), and GGGGS (SEQ ID NO: 7) sequences) that are present in at least 1, at least 2, at least 3, 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, or at least 14 copies within the linker.

[0167] B. Self-assembling polypeptide complexes

[0168] In one aspect, a self-assembling polypeptide complex comprising a plurality of fusion polypeptides disclosed herein is provided. Typically, the self-assembling polypeptide complex provided comprises (a) a plurality of first cytokine binding fusion polypeptides, each first cytokine binding fusion polypeptide comprising (1) a first cytokine binding portion connected to (2) a nanocage monomer or a subunit thereof, and (b) a plurality of Fc fusion polypeptides, each Fc fusion polypeptide comprising (1) an Fc polypeptide connected to (2) a nanocage monomer or a subunit thereof. In some embodiments, the self-assembling complex provided further comprises (c) a plurality of second cytokine binding fusion polypeptides, each second cytokine binding fusion polypeptide comprising (1) a second cytokine binding portion connected to (2) a nanocage monomer or a subunit thereof, wherein the second cytokine binding portion is different from the first cytokine binding portion. In certain embodiments, the self-assembling polypeptide complex further comprises a plurality of tumor binding fusion polypeptides, each tumor binding fusion polypeptide comprising (1) a tumor binding portion connected to (2) a nanocage monomer or a subunit thereof.

[0169] In some embodiments, the nanocage monomer is a ferritin monomer, and each fusion polypeptide within 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 any ferritin heavy chain, a subunit of a ferritin heavy chain, or other ferritin components capable of binding iron or capable of ferroxidase activity.

[0170] In some embodiments, the nanocage monomer or subunit thereof is a ferritin monomer subunit, and (a) each first cytokine binding fusion polypeptide comprises a ferritin monomer subunit that is a C-half ferritin, and each Fc fusion polypeptide comprises a ferritin monomer subunit that is an N-half ferritin; or (b) each first cytokine binding fusion polypeptide comprises a ferritin monomer subunit that is an N-half ferritin, and each Fc fusion polypeptide comprises a ferritin monomer subunit that is a C-half ferritin.

[0171] In some embodiments wherein the self-assembling polypeptide complex further comprises a plurality of second cytokine-binding fusion polypeptides, the nanocage monomer or subunit thereof is a ferritin monomer subunit, and (a) each second cytokine-binding fusion polypeptide comprises a ferritin monomer subunit that is a C-half ferritin, and each Fc-fusion polypeptide comprises a ferritin monomer subunit that is an N-half ferritin; or (b) each second cytokine-binding fusion polypeptide comprises a ferritin monomer subunit that is an N-half ferritin, and each Fc-fusion polypeptide comprises a ferritin monomer subunit that is a C-half ferritin.

[0172] 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, such as at least 26, at least 28, at least 30, at least 32 fusion polypeptides, at least 34 fusion polypeptides, at least 36 fusion polypeptides, at least 38 fusion polypeptides, at least 40 fusion polypeptides, at least 42 fusion polypeptides, at least 44 fusion polypeptides, at least 46 fusion polypeptides, or at least 48 fusion polypeptides. In some embodiments, the self-assembling polypeptide complex comprises about 32 fusion polypeptides.

[0173] 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 cytokine binding fusion polypeptides.

[0174] 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 Fc fusion polypeptides.

[0175] 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 fusion polypeptides different from the first cytokine binding fusion polypeptide and the Fc fusion polypeptide, e.g., a second cytokine binding fusion polypeptide and / or a tumor binding fusion polypeptide (e.g., a fusion polypeptide comprising a tumor binding portion linked to a nanocage monomer or a subunit thereof).

[0176] In some embodiments, the self-assembling polypeptide complex comprises a first cytokine binding fusion polypeptide to all other fusion polypeptides in a ratio of about 1:1, 11:13, 3:5, 1:2, 7:17, 1:3, 2:7, 5:19, 1:4, 1:5, 1:6, 1:7, 1:8, 1:12, 1:24.

[0177] In some embodiments, the self-assembling polypeptide complex comprises a first cytokine binding fusion polypeptide to an Fc fusion polypeptide at a ratio of about 1:1, 11:13, 3:5, 1:2, 7:17, 1:3, 2:7, 5:19, 1:4, 1:5, 1:6, 1:7, 1:8, 1:12, 1:24.

[0178] In some embodiments, the self-assembling polypeptide complex comprises a second cytokine binding fusion polypeptide to all other fusion polypeptides in a ratio of about 1:1, 11:13, 3:5, 1:2, 7:17, 1:3, 2:7, 5:19, 1:4, 1:5, 1:6, 1:7, 1:8, 1:12, 1:24.

[0179] In some embodiments, the self-assembling polypeptide complex comprises a second cytokine binding fusion polypeptide to an Fc fusion polypeptide at a ratio of about 1:1, 11:13, 3:5, 1:2, 7:17, 1:3, 2:7, 5:19, 1:4, 1:5, 1:6, 1:7, 1:8, 1:12, 1:24.

[0180] Pharmacokinetic characteristics

[0181] In certain embodiments, when administered to a subject in need thereof, the self-assembling polypeptide complex provided has one or more pharmacokinetic characteristics similar to a reference IgG molecule (e.g., an IgG molecule whose class matches the class of the Fc chain within the Fc polypeptide of the first fusion polypeptide within the self-assembling polypeptide complex).

[0182] effect

[0183] In certain embodiments, the disclosed self-assembling polypeptide complexes are capable of sequestering cytokines from the extracellular space and preventing binding to their cognate receptors, thereby abrogating signaling (eg, inflammatory signaling) mediated by the cytokine.

[0184] Any of the many assays known in the art can be used to assess the inhibitory effect of Multibody disclosed herein on intracellular signaling mediated by cytokines and their corresponding receptors in vitro. For example, in the context of IL-6 signaling, the assay for detecting phospho-STAT3 (pSTAT3) can be used to assess IL-6Rα activation. Another assay suitable for testing IL-6 signaling is a cell-based bioassay (e.g., Bio-Glo™ assay), which uses engineered cells expressing fluorescent proteins (e.g., firefly luciferase (luciferase) or FLuc) under the control of transcriptional regulators of one or more signals induced by IL-6 activating IL-6 receptors. In such assays, the luminosity (magnitude) of fluorescence is proportional to the intensity of IL-6 signaling, and can therefore be used to assess the impact of Multibody disclosed herein on IL-6 signaling. As will be appreciated by those skilled in the art, similar assays can be performed using different cytokine systems.

[0185] C. Treatment

[0186] In one aspect, methods useful for treating, ameliorating, or preventing a disease or condition are provided, generally comprising the step of administering a self-assembling polypeptide complex of the present disclosure (or a composition thereof) to a subject.

[0187] In some embodiments, the subject is a mammal, eg, a human.

[0188] 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.

[0189] The compositions can be formulated for administration by any of a variety of routes of administration, including systemic routes (eg, oral, inhalation, intranasal, intravenous, intraperitoneal, subcutaneous, or intramuscular administration).

[0190] In some embodiments, the administering step results in improvement of one or more clinical outcomes or metrics in the subject.

[0191] In some embodiments, the subject has cancer, e.g., a solid cancer or a blood cancer. In some embodiments, the administering step results in slowing or inhibiting the progression of the tumor, e.g., regression of the tumor. In some embodiments, the administering step results in complete regression of the tumor.

[0192] In some embodiments, the subject has an autoimmune disease or disorder.

[0193] In some embodiments, the administering step results in a reduction in one or more symptoms of the autoimmune disease or disorder, e.g., a reduction in the level of inflammation, a reduction in the frequency of inflammatory episodes, a reduction in the extent of one or more symptoms of the autoimmune disease or disorder, etc. In some embodiments, the administering step results in a complete elimination of symptoms of the autoimmune disease or disorder.

[0194] Example

[0195] Example 1. Construction and expression of anti-IL-6 Multibody

[0196] This example describes the generation of an anti-IL-6 multibody (MB) comprising a combination of a fusion protein comprising (1) a human ferritin light chain or a subunit thereof and (2) a single chain Fab (scFab) or a single chain Fc-dimer (scFc) linked via a linker (such as (Gly) as described herein). n -Ser) m amino acid linker) fusion.

[0197] Genes encoding the fusion proteins (1) anti-IL-6 scFab fused to the N-terminus of full-length ferritin, (2) anti-IL-6 scFab fused to the N-terminus of N-half ferritin, and (3) scFc fused to the N-terminus of C-half ferritin were prepared, mixed at a molar ratio of 2:1:1, and transiently transfected into ExpiCHO-S cells for the production and formation of anti-IL-6 MB. Figure 1B .

[0198] Three different monospecific Multabodies were prepared, each using the scFab of a different IL-6 antibody: siltuximab, clazakizumab, or olokizumab.

[0199] The scFc-ferritin fusion protein comprises an engineered IgG1 Fc chain containing "LLRAL" mutations (L234A, L235A, G236R, G237A, and A330L; wherein the amino acid residue at position P329 is proline (same as in the wild type)), as shown in Table 1 below. The numbering in Table 4 is according to the EU numbering scheme. This "LLRAL" combination of mutations renders the IgG1 Fc "silent" in effector activity, consistent with significantly reduced or no binding to Fcγ receptors (data not shown).

[0200] Table 4: Residues at certain positions within the IgG1 Fc chain used in the Multibody and the scFc-C_hFTL construct. SEQ ID NO

[0201] In the rows corresponding to the sets of engineered Fc chains, only residues at positions that differ from the wild-type residues are shown.

[0202]

[0203] Example 2. Anti-IL-6 Multibody Binds to IL-6 with High Affinity

[0204] The binding kinetics and affinity of the monospecific anti-IL-6 multibodies generated as described in Example 1 were tested using the Octet® Biolayer Interferometry (BLI) system and compared to IgG antibodies (siltuximab, clazakizumab, and olokizumab) derived from scFab displayed on multibodies. This method measures the binding response to the surface of a fiber-optic biosensor functionalized with an immobilized target moiety (e.g., IL-6).

[0205] If you can Figure 2A and 2D 、 Figure 2B and 2E and Figure 2C and 2F As can be seen, both anti-IL-6 Multibody and anti-IL-6 IgG bind with sub-picomolar affinity (K D <1.0×10 -12 ) binds to IL-6.

[0206] The relative binding and specificity of the anti-IL-6 Multibodies were further tested using an ELISA assay and compared against their corresponding IgGs. The results are shown in Figures 3A-3C In, EC 50 The values ​​are shown in Table 5 below. The anti-IL-6 Multibody exhibited approximately 3 to 4 times enhanced binding compared to the corresponding anti-IL-6 IgG. Both the anti-IL-6 Multibody and the corresponding anti-IL-6 IgG exhibited highly selective binding to IL-6, as can be seen from the almost complete absence of binding to surfaces functionalized with IL-11 or bovine serum albumin (BSA).

[0207] Table 5: EC values ​​of anti-IL-6 Multibody and IgG 50 Combine

[0208]

[0209] Example 3. Target binding of Multibodies determined by biolayer interferometry

[0210] The binding kinetics and affinity of exemplary anti-IL-6 Multibodies generated as described in Example 1 to IL-6 can be determined by biolayer interferometry (BLI). For example, the binding characteristics of the IgG antibody (from which the scFab within the IL-6 MB is derived) can also be determined for comparison.

[0211] For example, Ni-NTA biosensors can be coated with His-tagged antigens (e.g., IL-6). The coated biosensor can be immersed in a well containing a serial dilution of a Multibody (or antibody control) in a buffer such as phosphate-buffered saline (PBS) buffer, optionally containing Tween 20 and optionally containing bovine serum albumin (BSA) during the association phase and during the dissociation phase.

[0212] The biosensor can be regenerated between experiments, for example, by multiple applications of acidic glycine solution and by recharging in NiSO4.

[0213] Target binding can be based on the maximum associative binding response at the end of the associative phase, the dissociation rate (k off ), and / or the equilibrium dissociation constant (K) calculated using a 1:1 fitting model. D For a concentration of 20 nM Multabody, maximal associative binding responses less than 0.1 nm can be classified as “non-binding”.

[0214] Example 4. Binding of Multibodies to Fc Receptors Determined by Biolayer Interferometry

[0215] The binding kinetics and affinities of various Multibodies generated as described in Example 1 to various Fc receptors can be determined by biolayer interferometry.

[0216] The multibodies tested in this example contain polypeptides comprising one or more Fc chains. As controls for comparison, multibodies without Fc-containing polypeptides and / or IgG antibodies of the same class of Fc chains as those used in the multibodies can be used.

[0217] Binding to a variety of Fc receptors can be determined, for example, to human Fc receptors such as: human Fcγ receptor type I (hFcγRI), hFcγRIIa, hFcγRIIb, hFcγRIIIa, hFcγRIIIb, and human neonatal Fc receptor (hFcRn). To assess the feasibility and relevance of testing multibodies in animal models, binding to non-human Fc receptors, such as cynomolgus monkey Fc receptors (e.g., cynomolgus monkey FcγRI (cFcγRI), cFcγRIIa, cFcγRIIb, cFcγRIII, and cFcRn) and / or to mouse Fc receptors (e.g., mouse FcγRI (mFcγRI), mFcγRIIb, mFcγRIII, mFcγRIV, and mFcRn) can also be assessed.

[0218] The experiment can be performed similarly to that described in Example 3, except that His-tagged Fc receptors are coated onto Ni-NTA biosensors and titrated with varying concentrations of the test Multibody. To assess the potential of the Multibody to undergo endosomal recycling, binding to FcRn can be measured at pH 6.0 for association and at pH 7.4 for dissociation.

[0219] Example 5. Anti-IL-6 Multibody Modulates IL-6 Intracellular Signaling

[0220] Binding of IL-6 to its receptor, IL-6Rα, enables its association with the signaling receptor subunit gp130. Association of gp130 with the IL-6-IL-6Rα complex promotes gp130 dimerization, activating multiple intracellular signaling pathways, including the JAK-STAT pathway, which involves STAT3 phosphorylation. Therefore, phosphorylated STAT3 (pSTAT3) can be used as an indicator of IL-6Rα activation.

[0221] To test whether the anti-IL-6 multibodies of the present disclosure can interfere with IL-6 signaling, an in vitro functional assay was performed on HCT-15 colon tumor cells incubated with 50 ng / mL IL-6 in the presence or absence of 1 nM anti-IL-6 multibodies. Cell lysates were prepared and samples were evaluated by Western blotting using total STAT3 and pSTAT3 antibodies. Actin staining was used as a loading control. Figure 4 As shown in , pSTAT3 was detected in samples from cells incubated with IL-6 alone for as short as 5 minutes. However, in samples from cells incubated with both IL-6 and anti-IL-6 Multibody, pSTAT3 was not detected even after 15 minutes.

[0222] These results demonstrate that the anti-IL-6 Multibody is a potent antagonist of IL-6 signaling in tumor cells.

[0223] The antagonistic activity of the anti-IL-6 multibodies was further evaluated in an IL-6 bioassay using cells engineered with a luciferase transgene driven by a genetic element responsive to IL-6Rα downstream signaling. Figures 5A-5C As depicted in , the anti-IL-6 Multibody inhibited IL-6 signaling with enhanced potency compared to the corresponding parent anti-IL-6 IgG. Table 6 below shows the IC calculated from the above experimental results. 50 value.

[0224] Table 6: Inhibition of IL-6 signaling by anti-IL-6 Multibody and IgG

[0225]

[0226] The regulation of IL-6 signaling was further tested using an assay for IL-6 dependent pSTAT / STAT3 levels. The assay was performed on both HCT-15 and THP-1 cells (human leukemic monocytic cell lines). HCT-15 cells were plated at 2.5×106 cells per 6 cm dish, incubated in complete medium at 37°C, 5% CO2 for one day, and then starved overnight in 3 ml of serum-free medium at 37°C, 5% CO2 before treatment. THP-1 cells were plated at 4.5×106 cells per 6 cm dish in 3 ml of serum-free medium at 37°C, 5% CO2 before treatment. 6 The cells were starved overnight. MB or IgG were pre-incubated with IL-6 (Peprotech) for 10 minutes and then added to the cells to a final volume of 4 mL, with a final concentration of IL-6 of 50 ng / mL. The cells were incubated at 37°C, 5% CO2 for 15 minutes and then placed on ice. The cells were pelleted, washed with ice-cold PBS, and lysed with 150 μL of cold lysis buffer (Meso Scale Discovery). The lysate was centrifuged at 14,000 xg for 15 minutes, and the supernatant was collected. The protein concentration was determined by BCA assay kit (Pierce). pSTAT3 / STAT3 was determined using phospho-STAT3 and total STAT3 kits according to the manufacturer's instructions. Figure 6A and 6B As shown in , anti-IL-6 Multibody inhibited IL-6-induced STAT3 signaling with enhanced potency compared to the corresponding parental anti-IL-6 IgG, which is consistent with the results of the IL-6 bioassay.

[0227] Table 7 below shows the IC calculated from the above experimental results. 50 value.

[0228] Table 7: Inhibition of IL-6-induced STAT3 signaling by anti-IL-6 Multibody and IgG

[0229]

[0230] These results demonstrate that the anti-IL-6 Multibody is a potent inhibitor of IL-6 intracellular signaling with enhanced potency compared to the parental IgG.

[0231] Example 6. Pharmacokinetics of Multibody in Non-human Animal Models

[0232] The pharmacokinetics of exemplary anti-IL-6 Multibodies generated as described in Example 1 can be analyzed in one or more non-human animal models, such as mice and / or non-human primates (eg, cynomolgus monkeys).

[0233] Animals receive a single bolus injection of a Multibody or control molecule (e.g., IgG) by systemic administration (e.g., intravenously). Blood samples are collected at different time points, e.g., about 3 h, 24 h, 48 h, 72 h, 5 days, 7 days, 14 days, 21 days, and 28 days after injection.

[0234] Multiple-dose studies can also be conducted in which the first and second doses are separated by, for example, days or weeks.

[0235] Plasma samples can be obtained from blood samples and stored frozen until use.

[0236] In order to measure the level of Multibody or control molecules in plasma by enzyme-linked immunosorbent assay (ELISA), recombinant IL-6 is coated on a plate and placed overnight at 4°C. After washing twice (for example, using a solution such as phosphate buffered saline (PBS)-Tween-20), the plate is blocked with a bovine serum albumin (BSA) solution in PBS for 1 hour at room temperature, then washed twice. The plasma sample is diluted in PBS / Tween-20 / BSA solution, added to the wells, and incubated on a shaker at room temperature for 1 h. After another washing step, the bound molecule is detected by incubation with a diluted goat polyclonal anti-human Fc-HRP secondary antibody. After a further washing step, the substrate for detection is added, and the absorbance at 450 nm is read using a microplate reader. A calibration curve is prepared using the Multibody diluent in PBS / Tween-20 / BSA solution.

[0237] The half-life of the multibody in the animal can be calculated based on these measurements, and the half-life of the multibody can be compared to the half-life of an IgG molecule.

[0238] Example 7. Therapeutic Efficacy of Anti-IL6 Multibody in Xenograft Mouse Model

[0239] The therapeutic effects of exemplary Multabodies can be assessed in a colorectal cancer (CRC) xenograft model.

[0240] Mouse Model: BALB / c mice and C57BL / 6J mice can be used to establish CT26 and MC38 syngeneic mouse models, respectively. Cells can be harvested from cell culture flasks, washed, and resuspended in Matrigel at a concentration of 1×10 6 100 μL of cell suspension can be injected subcutaneously into the flank of each mouse.

[0241] Treatment: After tumors are established, mice can be administered an anti-IL-6 Multibody of the present disclosure or a control (e.g., an anti-IL-6 antibody (IgG), an IgG isotype control, and / or a control Multibody that does not bind to anti-IL-6) by, for example, intraperitoneal injection, according to a dosing schedule determined by the researcher.

[0242] Evaluation of therapeutic efficacy: Tumor growth, survival curves, and weight loss can be compared between treatment groups. Tumor length and width are measured weekly using calipers. A reduction in tumor growth and / or improved survival in animals treated with the Multibody (compared to animals treated with a control molecule) will indicate improved efficacy of the anti-IL-6 Multibody.

[0243] Example 8. Anti-IL-6 Multibody Potently Inhibits IL-6-Dependent Cell Proliferation

[0244] The ability of anti-IL-6 Multibodies (MBs) generated as described in Example 1 to inhibit IL-6-dependent cell proliferation was evaluated.

[0245] Cell proliferation assay. Cell proliferation was assessed using the CellTiter-Glo assay, which determines the number of viable cells in culture by quantifying the amount of ATP present. In this assay, the luminescent signal is proportional to the amount of ATP present, which in turn is directly proportional to the number of viable cells in culture.

[0246] INA-6 cells (which are IL-6 dependent cells) were maintained at 0.5-1.5×10 6 cells / mL. 2.5x10 4 Each well of a 96-well plate was plated with 75 μL of 90% RPMI 1640 + 10% FBS. MB or IgG was pre-incubated with IL-6 in 90% RPMI 1640 + 10% FBS for 10 minutes and then added to the cells to a final volume of 100 μL, with a final IL-6 concentration of 17.5 ng / mL. The cells were incubated at 37°C, 5% CO2 for three days. The final viable cell count was determined using CellTiter-Glo 2.0 reagent (Promega) according to the manufacturer's instructions. Proliferation was calculated by the fold change in signal compared to unstimulated cells (no IL-6).

[0247] Results: Anti-IL-6 Multibody potently inhibited cell proliferation with an IC 50 The IC values ​​of the corresponding parental IgG 50 Several times lower value ( Figure 7A and 7B and Table 8).

[0248] Table 8: Inhibition of cell proliferation by anti-IL-6 Multibody and IgG

[0249]

[0250] Example 9. Construction, expression and analysis of anti-IL-11 Multibody

[0251] This example describes the generation and analysis of anti-IL-11 Multibodies (MBs).

[0252] Construction and expression of anti-IL-11 Multibody

[0253] Anti-IL-11 multibodies were generated similarly to those described in Example 1, except that the scFab within some of the fusion proteins was derived from the variable region of an IL-11 antibody rather than an IL-6 antibody.

[0254] Evaluation of Anti-IL-11 Multibody Binding to IL-11

[0255] The binding of anti-IL-11 Multibody to IL-11 was assessed using enzyme-linked immunosorbent assay (ELISA). Figure 8 As shown in Table 9, in the same assay, anti-IL-11 Multibody (MB) bound IL-11 approximately 3-4 times better than the parental IgG control. Both anti-IL-11 MB and anti-IL-11 IgG exhibited minimal binding to IL-6.

[0256] Table 9: Binding of anti-IL-11 Multibody and IgG to IL-11

[0257]

[0258] Assessment of IL-11-induced modulation of STAT3 signaling

[0259] The modulation of IL-11 signaling was also tested using the assay for IL-11 dependent pSTAT / STAT3 levels. Panc-1 cells (pancreatic cancer cells) were plated at 2.5 x 10 6Cells were plated in complete medium in 6 cm culture dishes and incubated at 37°C, 5% CO2 for one day, then starved overnight in 3 ml of serum-free medium before treatment. MB or IgG were pre-incubated with IL-11 (AcroBiosystems) for 10 minutes and then added to the cells to a final volume of 4 mL, with a final concentration of IL-11 of 50 ng / mL. The cells were incubated at 37°C, 5% CO2 for 15 minutes and then placed on ice. The cells were washed with ice-cold PBS and lysed with 175 μL of cold lysis buffer. The lysate was centrifuged at 14,000 x g for 15 minutes, and the supernatant was collected. Protein concentration was determined using a BCA assay kit (Pierce). pSTAT3 / STAT3 was determined using a phospho-STAT3 and total STAT3 kit (Meso Scale Discovery) according to the manufacturer's instructions.

[0260] like Figure 9 As shown in Table 10, the anti-IL-11 Multibody exhibited superior potency in inhibiting pSTAT3 signaling compared to that exhibited by the parental IgG control.

[0261] Table 10: Inhibition of pSTAT3 signaling by anti-IL-11 Multibody and IgG

[0262]

[0263] Thus, these experiments demonstrated the successful generation of anti-IL-11 Multibodies that specifically bind to IL-11 and inhibit pSTAT3 signaling better than the parental anti-IL-11 IgG.

[0264] Example 10. Construction, expression and analysis of additional anti-IL-11 multibodies

[0265] Additional anti-IL-11 multibodies can be generated as described in Example 9, using, for example, Fabs from other IL-11 antibodies, examples of which are included in Tables 2A-2H.

[0266] Example 11. Pharmacokinetic Analysis of Anti-IL-11 Multibody

[0267] The pharmacokinetics (PK) of exemplary anti-IL-11 multibodies generated as described in Examples 9 and 10 can be analyzed in one or more non-human animal models such as mice and / or non-human primates (e.g., cynomolgus monkeys), similarly as described in Example 6.

[0268] Example 12. Therapeutic Efficacy of Anti-IL-11 Multibodies

[0269] The therapeutic effects of exemplary anti-IL-11 Multibodies can be assessed in a mouse cancer xenograft model, such as a colorectal cancer (CRC) xenograft model, similar to that described in Example 7.

[0270] Sequence Listing

[0271] Underlined within the fusion sequence indicates the linker sequence.

[0272] Bold within the fusion sequence indicates ferritin or ferritin subunit sequences.

[0273] Within the variable region sequences, underlining and bold together indicate complementarity determining region sequences.

[0274] Boxed and bold residues indicate residues that were mutated relative to a reference molecule, eg, relative to IgG1 Fc.

[0275] SEQ ID NO: 1 hFTL

[0276] MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD

[0277] SEQ ID NO: 2N_hFTL

[0278] SSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEW

[0279] SEQ ID NO: 3C_hFTL

[0280] GKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD

[0281] SEQ ID NO: 4IgG1 Fc

[0282] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0283] SEQ ID NO: 5IgG1 scFc

[0284]

[0285] SEQ ID NO: 6 Example GS linker

[0286] GGGS

[0287] SEQ ID NO: 7 Example GS linker

[0288] GGGGS

[0289] SEQ ID NOs: 8-368, as shown in Tables 1A-4D.

[0290] SEQ ID NO: 369scFc-C_hFTL IgG1 WT

[0291]

[0292] SEQ ID NO: 370scFc-C_hFTL IgG1 LLRAL

[0293]

[0294] SEQ ID NO: 371 Human IgG4 heavy chain constant region sequence

[0295] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0296] SEQ ID NO: 372 Human IgG1 heavy chain constant region sequence

[0297] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0298] SEQ ID NO: 373 Human IgG2 heavy chain constant region sequence

[0299] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0300] SEQ ID NO: 374 Human IgG3 heavy chain constant region sequence

[0301] ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

[0302] SEQ ID NO: 375 Human Igκ light chain constant region sequence

[0303] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0304] SEQ ID NO: 376 Human Igλ1 light chain constant region sequence

[0305] GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0306] SEQ ID NO: 377 Human Igλ2 light chain constant region sequence

[0307] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0308] SEQ ID NO: 378 Human Igλ3 light chain constant region sequence

[0309] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS

[0310] SEQ ID NO: 379 Human Igλ6 light chain constant region sequence

[0311] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVKVAWKADGSPVNTGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS

[0312] SEQ ID NO: 380 Human Igλ7 light chain constant region sequence

[0313] GQPKAAPSVTLFPPSSEELQANKATLVCLVSDFNPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAECS

[0314] Equivalents / Other Implementations

[0315] While the invention has been described in conjunction with specific embodiments thereof, it will be understood that the invention is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention which follow generally from the principles of the invention and which depart from the present disclosure and which come within known or customary practices in the art to which the invention pertains and which may be applied to the basic features herein before described.

Claims

1. A fusion polypeptide comprising: (1) a cytokine binding portion and (2) a nanocage monomer or a subunit thereof.

2. The fusion polypeptide of claim 1, wherein the cytokine binding portion comprises an antibody or an antigen-binding fragment thereof.

3. The fusion polypeptide of claim 2, wherein the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain variable region (V H ) and immunoglobulin light chain variable region (V L or V K ). The fusion polypeptide of claim 3 , wherein the antibody or antigen-binding fragment thereof comprises a Fab fragment. The fusion polypeptide of claim 4 , wherein the Fab fragment is a single-chain Fab fragment (scFab).

6. The fusion polypeptide of any one of claims 1 to 5, wherein the cytokine binding portion is capable of binding interleukin-6 (IL-6) or interleukin-11 (IL-11).

7. The fusion polypeptide of claim 6, wherein the cytokine binding portion comprises an antibody fragment comprising a heavy chain variable region (V H ) and light chain variable region (V L or V K ), wherein the V H and the V L or V K The CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of the IL-6 or IL-11 antibody have sequences that each differ from the CDRs of the IL-6 or IL-11 antibody by a maximum of two amino acids.

8. The fusion polypeptide according to claim 7, wherein the V H and the V L or V K The CDRs have sequences identical to those of the heavy and light chain CDRs of the IL-6 or IL-11 antibody, except for a total of one or two amino acid substitutions among all six CDRs.

9. The fusion polypeptide according to claim 8, wherein the V H and the V L or V K The CDRs are identical to those of the heavy and light chain CDRs of the IL-6 or IL-11 antibody.

10. The fusion polypeptide of claim 9, wherein the cytokine binding portion comprises an antibody fragment comprising heavy and light chain variable regions, the sequences of the heavy and light chain variable regions being at least 85% identical to the sequences of the heavy and light chain variable regions of an IL-6 or IL-11 antibody.

11. The fusion polypeptide of any one of claims 6 to 10, wherein the cytokine binding portion is capable of binding to an epitope of interleukin-6 (IL-6) that is involved in the binding of IL-6 to the IL-6 receptor (IL-6Rα).

12. The fusion polypeptide of any one of claims 6 to 10, wherein the cytokine binding portion is capable of binding to an epitope of IL-6 that is involved in the binding of IL-6 to glycoprotein 130 (gp130).

13. The fusion polypeptide of any one of claims 1-12, wherein the nanocage monomer is a ferritin monomer or a subunit thereof.

14. The fusion protein of claim 13, wherein the ferritin monomer is a human ferritin monomer.

15. The fusion protein of claim 13 or 14, wherein the ferritin monomer is a ferritin light chain.

16. The fusion polypeptide of any one of claims 1-15, wherein the cytokine binding moiety is linked to the nanocage monomer or subunit thereof via an amino acid linker.

17. The fusion polypeptide of any one of claims 1-16, wherein the cytokine binding moiety is attached via the N-terminus of the nanocage monomer or subunit thereof.

18. A self-assembling polypeptide complex, comprising: (a) a plurality of first cytokine binding fusion polypeptides, each first cytokine binding fusion polypeptide being a fusion polypeptide according to any one of claims 1 to 15; and (b) a plurality of Fc fusion polypeptides, each Fc fusion polypeptide comprising (1) an Fc polypeptide and (2) a nanocage monomer or a subunit thereof.

19. The self-assembling polypeptide complex according to claim 18, further comprising (c) a plurality of second cytokine-binding fusion polypeptides, each second cytokine-binding fusion polypeptide being a fusion polypeptide according to any one of claims 1 to 15, The first and second cytokine binding fusion polypeptides respectively comprise first and second cytokine binding portions capable of binding to different epitopes.

20. The self-assembling polypeptide complex of claim 19, wherein the first and second cytokine binding moieties are capable of binding different cytokines.

21. The self-assembling polypeptide complex of claim 20, wherein the first cytokine binding moiety is capable of binding to IL-6, and the second cytokine binding moiety is capable of binding to IL-11.

22. The self-assembling polypeptide complex according to any one of claims 18 to 21, further comprising (d) a plurality of tumor-binding fusion polypeptides, each tumor-binding fusion polypeptide comprising (1) a tumor-binding portion and (2) a nanocage monomer or a subunit thereof.

23. The self-assembling polypeptide complex according to any one of claims 18 to 22, wherein In each Fc fusion polypeptide, the Fc polypeptide is linked to the nanocage monomer or subunit thereof via an amino acid linker. The self-assembling polypeptide complex according to claim 23 , wherein the Fc polypeptide is linked via the N-terminus of the nanocage monomer or its subunit. The self-assembling polypeptide complex according to claim 24 , wherein the Fc polypeptide is linked via the C-terminus of the nanocage monomer or its subunit.

26. The self-assembling polypeptide complex according to any one of claims 22 to 25, wherein Within each tumor-binding fusion polypeptide, the tumor-binding moiety is linked to the nanocage monomer or subunit thereof via an amino acid linker.

27. The self-assembling polypeptide complex of claim 26, wherein the tumor-binding moiety is attached via the N-terminus of the nanocage monomer or its subunit.

28. The self-assembling polypeptide complex of any one of claims 22-27, wherein the tumor-binding moiety comprises an antibody or an antigen-binding fragment thereof.

29. The self-assembling polypeptide complex of claim 28, wherein the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain variable region (V H ) and immunoglobulin light chain variable region (V L or V K ).

30. The self-assembling polypeptide complex of claim 29, wherein the antibody or antigen-binding fragment thereof comprises a Fab fragment. The self-assembling polypeptide complex of claim 30 , wherein the Fab fragment is a single-chain Fab fragment (scFab).

32. The self-assembling polypeptide complex of any one of claims 28-31, wherein the tumor binding moiety is a PD-L1 binding moiety.

33. The self-assembling polypeptide complex of claim 32, wherein the tumor binding portion comprises a heavy chain variable region (V H ) and light chain variable region (V L or V K ) of the antibody fragment, wherein the V H and the V L or V K The CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of the PD-L1 antibody have sequences that each differ from the CDRs of the PD-L1 antibody by a maximum of two amino acids.

34. The self-assembling polypeptide complex according to claim 33, wherein said V H and the V L or V K The CDRs have sequences identical to those of the heavy and light chain CDRs of the PD-L1 antibody, except for a total of one or two amino acid substitutions among all six CDRs.

35. The self-assembling polypeptide complex according to claim 34, wherein said V H and the V L or V K The CDRs of are identical to those of the heavy and light chain CDRs of the PD-L1 antibody.

36. The self-assembling polypeptide complex of claim 32, wherein the tumor-binding portion comprises an antibody fragment comprising heavy and light chain variable regions, the sequences of the heavy and light chain variable regions being at least 85% identical to the sequences of the heavy and light chain variable regions of a PD-L1 antibody.

37. The self-assembling polypeptide complex of any one of claims 18-36, wherein each nanocage monomer within each Fc fusion polypeptide, each cytokine-binding fusion polypeptide, and / or each tumor-binding fusion polypeptide is a ferritin monomer or a subunit thereof.

38. The self-assembling polypeptide complex of claim 37, wherein the ferritin monomer is a human ferritin monomer.

39. The self-assembling polypeptide complex of claim 37 or 38, wherein the ferritin monomer is a ferritin light chain.

40. The self-assembling polypeptide complex of claim 39, which does not comprise any ferritin heavy chain or subunit of a ferritin heavy chain.

41. The self-assembling polypeptide complex of any one of claims 37-40, which does not comprise any iron-binding moiety.

42. A pharmaceutical composition comprising the self-assembling polypeptide complex according to any one of claims 18 to 41 and a pharmaceutically acceptable excipient.

43. Use of the self-assembling polypeptide complex of any one of claims 18 to 41 or the pharmaceutical composition of claim 42 for treating, ameliorating or preventing a disease or condition in a subject.

44. The use according to claim 43, wherein the subject is a mammal.

45. The use according to claim 44, wherein the subject is a human.

46. ​​The use according to any one of claims 43-45, wherein the disease or condition is cancer.

47. The use according to claim 46, wherein the cancer is a solid cancer.

48. The use according to claim 46, wherein the cancer is a blood cancer.

49. The use according to any one of claims 43-45, wherein the disease or condition is an autoimmune disease or condition.

50. A method for treating, ameliorating or preventing a disease or disorder, comprising administering the self-assembling polypeptide complex of any one of claims 18 to 41 or the pharmaceutical composition of claim 42 to a subject.

51. The method of claim 50, wherein the subject is a mammal.

52. The method of claim 51, wherein the subject is a human.

53. The method of any one of claims 50-52, wherein the disease or condition is cancer.

54. The method of claim 53, wherein the cancer is a solid cancer.

55. The method of claim 53, wherein the cancer is a hematological cancer.

56. The method of any one of claims 50-52, wherein the disease or condition is an autoimmune disease or condition.

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