Multivalent hematopoietic cell cement or activator

Abstract

A fusion polypeptide comprising: (1) a hematopoietic cell binding moiety and (2) a nanocage monomer or subunit thereof; a self-assembling polypeptide complex comprising such a fusion polypeptide. 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 / 433,467 (filed December 18, 2022), the entire contents of which are hereby incorporated by reference in their entirety for all purposes.

[0003] Sequence Listing

[0004] This application contains a sequence listing submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML file, created on December 15, 2023, is named "RBT051WO_SL.xml" and is 378 kilobytes in size. Background Art

[0005] Immuno-oncology is a therapeutic approach that harnesses the patient's own immune system to fight cancer. Bispecific T-cell engagers (BiTEs)—bispecific antibodies that recognize both T cells and tumor cells—are being developed as therapeutic agents to bring T cells into proximity with cancer cells, thereby promoting T-cell-mediated tumor cell killing. However, the development of BiTEs and similar therapies faces challenges, including safety concerns, such as those associated with cytokine release syndrome.

[0006] The innate immune system also plays a key role in tumor immunosurveillance and the generation of α-tumor immune responses. Therefore, similar approaches using bispecific antibodies to engage cells of the innate immune system (such as natural killer (NK) cells or myeloid cells) are also being developed to promote tumor cell killing. However, these approaches also face challenges, such as efficacy is related to the expression level of the target on innate immune cells and the highly immunosuppressive microenvironment of the tumor.

[0007] Therefore, there is a need for improved therapeutic agents in the field of immuno-oncology. SUMMARY OF THE INVENTION

[0009] The present invention addresses this need by providing self-assembling polypeptide complexes displaying IgG Fc fragments and one or both of (1) a tumor cell binding agent and (2) a hematopoietic cell (e.g., NK cell, T cell, or myeloid cell) binding agent. Such self-assembling polypeptide complexes can be used to promote hematopoietic cell-mediated tumor killing. In the provided self-assembling polypeptide complexes, the relative ratio, valency, and orientation of the binding agent and fragment are adjustable, thereby allowing for the modulation of the function and characteristics of the complex. Related fusion proteins, complexes, compositions, and methods are also disclosed.

[0010] In one aspect, a fusion polypeptide is provided comprising: (1) a hematopoietic cell binding portion and (2) a nanocage monomer or a subunit thereof.

[0011] In some embodiments, the hematopoietic cell binding moiety comprises an antibody or an antigen-binding fragment thereof.

[0012] 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 ).

[0013] In some embodiments, the antibody or antigen-binding fragment thereof comprises a Fab fragment.

[0014] In some embodiments, the Fab fragment is a single-chain Fab fragment (scFab).

[0015] In some embodiments, the fusion polypeptide comprises a lymphocyte binding portion.

[0016] In some embodiments, the lymphocyte binding moiety is a natural killer cell binding moiety or a T cell binding moiety.

[0017] In some embodiments, the lymphocyte binding moiety is a natural killer cell binding moiety.

[0018] In some embodiments, the natural killer cell binding moiety is a CD16a binding moiety, a NKp46 binding moiety, or a NKG2D binding moiety.

[0019] In some embodiments, the natural killer cell 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 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 each differ from the CDRs of the CD16a, NKp46 or NKG2D antibodies by a maximum of two amino acids.

[0020] In some embodiments, in addition to a total of one or two amino acid substitutions in all six CDRs, V H and V L or V K The CDRs of the antibody have sequences identical to those of the heavy and light chain CDRs of CD16a, NKp46 or NKG2D antibodies.

[0021] In some embodiments, V H and V L or V KThe CDRs of the antibodies are identical to the heavy and light chain CDRs of CD16a, NKp46 or NKG2D antibodies.

[0022] In some embodiments, the natural killer cell 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 a CD16a, NKp46, or NKG2D antibody.

[0023] In some embodiments, the lymphocyte binding moiety is a T cell binding moiety.

[0024] In some embodiments, the T cell binding moiety is a CD3 binding moiety.

[0025] In some embodiments, the T cell 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 V L or V K The CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3) of the CD3 antibody have sequences that each differ from the CDRs of the CD3 antibody by a maximum of two amino acids.

[0026] In some embodiments, in addition to a total of one or two amino acid substitutions in all six CDRs, V H and V L or V K The CDRs have sequences identical to those of the heavy and light chain CDRs of the CD3 antibody.

[0027] In some embodiments, V H and V L or V K The CDRs of the CD3 antibody are identical to the heavy and light chain CDRs of the CD3 antibody.

[0028] In some embodiments, the T cell binding portion comprises an antibody fragment comprising heavy and light chain variable regions having sequences at least 85% identical to the sequences of the heavy and light chain variable regions of a CD3 antibody.

[0029] In some embodiments, the fusion polypeptide comprises a myeloid cell binding portion.

[0030] In some embodiments, the myeloid cell binding moiety is a SIRPα binding moiety.

[0031] In some embodiments, the myeloid cell-binding portion comprises an antibody fragment comprising a heavy chain variable region (VH ) and light chain variable region (V L or V K ), where V H and V L or V K The CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of SIRPα have sequences that each differ from the CDRs of the SIRPα antibody by a maximum of two amino acids.

[0032] In some embodiments, in addition to a total of one or two amino acid substitutions in all six CDRs, V H and V L or V K The CDRs have sequences identical to those of the heavy and light chain CDRs of the SIRPα antibody.

[0033] In some embodiments, V H and V L or V K The CDRs of the SIRPα antibody are identical to the heavy and light chain CDRs of the SIRPα antibody.

[0034] In some embodiments, the natural killer cell binding portion comprises an antibody fragment comprising heavy and light chain variable regions having sequences at least 85% identical to the sequences of the heavy and light chain variable regions of a SIRPα antibody.

[0035] In some embodiments, the nanocage monomer is a ferritin monomer or a subunit thereof.

[0036] In some embodiments, the ferritin monomer is a human ferritin monomer.

[0037] In some embodiments, the ferritin monomer is a ferritin light chain.

[0038] In some embodiments, the hematopoietic cell-binding moiety is linked to the nanocage monomer or subunit thereof via an amino acid linker.

[0039] In some embodiments, the hematopoietic cell-binding moiety is attached via the N-terminus of the nanocage monomer or a subunit thereof.

[0040] In one aspect, a self-assembling polypeptide complex is provided, comprising: (a) a plurality of hematopoietic cell-binding fusion polypeptides, each hematopoietic cell-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.

[0041] In some embodiments, the self-assembling polypeptide complex further comprises (c) 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.

[0042] 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.

[0043] In some embodiments, the Fc polypeptide is linked via the N-terminus of the nanocage monomer or a subunit thereof.

[0044] In some embodiments, the Fc polypeptide is linked via the C-terminus of the nanocage monomer or a subunit thereof.

[0045] 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.

[0046] In some embodiments, the tumor-binding moiety is attached via the N-terminus of the nanocage monomer or a subunit thereof.

[0047] In one aspect, a self-assembling polypeptide complex is provided, comprising: (a) a plurality of hematopoietic cell-binding fusion polypeptides, each hematopoietic cell-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 linked via an amino acid linker to (2) the C-terminus of a nanocage monomer or a subunit thereof.

[0048] In one aspect, a self-assembling polypeptide complex is provided, comprising: (a) a plurality of tumor-binding fusion polypeptides, each comprising (1) a tumor-binding portion linked to (2) a nanocage monomer or a subunit thereof via an amino acid linker; and (b) a plurality of Fc fusion polypeptides, each comprising (1) an Fc polypeptide linked to the C-terminus of (2) a nanocage monomer or a subunit thereof via an amino acid linker.

[0049] In one aspect, a self-assembling polypeptide complex is provided, comprising: (a) a plurality of hematopoietic cell-binding fusion polypeptides, each hematopoietic cell-binding fusion polypeptide being a fusion polypeptide as disclosed herein; and (b) a plurality of tumor-binding fusion polypeptides, each tumor-binding fusion polypeptide comprising (1) a tumor-binding portion linked to (2) a nanocage monomer or subunit thereof via an amino acid linker; and (c) a plurality of Fc fusion polypeptides, each Fc fusion polypeptide comprising (1) an Fc polypeptide linked to the C-terminus of (2) a nanocage monomer or subunit thereof.

[0050] In some embodiments, an Fc polypeptide is linked to the C-terminus of the C-half nanocage monomer.

[0051] In some embodiments, the Fc polypeptide is linked to the C-terminus of the C-halferritin.

[0052] In some embodiments, the tumor-binding moiety comprises an antibody or an antigen-binding fragment thereof.

[0053] 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 ).

[0054] In some embodiments, the antibody or antigen-binding fragment thereof comprises a Fab fragment.

[0055] In some embodiments, the Fab fragment is a single-chain Fab fragment (scFab).

[0056] In some embodiments, the tumor binding moiety is a CD20 binding moiety or a CD37 binding moiety.

[0057] In some embodiments, the tumor binding moiety 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 V L or V K The CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of the CD20 or CD37 antibody have sequences that each differ from the CDRs of the CD20 or CD37 antibody by a maximum of two amino acids.

[0058] In some embodiments, in addition to a total of one or two amino acid substitutions in all six CDRs, V H and V L or V K The CDRs have sequences identical to those of the heavy and light chain CDRs of CD20 or CD37 antibodies.

[0059] In some embodiments, V H and V L or V K The CDRs of the antibody are identical to the heavy and light chain CDRs of the CD20 or CD37 antibody.

[0060] In some embodiments, the natural killer cell binding portion comprises an antibody fragment comprising heavy and light chain variable regions having sequences at least 85% identical to the sequences of the heavy and light chain variable regions of a CD20 or CD37 antibody.

[0061] In some embodiments, the nanocage monomer within the Fc fusion polypeptide and within the hematopoietic cell-binding fusion polypeptide and / or tumor-binding fusion polypeptide is each a ferritin monomer or a subunit thereof.

[0062] In some embodiments, the ferritin monomer is a human ferritin monomer.

[0063] In some embodiments, the ferritin monomer is a ferritin light chain.

[0064] In some embodiments, the self-assembling polypeptide complex does not comprise any ferritin heavy chain or subunit of a ferritin heavy chain.

[0065] In some embodiments, the self-assembling polypeptide complex does not comprise any iron-binding moiety.

[0066] In one aspect, a pharmaceutical composition comprising a self-assembling polypeptide complex as disclosed herein and a pharmaceutically acceptable excipient is provided.

[0067] 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.

[0068] 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.

[0069] In some embodiments, the subject is a mammal.

[0070] In some embodiments, the subject is a human.

[0071] In some embodiments, the disease or condition is cancer, eg, B-cell lymphoma.

[0072] In one aspect, a fusion polypeptide is provided comprising a contiguous full-length ferritin light chain having an N-terminus and a C-terminus, and (a) an antibody fragment fused to the N-terminus via a first amino acid linker and an Fc polypeptide fused to the C-terminus via a second amino acid linker; or (b) an antibody fragment fused to the C-terminus via a first amino acid linker and an Fc polypeptide fused to the N-terminus via a second amino acid linker.

[0073] In some embodiments, the antibody fragment is fused to the N-terminus via a first amino acid linker, and the Fc polypeptide is fused to the C-terminus via a second amino acid linker.

[0074] In some embodiments, the antibody fragment comprises an immunoglobulin heavy chain variable region (V H ) and immunoglobulin light chain variable region (V L or V K ).

[0075] In some embodiments, the antibody or antigen-binding fragment thereof comprises a Fab fragment.

[0076] In some embodiments, the Fab fragment is a single-chain Fab fragment (scFab).

[0077] In some embodiments, the antibody fragment is capable of binding to a tumor-associated antigen.

[0078] Also provided are a self-assembling polypeptide complex comprising any of the aforementioned fusion polypeptides, a pharmaceutical composition comprising such a self-assembling polypeptide complex and a pharmaceutically acceptable excipient, and a method for treating, ameliorating, or preventing a disease or condition, the method comprising administering such a self-assembling polypeptide complex or a pharmaceutical excipient to a subject.

[0079] Also provided are methods comprising the step of contacting tumor cells with a self-assembling polypeptide complex disclosed herein. In some embodiments, the contacting step results in apoptosis of the tumor cells. For example, tumor cells expressing CD37 can be contacted with a self-assembling polypeptide complex comprising a fusion protein comprising an α-CD37 antibody or a fragment thereof (such as a Fab). In some embodiments, the self-assembling polypeptide complex induces apoptosis with an EC50 value of less than 1 nM. In some embodiments, the contacting step results in antibody-dependent cellular cytotoxicity (ADCC) of the tumor cells. For example, for the self-assembling polypeptide complex, the EC50 value is less than 5 pM.

[0080] In some embodiments, the contacting step is performed in the presence of a complement protein and results in complement-dependent cytotoxicity (CDC) of the tumor cells, e.g., with an EC50 value of less than 0.4 nM for the self-assembling polypeptide complex. In some embodiments, the contacting step results in antibody-dependent cellular phagocytosis (ADCP) of the tumor cells, e.g., with an EC50 value of less than 18 pM for the self-assembling polypeptide complex. In some embodiments, the contacting step occurs in a mammal (e.g., a human) having a tumor.

[0081] Also provided is a self-assembling polypeptide complex as described herein, which, when in contact with a tumor cell, is capable of inducing an effect selected from the group consisting of:

[0082] (a) Apoptosis of tumor cells, EC50 value is less than 1 nM;

[0083] (b) ADCC of tumor cells, with an EC50 value of less than 1 nM;

[0084] (c) CDC of tumor cells: when the self-assembled polypeptide complex contacts tumor cells in the presence of complement, the EC50 value is less than 0.4 nM;

[0085] (d) ADCP, with an EC50 value of less than 0.1 nM, and

[0086] (e) any combination of the foregoing. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] The patent or application file contains at least one drawing printed in color. The Office will provide copies of the patent or patent application publication and the color drawing(s) upon request and payment of the necessary fee.

[0088] 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.

[0089] Figure 1B and 1C is a schematic representation of a set of fusion polypeptides that can together form an exemplary multivalent body (MB) of the present disclosure.

[0090] Figure 2A is a schematic representation of the fusion proteins used in Form 1 MBs.

[0091] Figure 2B Figure 2 shows the relationship between the size of a single Fc (including its FcγR binding and FcRn binding pockets) and the light chain ferritin complex within the MB of Form 1. Figure 2B Schematic diagram of the orientation of the Fab (lower part). The figure is not drawn to scale. For ease of illustration, the Fab is not depicted and only a single Fc is depicted.

[0092] Figure 3A is a schematic representation of the fusion proteins used in format 2 MBs.

[0093] Figure 3B Figure 2 shows the relationship between the size of a single Fc (including its FcγR binding and FcRn binding pockets) and the light chain ferritin complex ( Figure 2B Schematic diagram of the orientation of the Fab (lower part). The figure is not drawn to scale. For ease of illustration, the Fab is not depicted and only a single Fc is depicted.

[0094] Figure 4 Schematic diagram of a reporter gene assay for antibody-dependent cellular cytotoxicity (ADCC).

[0095] Figure 5 is a graph showing fold ADCC induction (y-axis) versus logarithmic concentration (x-axis) of various MBs: format 1 αCD37 / Fc MB, format 2 αCD37 / Fc MB, and format 2 control Fc MB.

[0096] Figure 6Depicted are graphs showing the percentage of single cells present within the FITC-Annexin-V+ gate determined by flow cytometry (y-axis) versus the concentration (x-axis) of Format 2 αCD37 / Fc MB (squares) or parental IgG (circles) incubated with target Ramos cells for 24 hours.

[0097] Figure 7 Depicted are graphs showing the percentage of cell-specific lysis (y-axis) versus the log concentration (x-axis) of Format 2 αCD37 / Fc MBs (squares) or control α-respiratory syncytial virus (RSV) MBs (circles) incubated with target Ramos cells and primary human NK cells for 4 hours.

[0098] Figure 8 is an ELISA graph showing absorbance measured at 450 nm (y-axis) versus the logarithmic concentration (x-axis) of Format 2 αCD37 / Fc MBs (triangles) or parental IgG (circles) coated on a 96-well plate and incubated with recombinant human C1q for 1 hour.

[0099] Figure 9 Depicted are graphs showing the percentage of single cells present within the propidium iodide (PI)+ gate determined by flow cytometry (y-axis) versus the logarithmic concentration (x-axis) of Format 2 αCD37 / Fc MB (squares) or parental IgG (circles) incubated with target Daudi cells for 2 hours in the presence of complement proteins.

[0100] Figure 10 Depicted are graphs showing the percentage of single cells present within the propidium iodide (PI)+ gate determined by flow cytometry (y-axis) versus the logarithmic concentration (x-axis) of Format 2 αCD37 / Fc MBs (squares) or control α-respiratory syncytial virus (RSV) MBs (circles) incubated with target Daudi cells for 2 hours in the presence of complement proteins.

[0101] Figure 11 Depicted are graphs showing the percentage of pHrodo Green AM / CellTrace Violet double-positive cells among all live single cells determined by flow cytometry (y-axis) versus the logarithmic concentration (x-axis) of Format 2 αCD37 / Fc MBs (squares) or control α-respiratory syncytial virus (RSV) MBs (circles) incubated with target Ramos and THP-1 cells for 1 hour.

[0102] Figure 12A is a schematic representation of the fusion protein used in Format 2 αCD37 / Fc MBs.

[0103] Figure 12Bis a schematic representation of the fusion proteins used in CD37 / NKp46 bispecific MBs.

[0104] Figures 13A-13C Depicted are graphs showing the percentage of cell-specific lysis (y-axis) versus the logarithmic concentration (x-axis) of CD37 / NKp46 bispecific MBs (squares) or control CD37 monospecific MBs (circles) incubated with target Ramos cells and primary human NK cells for 4 hours.

[0105] Figures 14A-14C Depicted are graphs showing the percentage of single cells present within the FITC-Annexin-V+ gate determined by flow cytometry versus the concentration (x-axis) of CD37 / NKp46 bispecific MBs (squares) or control CD37 monospecific MBs (circles) incubated with target Daudi cells for 24 hours. Detailed Description of the Invention

[0107] Disclosed herein are fusion polypeptides each comprising (1) a nanocage monomer or a subunit thereof and (2) an antibody fragment (e.g., a hematopoietic cell-binding agent or a tumor cell-binding agent) or an Fc polypeptide. The nanocage monomer or subunit thereof drives the self-assembly of the fusion polypeptide into a complex displaying one or more of the aforementioned cell-binding agents or tumor-binding agents and the Fc polypeptide.

[0108] definition

[0109] The terms "about" and "approximately" when used herein with respect to a value are used interchangeably and refer to a value similar to a reference value. Typically, one skilled in the art who is 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 referenced value.

[0110] As used herein, the terms "alter," "altered," "lower," "lowered," "increase," "increase," or "decrease," "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 known level or a level determined using an IgG that does not contain the referenced mutation in the Fc region.

[0111] 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 isolated 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 the effectiveness of the fragment can be screened in the same manner as with intact antibodies.

[0112] 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 interaction 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 studying the interacting entities or moieties in isolation or in a more complex system context (e.g., covalently or otherwise associated with a carrier entity and / or in a biological system or cell). As used herein, the phrase "non-binding" or "unbound" between two entities, or similar phrases, refers to 1) a lack of detectable binding or 2) binding below a set threshold, which corresponds to the absence of binding in an appropriate assay, such as 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, a maximum associative binding response of less than 0.1 nm to a biosensor loaded with 0.8 nm target after 180 seconds is classified as "no binding" when the test article is present at a concentration of 20 nM in an in vitro biolayer interferometry assay.

[0113] 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.

[0114] 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.

[0115] As used herein, the term "linker" is used to refer to an entity that connects two or more elements to form a multi-element medicament. For example, it is understood by those of ordinary skill in the art that a polypeptide whose structure includes two or more functional or organizational domains (e.g., a fusion polypeptide) typically includes a stretch of amino acids between these 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 that are associated with each other via 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.

[0116] As used herein, the term "multispecific" refers to the characteristic of having at least two binding sites to which at least two different binding partners, such as 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."

[0117] As used herein, the term "multivalent" refers to a feature of having at least two binding sites to which a binding partner, such as 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.

[0118] 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, fragments, and variants thereof of ferritin, apoferritin, encapsulin, sulfur oxygenase reductase (SOR), lumazine synthase, pyruvate dehydrogenase, carboxysome, vault proteins, GroEL, heat shock proteins, E2P coat protein, MS2 coat protein.

[0119] 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 to encompass not only polypeptides with complete sequences as described herein, but also 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 replacements without destroying activity. Therefore, any polypeptide that retains activity 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 at least one much higher (generally greater than 90% or even 95%, 96%, 97%, 98% or 99%) identity region in one or more highly conserved regions (generally encompassing at least 3-4 and often up to 20 or more amino acids) is encompassed within the associated term "polypeptide" used herein. Polypeptide can contain L-amino acids, D-amino acids or both, and can 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, the protein can comprise natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof.

[0120] As used herein, the term "specifically binds," "specifically binding," "binds specifically," or similar terms refers 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 -6 M 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 DIndicates 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 the target antigen but not to other antigens, such as distantly related family members of the antigen.

[0121] The term "self-assembly," when used in reference to a macromolecular complex (e.g., a polypeptide complex), refers to the spontaneous formation of a complex (e.g., a fusion polypeptide) when sufficient components of the complex to be formed are present. In some embodiments, the complex self-assembles under physiological conditions or in a buffer (e.g., a solution) corresponding to physiological conditions.

[0122] 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 related 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.

[0123] As used herein, the term "treatment" (also referred to as "treat" or "treating") refers to any administration of a therapy that partially or completely alleviates, ameliorates, alleviates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be for subjects who do not exhibit signs of the relevant disease, disorder, and / or condition, and / or for subjects who only exhibit early signs of the disease, disorder, and / or condition. Alternatively, or in addition, such treatment may be for subjects who exhibit one or more established signs of the relevant disease, disorder, and / or condition. In some embodiments, treatment may be for subjects who have been diagnosed with the relevant disease, disorder, and / or condition. In some embodiments, treatment may be directed to a subject 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.

[0124] A. Fusion polypeptide

[0125] In many 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 cell-binding agent (e.g., a hematopoietic cell-binding agent or a tumor cell-binding agent) or an Fc polypeptide, which may be connected via a linker, such as a linker described herein.

[0126] 1. Nanocage monomers and their subunits

[0127] In some embodiments, the nanocage monomer is a ferritin monomer.

[0128] The term "ferritin monomer" is used herein to refer to a single chain of ferritin that, in the presence of other ferritin chains, is capable of self-assembling into a polypeptide complex comprising a plurality of ferritin chains (e.g., 24 or more 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 having ferroxidase activity.

[0129] 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 containing the ferritin heavy chain.

[0130] 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.

[0131] As used herein, "full-length ferritin light chain" refers to a ferritin polypeptide that is capable of forming a four-helix bundle of two pairs of α-parallel α-helices connected by a loop and further comprises 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.

[0132] 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 the subunits together form a ferritin monomer that is, in turn, capable of self-assembling with other ferritin monomers to form a polypeptide complex.

[0133] 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, this half comprising the N-terminus of the ferritin chain. As used herein, the term "C-half ferritin" refers to about half of a ferritin chain, this half comprising the C-terminus of the ferritin chain. The exact point at which the ferritin chain can be split to form the N-half ferritin and the C-half ferritin can vary depending on the embodiment. For example, in the context of a ferritin monomer subunit based on a human ferritin light chain, the two halves can be separated at a point corresponding to a position between about position 75 and about position 100 of SEQ ID NO: 1 (or a substantial portion thereof). 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).

[0134] 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).

[0135] 2. Combined part

[0136] The binding moiety (eg, a hematopoietic cell binding moiety or a tumor binding moiety) typically comprises an antibody fragment.

[0137] 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 the Fab optionally connected by a linker (e.g., an amino acid linker as disclosed herein) is used.

[0138] In certain embodiments, an antibody fragment comprises a heavy chain variable region (e.g., V HIn 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 ).

[0139] In certain embodiments, the antibody fragment does not comprise any domain from the Fc region, for example, does not comprise any constant heavy (CH2 or CH3) domain. In some embodiments, the antibody fragment is a fully human or humanized antibody or an antibody fragment derived from a fully human or humanized antibody. In some embodiments, the antibody fragment is a chimeric antibody or an antibody fragment 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, an IgG1 antibody, an IgG2 antibody, or an IgG4 antibody. In some embodiments, the antibody fragment is obtained or derived from an agonist antibody, for example, an agonist humanized antibody.

[0140] In embodiments using multiple types of fusion polypeptides with antibody fragments, the antibody fragments in the multiple types of fusion polypeptides can be capable of binding to the same epitope on a given antigen (e.g., an antigen on a hematopoietic cell 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.

[0141] A. Hematopoietic cell binding part

[0142] The hematopoietic cell binding moiety is typically capable of binding (eg, specifically binding) to an antigen on a hematopoietic cell (eg, a lymphocyte (such as a natural killer (NK) cell or T cell) or a myeloid cell).

[0143] Lymphocyte binding fraction

[0144] In some embodiments, the hematopoietic cell binding moiety is a lymphocyte binding moiety, e.g., a natural killer cell binding moiety or a T cell binding moiety.

[0145] NK cell binding moiety

[0146] In some embodiments, the natural killer cell binding portion is capable of binding (e.g., specifically binds) CD16a, NKp46, or NKG2D.

[0147] In some embodiments, the natural killer cell 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 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., each differs by at most one or two amino acids) to the sequences of the heavy and light chain CDRs of a CD16a, NKp46, or NKG2D antibody (e.g., a human or humanized CD16a, NKp46, or NKG2D antibody).

[0148] In some embodiments, the natural killer cell binding portion comprises a heavy chain variable region (V H ) and light chain variable region (V L or V K ) wherein, in addition to one or two amino acid substitutions in all six CDRs, V H and V L or V K The CDRs have sequences identical to those of the heavy and light chain CDRs of a CD16a, NKp46, or NKG2D antibody (eg, a human or humanized CD16a, NKp46, or NKG2D antibody).

[0149] In some embodiments, the natural killer cell 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 V H and V L or V K The CDRs of the antibody are identical to the heavy and light chain CDRs of a CD16a, NKp46 or NKG2D antibody (eg, a human or humanized CD16a, NKp46 or NKG2D antibody).

[0150] In some embodiments, the natural killer cell 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 a CD16a, NKp46, or NKG2D antibody (e.g., a human or humanized CD16a, NKp46, or NKG2D antibody). In some embodiments, the natural killer cell 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 have sequences identical to those of the heavy and light chain variable regions of a CD16a, NKp46 or NKG2D antibody (e.g., a human or humanized CD16a, NKp46 or NKG2D antibody).

[0151] Non-limiting examples of CD16a antibodies include their heavy chain variable region, light chain variable region, and CDR sequences as shown in Tables 1A (VH1 / VL1) and 1B (VH2 / VL1).

[0152] Non-limiting examples of NKp46 antibodies include their heavy chain variable region, light chain variable region and CDR sequences as shown in Tables 2A (clone 1), 2B (clone 3), 2C (NKp46-1 H1L1), 2D (NKp46-2 H1L1), 2E (NKp46-3 H1L1), 2F (NKp46-4 H1L2) and 2G (09).

[0153] Non-limiting examples of NKG2D antibodies include tesnatilimab, and the heavy chain variable region, light chain variable region, and CDR sequences thereof are shown in Tables 3A (A49), 3B (E79), and 3C (tesnatilimab).

[0154] Table 1A: CD16A antibody sequences (VH1 / VL1)

[0155]

[0156] Table 1B: CD16A antibody sequences (VH2 / VL1)

[0157]

[0158] Table 2A: NKp46 antibody sequence (clone 1)

[0159]

[0160] Table 2B: NKp46 antibody sequence (clone 3)

[0161]

[0162] Table 2C: NKp46 antibody sequence (NKp46-1 H1L1)

[0163]

[0164] Table 2D: NKp46 antibody sequence (NKp46-2 H1L1)

[0165]

[0166] Table 2E: NKp46 antibody sequence (NKp46-3 H1L1)

[0167]

[0168] Table 2F: NKp46 antibody sequence (NKp46-4 H1L2)

[0169]

[0170] Table 2G: NKp46 antibody sequences (09)

[0171]

[0172] Table 3A: NKG2D antibody sequence (A49)

[0173]

[0174] Table 3B: NKG2D antibody sequence (E79)

[0175]

[0176] Table 3C: NKG2D Antibody Sequences (Telazolimab)

[0177]

[0178] T cell binding moiety

[0179] In some embodiments, the T cell binding portion is capable of binding (e.g., specifically binds) CD3.

[0180] In some embodiments, the T cell 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 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 CD3 antibody (e.g., a human or humanized CD3 antibody).

[0181] In some embodiments, the T cell binding portion is an antibody fragment comprising a heavy chain variable region (V H ) and light chain variable region (V L or V K ), wherein in addition to a total of one or two amino acid substitutions in all six CDRs, V H and V L or V K The CDRs have sequences identical to those of the heavy and light chain CDRs of a CD3 antibody (eg, a human or humanized CD3 antibody).

[0182] In some embodiments, the T cell binding portion is 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 V L or V K The CDRs of the antibody are identical to the heavy and light chain CDRs of a CD3 antibody (eg, a human or humanized CD3 antibody).

[0183] In some embodiments, the T cell 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 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 CD3 antibody (e.g., a human or humanized CD3 antibody). In some embodiments, the T cell binding portion is an antibody fragment comprising the heavy and light chain variable regions (e.g., V H and V L or V K ), the heavy and light chain variable regions have sequences identical to those of the heavy and light chain variable regions of a CD3 antibody (e.g., a human or humanized CD3 antibody).

[0184] Non-limiting examples of CD3 antibodies include otelixizumab, muromonab-CD3, teplizumab, and visilizumab. Table 4 depicts the heavy chain variable region, light chain variable region, and CDR sequences in teplizumab.

[0185] Table 4: CD3 antibody sequences (Teplizumab)

[0186]

[0187] Myeloid cell binding fraction

[0188] In some embodiments, the hematopoietic cell binding moiety is a myeloid cell binding moiety.

[0189] In some embodiments, the myeloid cell-binding moiety is capable of binding (eg, specifically binds) SIRPα.

[0190] In some embodiments, the myeloid cell-binding moiety is 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 V L or V K The CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of the SIRPα antibody have sequences similar to (e.g., each differs by at most one or two amino acids) the CDRs of a SIRPα antibody (e.g., a human or humanized SIRPα antibody).

[0191] In some embodiments, the myeloid cell-binding moiety is an antibody fragment comprising a heavy chain variable region (V H ) and light chain variable region (V L or V K ), wherein in addition to a total of one or two amino acid substitutions in all six CDRs, V H and V L or V K The CDRs have sequences identical to those of the heavy and light chain CDRs of a SIRPα antibody (eg, a human or humanized SIRPα antibody).

[0192] In some embodiments, the myeloid cell-binding moiety is 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 V L or V K The CDRs of the SIRPα antibody are identical to the heavy and light chain CDRs of a SIRPα antibody (eg, a human or humanized SIRPα antibody).

[0193] In some embodiments, the myeloid cell-binding moiety 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 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 SIRPα antibody (e.g., a human or humanized SIRPα antibody). In some embodiments, the myeloid cell-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 chain and light chain variable regions have sequences identical to those of the heavy chain and light chain variable regions of a SIRPα antibody (eg, a human or humanized SIRPα antibody).

[0194] Non-limiting examples of SIRPα antibodies include their heavy chain variable region, light chain variable region, and CDR sequences as shown in Tables 5A (1H9) and 5B (3C2).

[0195] Table 5A: SIRPα antibody sequence (1H9)

[0196]

[0197] Table 5B: SIRPα antibody sequences (3C2)

[0198]

[0199] B. Tumor-binding moiety

[0200] Tumor binding moieties are generally capable of binding (e.g., specifically binding) to tumor-associated antigens (e.g., tumor-specific antigens) expressed on tumor cells or cells supporting tumors (e.g., stromal cells). In some embodiments, the tumor binding moiety is capable of binding (e.g., specifically binding) to CD20 or CD37.

[0201] In some embodiments, the tumor binding moiety 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 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 CD20 or CD37 antibody (e.g., a human or humanized CD20 or CD37 antibody).

[0202] In some embodiments, the tumor binding moiety comprises an antibody fragment comprising a heavy chain variable region (V H ) and light chain variable region (V L or V K ), wherein in addition to a total of one or two amino acid substitutions in all six CDRs, V H and V L or V K The CDRs have sequences identical to those of the heavy and light chain CDRs of a CD20 or CD37 antibody (e.g., a human or humanized CD20 or CD37 antibody).

[0203] In some embodiments, the tumor binding moiety 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 V L or V K The CDRs of the antibody are identical to the heavy and light chain CDRs of a CD20 or CD37 antibody (e.g., a human or humanized CD20 or CD37 antibody).

[0204] In some embodiments, the tumor binding moiety comprises 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 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 CD20 or CD37 antibody (e.g., a human or humanized CD20 or CD37 antibody). In some embodiments, the tumor-binding portion is an antibody fragment comprising the heavy and light chain variable regions (e.g., V H and V L or V K ), the heavy and light chain variable regions have sequences identical to those of the heavy and light chain variable regions of a CD20 or CD37 antibody (e.g., a human or humanized CD20 or CD37 antibody).

[0205] Non-limiting examples of CD20 antibodies include rituximab, ofatumumab, veltuzumab, and ocrelizumab. Tables 6A and 6B depict the heavy chain variable region, light chain variable region, and CDR sequences of rituximab and ofatumumab, respectively.

[0206] Non-limiting examples of CD37 antibodies include naratuximab, lilotomab, AGS67C, and otlertuzumab. Tables 7A, 7B, 7C, and 7D depict the heavy chain variable region, light chain variable region, and CDR sequences of naratuximab, lilotomab, AGS67C, and otlertuzumab, respectively.

[0207] Table 6A: CD20 Antibody Sequences (Rituximab)

[0208]

[0209] Table 6B: CD20 Antibody Sequences (Ofatumumab)

[0210]

[0211] Table 7A: CD37 Antibody Sequences (Natuximab)

[0212]

[0213] Table 7B: CD37 antibody sequences (rilotomab)

[0214]

[0215] Table 7C: CD37 antibody sequence (AGS67C)

[0216]

[0217] Table 7D: CD37 antibody sequence (oletuzumab)

[0218]

[0219] Table 7E: CD37 antibody sequence (016-H5L2)

[0220]

[0221] 3. Fc peptide

[0222] 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.

[0223] An IgG Fc chain (eg, an IgG1 Fc chain) typically contains two constant heavy domains (CH2 and CH3) and a hinge region connected to the CH2 domain.

[0224] 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.

[0225] 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.

[0226] 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 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 identical to the heavy chain constant region of SEQ ID NO: 426, 427, 428, or 429.

[0227] 4. Connector

[0228] In certain embodiments, linkers are used within fusion polypeptides and / or within single-chain molecules such as scFc. In some embodiments, linkers are amino acid linkers. For example, linkers as used herein may comprise from about 1 to about 100 amino acid residues, for example, from about 1 to about 70, from about 2 to about 70, from about 1 to about 30, or from about 2 to about 30 amino acid residues. In some embodiments, linkers comprise 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.

[0229] In certain embodiments, the linker comprises a glycine-serine sequence, e.g., (G n S) m sequences (e.g., GGS, GGGS (SEQ ID NO: 6), and GGGGS (SEQ ID NO: 7) sequences), which 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.

[0230] B. Self-assembling polypeptide complexes

[0231] In one aspect, self-assembling polypeptide complexes ("multivalents" or "MBs") comprising a plurality of fusion polypeptides disclosed herein are provided. Generally, the self-assembling polypeptide complexes provided comprise (a) a plurality of hematopoietic cell-binding fusion polypeptides, each hematopoietic cell-binding fusion polypeptide comprising (1) a hematopoietic cell-binding moiety linked to (2) a nanocage monomer or subunit thereof, and (b) a plurality of Fc fusion polypeptides, each Fc fusion polypeptide comprising (1) an Fc polypeptide linked to (2) a nanocage monomer or subunit thereof, wherein the Fc polypeptide comprises an Fc chain.

[0232] In some 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 linked to (2) a nanocage monomer or a subunit thereof.

[0233] 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.

[0234] In some embodiments, the nanocage monomer or subunit thereof is a ferritin monomer subunit, and (a) each hematopoietic cell-binding fusion polypeptide comprises a ferritin monomer subunit that is the C half-ferritin, and each Fc-fusion polypeptide comprises a ferritin monomer subunit that is the N half-ferritin; or (b) each hematopoietic cell-binding fusion polypeptide comprises a ferritin monomer subunit that is the N half-ferritin, and each Fc-fusion polypeptide comprises a ferritin monomer subunit that is the C half-ferritin.

[0235] 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.

[0236] 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 hematopoietic cell-binding fusion polypeptides.

[0237] 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.

[0238] 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.

[0239] In some embodiments, the self-assembling polypeptide complex comprises a ratio of hematopoietic cell binding fusion polypeptide to all other fusion polypeptides 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.

[0240] In some embodiments, the self-assembling polypeptide complex comprises a ratio of hematopoietic cell binding fusion polypeptide to Fc fusion polypeptide 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.

[0241] Pharmacokinetic characteristics

[0242] In certain embodiments, when administered to a subject in need thereof, the provided self-assembling polypeptide complex has one or more pharmacokinetic characteristics that are similar to the pharmacokinetic characteristics of a reference IgG molecule (e.g., an IgG molecule whose class matches the class of the Fc chain within the Fc polypeptide of the Fc fusion polypeptide within the self-assembling polypeptide complex).

[0243] Effect

[0244] In certain embodiments, provided self-assembling polypeptide complexes are capable of inducing antibody-dependent cellular cytotoxicity (ADCC) upon contact with target cells, eg, killing target cells (eg, tumor cells).

[0245] ADCC can be assessed in vitro using any of a variety of assays, for example, cell-based assays that employ effector cells (e.g., NK cells, peripheral blood mononuclear cells), target cells (e.g., tumor cells), and a reporter that indicates target cell lysis and / or activation within the effector cells.

[0246] C. Methods of Treatment and Access to Tumor Cells

[0247] 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.

[0248] In some embodiments, the subject is a mammal, eg, a human.

[0249] 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.

[0250] 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).

[0251] In some embodiments, the administering step results in an improvement in one or more clinical outcomes or metrics in the subject.

[0252] For example, in some embodiments, the subject has cancer, eg, B cell lymphoma.

[0253] In some embodiments, the administering step results in slowing or inhibiting the progression of a tumor, e.g., regression of a tumor. In some embodiments, the administering step results in complete regression of a tumor.

[0254] Contact method

[0255] The present disclosure also provides methods of contacting a self-assembling polypeptide complex disclosed herein with a tumor cell. In some embodiments, the contacting step occurs in a mammalian body (eg, a human body).

[0256] In some embodiments, the contacting step induces apoptosis in the tumor cells. In some embodiments, apoptosis is induced at an EC of less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM, or less than 0.05 nM. 50 In some embodiments, the tumor cells express CD37, and the self-assembling polypeptide complex comprises a fusion protein comprising an α-CD37 Fab.

[0257] In some embodiments, the contacting step is performed in the presence of hematopoietic cells, such as NK cells, and induces antibody-dependent cellular cytotoxicity (ADCC) of the tumor cells. In some embodiments, ADCC is performed with an EC of less than 11 nM, less than 500 pM, less than 100 pM, less than 50 pM, less than 10 pM, less than 5 pM, less than 4 pM, less than 3 pM, less than 2 pM, less than 1 pM, and less than 0.5 pM. 50 Value (for self-assembling polypeptide complexes) induced.

[0258] In some embodiments, the contacting step is performed in the presence of a complement protein and results in complement dependent cytotoxicity (CDC) of the tumor cells. In some embodiments, the CDC is performed at an EC of less than 10 nM, less than 1.0 nM, less than 0.50 nM, less than 0.40 nM, less than 0.30 nM, less than 0.20 nM, or less than 0.15 nM. 50 Value (for self-assembling polypeptide complexes) induced.

[0259] In some embodiments, the contacting step is performed in the presence of hematopoietic cells, such as myeloid cells, and results in antibody-dependent cellular cytotoxicity (ADCP) of the tumor cells. In some embodiments, ADCP is performed at an EC of less than 100 pM (0.1 nM), less than 50 pM, less than 25 pM, less than 20 pM, less than 18 pM, less than 15 pM, or less than 10 pM. 50 Value (for self-assembling polypeptide complexes) induced. Example

[0260] Example 1. Construction and expression of immune cell engaging or activating multivalent bodies (MBs)

[0261] This example describes the generation of immune cell engaging or activating multivalent bodies (MBs) that are self-assembled from a plurality of at least two types of ferritin-containing fusion polypeptides. Specifically, the MBs comprise:

[0262] (1) One or both of the following:

[0263] (a) tumor-binding fusion polypeptides, each comprising a human ferritin light chain or a subunit thereof, and an antibody fragment capable of binding (e.g., specifically binding) to a tumor cell; and

[0264] (b) an immune cell-binding fusion polypeptide comprising a human ferritin light chain or a subunit thereof and an antibody fragment capable of binding (e.g., specifically binding) to an immune cell; and

[0265] (2) An Fc fusion polypeptide comprising a human ferritin light chain or a subunit thereof and an Fc polypeptide.

[0266] A set of constructs encoding the following fusion proteins was prepared, mixed at a predetermined molar ratio, and then transiently transfected into cells expressing the encoded polypeptides. The following are non-limiting examples of sets of fusion polypeptides that can be used to generate MBs that engage or activate immune cells.

[0267] Group 1

[0268] (1) a single-chain Fab (scFab) of an antibody capable of binding (e.g., specifically binding) to an immune cell, the scFab being fused to a light chain of human ferritin via a linker; and

[0269] (2) Single-chain Fc (scFc), which is fused to the light chain of human ferritin via a linker.

[0270] Group 2

[0271] (1) a scFab of an antibody capable of binding (e.g., specifically binding) to an immune cell, wherein the scFab is fused to the N-half of the light chain of human ferritin via a linker,

[0272] (2) a scFab of an antibody capable of binding (e.g., specifically binding) to a tumor-associated antigen, the scFab being fused to the C half of the light chain of human ferritin via a linker, and

[0273] (3) scFc, which is fused to the light chain of human ferritin via a linker,

[0274] The ratio of polypeptides (1) and (2) is 1:1.

[0275] Group 3

[0276] (1) an scFab of an antibody capable of binding (e.g., specifically binding) to an immune cell, wherein the scFab is fused to the C half of the light chain of human ferritin via a linker,

[0277] (2) a scFab of an antibody capable of binding (e.g., specifically binding) to a tumor-associated antigen, the scFab being fused to the N-half of the light chain of human ferritin via a linker, and

[0278] (3) scFc, which is fused to the light chain of human ferritin via a linker,

[0279] The ratio of polypeptides (1) and (2) is 1:1.

[0280] Group 4

[0281] (1) an scFab of an antibody capable of binding (e.g., specifically binding) to an immune cell, wherein the scFab is fused to a light chain of human ferritin via a linker;

[0282] (2) a scFab of an antibody capable of binding (e.g., specifically binding) to a tumor-associated antigen, the scFab being fused to the N-half of the light chain of human ferritin via a linker; and

[0283] (3) scFc, which is fused to the C half of the light chain of human ferritin via a linker,

[0284] The ratio of polypeptides (2) and (3) is 1:1.

[0285] Group 5

[0286] (1) an scFab of an antibody capable of binding (e.g., specifically binding) to an immune cell, wherein the scFab is fused to a light chain of human ferritin via a linker;

[0287] (2) a scFab of an antibody capable of binding (e.g., specifically binding) to a tumor-associated antigen, the scFab being fused to the C half of the light chain of human ferritin via a linker; and

[0288] (3) scFc, which is fused to the N half of the light chain of human ferritin via a linker,

[0289] The ratio of polypeptides (2) and (3) is 1:1.

[0290] Group 6

[0291] (1) an scFab of an antibody capable of binding (e.g., specifically binding) to an immune cell, wherein the scFab is fused to the N half of the light chain of human ferritin via a linker;

[0292] (2) a scFab of an antibody capable of binding (e.g., specifically binding) to a tumor-associated antigen, the scFab being fused to a light chain of human ferritin via a linker; and

[0293] (3) scFc, which is fused to the C half of the light chain of human ferritin via a linker,

[0294] The ratio of polypeptides (2) and (3) is 1:1.

[0295] Group 7

[0296] (1) an scFab of an antibody capable of binding (e.g., specifically binding) to an immune cell, wherein the scFab is fused to the C half of the light chain of human ferritin via a linker;

[0297] (2) a scFab of an antibody capable of binding (e.g., specifically binding) to a tumor-associated antigen, the scFab being fused to a light chain of human ferritin via a linker; and

[0298] (3) scFc, which is fused to the N half of the light chain of human ferritin via a linker,

[0299] The ratio of polypeptides (1) and (3) is 1:1.

[0300] Group 8

[0301] (1) an scFab of an antibody capable of binding (e.g., specifically binding) to a tumor-associated antigen, the scFab being fused to a light chain of human ferritin via a linker; and

[0302] (2) scFc, which is fused to the light chain of human ferritin via a linker.

[0303] Group 9

[0304] (1) a scFab of an antibody capable of binding (e.g., specifically binding) to a tumor-associated antigen, the scFab being fused to the C half of the light chain of human ferritin via a linker, and

[0305] (2) scFc, which is fused to the N-half of the light chain of human ferritin via a linker.

[0306] The ratio of polypeptides (1) and (2) is 1:1.

[0307] Group 10

[0308] (1) a scFab of an antibody capable of binding (e.g., specifically binding) to a tumor-associated antigen, the scFab being fused to the N-half of the light chain of human ferritin via a linker, and

[0309] (2) scFc, which is fused to the C half of the light chain of human ferritin via a linker.

[0310] The ratio of polypeptides (1) and (2) is 1:1.

[0311] Group 11

[0312] (1) an scFab of an antibody capable of binding (e.g., specifically binding) to a tumor-associated antigen, wherein the scFab is fused to a light chain of human ferritin via a linker;

[0313] (2) an scFab of an antibody capable of binding (e.g., specifically binding) to an immune cell, wherein the scFab is fused to the light chain of human ferritin via a linker;

[0314] (3) a scFab of an antibody capable of binding (e.g., specifically binding) to a tumor-associated antigen, the scFab being fused to the N-half of the light chain of human ferritin via a linker; and

[0315] (4) scFc, which is fused to the C half of the light chain of human ferritin via a linker,

[0316] The ratio of polypeptides (3) and (4) is 1:1.

[0317] Group 12

[0318] (1) an scFab of an antibody capable of binding (e.g., specifically binding) to a tumor-associated antigen, wherein the scFab is fused to a light chain of human ferritin via a linker;

[0319] (2) an scFab of an antibody capable of binding (e.g., specifically binding) to an immune cell, wherein the scFab is fused to the light chain of human ferritin via a linker;

[0320] (3) a scFab of an antibody capable of binding (e.g., specifically binding) to an immune cell, the scFab being fused to the N-half of the light chain of human ferritin via a linker; and

[0321] (4) scFc, which is fused to the C half of the light chain of human ferritin via a linker,

[0322] The ratio of polypeptides (3) and (4) is 1:1.

[0323] Group 13

[0324] (1) an scFab of an antibody capable of binding (e.g., specifically binding) to a tumor-associated antigen, the scFab being fused to a light chain of human ferritin via a linker; and

[0325] (2) scFab of an antibody capable of binding (eg, specifically binding) to a tumor-associated antigen, wherein the scFab is fused to one end of the light chain of human ferritin via a linker, and the scFc is fused to the other end of the light chain of human ferritin via a linker.

[0326] Group 14

[0327] (1) an scFab of an antibody capable of binding (e.g., specifically binding) to an immune cell, the scFab being fused to a light chain of human ferritin via a linker; and

[0328] (2) scFab of an antibody capable of binding (eg, specifically binding) to an immune cell, wherein the scFab is fused to one end of the light chain of human ferritin via a linker, and the scFc is fused to the other end of the light chain of human ferritin via a linker.

[0329] Group 15

[0330] (1) an scFab of an antibody capable of binding (e.g., specifically binding) to a tumor-associated antigen, the scFab being fused to a light chain of human ferritin via a linker; and

[0331] (2) scFab of an antibody capable of binding (eg, specifically binding) to an immune cell, wherein the scFab is fused to one end of the light chain of human ferritin via a linker, and the scFc is fused to the other end of the light chain of human ferritin via a linker.

[0332] Group 16

[0333] (1) an scFab of an antibody capable of binding (e.g., specifically binding) to an immune cell, the scFab being fused to a light chain of human ferritin via a linker; and

[0334] (2) scFab of an antibody capable of binding (eg, specifically binding) to a tumor-associated antigen, wherein the scFab is fused to one end of the light chain of human ferritin via a linker, and the scFc is fused to the other end of the light chain of human ferritin via a linker.

[0335] Upon expression, the encoded polypeptides self-assemble to form immune cell-engaging or activating MBs.

[0336] Example 2. Target binding of MBs as determined by biolayer interferometry

[0337] The binding kinetics and affinity of exemplary immune cell-engaging MBs generated as described in Example 1 to antigens on immune cells and / or tumor-associated antigens can be measured by biolayer interferometry (BLI). For example, the binding characteristics of IgG antibodies (from which the scFab within the immune cell-engaging MBs is derived) can also be measured for comparison.

[0338] For example, Ni-NTA biosensors can be coated with His-tagged antigens (e.g., the extracellular domain of an antigen on an immune cell or the extracellular domain of a tumor-associated antigen). The coated biosensor can be immersed in a well containing a serial dilution of MB (or antibody control) in a buffer such as phosphate-buffered saline (PBS) buffer containing Tween 20 and optionally bovine serum albumin (BSA) during the association phase and the dissociation phase.

[0339] The biosensor can be regenerated between experiments, for example, by multiple applications of glycine solution and by recharging in NiSO4.

[0340] Target binding can be based on the maximum associative binding response at the end of the association 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 multivalent, a maximal associative binding response of less than 0.1 nM can be classified as "non-binding".

[0341] Example 3. Binding of MB to Fc receptors as determined by biolayer interferometry

[0342] The binding kinetics and affinities of various MBs generated as described in Example 1 to various Fc receptors were determined by biolayer interferometry.

[0343] The MBs tested in this example contained polypeptides comprising one or more Fc chains. As controls for comparison, MBs without Fc-containing polypeptides and / or IgG antibodies of the same class as the Fc chains used in the MBs can be used.

[0344] Binding to various Fc receptors can be measured, for example, binding 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 MB in animal models, binding to non-human Fc receptors can also be assessed, such as cynomolgus monkey Fc receptors (e.g., cynomolgus monkey FcγRI (cFCγRI), cFcγRIIa, cFcγRIIb, cFcγRIII, and cFcRn) and / or mouse Fc receptors (e.g., mouse FcγRI (mFcγRI), mFcγRIIb, mFcγRIII, mFcγRIV, and mFcRn).

[0345] The experiment can be performed similarly to that described in Example 2, except that His-tagged Fc receptors are coated onto Ni-NTA biosensors and titrated with different concentrations of test MBs. To assess the potential of MBs to undergo endosomal recycling, binding to FcRn can be measured at pH 6.0 for association and at pH 7.4 for dissociation.

[0346] Example 4. In vitro evaluation of tumor cytotoxicity promoted by natural killer (NK) cell engagement of MBs

[0347] The ability of natural killer (NK) cell-engaged MBs to promote killing of B cell lymphoma cells expressing CD20 and CD37 can be assessed. MBs were generated as described in Example 1 using scFabs of antibodies against NKG2D (which is expressed on natural killer cells and some subsets of T cells) and scFabs of α-CD20 or α-CD37.

[0348] Cytotoxicity is assessed using a co-culture assay in which (1) human natural killer cells (e.g., primary NK cells, available from StemCell Technologies) are co-cultured with (2) B cell lymphoma target cells. For B cell lymphoma cells, CD20+, CD37+ Raji and / or Daudi B cell lymphoma cell lines (e.g., commercially available from ATCC (Manassas, VA)) can be used.

[0349] B cell lymphoma target cells are co-cultured with human natural killer cells (e.g., at an effector cell to target cell ratio of about 1:1) and treated with serially diluted MBs or a control molecule (e.g., a control multivalent (α-respiratory syncytial virus (RSV)). After incubation at about 37°C for at least about 3 hours, the cell viability of the B cell lymphoma target cells is measured (e.g., using a kit such as Invitrogen Calcein-AM™). Calcein-AM is a cell-permeable dye used as a marker to indicate cell viability.

[0350] Increased calcein release in samples incubated with NK cell-engaging MBs compared to calcein release in samples incubated with a control molecule indicates that NK cell-engaging MBs are more effective in killing B cell lymphoma target cells.

[0351] Example 5. In vitro evaluation of the promotion of antibody-dependent cellular cytotoxicity by natural killer (NK) cell engagement of MBs

[0352] To assess the ability of wild-type Fc molecules present on MBs to induce antibody-dependent cellular cytotoxicity (ADCC), tumor-bound MBs containing wild-type IgG1 Fc were generated and assayed in an in vitro ADCC reporter.

[0353] Multivalent body generation: MBs containing CD37 Fab and wild-type IgG1 Fc were generated similarly to those described in Example 1 in the following format:

[0354] Format 1: Construct MBs containing the following fusion proteins. (See Figure 2A )

[0355] (1) scFab of CD37 antibody, which is expressed via (GGGS) n The linker was fused to the N-terminus of the human ferritin light chain;

[0356] (2) scFab of CD37 antibody, which is expressed via (GGGS) n a linker fused to the N-terminus of the N-terminal half of the human ferritin light chain; and

[0357] (3) scFc (IgG1) via (GGGS) n The linker is fused to the N-terminus of the C-terminal half of the human ferritin light chain.

[0358] The ratio of polypeptides (2) and (3) is 1:1.

[0359] In the resulting Form 1 MBs, the Fc is oriented such that the FcγR binding pocket is distal to the ferritin core of the MBs (see Figure 2B ).

[0360] Format 2: Construct MBs containing the following fusion proteins. (See Figure 3A )

[0361] (1) scFab of CD37 antibody, which is expressed via (GGGS) n A linker fused to the N-terminus of the human ferritin light chain; and

[0362] (2) Human ferritin light chain fused to:

[0363] -scFab of CD37 antibody (via (GGGS) n Linker fused to the N-terminus of human ferritin light chain)

[0364] -scFc(IgG1)(via (GGGS) n The linker was fused to the C-terminus of the human ferritin light chain).

[0365] In the resulting Form 2 MBs, the Fc is oriented such that the FcγR binding pocket faces the ferritin core of the MBs (see Figure 3B ).

[0366] ADCC function: The ability of α-CD37 / Fc MB to induce antibody-dependent cellular cytotoxicity (ADCC) was then tested in an in vitro assay using Ramos target cells and ADCC bioassay effector cells. Successful induction of ADCC via the Fc of the test molecule (such as the multivalents disclosed herein) leads to NFAT activation in effector cells, which in this assay system results in luciferase expression driven by the NFAT response element. (See Figure 4 . ) Therefore, the luciferase signal in this assay is indicative of ADCC function.

[0367] Format 2α-CD37 / Fc MB demonstrated the ability to induce ADCC, EC 50 was approximately 4.2 pM. In contrast, Form 1 α-CD37 / Fc MB did not induce ADCC in this assay. (See Figure 5 . )

[0368] Thus, this example demonstrates that certain MBs of the present disclosure can induce ADCC in vitro.

[0369] Example 6. In vitro evaluation of tumor cytotoxicity promoted by T cell engagement of MBs

[0370] The ability of T cell-engaged MBs to promote killing of B cell lymphoma cells expressing CD20 and CD37 can be assessed. MBs were generated as described in Example 1 using scFabs of antibodies against CD3 (which is expressed on T cells) and scFabs of α-CD20 or α-CD37.

[0371] Cytotoxicity was assessed using a co-culture assay in which (1) human peripheral blood mononuclear cells (PBMCs) or purified human T cells were co-cultured with (2) B cell lymphoma target cells. PBMCs were obtained from healthy donor blood using, for example, a Ficoll-Paque Plus kit (GE Healthcare), and CD8+ T cells were purified using a T cell isolation kit (e.g., from StemCell Technologies). For B cell lymphoma cells, CD20+, CD37+ Raji and / or Daudi B cell lymphoma cell lines (e.g., commercially available at ATCC (Manassas, VA)) were used.

[0372] B cell lymphoma target cells are co-cultured with (1) PBMCs (e.g., at an effector cell to target cell ratio of about 25-30:1) or (2) IL-2 activated PBMCs (e.g., at an effector cell to target cell ratio of about 15:1) and treated with serial dilutions of MBs or control molecules (e.g., bispecific α-CD3 / α-CD20 and / or bispecific α-CD3 / α-CD37). After incubation at about 37°C overnight, lactate dehydrogenase (LDH) release is measured in B cell lymphoma target cells (e.g., using a kit such as Promega's LDH-Glo™). LDH release is used as a marker to indicate target cell killing.

[0373] Increased LDH release in samples incubated with T cell-engaging MBs compared to LDH release in samples incubated with a control molecule indicates that the T cell-engaging MBs are more effective in killing B cell lymphoma target cells.

[0374] For experiments with purified T cells, B cell lymphoma target cells were pre-labeled with carboxyfluorescein succinimidyl ester (CFSE) dye or CellTrace fluorescent stain (ThermoFisher Scientific) to distinguish B cell lymphoma target cells from T cells. Labeled B cell lymphoma target cells were co-cultured with purified T cells and incubated at about 37°C for at least about two hours. Cell killing was assessed by staining the cells with propidium iodide (PI), which stains dead cells, and then evaluating the staining by flow cytometry.

[0375] Increased PI staining in samples incubated with T cell-engaging MBs compared to PI staining in samples incubated with a control molecule indicates that the T cell-engaging MBs are more effective in killing B cell lymphoma target cells.

[0376] Example 7. Pharmacokinetics of MB in non-human animal models

[0377] The pharmacokinetics of exemplary immune cell-binding MBs 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).

[0378] Animals receive a single bolus injection of a multivalent 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, 15 days, and 21 days after injection.

[0379] Multiple dose studies can also be performed in which the first and second doses are separated by, for example, days or weeks.

[0380] Plasma samples can be obtained from blood samples and stored frozen until use.

[0381] To measure the levels of MB or a control molecule in plasma by enzyme-linked immunosorbent assay (ELISA), recombinant tumor-associated antigens (e.g., CD20 or CD37) are coated onto plates and incubated overnight at 4°C. After two washes (e.g., with a solution such as phosphate-buffered saline (PBS)-Tween-20), the plates are blocked with a bovine serum albumin (BSA) solution in PBS for 1 hour at room temperature and then washed twice. Plasma samples are diluted in a PBS / Tween-20 / BSA solution, added to the wells, and incubated on a shaker at room temperature for 1 hour. After another wash step, bound molecules are detected by incubation with a diluted goat polyclonal α-human Fc-HRP secondary antibody. After a further wash 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 dilutions of MB in PBS / Tween-20 / BSA solution.

[0382] The half-life of MB in animals can be calculated based on these measurements, and the half-life of MB can be compared to the half-life of IgG molecules.

[0383] Example 8. Therapeutic Effect of Immune Cell Engagement with MB in a Xenograft Mouse Model

[0384] The efficacy of exemplary MB treatments can be evaluated in B cell lymphoma xenograft models. Daudi and Raji human Burkitt lymphoma cell lines (commercially available from ATCC) were grown as suspension cultures. For solid tumor models, approximately 5×10 6 Raji cells or Daudi cells were injected subcutaneously into the flank of SCID mice. Tumor volume was measured twice a week using a caliper (solid tumor model). For the disseminated tumor model, approximately 1.5 × 10 7 Daudi cells or approximately 2.5 × 10 6Raji cells are injected intravenously into SCID mice via the tail vein. Mice are examined daily for hind leg paralysis and weighed weekly. MBs generated as described in Example 1 (or control MBs (e.g., MBs without a tumor-binding moiety and / or a tumor-binding IgG1 control antibody)) are injected at several time points after tumor cell inoculation (e.g., 1 day, 5 days, 10 days, and 15 days). Various doses of MBs can be tested. Animals are sacrificed when they have lost 20% of their initial body weight, when tumors have reached maximum tumor volume, or when they develop hind leg paralysis.

[0385] Tumor growth, survival curves, and weight loss can be compared between treatment groups.Reduction in tumor growth and / or improved survival in animals treated with MB (compared to animals treated with a control molecule) indicates improved efficacy of immune cell-engaging MB.

[0386] Example 9. α-CD37 MB induces tumor cell apoptosis through CD37 receptor aggregation

[0387] The ability of αCD37 / Fc Mb (version 2) (generated as described in Example 5) to induce apoptosis in CD37+ tumor cells via CD37 receptor aggregation was assessed. α-CD37 / Fc MB was generated as described in Example 5.

[0388] The apoptotic activity of α-CD37 IgG and α-CD37 / Fc MBs was determined using an apoptosis assay.

[0389] Apoptosis assay. Daudi or Ramos cells are used as CD37+ target tumor cells, and Annexin-V staining is used to identify apoptotic cells. Target cells are incubated in 96-well U-shaped bottom plates with serial dilutions of test IgG or MB at 37°C, 5% CO2 for 24 hours. At the end of the incubation period, flow cytometry is performed to assess the viability of target cells. Cells are washed with cooled phosphate-buffered saline (PBS) and stained with live / dead (L / D) violet viability dye on ice for 30 minutes. After L / D staining, cells are washed and then stained on ice for 15 minutes with FITC-conjugated annexin-V dye. FITC signals are measured on a CytoFLEX flow cytometer (Beckman Coulter). FlowJo software is used for data analysis. The FITC-Annexin-V+ gate is determined based on fluorescence minus one (FMO) and no treatment control, and the FITC-Annexin-V+ cell % is calculated according to the % of individual cells present in the FITC-Annexin-V+ gate.

[0390] Results. Increased fluorescence was observed in target cell samples incubated with αCD37 / Fc MB compared to that observed in target cell samples incubated with parental IgG. ( Figure 6These results indicate that all tested αCD37 / Fc MBs induced potent tumor cell apoptosis and exhibited superior tumor cell killing compared to the parental IgG. Without wishing to be bound by any particular theory, this increase in apoptotic capacity may be due to enhanced receptor aggregation achieved by the multivalency of the MBs.

[0391] αCD37 / Fc Mb demonstrated that EC 50 The ability to induce apoptosis (see Table 8).

[0392] Table 8: Half maximal effective concentration (EC) of CD37 IgG and MB on Daudi cell apoptosis 50 )

[0393] Example 10. Natural Killer (NK) Cell Engagement with MB Promotes Antibody-Dependent Cellular Cytotoxicity in Vitro

[0394] The ability of αCD37 / Fc MBs (version 2) generated as described in Example 5 to induce antibody-dependent cellular cytotoxicity (ADCC) was evaluated in an in vitro cytotoxicity assay. Parental IgG and control MBs (using α-RSV Fab) were used as controls.

[0395] ADCC assay. Ramos cells are used as target cells, and primary human NK cells (STEMCELL Technologies) are used as effector cells. The day before the experiment, frozen primary human NK cells were thawed and in RPMI-1640 culture medium containing 20% ​​fetal bovine serum (FBS) and 5 U / ml human IL-2, left to stand overnight at 37°C, 5% CO2. On the day of the experiment, target cells were labeled for 30 minutes at 37°C, 5% CO2 with 10 mM calcein-AM dye. The labeled target cells were then washed twice with RPMI-1640 culture medium. Target cells and effector cells were adjusted to appropriate cell concentrations. Assay plates were assembled by combining target cells, effector cells, and test IgG or MB serial dilutions into 96-well U-bottom plates so that the ratio of effector cells to target cells was 1:1 (10,000 cells per well). Assay plates were incubated at 37°C, 5% CO2 for 4 hours. The fluorescent signal from the released Calcein-AM dye is then measured by a microplate reader at Ex 488 / Em 530. Increased Calcein release (measured as increased fluorescent signal in the sample) indicates more efficiency in killing target cells.

[0396] Results. Increased fluorescence (indicating more efficient killing) was observed in target cell samples incubated with αCD37 / Fc MBs compared to the fluorescence observed in target cell samples incubated with control MBs ( Figure 7 These results indicate that all tested αCD37 / Fc MBs induced potent tumor cell ADCC.

[0397] No increase in fluorescence was observed in target cell samples incubated with control α-RSV MBs containing an active wild-type Fc. (See Figure 7 These results indicate that MBs do not induce nonspecific activation of ADCC in the absence of target.

[0398] Table 9 shows the calculated EC values ​​for ADCC activity induced by αCD37 / Fc MB or by parental IgG. 50 value.

[0399] Table 9: Half maximal effective concentration (EC) of CD37 IgG and MB for ADCC of Ramos cells 50 )

[0400]

[0401] Example 11. Evaluation of in vitro C1q binding and complement-dependent cytotoxicity promoted by MBs engaging human complement

[0402] C1q binding was assessed by ELISA.

[0403] The αCD37 / Fc Mb (version 2) generated as described in Example 5 was evaluated for its ability to bind C1q as determined by ELISA. Parental IgG was used as a control.

[0404] Serial dilutions of test IgG and MB were coated onto 96-well plates overnight at 4°C. The next day, the plates were blocked with 3% bovine serum albumin (BSA) for 1 hour at room temperature. After washing out the blocking solution, 2 μg / ml recombinant human C1q (Complement Technology Inc.) was added to the plates and incubated at room temperature for 1 hour to allow binding to the coated test targets. C1q binding was then detected using a TMB / HRP reaction using an HRP-conjugated sheep-α-human C1q secondary antibody.

[0405] The binding of C1q protein to α-CD37 / FcMB and parental IgG was determined (see Figure 8 ).

[0406] Complement-dependent cytotoxicity (CDC) activity was assessed by CDC assay.

[0407] The ability of αCD37 / Fc MBs to induce complement-dependent cytotoxicity (CDC) was also assessed in an in vitro CDC assay. Parental IgG and control MBs (using α-respiratory syncytial virus (RSV Fab) were used as controls.

[0408] Daudi cells were used as target cells, and normal human serum (Complement Technology) was the source of human complement proteins. Target cells were incubated with a serial dilution of test IgG / MB and normal human serum (final concentration of 20%) in a 96-well U-bottom plate at 37°C, 5% CO2 for 2 hours. After the incubation period, flow cytometry was performed to measure the viability of the target cells. The cells were washed and stained with propidium iodide (PI) viability dye on ice for 10 minutes. PI fluorescence signals were measured on a CytoFLEX flow cytometer (Beckman Coulter). FlowJo software was used for data analysis. The PI+ gate was determined based on FMO and untreated controls, and the PI+ cell % was calculated based on the % of individual cells present within the PI+ gate. PI is a fluorescent DNA-binding dye that can freely penetrate the cell membrane of dead or dying cells (but excluded from living cells). Therefore, an increased PI fluorescence signal in the sample indicates a more effective killing of target cells.

[0409] Results. Increased PI fluorescence signal (indicating more efficient killing) was observed in target cell samples incubated with αCD37 / Fc MBs compared to the PI fluorescence signal observed in target cell samples incubated with parental IgG, or compared to control MBs ( Figure 10 These results indicate that all tested α-CD37 / Fc MBs induced more potent CDC activity compared to parental IgG or control MBs.

[0410] No increase in PI fluorescence was observed in target cell samples incubated with control MBs containing active wild-type Fc. Figure 10 These results indicate that MBs do not induce nonspecific activation of CDC in the absence of target.

[0411] Table 10 shows the calculated EC values ​​for CDC activity induced by α-CD37 / Fc MB or by parental IgG. 50 value.

[0412] Table 10: Half maximal effective concentration (EC) of CD37 IgG and MB for CDC of Daudi cells 50 )

[0413]

[0414] Example 12. Evaluation of in vitro antibody-dependent cellular phagocytosis promoted by macrophage-engaged MBs

[0415] The ability of αCD37 / Fc MBs (Format 2) generated as described in Example 5 to induce antibody-dependent cellular phagocytosis (ADCP) was assessed in an in vitro ADCP assay. Parental IgG or control MBs (using α-RSV Fab) were used as controls.

[0416] ADCP assay. Using Ramos cells as target cells and THP-1 cells as effector cells, the ADCP activity of IgG and MB was determined by ADCP assay. Target cells were labeled with pHrodo Green AM intracellular pH indicator dye (Invitrogen), and effector cells were labeled with CellTrace Violet dye. After washing the labeled cells, the assay plate was assembled by combining the labeled target cells, labeled effector cells, and serial dilutions of the test IgG / MB into a 96-well U-bottom plate. The samples were incubated at 37°C, 5% CO2 for 1 h. After incubation, the samples were stained with L / D far-red viability dye on ice for 15 minutes before analysis on a CytoFLEX flow cytometer (Beckman Coulter). Data analysis was performed using FlowJo software, and pHrodo Green AM and CellTrace Violet+ / - gates were determined based on FMO and untreated controls. ADCP activity was assessed based on the percentage of pHrodo Green AM / CellTrace Violet double-positive cells in all living single cells.

[0417] Results. A higher percentage of pHrodo Green AM / CellTrace Violet double-positive cells (among all live single cells) was observed in target cell samples incubated with αCD37 / Fc MBs compared to the pHrodo Green AM / CellTrace Violet double-positive cells observed in target cell samples incubated with control MBs ( Figure 11 These results indicate that all tested αCD37 / Fc MBs induced potent tumor cell ADCP compared to control α-respiratory syncytial virus (RSV) MBs.

[0418] In target cell samples incubated with control MBs containing active wild-type Fc, no increase in the percentage of pHrodo Green AM / CellTrace Violet double-positive cells was observed among all live single cells ( Figure 11 These results indicate that MBs do not induce nonspecific activation of ADCP in the absence of target.

[0419] Table 11 shows the calculated EC values ​​for ADCP activity induced by αCD37 / Fc MB or by parental IgG. 50 value.

[0420] Table 11: Half maximal effective concentration (EC) of ADCP of CD37 IgG and MB on Ramos cells 50 )

[0421]

[0422] Example 13. Evaluation of bispecific MB formats

[0423] Generation of MB.

[0424] This example describes the generation of bispecific MBs that can target both the CD37 tumor antigen and the NKp46 NK cell activating receptor. The binding properties, ADCC activity, and apoptotic activity of these bispecific MBs were also evaluated compared to control CD37 monospecific MBs.

[0425] Monospecific format: αCD37 / Fc MB (Format 2) was generated as described in Example 5. (See Figure 12A ).

[0426] Bispecific format: Similar to that described in Example 1, bispecific MBs containing CD37 Fab, NKp46 Fab, and wild-type IgG1 Fc were generated in the following format. (See Figure 12B ).

[0427] (1) scFab of CD37 antibody, which is expressed via (GGGS) n A linker fused to the N-terminus of the human ferritin light chain; and

[0428] (2) Human ferritin light chain fused to:

[0429] -scFab of NKp46 antibody (via (GGGS) n Linker fused to the N-terminus of human ferritin light chain)

[0430] -scFc(wt IgG1)(via (GGGS) n The linker was fused to the C-terminus of the human ferritin light chain).

[0431] The A-CD37 Fab and α-NKp46 Fab listed in Table 12 were used for the bispecific CD37 / NKp46 MB.

[0432] Table 12: A-CD37 and A-NKp46 Fab used in bispecific MB format

[0433]

[0434] In vitro binding of CD37 / NKp46 MB to recombinant human NKp46 was assessed by biolayer interferometry.

[0435] The binding kinetics and affinity of MBs to recombinant human NKp46 (hNKp46) were determined by biolayer interferometry (BLI) using an Octet RED96 instrument. Ni-NTA biosensors were coated with hNKp46-His (the extracellular domain of hNKp46 with a C-terminal polyhistidine tag) to achieve a signal response of 0.8 nm. The coated biosensors were immersed in wells containing serial dilutions of the test MBs (20-10-5-2.5-1.25-0.63 nM) in PBS-0.02% T-0.01% BSA (PBS supplemented with 0.02% (v / v) Tween 20 and 0.01% (w / v) BSA) for 180 seconds (association phase), followed by immersion in PBS-0.02% T-0.01% BSA for 180 seconds (dissociation phase). All measurements were performed at 30°C in PBS-0.02% T-0.01% BSA, pH 7.4, with shaking at 1000 rpm and real-time monitoring. The biosensor was regenerated between experiments by applying 10 mM glycine, pH 1.7, for 5 seconds four times, followed by replenishing with 10 mM NiSO4 for 1 minute. Target binding was based on the maximum associative binding response at the end of the association phase, the dissociation rate (k off ) and / or the equilibrium dissociation constant (K) calculated using a 1:1 fitting model. D When testing 20 nM of the multivalent, maximal associative binding responses less than 0.1 nM were classified as “non-binding”.

[0436] All CD37 / NKp46 bispecific MBs exhibited binding in the picomolar or sub-picomolar range (see Table 13).

[0437] Table 13: K of CD37 / NKp46 bispecific MBs D 、k on and k dis value

[0438]

[0439] In vitro evaluation of antibody-dependent cellular cytotoxicity promoted by natural killer (NK) cell-engaging bispecific MBs.

[0440] The ability of CD37 / NKp46 bispecific MBs containing wild-type IgG1 Fc to induce antibody-dependent cellular cytotoxicity (ADCC) was assessed in an in vitro ADCC assay similar to that described in Example 10, in which Ramos cells were used as target cells and primary human NK cells (STEMCELL Technologies) were used as effector cells.

[0441] Increased fluorescence (indicating more efficient target cell killing) was observed in target cell samples incubated with CD37 / NKp46 bispecific MBs compared to the fluorescence observed in target cell samples incubated with monospecific CD37 MBs ( Figures 13A-13C These results indicate that all tested CD37 / NKp46 bispecific MBs induced potent tumor cell ADCC and enhanced NK-mediated tumor cell killing relative to tumor cell ADCC and NK-mediated tumor cell killing induced by monospecific CD37 MBs.

[0442] Table 14 shows the calculated EC values ​​for ADCC activity induced by monospecific CD37 MB or CD37 / NKp46 bispecific MB. 50 value.

[0443] Table 14: Half maximal effective concentration (EC) of monospecific and bispecific MBs for ADCC of Ramos cells 50 )

[0444]

[0445] Evaluation of in vitro tumor cell apoptosis induced by NK cell-engaged bispecific MBs that aggregate the CD37 receptor

[0446] An assay similar to that described in Example 9 was used to assess the ability of CD37 / NKp46 bispecific MBs to induce apoptosis in CD37+ tumor cells via CD37 receptor aggregation, with Daudi cells as target cells and Annexin-V staining used to identify apoptotic cells.

[0447] CD37 / NKp46 bispecific MBs showed comparable apoptotic activity to CD37 monospecific MBs (see Figures 14A-14C ).

[0448] Table 15 shows the calculated EC values ​​of apoptotic activity induced by CD37 monospecific MBs or CD37 / NKp47 bispecific MBs. 50 value.

[0449] Table 15: Half maximal effective concentration (EC) of monospecific and bispecific MBs on apoptosis of Daudi cells 50 )

[0450]

[0451] Sequence Listing

[0452] Underlined within the fusion sequence indicates the linker sequence.

[0453] Bold within the fusion sequence indicates ferritin or ferritin subunit sequences.

[0454] Within the variable region sequences, underlining and bold together indicate complementarity determining region sequences.

[0455] Boxed and bolded residues represent residues relative to a reference molecule, e.g., relative to an IgG1 Fc mutant.

[0456] SEQ ID NO: 1 hFTL

[0457] MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD

[0458] SEQ ID NO: 2N_hFTL

[0459] MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEW

[0460] SEQ ID NO: 3C_hFTL

[0461] GKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD

[0462] SEQ ID NO: 4IgG1 Fc

[0463] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0464] SEQ ID NO: 5IgG1 scFc

[0465]

[0466] SEQ ID NO: 6 Example GS linker

[0467] GGGS

[0468] SEQ ID NO: 7 Example GS linker

[0469] GGGGS

[0470] SEQ ID NOs: 8-425, as shown in Tables 1A-8D.

[0471] SEQ ID NO: 426 Human IgG4 heavy chain constant region sequence

[0472] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0473] SEQ ID NO: 427 Human IgG1 heavy chain constant region sequence

[0474] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0475] SEQ ID NO: 428 Human IgG2 heavy chain constant region sequence

[0476] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0477] SEQ ID NO: 429 Human IgG3 heavy chain constant region sequence

[0478] ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

[0479] SEQ ID NO: 430 Human Igκ light chain constant region sequence

[0480] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0481] SEQ ID NO: 431 Human Igλ1 light chain constant region sequence

[0482] GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0483] SEQ ID NO: 432 Human Igλ2 light chain constant region sequence [[ID=id=17]]

[0484] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0485] SEQ ID NO: 433 Human Igλ3 light chain constant region sequence

[0486] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS

[0487] SEQ ID NO: 434 Human Igλ6 light chain constant region sequence

[0488] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVKVAWKADGSPVNTGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS

[0489] SEQ ID NO: 435 Human Igλ7 light chain constant region sequence

[0490] GQPKAAPSVTLFPPSSEELQANKATLVCLVSDFNPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAECS

[0491] Equivalents / Other Implementations

[0492] 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 in accordance with the principles of the invention in general and including departures from the present disclosure that come within known or customary practice 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 hematopoietic cell binding portion and (2) a nanocage monomer or a subunit thereof.

2. The fusion polypeptide of claim 1, wherein the hematopoietic cell 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 ).

4. 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 1-5, comprising a lymphocyte binding portion.

7. The fusion polypeptide of claim 6, wherein the lymphocyte binding portion is a natural killer cell binding portion or a T cell binding portion.

8. The fusion polypeptide of claim 7, wherein the lymphocyte binding portion is a natural killer cell binding portion.

9. The fusion polypeptide of claim 8, wherein the natural killer cell binding portion is a CD16a binding portion, a NKp46 binding portion, or a NKG2D binding portion.

10. The fusion polypeptide of claim 9, wherein the natural killer cell 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 antibody have sequences that each differ from the CDRs of the CD16a, NKp46 or NKG2D antibodies by a maximum of two amino acids.

11. The fusion polypeptide of claim 10, wherein in addition to one or two amino acid substitutions in all six CDRs, the V H and the V L or V K The CDRs of the antibody have sequences identical to those of the heavy and light chain CDRs of CD16a, NKp46 or NKG2D antibodies.

12. The fusion polypeptide of claim 11, wherein the V H and the V L or V K The CDRs of the antibody are identical to the heavy and light chain CDRs of the CD16a, NKp46 or NKG2D antibody.

13. The fusion polypeptide of claim 9, wherein the natural killer cell 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 CD16a, NKp46, or NKG2D antibody.

14. The fusion polypeptide of claim 7, wherein the lymphocyte binding portion is a T cell binding portion.

15. The fusion polypeptide of claim 14, wherein the T cell binding portion is a CD3 binding portion.

16. The fusion polypeptide of claim 15, wherein the T cell binding portion comprises a heavy chain variable region (V H ) and light chain variable region (V L or V K ), wherein 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 CD3 antibody have sequences that each differ from the CDRs of the CD3 antibody by a maximum of two amino acids.

17. The fusion polypeptide of claim 16, wherein in addition to one or two amino acid substitutions in all six CDRs, 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 CD3 antibody.

18. The fusion polypeptide of claim 17, wherein the V H and the V L or V K The CDRs are identical to the heavy and light chain CDRs of the CD3 antibody.

19. The fusion polypeptide of claim 15, wherein the T cell 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 the CD3 antibody.

20. The fusion polypeptide of any one of claims 1-5, comprising a myeloid cell binding portion. The fusion polypeptide of claim 20 , wherein the myeloid cell binding moiety is a SIRPα binding moiety.

22. The fusion polypeptide of claim 21, wherein the myeloid cell binding portion comprises a heavy chain variable region (V H ) and light chain variable region (V L or V K ), wherein 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 SIRPα have sequences that each differ from the CDRs of the SIRPα antibody by a maximum of two amino acids.

23. The fusion polypeptide of claim 22, wherein in addition to having one or two amino acid substitutions in all six CDRs, 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 SIRPα antibody.

24. The fusion polypeptide of claim 23, wherein the V H and the V L or V K The CDRs of the SIRPα antibody are identical to the heavy and light chain CDRs of the SIRPα antibody.

25. The fusion polypeptide of claim 21, wherein the natural killer cell 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 SIRPα antibody.

26. The fusion polypeptide of any one of claims 1-25, wherein the nanocage monomer is a ferritin monomer or a subunit thereof.

27. The fusion protein of claim 26, wherein the ferritin monomer is a human ferritin monomer.

28. The fusion protein of claim 26 or 27, wherein the ferritin monomer is a ferritin light chain.

29. The fusion polypeptide of any one of claims 1-28, wherein the hematopoietic cell binding moiety is linked to the nanocage monomer or subunit thereof via an amino acid linker.

30. The fusion polypeptide of any one of claims 1-29, wherein the hematopoietic cell binding moiety is attached via the N-terminus of the nanocage monomer or subunit thereof.

31. A self-assembling polypeptide complex, comprising: (a) a plurality of hematopoietic cell-binding fusion polypeptides, each hematopoietic cell-binding fusion polypeptide being a fusion polypeptide according to any one of claims 1 to 30; 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.

32. The self-assembling polypeptide complex of claim 31, further comprising (c) 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.

33. The self-assembling polypeptide complex according to claim 31 or 32, wherein In each Fc fusion polypeptide, the Fc polypeptide is linked to the nanocage monomer or subunit thereof via an amino acid linker.

34. The self-assembling polypeptide complex of claim 33, wherein the Fc polypeptide is linked via the N-terminus of the nanocage monomer or subunit thereof.

35. The self-assembling polypeptide complex of claim 33, wherein the Fc polypeptide is linked via the C-terminus of the nanocage monomer or a subunit thereof.

36. The self-assembling polypeptide complex according to any one of claims 32 to 36, 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.

37. The self-assembling polypeptide complex of claim 36, wherein the tumor-binding moiety is attached via the N-terminus of the nanocage monomer or its subunit.

38. A self-assembling polypeptide complex, comprising: (a) a plurality of hematopoietic cell-binding fusion polypeptides, each hematopoietic cell-binding fusion polypeptide being a fusion polypeptide according to any one of claims 1 to 30; and (b) a plurality of Fc fusion polypeptides, each Fc fusion polypeptide comprising (1) an Fc polypeptide linked to the C-terminus of (2) a nanocage monomer or a subunit thereof via an amino acid linker.

39. A self-assembling polypeptide complex, comprising: (a) a plurality of tumor-binding fusion polypeptides, each comprising (1) a tumor-binding moiety linked to (2) a nanocage monomer or a subunit thereof via an amino acid linker; and (b) a plurality of Fc fusion polypeptides, each Fc fusion polypeptide comprising (1) an Fc polypeptide linked to the C-terminus of (2) a nanocage monomer or a subunit thereof via an amino acid linker.

40. A self-assembling polypeptide complex, comprising: (a) a plurality of hematopoietic cell-binding fusion polypeptides, each hematopoietic cell-binding fusion polypeptide being a fusion polypeptide according to any one of claims 1 to 30; and (b) a plurality of tumor-binding fusion polypeptides, each comprising (1) a tumor-binding moiety linked to (2) a nanocage monomer or a subunit thereof via an amino acid linker; and (c) a plurality of Fc fusion polypeptides, each Fc fusion polypeptide comprising (1) an Fc polypeptide linked to the C-terminus of (2) a nanocage monomer or a subunit thereof.

41. The self-assembling polypeptide complex of any one of claims 39-41, wherein the Fc polypeptide is linked to the C-terminus of the C-half nanocage monomer.

42. The self-assembling polypeptide complex of claim 41, wherein the Fc polypeptide is linked to the C-terminus of C-halferritin.

43. The self-assembling polypeptide complex of any one of claims 32-37 or 39-43, wherein the tumor-binding moiety comprises an antibody or an antigen-binding fragment thereof.

44. The self-assembling polypeptide complex of claim 43, 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 ).

45. The self-assembling polypeptide complex of claim 44, wherein the antibody or antigen-binding fragment thereof comprises a Fab fragment.

46. ​​The self-assembling polypeptide complex of claim 45, wherein the Fab fragment is a single-chain Fab fragment (scFab).

47. The self-assembling polypeptide complex of any one of claims 43-46, wherein the tumor binding moiety is a CD20 binding moiety or a CD37 binding moiety.

48. The self-assembling polypeptide complex of claim 47, wherein the tumor binding portion comprises a heavy chain variable region (V H ) and light chain variable region (V L or V K ), wherein 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 CD20 or CD37 antibody have sequences that each differ from the CDRs of the CD20 or CD37 antibody by a maximum of two amino acids.

49. The self-assembling polypeptide complex of claim 48, wherein in addition to one or two amino acid substitutions in all six CDRs, 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 CD20 or CD37 antibodies.

50. The self-assembling polypeptide complex of claim 49, wherein said V H and the V L or V K The CDRs are identical to the heavy and light chain CDRs of the CD20 or CD37 antibody.

51. The self-assembling polypeptide complex of claim 47, wherein the natural killer cell 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 CD20 or CD37 antibody.

52. The self-assembling polypeptide complex of any one of claims 31-51, wherein the nanocage monomers within the Fc fusion polypeptide and within the hematopoietic cell-binding fusion polypeptide and / or the tumor-binding fusion polypeptide are each ferritin monomers or subunits thereof.

53. The self-assembling polypeptide complex of claim 52, wherein the ferritin monomer is a human ferritin monomer.

54. The self-assembling polypeptide complex of claim 52 or 53, wherein the ferritin monomer is a ferritin light chain.

55. The self-assembling polypeptide complex of claim 54, which does not comprise any ferritin heavy chain or subunit of a ferritin heavy chain.

56. The self-assembling polypeptide complex of any one of claims 52-55, which does not comprise any iron-binding moiety.

57. A pharmaceutical composition comprising the self-assembling polypeptide complex according to any one of claims 31 to 56 and a pharmaceutically acceptable excipient.

58. A method for treating, ameliorating or preventing a disease or condition, the method comprising administering to a subject the self-assembling polypeptide complex of any one of claims 31 to 56 or the pharmaceutical composition of claim 57.

59. The method of claim 58, wherein the subject is a mammal.

60. The method of claim 59, wherein the subject is a human.

61. The method of claim 60, wherein the disease or condition is cancer.

62. The method of claim 61, wherein the cancer is B-cell lymphoma.

63. A fusion polypeptide comprising a continuous full-length ferritin light chain having an N-terminus and a C-terminus, and (a) an antibody fragment fused to the N-terminus via a first amino acid linker and an Fc polypeptide fused to the C-terminus via a second amino acid linker; or (b) an antibody fragment fused to the C-terminus via a first amino acid linker and an Fc polypeptide fused to the N-terminus via a second amino acid linker.

64. The fusion polypeptide of claim 63, wherein the antibody fragment is fused to the N-terminus via a first amino acid linker and an Fc polypeptide is fused to the C-terminus via a second amino acid linker.

65. The fusion polypeptide of claim 63 or 64, wherein the antibody fragment comprises an immunoglobulin heavy chain variable region (V H ) and immunoglobulin light chain variable region (V L or V K ).

66. The fusion polypeptide of claim 65, wherein the antibody or antigen-binding fragment thereof comprises a Fab fragment.

67. The fusion polypeptide of claim 66, wherein the Fab fragment is a single-chain Fab fragment (scFab).

68. The fusion polypeptide of any one of claims 63-67, wherein the antibody fragment is capable of binding to a tumor-associated antigen.

69. A self-assembling polypeptide complex comprising the fusion polypeptide of any one of claims 63-68.

70. A pharmaceutical composition comprising the self-assembling polypeptide complex of claim 69 and a pharmaceutically acceptable excipient.

71. A method for treating, ameliorating or preventing a disease or condition, the method comprising administering the self-assembling polypeptide complex of claim 69 or the pharmaceutical composition of claim 70 to a subject.

72. A method comprising the step of contacting a tumor cell with the self-assembling polypeptide complex of any one of claims 32-37 and 39-56.

73. The method of claim 72, wherein the contacting step results in apoptosis of the tumor cells.

74. The method of claim 73, wherein the tumor cells express CD37 and the self-assembling polypeptide complex comprises a fusion protein comprising α-CD37 Fab.

75. The method of claim 73 or 74, wherein the self-assembling polypeptide complex has an EC of less than 1 nM. 50 Induces apoptosis.

76. The method of any one of claims 72-75, wherein the contacting step results in antibody-dependent cellular cytotoxicity (ADCC) of the tumor cells.

77. The method of claim 76, wherein the self-assembling polypeptide complex has an EC of less than 1 nM. 50 Values ​​induce ADCC.

78. The method of any one of claims 72-77, wherein the contacting step is performed in the presence of a complement protein and results in complement dependent cytotoxicity (CDC) of the tumor cells.

79. The method of claim 78, wherein the self-assembling polypeptide complex has an EC of less than 0.4 nM. 50 Value induced CDC.

80. The method of any one of claims 72-79, wherein the contacting step results in antibody-dependent cellular phagocytosis (ADCP) of the tumor cells.

81. The method of claim 80, wherein the self-assembling polypeptide complex has an EC of less than 0.1 nM. 50 Values ​​for inducing ADCP.

82. The method of any one of claims 72-80, wherein the contacting step occurs in a mammal having a tumor.

83. The method of claim 82, wherein the mammal is a human.

84. The self-assembling polypeptide complex of any one of claims 32-37 and 39-56, wherein when contacted with a tumor cell, the self-assembling polypeptide complex is capable of inducing an effect selected from the group consisting of: (a) Apoptosis of the tumor cells, EC 50 Values ​​less than 1 nM; (b) ADCC, EC of the tumor cells 50 Values ​​less than 1 nM; (c) CDC of the tumor cells, when the self-assembling polypeptide complex contacts the tumor cells in the presence of complement, EC 50 values ​​less than 0.4 nM; (d)ADCP, EC 50 Values ​​less than 0.1 nM; and (e) any combination of the foregoing.

85. Use of the self-assembling polypeptide complex of any one of claims 31-56 or 69 or the pharmaceutical composition of claim 57 or 70 for treating, ameliorating or preventing a disease or condition in a subject.

86. The use of claim 85, wherein the subject is a mammal.

87. The use of claim 86, wherein the subject is a human.

88. The use of any one of claims 85-87, wherein the disease or condition is cancer.

89. The use of claim 88, wherein the cancer is B-cell lymphoma.

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