Formulation of DR5-binding polypeptides

Abstract

Formulations of DR5 binding polypeptides are provided herein. Uses of the DR5 binding polypeptides are also provided.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 151,131, filed February 19, 2021, which is incorporated by reference in its entirety for all purposes.

[0002] The present invention relates to formulations of DR5 binding proteins and methods of using the formulations, including, but not limited to, methods for treating cancer. [Background technology]

[0003] DR5 is a member of the TNF receptor superfamily (TNFRSF) and is a cell surface receptor of the TNF receptor superfamily that binds to TNF-related apoptosis-inducing ligand (TRAIL). TRAIL has evolved to play an important role in mammalian development and host defense by selectively eliminating unwanted, infected, and malignant cells from healthy cell populations. TRAIL induces cell death by caspase-dependent apoptosis upon binding to TNF receptor family members DR4 or DR5. DR5 appears to be the primary receptor on tumor cells that drives the tumor-biasing activity observed in the TRAIL pathway. DR5 is activated by the natural ligand TRAIL, which brings three DR5 receptors into close proximity, activating intracellular caspase-8 and initiating the activation of other cell death-inducing caspases such as caspase-9 and caspase-3. Thus, initiation of this cell death pathway requires clustering of DR5 receptors for efficient cell death. Summary of the Invention

[0004] Provided herein are stable liquid and lyophilized pharmaceutical formulations of multivalent DR5 binding polypeptides capable of agonizing DR5 signaling to mediate direct cell death, and uses of the pharmaceutical formulations for the treatment of cancer. [Means for solving the problem]

[0005] Embodiment 1. A pharmaceutical formulation comprising a DR5-binding polypeptide, the formulation comprising 20 mg / mL to 70 mg / mL of the DR5-binding polypeptide, 5 mM to 20 mM histidine, 7% to 10% (w / v) sucrose and 0.1% to 0.8% poloxamer P188, and having a pH of 5.3 to 6.7, the DR5-binding polypeptide comprising at least one VHH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:1, a CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a CDR3 comprising the amino acid sequence of SEQ ID NO:3. Embodiment 2. The pharmaceutical formulation of embodiment 1, comprising 30 mg / mL to 60 mg / mL of the DR5 binding polypeptide. Embodiment 3. The pharmaceutical formulation of embodiment 1, comprising 50 mg / mL of a DR5 binding polypeptide. Embodiment 4. The pharmaceutical formulation of any one of embodiments 1 to 3, comprising 7 mM to 15 mM histidine. Embodiment 5. The pharmaceutical formulation of any one of embodiments 1 to 3, comprising 10 mM histidine. Embodiment 6 The pharmaceutical formulation of any one of embodiments 1-5, wherein the histidine is histidine HCl. Embodiment 7. The pharmaceutical formulation of any one of embodiments 1-6, comprising 8%-9% sucrose. Embodiment 8. The pharmaceutical formulation of any one of embodiments 1-7, comprising 8% sucrose or 9% sucrose. Embodiment 9. The pharmaceutical formulation of any one of embodiments 1 to 8, comprising 0.2% to 0.4% poloxamer P188. Embodiment 10. The pharmaceutical formulation of any one of embodiments 1 to 9, comprising 0.2% poloxamer P188. Embodiment 11. The pharmaceutical formulation of any one of embodiments 1 to 10, comprising 1 mM to 10 mM, 2 mM to 8 mM, 3 mM to 7 mM, or 4 mM to 6 mM methionine. Embodiment 12. The pharmaceutical formulation of any one of embodiments 1 to 11, comprising 5 mM methionine. Embodiment 13. The pharmaceutical formulation of any one of embodiments 1 to 12, wherein the pH of the formulation is 5.4 to 6.6, 5.5 to 6.5, 5.6 to 6.4, 5.7 to 6.3, or 5.8 to 6.2. Embodiment 14 The pharmaceutical formulation of any one of embodiments 1 to 13, wherein the pH of the formulation is about 6. Embodiment 15. The pharmaceutical formulation of any one of embodiments 1-14, wherein the formulation comprises 50 mg / mL of DR5 binding polypeptide, 10 mM histidine HCl, 8% sucrose and 0.2% poloxamer P188, and the pH of the formulation is about 6. Embodiment 16. The pharmaceutical formulation of embodiment 15, wherein the formulation consists essentially of 50 mg / mL DR5 binding polypeptide, 10 mM histidine HCl, 8% sucrose, 0.2% poloxamer P188 and water, and wherein the pH of the formulation is about 6. Embodiment 17. The pharmaceutical formulation of any one of embodiments 1 to 16, wherein the DR5 binding polypeptide comprises a VHH domain comprising the amino acid sequence of SEQ ID NO:4. Embodiment 18 The pharmaceutical formulation of any one of embodiments 1 to 17, wherein the DR5 binding polypeptide comprises an Fc region. Embodiment 19 The pharmaceutical formulation of embodiment 18, wherein the Fc region comprises the amino acid sequence of SEQ ID NO:6. Embodiment 20. The pharmaceutical formulation of any one of embodiments 1 to 19, wherein the DR5 binding polypeptide has the structure VHH-linker-VHH-linker-Fc. Embodiment 21. The pharmaceutical formulation of embodiment 20, wherein the VHH-linker-VHH comprises the amino acid sequence of SEQ ID NO:5. Embodiment 22 The pharmaceutical formulation of any one of embodiments 1 to 21, wherein the DR5 binding polypeptide comprises the amino acid sequence of SEQ ID NO:7. Embodiment 23. The pharmaceutical formulation of any one of embodiments 1 to 21, wherein the DR5 binding polypeptide consists of the amino acid sequence of SEQ ID NO:7. Embodiment 24. A lyophilized formulation comprising a DR5 binding polypeptide, the DR5 binding polypeptide comprising at least one VHH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3, the lyophilized formulation being reconstituted in water to form an aqueous formulation, the aqueous formulation comprising 20 mg / mL to 70 mg / mL of the DR5 binding polypeptide, 5 mM to 20 mM histidine, 7% to 10% sucrose, and 0.1% to 0.5% poloxamer P188, and having a pH of 5.3 to 6.7. Embodiment 25. The lyophilized formulation of embodiment 24, wherein when the formulation is reconstituted in water to form an aqueous formulation, the aqueous formulation comprises between 30 mg / mL and 60 mg / mL of the DR5 binding polypeptide. Embodiment 26 The lyophilized formulation of embodiment 24, wherein upon reconstitution of the formulation in water to form an aqueous formulation, the aqueous formulation comprises 50 mg / mL of the DR5 binding polypeptide. Embodiment 27. The lyophilized formulation of any one of embodiments 24-26, wherein upon reconstitution of the formulation in water to form an aqueous formulation, the aqueous formulation comprises between 7 mM and 15 mM histidine. Embodiment 28. The lyophilized formulation of any one of embodiments 24-26, wherein upon reconstitution of the formulation in water to form an aqueous formulation, the aqueous formulation comprises 10 mM histidine. Embodiment 29. The lyophilized formulation of any one of embodiments 24 to 28, wherein the histidine is histidine HCl. Embodiment 30. The lyophilized formulation of any one of embodiments 24 to 29, wherein when the lyophilized formulation is reconstituted in water to form an aqueous formulation, the aqueous formulation comprises 8% to 9% sucrose. Embodiment 31. The lyophilized formulation of any one of embodiments 24-30, wherein upon reconstitution of the formulation in water to form an aqueous formulation, the aqueous formulation comprises 8% sucrose or 9% sucrose. Embodiment 32. The lyophilized formulation of any one of embodiments 24 to 31, wherein when the formulation is reconstituted in water to form an aqueous formulation, the aqueous formulation comprises 0.2% to 0.4% poloxamer P188. Embodiment 33. The lyophilized formulation of any one of embodiments 24 to 32, wherein upon reconstitution of the formulation in water to form an aqueous formulation, the aqueous formulation comprises 0.2% poloxamer P188. Embodiment 34. The lyophilized formulation of any one of embodiments 24 to 33, wherein upon reconstitution of the formulation in water to form an aqueous formulation, the aqueous formulation comprises between 1 mM and 10 mM, between 2 mM and 8 mM, between 3 mM and 7 mM, or between 4 mM and 6 mM methionine. Embodiment 35. The lyophilized formulation of any one of embodiments 24-33, wherein upon reconstitution of the formulation in water to form an aqueous formulation, the aqueous formulation comprises 5 mM methionine. Embodiment 36. The lyophilized formulation of any one of embodiments 24 to 35, wherein when the formulation is reconstituted in water to form an aqueous formulation, the aqueous formulation has a pH of 5.4 to 6.6, 5.5 to 6.5, 5.6 to 6.4, 5.7 to 6.3, or 5.8 to 6.2. Embodiment 37. The lyophilized formulation of any one of embodiments 24-35, wherein upon reconstitution of the formulation in water to form an aqueous formulation, the aqueous formulation has a pH of about 6. Embodiment 38. The lyophilized formulation of any one of embodiments 24 to 37, wherein when the formulation is reconstituted in water to form an aqueous formulation, the aqueous formulation comprises 50 mg / mL of the DR5 binding polypeptide, 10 mM histidine HCl, 8% sucrose, and 0.2% poloxamer P188, and has a pH of about 6. Embodiment 39. The lyophilized formulation of embodiment 28, wherein when the formulation is reconstituted in water to form an aqueous formulation, the aqueous formulation consists essentially of 50 mg / mL of the DR5 binding polypeptide, 10 mM histidine HCl, 8% sucrose, 0.2% poloxamer P188 and water, and wherein the pH of the formulation is about 6. Embodiment 40. The lyophilized formulation of any one of embodiments 24 to 39, wherein the DR5 binding polypeptide comprises a VHH domain comprising the amino acid sequence of SEQ ID NO:4. Embodiment 41 The lyophilized formulation of any one of embodiments 24 to 40, wherein the DR5 binding polypeptide comprises an Fc region. Embodiment 42 The lyophilized formulation of embodiment 41, wherein the Fc region comprises the amino acid sequence of SEQ ID NO:6. Embodiment 43. The lyophilized formulation of any one of embodiments 24 to 42, wherein the DR5 binding polypeptide has the structure VHH-linker-VHH-linker-Fc. Embodiment 44. The lyophilized formulation of embodiment 43, wherein the VHH-linker-VHH comprises the amino acid sequence of SEQ ID NO:5. Embodiment 45. The lyophilized formulation of any one of embodiments 24 to 44, wherein the DR5 binding polypeptide comprises the amino acid sequence of SEQ ID NO:7. Embodiment 46 The lyophilized formulation of any one of embodiments 24 to 44, wherein the DR5 binding polypeptide consists of the amino acid sequence of SEQ ID NO:7. Embodiment 47. A lyophilized formulation formed by lyophilizing the pharmaceutical formulation of any one of embodiments 1-23. Embodiment 48. The freeze-dried formulation of any one of embodiments 24 to 47, wherein after storage at 2°C to 8°C for up to 3 months, up to 6 months, up to 9 months, up to 12 months or more than 12 months, the freeze-dried formulation is an off-white, uniform, fine cake free of any visible impurities. Embodiment 49. The lyophilized formulation of any one of embodiments 24 to 48, wherein after storage at 2°C to 8°C for up to 3 months, up to 6 months, up to 9 months, up to 12 months, or more than 12 months, the aqueous formulation is substantially free of visible particles when the formulation is reconstituted in water to form an aqueous formulation. Embodiment 50. The lyophilized formulation of any one of embodiments 24-49, wherein following storage at 2°C to 8°C for up to 3 months, up to 6 months, up to 9 months, up to 12 months or more than 12 months, when the formulation is reconstituted in water to form an aqueous formulation, less than 3% or less than 2% of the DR5 binding polypeptide present in the aqueous formulation is aggregated as measured by size exclusion chromatography. Embodiment 51. The lyophilized formulation of embodiment 50, wherein less than 1% or less than 0.5% of the DR5 binding polypeptide present in the aqueous formulation is degraded as measured by size exclusion chromatography. Embodiment 52. A pharmaceutical formulation formed by reconstituting the lyophilized formulation of any one of embodiments 24-51. Embodiment 53. A method of treating cancer, comprising administering to a subject having cancer the pharmaceutical formulation of any one of embodiments 1-23 and 52. Embodiment 54. The method of embodiment 53, wherein the cancer is chondrosarcoma, mesothelioma, Ewing's sarcoma, colorectal cancer, or pancreatic adenocarcinoma. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] The embodiments presented herein relate to formulations of DR5 binding polypeptides and their use in various methods of treating, for example, cancer.

[0007] Definitions and Various Embodiments The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0008] All references cited in this specification, including patent applications, patent publications, and Genbank accession numbers, are incorporated herein by reference to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference in its entirety.

[0009] The techniques and procedures described or referenced herein are generally well understood and generally described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel, et al. eds., (2003)), the series METHODS IN ENZYMOLOGY (Academic Press, Inc.), PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)), Oligonucleotide Synthesis (MJ Gait, ed., 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press, Animal Cell Culture (RI Freshney, ed., 1987), Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell eds., 1993-8) J. Wiley and Sons, Handbook of Experimental Immunology (DM Weir and CCBlackwell, eds.), Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987), PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994), Current Protocols in Immunology (JE Coligan et al., eds., 1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (CA Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989), Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using The antibodies are used using routine methodologies by those skilled in the art, such as the widely used methodologies described in Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995), and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993) and their latest editions.

[0010] Unless otherwise specified, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context or expressly indicated otherwise, singular terms shall include the plural and plural terms shall include the singular. In the event of discrepancies in definitions among various sources or references, the definitions set forth herein shall prevail.

[0011] Generally, the numbering of residues in an immunoglobulin heavy chain is that of the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0012] Embodiments of the invention described herein are understood to include "consisting of" and / or "consisting essentially of" embodiments. As used herein, the singular forms "a", "an", and "the" include plural references unless otherwise indicated. Use of the term "or" herein is not to be construed as implying that options are mutually exclusive.

[0013] In this application, the use of "or" means "and / or" unless expressly stated otherwise or understood by a person skilled in the art. In the context of a multiple dependent claim, the use of "or" refers to more than one previous independent or dependent claim.

[0014] The phrases "reference sample", "reference cell", or "reference tissue" refer to a sample with at least one known characteristic that can be used as a comparison with a sample with at least one unknown characteristic. In some embodiments, a reference sample can be used as a positive or negative indicator. A reference sample can be used to establish, for example, the level of protein and / or mRNA present in a healthy tissue relative to the level of protein and / or mRNA present in a sample with an unknown characteristic. In some embodiments, the reference sample is a sample from the same subject, but from a part of the subject different from the part being tested. In some embodiments, the reference sample is a sample from a tissue area surrounding or adjacent to the cancer. In some embodiments, the reference sample is not from the subject being tested, but from a subject known to have or not have a disorder of interest (e.g., a particular cancer or DR5-associated disorder). In some embodiments, the reference sample is from the same subject, but at a time before the subject developed cancer. In some embodiments, the reference sample is a sample from a benign cancer sample from the same subject or a different subject. When a negative reference sample is used for comparison, the expression level or amount of the molecule of interest in the negative reference sample indicates a level at which one skilled in the art would recognize, in view of the present disclosure, that the molecule is absent and / or at a low level.When a positive reference sample is used for comparison, the expression level or amount of the molecule of interest in the positive reference sample indicates a level at which one skilled in the art would recognize, in view of the present disclosure, that the molecule is present at a certain level.

[0015] The terms "benefit", "clinical benefit", "responsiveness" and "therapeutic response" as used herein in the context of benefiting from or responding to the administration of a therapeutic agent can be measured by assessing various endpoints, such as a degree of inhibition of disease progression, including slowing and complete halt, a reduction in the number of disease episodes and / or symptoms, a reduction in lesion size, an inhibition (i.e., reduction, slowing, or complete halt) of disease cell invasion into adjacent peripheral organs and / or tissues, an inhibition (i.e., reduction, slowing, or complete halt) of disease spread, a degree of relief of one or more symptoms associated with the disorder, a disease-free presentation after treatment, such as an increase in the length of progression-free survival, an increase in overall survival, a higher response rate, and / or a reduction in mortality at a given time point after treatment. A "non-responsive" or "non-responsive" subject or cancer is one that does not meet the above criteria of "responding".

[0016] The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably and may refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and / or non-natural nucleotides, including, but not limited to, DNA, RNA, and PNA. A "nucleic acid sequence" refers to the linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0017] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. This definition includes both full-length proteins and fragments thereof. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of this disclosure, "polypeptide" refers to proteins that contain modifications (generally conservative in nature), such as deletions, additions, and substitutions to the native sequence, so long as the protein maintains a desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as by mutations of the host that produces the protein or by errors due to PCR amplification.

[0018] As used herein, the terms "DR5", "death receptor 5" and "TNFRSF10B" refer to any naturally occurring mature DR5 resulting from processing of a DR5 precursor in a cell. The term includes DR5 from any vertebrate origin, including mammals such as primates (e.g., humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. The term also includes naturally occurring variants of DR5, such as splice variants or allelic variants. A non-limiting exemplary precursor human DR5 amino acid sequence is shown, for example, in NCBI Accession No. NP_003833.4. See SEQ ID NO: 8. A non-limiting exemplary precursor human DR5 amino acid sequence is shown, for example, in SEQ ID NO: 9.

[0019] The term "specifically binds" to an antigen or epitope is a term well understood in the art, and methods for determining such specific binding are also known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, with a longer duration and / or with a higher affinity with a particular cell or substance than it reacts or associates with another cell or substance. A single domain antibody (sdAb) or VHH-containing polypeptide "specifically binds" or "preferentially binds" to a target if it binds with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other substances. For example, an sdAb or VHH-containing polypeptide that specifically or preferentially binds to the DR5 epitope is an sdAb or VHH-containing polypeptide that binds this epitope with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other DR5 epitopes or non-DR5 epitopes. It is also understood by interpreting this definition that, for example, an sdAb or VHH-containing polypeptide that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding. "Specificity" refers to the ability of a binding protein to selectively bind to an antigen.

[0020] The term "inhibition" or "inhibiting" refers to a decrease or cessation of any phenotypic characteristic, or a decrease or cessation of the incidence, extent, or likelihood of that characteristic. "Reduce" or "inhibit" refers to a decrease, reduction, or cessation of an activity, function, and / or amount compared to a reference. In some embodiments, "reduce" or "inhibit" refers to the ability to cause an overall decrease of 10% or more. In some embodiments, "reduce" or "inhibit" refers to the ability to cause an overall decrease of 50% or more. In some embodiments, "reduce" or "inhibit" refers to the ability to cause an overall decrease of 75%, 85%, 90%, 95% or more. In some embodiments, the amount is inhibited or reduced over a period of time relative to a control over the same period of time.

[0021] As used herein, the term "epitope" refers to a site on a target molecule (e.g., an antigen such as a protein, nucleic acid, carbohydrate, or lipid) to which an antigen-binding molecule (e.g., an sdAb or VHH-containing polypeptide) binds. Epitopes often comprise chemically active surface arrangements of molecules such as amino acids, polypeptides, or sugar side chains, and have specific three-dimensional structural features and specific charge characteristics. Epitopes can be formed from both contiguous and / or juxtaposed non-contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) of a target molecule. Epitopes formed from contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. Epitopes can include, but are not limited to, at least 3, at least 5, or 8-10 residues (e.g., amino acids or nucleotides). In some embodiments, an epitope is less than 20 residues (e.g., amino acids or nucleotides), less than 15 residues, or less than 12 residues in length. Two antibodies may bind to the same epitope in an antigen if they exhibit competitive binding to the antigen. In some embodiments, an epitope may be specified by a certain minimum distance to the CDR residues on the antigen-binding molecule. In some embodiments, an epitope may be specified by the above distance and further limited to those residues involved in binding (e.g., hydrogen bonding) between the antigen-binding molecule residues and the antigen residues. An epitope may also be specified by a variety of scans. For example, an alanine scan or an arginine scan may indicate one or more residues with which an antigen-binding molecule may interact. Unless explicitly indicated, a set of residues as an epitope does not exclude other residues from being part of the epitope for a particular antigen-binding molecule. Rather, the existence of such a set indicates a minimum epitope sequence (or set of types). Thus, in some embodiments, the set of residues identified as an epitope is not an exclusive list of residues for the epitope on the antigen, but rather represents a minimal epitope associated with the antigen.

[0022] A "non-linear epitope" or "conformational epitope" comprises non-contiguous polypeptides, amino acids and / or sugars within an antigenic protein to which an epitope-specific antigen-binding molecule binds. In some embodiments, at least one residue is non-contiguous with other represented residues of the epitope, although one or more residues may be contiguous with other residues.

[0023] A "linear epitope" comprises a continuous polypeptide, amino acid and / or sugar in an antigenic protein to which an antigen-binding molecule specific for the epitope binds. Note that in some embodiments, not all of the residues in a linear epitope need to be directly bound (or involved in binding) by an antigen-binding molecule. In some embodiments, a linear epitope can be derived from immunization with a peptide that essentially consists of the sequence of the linear epitope, or from a structural section of a protein that is relatively isolated from the rest of the protein (so that the antigen-binding molecule can interact, at least primarily, with just that sequence section).

[0024] The term "antibody" is used in the broadest sense and includes various polypeptides, including but not limited to conventional antibodies (typically comprising at least one heavy chain and at least one light chain), single domain antibodies (sdAbs comprising at least one VHH domain and an Fc region), VHH-containing polypeptides (polypeptides comprising at least one VHH domain), and antibody-like antigen-binding domains, including fragments of any of the above, so long as they exhibit the desired antigen-binding activity. In some embodiments, the antibody comprises a dimerization domain. Such dimerization domains include but are not limited to a heavy chain constant domain (comprising CH1, hinge, CH2, and CH3, where CH1 is typically paired with a light chain constant domain CL, while the hinge mediates dimerization) and an Fc region (comprising hinge, CH2, and CH3, where the hinge mediates dimerization).

[0025] The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species such as camel (including llama), shark, mouse, human, cynomolgus monkey, etc.

[0026] As used herein, the term "antigen-binding domain" refers to a portion of an antibody sufficient to bind to an antigen. In some embodiments, the antigen-binding domain of a conventional antibody comprises three heavy chain CDRs and three light chain CDRs. Thus, in some embodiments, the antigen-binding domain comprises a heavy chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 required to maintain binding to the antigen, and a light chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 required to maintain binding to the antigen. In some embodiments, the antigen-binding domain of an sdAb or VHH-containing polypeptide comprises three CDRs of a VHH domain. Thus, in some embodiments, the antigen-binding domain of an sdAb or VHH-containing polypeptide comprises a VHH domain comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 required to maintain binding to the antigen.

[0027] As used herein, the term "VHH" or "VHH domain" or "VHH antigen-binding domain" refers to the antigen-binding portion of a single domain antibody, such as a camelid antibody or a shark antibody. In some embodiments, a VHH comprises three CDRs and four framework regions, designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, a VHH may be truncated at the N-terminus or C-terminus to include only partial FR1 and / or FR4, or to lack one or both of these framework regions, so long as the VHH substantially retains antigen binding and specificity.

[0028] The terms "single domain antibody" and "sdAb" are used interchangeably herein to refer to an antibody comprising at least one monomer domain, such as a VHH domain, without a light chain and an Fc region. In some embodiments, an sdAb is a dimer of two polypeptides, each polypeptide comprising at least one VHH domain and an Fc region. As used herein, the terms "single domain antibody" and "sdAb" refer to a polypeptide comprising multiple VHH domains, e.g., a polypeptide comprising the structure VHH 1 -VHH 2 -Fc or VHH 1 -VHH 2 -VHH 3 - Fc, where VHH 1 , V.H.H. 2 , and VHH 3 The term encompasses polypeptides in which the amino acid sequence may be the same or different.

[0029] The term "VHH-containing polypeptide" refers to a polypeptide comprising at least one VHH domain. In some embodiments, a VHH polypeptide comprises two, three, or four or more VHH domains, where each VHH domain may be the same or different. In some embodiments, a VHH-containing polypeptide comprises an Fc region. In some such embodiments, a VHH-containing polypeptide may be referred to as an sdAb. Furthermore, in some such embodiments, a VHH polypeptide may form a dimer. Non-limiting structures of a VHH-containing polypeptide, also referred to as an sdAb, include a VHH 1 -Fc, VHH 1 -VHH 2 -Fc, and VHH 1 -VHH 2 -VHH 3 - Fc, where VHH 1 , V.H.H. 2 , and VHH 3may be the same or different. In some embodiments of such structures, a VHH may be linked to another VHH by a linker, or a VHH may be linked to an Fc by a linker. In some such embodiments, the linker comprises 1-20 amino acids, preferably 1-20 amino acids consisting primarily of glycines and optionally serine. In some embodiments, when the VHH-containing polypeptide comprises an Fc, it forms a dimer. Thus, the structure VHH 1 -VHH 2 -Fc is considered to be tetravalent if it forms a dimer (i.e., the dimer has four VHH domains). Similarly, the structure VHH 1 -VHH 2 -VHH 3 -Fc is considered to be hexavalent if it forms a dimer (i.e., the dimer has six VHH domains).

[0030] The term "monoclonal antibody" refers to an antibody (including sdAb or VHH-containing polypeptide) of a substantially homogeneous antibody population. That is, the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Thus, a sample of monoclonal antibodies is capable of binding to the same epitope on the antigen. The modifier "monoclonal" indicates the nature of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be made by recombinant DNA methods such as those described in U.S. Pat. No. 4,816,567. The monoclonal antibodies can also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554, for example.

[0031] The term "CDR" refers to a complementarity determining region defined by at least one specific manner to one skilled in the art. In some embodiments, the CDRs may be defined according to any of the Chothia numbering scheme, the Kabat numbering scheme, a combination of Kabat and Chothia, the AbM definition, and / or the contact definition. A VHH comprises three CDRs designated CDR1, CDR2, and CDR3.

[0032] As used herein, the term "heavy chain constant region" refers to at least three heavy chain constant domains, i.e., H 1, hinge, C H 2, and C H3. Of course, deletions and modifications that do not alter function within a domain are included within the scope of the term "heavy chain constant region" unless otherwise specified. Non-limiting exemplary heavy chain constant regions include gamma, delta, and alpha. Non-limiting exemplary heavy chain constant regions also include epsilon and mu. Each heavy chain constant region corresponds to one antibody isotype. For example, an antibody comprising a gamma constant region is an IgG antibody, an antibody comprising a delta constant region is an IgD antibody, and an antibody comprising an alpha constant region is an IgA antibody. Furthermore, an antibody comprising a mu constant region is an IgM antibody, and an antibody comprising an epsilon constant region is an IgE antibody. Certain isotypes may be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 antibodies (gamma constant region, alpha constant region, mu ... 1 constant region), IgG2 antibody (γ 2 constant region), IgG3 antibody (γ 3 constant region), and IgG4 antibody (γ 4 The IgA antibody includes, but is not limited to, IgA1 antibody (α 1 constant region) and IgA2 antibody (α 2 constant region), and IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0033] As used herein, "Fc region" refers to a portion of a heavy chain constant region comprising CH2 and CH3. In some embodiments, the Fc region comprises a hinge, CH2, and CH3. In various embodiments, when the Fc region comprises a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region can be of any antibody heavy chain constant region isotype discussed herein. In some embodiments, the Fc region is IgG1, IgG2, IgG3, or IgG4.

[0034] As used herein, an "acceptor human framework" refers to a heavy chain variable domain (V) derived from a human immunoglobulin framework or a human consensus framework, as discussed herein. H) framework amino acid sequence. The acceptor human framework derived from a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence or can comprise amino acid sequence changes. In some embodiments, the number of amino acid changes is less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or less than 4, or less than 3 across all human frameworks within a single antigen-binding domain such as a VHH.

[0035] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody, such as an sdAb or a VHH-containing polypeptide) and its binding partner (e.g., an antigen). The affinity or apparent affinity of a molecule X for its partner Y is generally expressed as a function of the dissociation constant (K D ) or K D(見かけ) Affinity can be expressed by any of the conventional methods known in the art, including those described herein (e.g., ELISA K D , KinExA, flow cytometry, and / or surface plasmon resonance devices, etc. Such methods include, but are not limited to, BIAcore™, Octet™, or methods involving flow cytometry.

[0036] As used herein, "K D As used herein, the term "K" refers to the equilibrium dissociation constant of an antigen-binding molecule / antigen interaction. D When the term "K" is used, it includes D and K D(見かけ) Includes:

[0037] In some embodiments, the K of the antigen binding molecule D is measured by flow cytometry using antigen-expressing cell lines and fitting the mean fluorescence measured at each antibody concentration to a nonlinear one-site binding equation (Graphpad's Prism Software). In some such embodiments, KD is K D(見かけ) It is.

[0038] The term "biological activity" refers to any one or more biological properties of a molecule (whether naturally occurring as found in vivo or provided or enabled by recombinant means), including, but not limited to, binding to a ligand, induction or increase in cell proliferation, and induction or increase in expression of a cytokine.

[0039] An "agonist" or "activating" antibody is an antibody that increases and / or activates the biological activity of a target antigen. In some embodiments, an agonist antibody binds to an antigen and increases its biological activity by at least about 20%, 40%, 60%, 80%, 85% or more.

[0040] An "antagonist," "blocking," or "neutralizing" antibody is an antibody that inhibits, reduces and / or inactivates the biological activity of a target antigen. In some embodiments, a neutralizing antibody binds to an antigen and reduces its biological activity by at least about 20%, 40%, 60%, 80%, 85%, 90%, 95%, 99% or more.

[0041] An "affinity matured" sdAb or VHH containing polypeptide refers to an sdAb or VHH containing polypeptide which has one or more modifications in one or more CDRs which result in an improvement in the affinity of the sdAb or VHH containing polypeptide for its antigen compared to a parent sdAb or VHH containing polypeptide which does not possess such modifications.

[0042] As used herein, "humanized VHH" refers to a VHH in which one or more framework regions have been substantially replaced with human framework regions. In some cases, certain framework region (FR) residues of a human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized VHH may contain residues that are not found in the original VHH or in the human framework sequence, but are included to further improve and optimize the performance of the sdAb or VHH-containing polypeptide. In some embodiments, a humanized sdAb or VHH-containing polypeptide comprises a human Fc region. As will be appreciated, a humanized sequence may be identified by its primary sequence and does not necessarily indicate the process by which the antibody was made.

[0043] An "effector-positive Fc region" has an "effector function" of a native sequence Fc region. Exemplary "effector functions" include Fc receptor binding, Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Such effector functions generally require combining the Fc region with a binding domain (e.g., an antibody variable domain) and can be assessed using a variety of assays.

[0044] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes), native sequence human IgG2 Fc regions, native sequence human IgG3 Fc regions, and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0045] A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by at least one amino acid modification. In some embodiments, a "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by at least one amino acid modification, but that retains at least one effector function of the native sequence Fc region. In some embodiments, a variant Fc region has at least one amino acid substitution, e.g., about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, in the native sequence Fc region or the Fc region of the parent polypeptide compared to the native sequence Fc region or the Fc region of the parent polypeptide. In some embodiments, a variant Fc region herein has at least about 80% sequence identity, at least about 90% sequence identity, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the native sequence Fc region and / or the Fc region of the parent polypeptide.

[0046] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, the FcγR is a native human FcR. In some embodiments, the FcR binds IgG antibodies (gamma receptors), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences but differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, e.g., in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein. For example, the term "Fc receptor" or "FcR" also includes the fetal receptor FcRn, which plays a role in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and regulating immunoglobulin homeostasis.Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004 / 92219 (Hinton et al.)).

[0047] As used herein, the terms "substantially similar" or "substantially the same" refer to a sufficiently high degree of similarity between two or more numerical values ​​such that one of skill in the art would consider the difference between the two or more values ​​to have little or no biological and / or statistical significance within the context of the biological characteristic measured by the values. In some embodiments, two or more substantially similar values ​​differ by no more than the approximate value of any one of 5%, 10%, 15%, 20%, 25%, or 50%.

[0048] A polypeptide "variant" refers to a biologically active polypeptide having at least about 80% amino acid sequence identity with a native sequence polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N-terminus or C-terminus of the polypeptide. In some embodiments, a variant has at least about 80% amino acid sequence identity. In some embodiments, a variant has at least about 90% amino acid sequence identity. In some embodiments, a variant has at least about 95% amino acid sequence identity with a native sequence polypeptide.

[0049] As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide, or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

[0050] Amino acid substitutions can include, but are not limited to, replacing one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 1. Amino acid substitutions can be introduced into a subject antibody and the products screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0051] TIFF2024506931000001.tif111170

[0052] Amino acids can be grouped according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

[0053] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0054] The term "vector" is used to describe a polynucleotide that can be manipulated to include a cloned polynucleotide or polynucleotides that can be propagated in a host cell. A vector can include one or more of the following elements: an origin of replication, one or more regulatory sequences that regulate the expression of a polypeptide of interest (such as, for example, a promoter and / or enhancer), and / or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, such as, for example, β-galactosidase). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0055] "Host cell" refers to a cell that can be or has been the recipient of a vector or isolated polynucleotide. Host cells can be prokaryotic or eukaryotic. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells, fungal cells, such as yeast, plant cells, and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6™ cells (Crucell), and 293 and CHO cells, and their derivatives, such as 293-6E cells, CHO-DG44 cells, CHO-K1 cells, CHO-S cells, and CHO-DS cells. Host cells include the progeny of a single host cell, although the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutations. Host cells include cells transfected in vivo with a polynucleotide(s) provided herein.

[0056] As used herein, the term "isolated" refers to a molecule that is separated from at least some of the components that it is typically found or produced with in nature. For example, a polypeptide is referred to as "isolated" when it is separated from at least some of the components of the cell that produced it. If the polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to "isolate" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" when it is not part of a larger polynucleotide that it is typically found in nature (e.g., in the case of a DNA polynucleotide, genomic DNA or mitochondrial DNA, etc.), or when it is separated from at least some of the components of the cell that produced it, for example, in the case of an RNA polynucleotide. Thus, a DNA polynucleotide contained in a vector within a host cell can be referred to as "isolated".

[0057] The terms "individual" and "subject" are used interchangeably herein to refer to animals, e.g., mammals. In some embodiments, methods of treating mammals are provided, including, but not limited to, humans, rodents, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, laboratory mammals, mammalian livestock, mammalian sport animals, and mammalian pets. In some instances, "individual" or "subject" refers to an individual or subject in need of treatment for a disease or disorder. In some embodiments, the subject to be treated may be a patient, which means the subject has been identified as having or being at sufficient risk of suffering from a disorder relevant to the treatment.

[0058] As used herein, "disease" or "disorder" refers to a condition for which treatment is needed and / or desired.

[0059] The terms "tumor cell," "cancer cell," "cancer," "tumor," and / or "neoplasm" are used interchangeably herein, unless otherwise specified, to refer to a cell (or cells) that exhibit uncontrolled proliferation and / or abnormally increased cell survival and / or inhibited apoptosis that interferes with the normal functioning of bodily organs and systems. This definition includes benign and malignant cancers, polyps, hyperplasias, and occult tumors or micrometastases.

[0060] The terms "cancer" and "tumor" include solid cancers and hematological / lymphatic cancers, as well as malignant, premalignant, and benign tumors, such as dysplasia. Exemplary cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, chondrosarcoma, Ewing's sarcoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, and ovarian cancer. cancer) (including gastrointestinal cancer), glioblastoma, hepatocellular carcinoma, intraepithelial neoplasia, renal or kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), melanoma, myeloma, neuroblastoma, oral cancer (lips, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer (e.g., pancreatic adenocarcinoma), prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, mesothelioma, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer cancer), testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancer, vulvar cancer, lymphomas including Hodgkin lymphoma and non-Hodgkin lymphoma, and B-cell lymphomas (low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-dividing cell NHL, bulky disease NH These include lymphomas including L, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, as well as post-transplant lymphoproliferative disorders (PTLD), and the abnormal blood vessel growth associated with phacomas, edema (such as that associated with brain tumors), and Meigs' syndrome.

[0061] In some embodiments, "increase" or "decrease" refers to a statistically significant increase or decrease, respectively. As will be clear to the skilled artisan, "modulation" can also include causing a change (which can be either an increase or a decrease) in the affinity, avidity, specificity and / or selectivity of a target or antigen to one or more of its ligands, binding partners, partners that associate into homo- or heteromultimeric forms or substrates, causing a change (which can be either an increase or a decrease) in the sensitivity of the target or antigen to one or more conditions (pH, ionic strength, presence of cofactors, etc.) in the medium or environment in which the target or antigen is present, and / or cell proliferation or cytokine production, compared to the same conditions except in the presence of the test agent. This can be determined in any suitable manner and / or using any suitable assay known per se or described herein, depending on the target involved.

[0062] As used herein, "treatment" is an approach to obtain beneficial or desired clinical results. As used herein, "treatment" covers any administration or application of a therapeutic agent for a disease in a mammal, including a human. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, reduction in the extent of the disease, prevention or delay of disease progression (e.g., metastasis, e.g., to the lungs or lymph nodes), prevention or delay of disease recurrence, delay or slowing of disease progression, amelioration of disease status, inhibition of disease or disease progression, inhibition or slowing of disease or its progression, arrest of its development, and remission (whether partial or total). "Treatment" also includes reduction of pathological consequences of proliferative diseases. The methods provided herein contemplate any one or more of these aspects of treatment. In accordance with the above, the term treatment does not require 100 percent elimination of all aspects of the disorder.

[0063] By "ameliorate" is meant that one or more symptoms are lessened or improved compared to when the therapeutic agent is not administered. "Amelioration" also includes a shortening or reduction in the duration of the symptoms.

[0064] The term "anti-cancer agent" is used herein in its broadest sense to refer to an agent used to treat one or more cancers. Exemplary classes of such agents include, but are not limited to, chemotherapeutic agents, anti-cancer biologics (such as cytokines, receptor extracellular domain-Fc fusions, and antibodies), radiotherapy agents, CAR-T therapy agents, therapeutic oligonucleotides (such as antisense oligonucleotides and siRNAs), and oncolytic viruses.

[0065] The term "biological sample" refers to a quantity of a substance from a living or formerly living organism, including, but not limited to, blood (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

[0066] The term "control" or "reference" refers to a composition known to be free of the analyte (a "negative control") or a composition known to contain the analyte (a "positive control"). A positive control may contain a known concentration of the analyte.

[0067] As used herein, "delaying the onset of disease" means delaying, preventing, slowing, retarding, stabilizing, inhibiting, and / or prolonging the onset of a disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and / or the individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, late-stage cancer, such as the onset of metastases, can be delayed.

[0068] As used herein, "prevention" includes providing prevention against the occurrence or recurrence of a disease in a subject who may have a predisposition to the disease, but has not yet been diagnosed with the disease. Unless otherwise indicated, the terms "reduce," "inhibit," or "prevent, prevent" do not indicate or require complete prevention over the entire period of time, but only prevention over the period being measured.

[0069] A "therapeutically effective amount" of a substance / molecule, agonist or antagonist may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule, agonist or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the substance / molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects. A therapeutically effective amount may be delivered in one or more administrations. A therapeutically effective amount refers to an amount effective to achieve a desired therapeutic and / or prophylactic result at the required dosages for the required time.

[0070] The terms "pharmaceutical formulation" and "pharmaceutical composition" are used interchangeably and refer to a preparation that is in a form that allows the biological activity of the active ingredient(s) to be effective and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered. Such formulations may be sterile.

[0071] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation aid, or carrier conventional in the art used with therapeutic agents that together comprise a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to a recipient at the dosage and concentration used and is compatible with other ingredients of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation in which it is used.

[0072] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0073] The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents where at least a portion of the administration overlaps in time, or where the administration of one therapeutic agent falls within a short period of time relative to the administration of the other therapeutic agent, or where the therapeutic effects of both therapeutic agents overlap over at least some period of time.

[0074] The term "sequentially" is used herein to refer to the administration of two or more therapeutic agents that do not overlap in time or where the therapeutic effects of the therapeutic agents do not overlap.

[0075] As used herein, "in combination with" refers to the administration of one therapy in addition to another. Thus, "in combination with" refers to the administration of one therapy before, during, or after the administration of another therapy to an individual.

[0076] The term "package insert" is used to refer to instructions typically included in commercial packaging for a therapeutic product, which contain information regarding directions, usage, dosage, administration, concomitant therapy, contraindications and / or warnings regarding the use of such therapeutic product.

[0077] An "article of manufacture" is any manufacture (e.g., package or container) or kit that contains at least one reagent, e.g., a pharmaceutical agent for treating a disease or disorder (e.g., cancer), or a probe that specifically detects a biomarker described herein. In some embodiments, the article of manufacture or kit is advertised, delivered, or sold as a unit for performing a method described herein.

[0078] The terms "label" and "detectable label" refer to a moiety that, for example, when attached to an antibody or antigen, renders the reaction (e.g., binding) between members of a specific binding pair detectable. A labeled member of a specific binding pair is said to be "detectably labeled." Thus, the term "labeled binding protein" refers to a protein that incorporates a label that provides for the identification of the binding protein. In some embodiments, the label is a detectable marker that can generate a signal that is detectable visually or by instrumental means, for example, the incorporation of a radiolabeled amino acid or the attachment of a biotinyl moiety to the polypeptide that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or an enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C. 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153 Sm), chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorophores), enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), and magnetic agents such as gadolinium chelates. Representative examples of labels commonly used in immunoassays include moieties that emit light, e.g., acridinium compounds, and moieties that emit fluorescence, e.g., fluorescein. In this regard, the moiety itself may not be detectably labeled, but may become detectable upon reaction with yet another moiety.

[0079] Exemplary DR5 Binding Polypeptides Provided herein is a formulation of a DR5 binding polypeptide. In various embodiments, the DR5 binding polypeptide comprises at least one VHH domain comprising a CDR1 comprising the sequence of SEQ ID NO:1, a CDR2 comprising the sequence of SEQ ID NO:2, and a CDR3 comprising the sequence of SEQ ID NO:3. In some embodiments, at least one VHH domain is humanized. In some embodiments, the DR5 binding polypeptide comprises at least one VHH domain comprising the amino acid sequence of SEQ ID NO:4.

[0080] In some embodiments, the DR5 binding polypeptide comprises at least one VHH domain that binds to DR5 and an Fc region. In some embodiments, the DR5 binding polypeptide provided herein comprises two VHH domains that bind to DR5 and an Fc region. In some embodiments, the Fc region mediates dimerization of the DR5 binding polypeptide under physiological conditions, thus forming a dimer and doubling the number of DR5 binding sites. For example, a DR5 binding polypeptide comprising two VHH domains that bind to DR5 and an Fc region is divalent as a monomer, but under physiological conditions, the Fc region can mediate dimerization, such that the DR5 binding polypeptide exists as a tetravalent dimer under such conditions.

[0081] In some embodiments, the DR5 binding polypeptide comprises the structure VHH-linker-VHH-linker-Fc. In some embodiments, the VHH-linker-VHH portion of the DR5 binding polypeptide comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the Fc comprises a hinge. In some such embodiments, the Fc comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the DR5 binding polypeptide comprises the amino acid sequence of SEQ ID NO:7, which comprises two VHH domains and an Fc region.

[0082] In some embodiments, the VHH domain that binds DR5 may be humanized. Humanized antibodies (such as sdAbs or VHH-containing polypeptides) are useful as therapeutic molecules because they reduce or eliminate human immune responses to non-human antibodies that may result in immune responses to antibody therapeutics and reduce the efficacy of the therapeutics. Generally, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. The humanized antibody also optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0083] Humanized antibodies and methods for making them are reviewed, e.g., in Almagro and Fransson, (2008) Front. Biosci. 13: 1619-1633, and are described, e.g., in Riechmann et al., (1988) Nature 332:323-329, Queen et al., (1989) Proc. Natl Acad. Sci. USA 86: 10029-10033, U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409, Kashmiri et al., (2005) Methods 36:25-34, Padlan, (1991) Mol. Immunol. 28:489-498 (describing "resurfacing"), Dall'Acqua et al., (2005) Methods 36:43-60 (describing "FR shuffling"), as well as Osbourn et al., (2005) Methods 36:61-68 and Klimka et al., (2000) Br. J. Cancer, 83:252-260 (describing a "guided selection" approach to FR shuffling).

[0084] Human framework regions that may be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. (1993) J. Immunol. 151:2296), framework regions derived from consensus sequences of human antibodies of particular subgroups of heavy chain variable regions (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285, and Presta et al. (1993) J. Immunol, 151:2623), human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633), and framework regions obtained from screening of FR libraries (see, e.g., Baca et al., (1997) J. Biol. Chem. 272: 10678-10684, and Rosok et al., (1996) J. Biol. Chem. 271:22611-22618). Typically, the FR regions of a VHH are replaced with human FR regions to generate a humanized VHH. In some embodiments, certain FR residues of the human FR are replaced to improve one or more properties of the humanized VHH. A VHH domain having such replaced residues is also referred to herein as "humanized".

[0085] In various embodiments, the Fc region comprised in the DR5 binding polypeptide is a human Fc region or is derived from a human Fc region.

[0086] In some embodiments, the Fc region included in the DR5 binding polypeptide is derived from a human Fc region and includes a three amino acid deletion in the lower hinge corresponding to IgG1 E233, L234, and L235, referred to herein as "Fc xELL". Fc xELL polypeptides do not bind FcγR and are therefore referred to as "effector silent" or "effector null", however, in some embodiments, the xELL Fc region binds to FcRn, with associated transcytosis associated with extended half-life and FcRn mediated recycling. In some embodiments, the Fc region is a human IgG1 xELL Fc region.

[0087] Polypeptide Expression and Production Nucleic acid molecules are provided that comprise a polynucleotide encoding a DR5 binding polypeptide. In some embodiments, the nucleic acid molecule may also encode a leader sequence that directs secretion of the DR5 binding polypeptide, and the leader sequence is typically cleaved so that it is not present in the secreted polypeptide. The leader sequence may be the native heavy chain (or VHH) leader sequence, or may be another heterologous leader sequence.

[0088] The nucleic acid molecule can be constructed using recombinant DNA techniques routine in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0089] Vectors are provided that contain a nucleic acid encoding a DR5 binding polypeptide as described herein. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like. In some embodiments, a vector is selected that is optimized for expression of the polypeptide in a desired cell type, such as a CHO cell or CHO-derived cell, or an NSO cell. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0090] In some embodiments, DR5 binding polypeptides may be expressed in prokaryotic cells, such as bacterial cells, or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression may be performed, for example, according to procedures known in the art. Exemplary eukaryotic cells that may be used to express the polypeptide include, but are not limited to, COS cells, including COS7 cells, 293 cells, including 293-6E cells, CHO cells, including CHO-S, DG44, Lec13 CHO cells, and FUT8 CHO cells, PER.C6™ cells (Crucell), and NSO cells. In some embodiments, DR5 binding polypeptides may be expressed in yeast. See, for example, US Patent Application Publication No. 2006 / 0270045. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to a polypeptide. For example, in some embodiments, CHO cells produce polypeptides with higher levels of sialylation than the same polypeptides produced in 293 cells.

[0091] Introduction of one or more nucleic acids (e.g., vectors) into a desired host cell can be accomplished by any method, including, but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, etc. Non-limiting exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3 rd Cold Spring Harbor Laboratory Press, ed., 2001. The nucleic acid may be transiently or stably transfected into the desired host cell according to any suitable method.

[0092] Host cells comprising any of the nucleic acids or vectors described herein are also provided. In some embodiments, host cells expressing the DR5 binding polypeptides described herein are provided. The DR5 binding polypeptides expressed in the host cells can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrix or hydrophobic interaction chromatography. Suitable affinity ligands include agents that bind to the ROR1 ECD and Fc region. For example, Protein A, Protein G, Protein A / G, or antibody affinity columns can be used to purify DR5 binding polypeptides comprising an Fc region by binding to the Fc region. Hydrophobic interaction chromatography, e.g., butyl or phenyl columns, can also be suitable for purifying some polypeptides, such as antibodies. Ion exchange chromatography (e.g., anion exchange and / or cation exchange chromatography) can also be suitable for purifying some polypeptides, such as antibodies. Mixed mode chromatography (e.g., reversed phase / anion exchange, reversed phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, etc.) can also be suitable for purifying some polypeptides, such as antibodies. Many methods for purifying polypeptides are known in the art.

[0093] In some embodiments, the DR5 binding polypeptide is produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009), Spirin, Trends Biotechnol. 22: 538-45 (2004), and Endo et al., Biotechnol. Adv. 21: 695-713 (2003).

[0094] In some embodiments, a DR5 binding polypeptide produced by the above method is provided. In some embodiments, the DR5 binding polypeptide is produced in a host cell. In some embodiments, the DR5 binding polypeptide is produced in a cell-free system. In some embodiments, the DR5 binding polypeptide is purified. In some embodiments, a cell culture medium comprising the DR5 binding polypeptide is provided.

[0095] In some embodiments, a composition is provided that comprises the antibody produced by the above method.In some embodiments, the composition comprises a DR5 binding polypeptide produced in a host cell.In some embodiments, the composition comprises a DR5 binding polypeptide produced in a cell-free system.In some embodiments, the composition comprises a purified DR5 binding polypeptide.

[0096] Pharmaceutical formulations of DR5 binding polypeptides In some embodiments, the pharmaceutical formulation of the DR5 binding polypeptide is an aqueous liquid formulation. In other embodiments, the formulation is lyophilized. In either case, the formulation comprises a DR5 binding polypeptide. In some embodiments, the DR5 binding polypeptide comprises at least one VHH domain comprising CDR1 comprising the amino acid sequence of SEQ ID NO:1, CDR2 comprising the amino acid sequence of SEQ ID NO:2, and CDR3 comprising the amino acid sequence of SEQ ID NO:3. In some embodiments, at least one VHH domain is humanized. In some embodiments, the DR5 binding polypeptide comprises at least one VHH domain comprising the amino acid sequence of SEQ ID NO:4. In some embodiments, the DR5 binding polypeptide comprises the structure VHH-linker-VHH-linker-Fc. In some embodiments, the VHH-linker-VHH portion of the DR5 binding polypeptide comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the Fc comprises a hinge. In some such embodiments, the Fc comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the DR5 binding polypeptide comprises the amino acid sequence of SEQ ID NO:7, which comprises two VHH domains and an Fc region.

[0097] In some embodiments, the concentration of the DR5 binding polypeptide in an aqueous formulation herein or in a reconstituted lyophilized formulation described herein is, for example, 20 mg / mL to 70 mg / mL, for example, 30 mg / mL to 60 mg / mL, 20 mg / mL to 60 mg / mL, 20 mg / mL to 50 mg / mL, 20 mg / mL to 40 mg / mL, 30 mg / mL to 70 mg / mL, 30 mg / mL to 50 mg / mL, 30 mg / mL to 40 mg / mL, 50 mg / mL to 70 mg / mL, or 50 mg / mL to 60 mg / mL. In some embodiments, the formulation comprises 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, or 70 mg / mL of the DR5 binding polypeptide. In some embodiments, the formulation comprises 50 mg / mL of the DR5 binding polypeptide.

[0098] In some embodiments, the pH of the formulation is 5.3 to 6.7, for example, 5.4 to 6.6, 5.5 to 6.5, 5.6 to 6.4, 5.6 to 6.5, 5.7 to 6.5, 5.8 to 6.5, 5.9 to 6.5, 6 to 6.5, 5.5 to 6.4, 5.5 to 6.3, 5.5 to 6.2, 5.5 to 6.1, 5.5 to 6, 5.8 to 6.2, 5.8 to 6, 5.9 to 6, 5.9 to 6.1, 6 to 6.1, or 6 to 6.2. In some embodiments, the pH of the formulation is about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5. In some embodiments, the pH of the formulation is about 6. In some embodiments, the pH of the formulation is between 5.5 and 6.5.

[0099] In some embodiments, the formulation comprises histidine, the concentration of which may be, for example, 5 mM to 20 mM histidine, for example, 5 mM to 15 mM, 7 mM to 15 mM, 5 mM to 12 mM, 7 mM to 12 mM. In some embodiments, the formulation comprises 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM histidine. In some embodiments, the formulation comprises 10 mM histidine. In some embodiments, the histidine is histidine HCl.

[0100] In some embodiments, the formulation comprises sucrose at a concentration of, for example, 7%-10% (w / v), such as 7%-9%, 8%-10%, 8%-9%, 7%, 8%, 9% or 10% (w / v). In some embodiments, the formulation comprises 8% sucrose. In some embodiments, the formulation comprises 9% sucrose.

[0101] In some embodiments, the formulation comprises poloxamer 188 (P188) at a concentration of, for example, 0.1% to 0.8%, for example, 0.1% to 0.5%, for example, 0.2% to 0.4%, 0.2% to 0.5%, 0.3% to 0.5%, 0.3% to 0.4%, 0.1% to 0.2%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7% or 0.8%. In some embodiments, the formulation comprises 0.2% poloxamer P188.

[0102] A non-limiting exemplary formulation comprises 50 mg / mL DR5 binding polypeptide, 10 mM histidine HCl, 8% sucrose and 0.2% poloxamer P188, with the pH of the formulation being about 6. In some embodiments, the DR5 binding polypeptide comprises at least one VHH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:1, a CDR2 comprising the amino acid sequence of SEQ ID NO:2 and a CDR3 comprising the amino acid sequence of SEQ ID NO:3. In some embodiments, at least one VHH domain is humanized. In some embodiments, the DR5 binding polypeptide comprises at least one VHH domain comprising the amino acid sequence of SEQ ID NO:4. In some embodiments, the DR5 binding polypeptide comprises the structure VHH-linker-VHH-linker-Fc. In some embodiments, the VHH-linker-VHH portion of the DR5 binding polypeptide comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the Fc comprises a hinge. In some such embodiments, the Fc comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the DR5 binding polypeptide comprises the amino acid sequence of SEQ ID NO:7, which comprises two VHH domains and an Fc region.

[0103] In some embodiments, the pharmaceutical formulation of the DR5 binding polypeptide is provided in lyophilized form. Methods for lyophilizing protein-containing drug products are known in the art. In some embodiments, the lyophilized formulation provided herein is stable for at least or up to 3 months, at least or up to 6 months, at least or up to 9 months, at least or up to 12 months, or more than 12 months when stored at 2°C to 8°C. In some such embodiments, the lyophilized formulation retains its cake characteristics during storage. In some embodiments, the lyophilized formulation is a uniform, fine cake with an off-white color that is free of any visible impurities.

[0104] In some embodiments, upon reconstitution of the lyophilized formulation after storage, the reconstituted aqueous formulation is substantially free of visible particles. In some embodiments, less than 3% or less than 2% of the DR5 binding polypeptide present in the aqueous formulation is aggregated and / or less than 1% or less than 0.5% of the DR5 binding polypeptide present in the aqueous formulation is degraded. The presence of aggregated and / or degraded DR5 binding polypeptide can be measured, for example, using size exclusion chromatography.

[0105] The pharmaceutical formulation of the DR5 binding polypeptide disclosed herein can be provided in unit dosage form or stored in a suitable form to provide more than one unit dose.The pharmaceutical formulation should be compatible with its intended route of administration.Lyophilized formulations are typically reconstituted in a solution before administration or use, while aqueous formulations can be "ready to use", meaning that they are administered directly without first being diluted, or they can be diluted with saline or another solution before use.

[0106] The pharmaceutical formulation is preferably sterile. Sterilization can be achieved by any suitable method, for example, filtration through a sterile filtration membrane. If the composition is lyophilized, sterilization by filtration can be performed before or after lyophilization and reconstitution. In various embodiments, a lyophilized formulation is provided, which can be reconstituted to form the liquid pharmaceutical formulation provided herein.

[0107] Exemplary methods of treating disease with DR5 binding polypeptide formulations In some embodiments, methods are provided for treating a disease in an individual comprising administering a pharmaceutical formulation comprising a DR5 binding polypeptide, hi some embodiments, methods are provided for treating cancer in an individual.

[0108] In some embodiments, the method comprises administering to an individual an effective amount of a pharmaceutical formulation comprising a DR5 binding polypeptide as provided herein. Such a method of treatment can be a method of treatment in a human or an animal. In some embodiments, a method of treatment in a human is provided. Non-limiting exemplary cancers that may be treated with the DR5 binding polypeptides provided herein include basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, chondrosarcoma, Ewing's sarcoma, colorectal cancer (colon cancer), connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, renal or kidney cancer, laryngeal cancer, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer such as pancreatic adenocarcinoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, mesothelioma, salivary gland cancer, sarcoma, skin cancer, and the like. These include skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancer and vulvar cancer, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-dividing cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, and chronic myeloblastic leukemia.

[0109] The pharmaceutical formulation may be administered to the subject as needed. The frequency of administration may be determined by a person skilled in the art, such as the attending physician, based on considerations such as the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the subject being treated, etc. In some embodiments, an effective dose of the DR5 binding polypeptide is administered to the subject one or more times. In some embodiments, an effective dose of the DR5 binding polypeptide is administered to the subject daily, twice weekly, weekly, biweekly, monthly, etc. An effective dose of the DR5 binding polypeptide is administered to the subject at least once. In some embodiments, an effective dose of the DR5 binding polypeptide may be administered multiple times, including multiple times over at least one month, at least six months, or at least one year.

[0110] In some embodiments, the pharmaceutical formulation is administered in an amount effective to treat (including prevent) cancer. The therapeutically effective amount typically depends on the weight of the subject being treated, the physical or health condition of the subject, the extent of the condition being treated, or the age of the subject being treated. In general, the antibody may be administered in an amount ranging from about 0.05 mg / kg body weight to about 100 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 10 μg / kg body weight to about 100 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 50 μg / kg body weight to about 5 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 100 μg / kg body weight to about 10 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 100 μg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 0.5 mg / kg body weight to about 10 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 0.05 mg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 0.05 mg / kg body weight to about 10 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 5 mg / kg body weight or less, e.g., less than 4 mg / kg, less than 3 mg / kg, less than 2 mg / kg, or less than 1 mg / kg of antibody.

[0111] In some embodiments, the DR5 binding polypeptide may be administered in vivo by a variety of routes, including but not limited to, intramuscular, intravenous, intraarterial, parenteral, intraperitoneal, or subcutaneous. Depending on the intended use, an appropriate formulation and route of administration may be selected.

[0112] Combination therapy The DR5 binding polypeptide may be administered alone or in combination with other therapeutic modalities, such as other anti-cancer agents. The DR5 binding polypeptide may be provided prior to, substantially simultaneously with, or subsequent to (i.e., simultaneously or sequentially with) other therapeutic modalities. In some embodiments, the therapeutic methods described herein may further comprise administering radiotherapy, chemotherapy, vaccination, targeted tumor therapy, CAR-T therapy, oncolytic virus therapy, cancer immunotherapy, cytokine therapy, surgical resection, chromatin modification, resection, cryotherapy, antisense agents against tumor targets, siRNA agents against tumor targets, microRNA agents against tumor targets, or anti-cancer / anti-tumor agents, or biologics such as antibodies, cytokines, or receptor extracellular domain-Fc fusions.

[0113] In some embodiments, a DR5 binding polypeptide provided herein is given simultaneously with a second therapeutic agent, e.g., a PD-1 / PD-L1 therapy. Examples of PD-1 / PD-L1 therapies include nivolumab (BMS), pidilizumab (CureTech, CT-011), pembrolizumab (Merck), durvalumab (Medimmune / AstraZeneca), atezolizumab (Genentech / Roche), avelumab (Pfizer), AMP-224 (Amplimmune), BMS-936559, AMP-514 (Amplimmune), MDX-1105 (Merck), TSR-042 (Tesaro / AnaptysBio, ANB-011), STI-A1010 (Sorrento Therapeutics), STI-A1110 (Sorrento Therapeutics), and other agents directed against programmed death-1 (PD-1) or programmed death-ligand 1 (PD-L1).

[0114] In some embodiments, the DR5 binding polypeptides provided herein are administered simultaneously with an immunostimulatory agent, such as an agonist of a member of the tumor necrosis factor receptor superfamily (TNFRSF) or a member of the B7 family. Non-limiting examples of immunostimulatory TNFRSF members include OX40, GITR, 41BB, CD27, and HVEM. Non-limiting examples of B7 family members include CD28 and ICOS. Thus, in some embodiments, the CD8 binding polypeptides provided herein are administered simultaneously with an agonist of OX40, GITR, 41BB, CD27, HVEM, CD28, and / or ICOS, such as an agonist antibody.

[0115] In some embodiments, the DR5 binding polypeptides provided herein are given concomitantly with CAR-T (chimeric antigen receptor T cell) therapy, oncolytic virus therapy, cytokine therapy, and / or agents targeting other checkpoint molecules such as VISTA, gpNMB, B7H3, B7H4, HHLA2, CTLA4, TIGIT, etc.

[0116] kit Also provided are articles of manufacture and kits comprising any of the formulations provided herein and suitable packaging. In some embodiments, the invention includes a kit comprising (i) a formulation comprising a DR5 binding polypeptide, and (ii) instructions for using the kit to administer the formulation to an individual.

[0117] Suitable packaging for the compositions described herein are known in the art and include, for example, vials (e.g., sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles of manufacture may be further sterilized and / or sealed. Unit dosage forms comprising the compositions described herein are also provided. These unit dosage forms may be stored in suitable packaging in single or multiple unit dosage forms, and may also be further sterilized and sealed. Instructions provided in the kits of the invention are typically written instructions on a label or insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. Instructions for use of the antibody generally include information regarding dosage, administration schedule, and route of administration for the intended therapeutic or industrial use. The kit may further include instructions for selecting an appropriate individual treatment.

[0118] The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Kits may also be provided that contain a sufficient dose of the molecules disclosed herein to provide effective treatment to an individual for an extended period of time, such as any approximate period of 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months or more. Kits may also contain multiple unit doses of the molecules and instructions for use, and may be packaged in an amount sufficient for storage and use in pharmacies, such as hospital pharmacies and compounding pharmacies. In some embodiments, the kits include a dried (e.g., lyophilized) composition that can be reconstituted, resuspended, or rehydrated to form a generally stable aqueous solution of the antibody. EXAMPLES

[0119] The examples discussed below are intended to be purely illustrative of the present invention and should not be considered as limiting the present invention in any way. These examples are not intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0120] Example 1: Development of the initial lyophilized INBRX-109 formulation The Phase 1 formulation of INBRX-109 contained 20 mg / mL INBRX-109, 20 mM sodium acetate, 263 mM sucrose, and 0.20% poloxamer 188, at pH 5.0. The formulation was found to develop visible proteinaceous particles after 6 months at 5°C when stored in liquid state. Therefore, the clinical product was frozen to prevent particle formation. Therefore, a lyophilized formulation was developed for the next clinical phase.

[0121] The objective was to develop a lyophilized formulation that was substantially free of visible particles and showed minimal increase in high molecular weight (HMW) species by size exclusion (SEC) chromatography both after reconstitution and during storage at 5°C for 2 years.

[0122] Although the Phase 1 formulation contained 20 mM acetate, a lyophilized formulation containing histidine was developed and tested first because histidine is more compatible with the freeze-drying process. The initial lyophilized formulation contained 20 mM histidine buffer (pH 6.0). The 20 mg / ml INBRX-109 formulation contained 0.2% poloxamer P188, and the 40 mg / ml INBRX-109 formulation contained 0.4% poloxamer P188. Three different lyoprotectants were tested at various % (w / v) concentrations: sucrose, trehalose, or sucrose and mannitol. The formulations were filled in glass vials at 2 ml. Changes in soluble aggregates after one freeze / thaw cycle and after lyophilization, as well as after one month of storage of the lyophilized formulation at 40°C or 50°C, were determined by SEC analysis using a TSK gel G3000SWxl column (30 cm x 7.8 mm, 5 μm) and an Agilent HPLC. The mobile phase was 50 mM sodium phosphate buffer, 300 mM NaCl (pH 6.8). The flow rate was 1.0 mL / min. Samples were diluted to 1 mg / mL, sterile filtered through a 0.22 μm PES membrane if there were visible particles, injected into the HPLC column in a volume of 10 μL, and detected at 280 nm. The results are shown in Table 1.

[0123] TIFF2024506931000002.tif254170

[0124] As shown in Table 1, sucrose was superior to trehalose as a lyoprotectant in this formulation (compare 1A-3A vs. 4A-6A; 1B-3B vs. 4B-6B), and higher concentrations of sucrose were more stable (compare 4A vs. 6A; 4B vs. 6B).

[0125] Example 2: Development of further lyophilized INBRX-109 formulations A series of additional formulations were tested using 50 mg / mL INBRX-109, 10 mM histidine (pH 6) and 0.2% poloxamer P188, all of which contained 5 mM methionine and either trehalose or sucrose, as shown in Table 2.

[0126] TIFF2024506931000003.tif155170

[0127] Prior to lyophilization, each formulation in Table 2 was assayed for the appearance of visible particles and by SEC. After lyophilization, the formulations were assayed for cake appearance and reconstitution time both at time 0 and after 1 week storage at 40° C., and the reconstituted formulations were assayed for the appearance of visible particles and by SEC and HIAC. For appearance testing, the formulations were tested against a black and white background using a YB-2 light box. The lyophilization cycle used in this study is shown in Table 3.

[0128] TIFF2024506931000004.tif54170

[0129] The results of the appearance test are shown in Table 4. So = slightly milky; FVP = no visible particles.

[0130] TIFF2024506931000005.tif124170

[0131] All formulations appeared slightly milky and free of visible particles before and after lyophilization and after one month of storage in the lyophilized state.

[0132] As shown in Table 5, SEC analysis of lyophilized formulations at T0 showed minimal increases after lyophilization for all formulations. For sucrose-containing formulations, no changes in HMW were observed after 1 week of storage at 40 °C, however, poorer performance of the SEC column during analysis of trehalose formulations (F1-F5) at the 1 week time point may have resulted in an apparent increase in HMW for these samples. Formulations containing poloxamer in the concentration range of 0.2%-0.8% showed similar changes in HMW after storage (F2 vs. F6).

[0133] TIFF2024506931000006.tif120170

[0134] To semi-quantify the relative cake appearance, a "visual appearance" score was developed, where a score of "0" is a cake with very poor appearance (crumbs, cracks, meltback, etc.) and a score of "10" is a nice solid cake with no objectionable appearance (solid cake with no cracks, meltback, collapse, etc.). Table 6 shows the cake appearance and visual appearance scores at time point 0.

[0135] TIFF2024506931000007.tif103170

[0136] The appearance of each formulation was then determined after reconstitution with water to 50 mg / ml INBRX-109 and storage for up to 30 hours at 25° C. The results are shown in Table 7. PO=particles observed; FVP=no visible particles; (+)=more particles observed; (-)=fewer particles observed.

[0137] TIFF2024506931000008.tif60170

[0138] It was found that trehalose-containing formulations (F1-F4) were more likely to form particles, showing the presence of visible particles after only 4 hours of storage (F1) and by 24 hours of storage for all trehalose-containing formulations. It was also found that the concentration of poloxamer did not change the time to particle formation, as both F5 and F6 (0.8% poloxamer) showed visible particles after 4 hours, as did F1 (0.2% poloxamer). Sucrose-containing formulations were superior to trehalose in time to particle formation. Formulation F7 (260 mM sucrose) formed particles at 24 hours versus 4 hours for F1 (260 mM trehalose), and F9 (200 mM sucrose) was free of particles at 30 hours compared to F3 (200 mM trehalose), which did not contain particles until the 18 hour time point.

[0139] Example 3: Stability of formulations before lyophilization The formulations were evaluated for physical stability (aggregation by SEC and visible particle appearance) during short-term storage as liquid drug substance prior to lyophilization. A series of new formulations of protein at 25 mg / ml or 50 mg / ml in 20 mM histidine HCl buffer (pH 6.0) were prepared using either poloxamer or polysorbate 80 as the non-ionic surfactant. A control formulation in 20 mM sodium acetate buffer (pH 5.0) containing 8% sucrose with poloxamer was also prepared and tested. The formulations were stored at 25°C for up to 3 days and observed for particle formation. The formulations are shown in Table 8 and the results in Table 9. FVP = no visible particles.

[0140] TIFF2024506931000009.tif59170

[0141] TIFF2024506931000010.tif42170

[0142] As shown in Table 9, when stored at 25° C., these formulations remained particle-free for up to 3 days, indicating that the histidine formulations with sucrose and either poloxamer or polysorbate 80 were as stable as the control formulation containing acetate. Both the 20 mg / ml and 50 mg / ml formulations were stable under these conditions.

[0143] Changes in high molecular weight (HMW) aggregates were also assayed by SEC after 1 and 3 days at 25° C. As shown in Table 10, HMW aggregate formation was similar for formulations containing sodium acetate buffer (pH 5.0) and histidine HCl buffer (pH 6.0) when stored in liquid state at 25° C. for up to 3 days. As expected, the 50 mg / ml formulations had higher rates of aggregation compared to the 20 mg / ml formulations (bold; F11, F13, and F15). LMW = low molecular weight species.

[0144] TIFF2024506931000011.tif67170

[0145] Example 4: Various concentrations of sucrose and INBRX-109 Additional formulations were developed to optimize the concentration of sucrose and evaluate a protein concentration of 25 mg / ml. These formulations also contained methionine, which may act as a stabilizer. The formulations are shown in Table 11.

[0146] TIFF2024506931000012.tif44170

[0147] The three formulations were assayed for appearance before lyophilization, at time 0 after lyophilization (assayed after reconstitution), after 4 weeks of storage at 40° C. for the lyophilized formulation (assayed after reconstitution), after three freeze / thaw cycles for the liquid formulation, and after 2 or 4 weeks of storage at 25° C. for the liquid formulation. The results are shown in Table 12. SO-slightly opalescent; FVP=no visible particles.

[0148] TIFF2024506931000013.tif86170

[0149] As shown in Table 12, all three formulations were free of visible particles when observed by standard testing procedures before and after lyophilization, after four weeks of storage at 40° C. in the lyophilized state, after three freeze-thaw cycles, and after two and four weeks of storage at 25° C. in liquid form. When observed using more detailed testing procedures (USP <790> ), formulations containing 9% sucrose (F17 and F18) showed about 10 very small particles at time 0 after lyophilization, and all three formulations showed these small particles upon close inspection after the longest stress conditions of 4 weeks at 25° C. in liquid form.

[0150] The visual appearance of all lyophilized formulations was acceptable, appearing as white to off-white solid cakes with no evidence of physical collapse, shrinkage or cracking.

[0151] The formulations were also tested for lyophilization moisture content, reconstitution time, and retained potency after 4 weeks of storage at 40° C. as a lyophilized formulation, after 4 weeks of storage at 25° C. as a liquid formulation, and after three freeze / thaw cycles. As shown in Table 13, all three formulations were within acceptable limits.

[0152] TIFF2024506931000014.tif60170

[0153] The formulations were also assayed for HMW aggregate formation by SEC. All three formulations were found to be stable as measured by HMW aggregate formation before lyophilization, after lyophilization, and after 4 weeks of storage at 40° C. as lyophilized formulations. The results are shown in Table 14.

[0154] TIFF2024506931000015.tif254170

[0155] As shown in Table 15, in the liquid state, the 50 mg / ml formulation showed a slight increase in HMW aggregation as measured by SEC after 4 weeks at 25° C., with no change observed after three freeze / thaw cycles or after 4 weeks of storage at 25° C.

[0156] TIFF2024506931000016.tif254170

[0157] The formulations were then assayed for the presence and number of subvisible particles per milliliter using High Accuracy Fluid Particle Counting (HIAC) prior to lyophilization, at time 0 after lyophilization, and after 4 weeks of storage of the lyophilized formulations at 40° C. Particles were measured and counted using the HIAC instrument, and reported values ​​were the average of triplicate measurements for each sample. The results are shown in Table 16.

[0158] TIFF2024506931000017.tif71170

[0159] All three formulations showed very low subvisible particles per milliliter after lyophilization and after 4 weeks of storage at 40°C in the lyophilized state (acceptable limits for subvisible particles are less than 6000 particles / container (1000 particles / mL) for particles 10 μm and greater and less than 600 particles / container (100 particles / mL) for sizes 25 μm and greater).

[0160] Table 17 shows the number of subvisible particles in the liquid formulation under various conditions.

[0161] TIFF2024506931000018.tif254170

[0162] Subvisible particles were also within acceptable limits in the liquid formulation under the conditions tested, demonstrating that the formulation exhibits good physical stability.

[0163] The size distribution of the proteins in the formulations (aggregates (HMW), non-reduced monomers (main), fragments (LMW)) was measured by non-reducing CE-SDS (capillary electrophoresis with SDS denaturant). As shown in Table 18, the size distribution was found to be essentially unchanged after lyophilization and after storage of the lyophilized formulations at 40° C. for 4 weeks, indicating that the proteins are not physically degraded in these formulations.

[0164] TIFF2024506931000019.tif70170

[0165] The protein also underwent little physical degradation under the conditions assayed and was stable in the three formulations in the liquid state, as shown in Table 19.

[0166] TIFF2024506931000020.tif254170

[0167] The charge profile (% acidic species, % basic species, % monomer) of the proteins in the formulations was assayed by capillary isoelectric focusing (cIEF). Acidic species have a lower pI (isoelectric point) than the main peak and may include molecules with increased levels of deamidation of asparagine (Asn) and / or glutamic acid (Gln). Basic species have a higher pI relative to the main peak and may include molecules with C-terminal lysine and / or increased succinimide formation at aspartic acid (Asp) residues. As shown in Table 20, the charge profile was essentially unchanged after lyophilization and after storage of the lyophilized formulations at 40° C. for 4 weeks. These results indicate that the formulations protect against chemical modification of the proteins under these conditions.

[0168] TIFF2024506931000021.tif86170

[0169] The formulation also did not show any significant change in the charge profile of the protein in the liquid state under the conditions tested, as shown in Table 21. Thus, the formulation protects against chemical modification of the protein in the liquid state, indicating that the formulation is stable enough to allow manufacturing steps prior to lyophilization of the drug product.

[0170] TIFF2024506931000022.tif254170

[0171] Based on the test results, a final formulation (pH 6.0) of 50 mg / ml INBRX-109, 10 mM histidine HCl, 8% sucrose, and 0.2% poloxamer 188 was selected. 8% sucrose was selected over 9% sucrose based on initial appearance results where the 8% sucrose formulation (F8) was free of particles for an extended period of time after reconstitution compared to the 9% sucrose formulation (F7).

[0172] Batches of the final formulation of INBRX-109 were made and tested for various properties under various conditions as shown in Table 22.

[0173] TIFF2024506931000023.tif139170

[0174] Product quality attributes of the final formulation demonstrated that the formulation prevented the formation of visible particles both after reconstitution and storage of the lyophilized drug product, and maintained the physical and chemical stability and potency of INBRX-109.

[0175] Example 5: Stability of INBRX-109 drug product in formulation The stability of pilot-scale manufactured batches of INBRX-109 lyophilized drug product after storage at several temperatures was evaluated. The lyophilized product was reconstituted with sterile water to form an aqueous formulation (pH 6.0) containing 50 mg / ml INBRX-109, 10 mM histidine HCl, 8% sucrose, and 0.2% poloxamer 188.

[0176] The results of analytical testing of the reconstituted product are shown in Tables 23 and 24. INBRX-109 in the lyophilized drug product retained its physical, chemical (size distribution by SEC, CE-SDS, charge variant profile by iCIEF) and biological (potency) properties after 9 months of storage at the intended storage conditions of 2° C.-8° C., and for extended periods at 25° C. and 40° C., temperatures used to accelerate degradation of the product. Under these storage conditions, the lyophilized drug product remained substantially free of visible particles. In Tables 23 and 24, "SVP" means subvisible particles, "SEC" means size exclusion chromatography, "cIEF" means capillary isoelectric focusing, "CE-SDS-NR" means capillary electrophoresis-sodium dodecyl sulfate-non-reduced, "CE-SDS-R" means capillary electrophoresis-sodium dodecyl sulfate-reduced, "# / C" means particles / container, "HMW" means high molecular weight species, "Main" means main peak, "LMW" means low molecular weight species, "mOsm" means milliosmoles, "PFVP" means substantially free of visible particles, and "RH" means relative humidity.

[0177] TIFF2024506931000024.tif254170

[0178] TIFF2024506931000025.tif90170

[0179] The results of analytical testing of the lyophilized product prior to reconstitution are shown in Table 25. The data indicate that the lyophilized drug product retained its stability over time and across several temperatures, as very little change was detected in the physical appearance, reconstitution time, and moisture content of the drug product. "RH" means relative humidity, "NT" means not tested, and "reconstitution time" is the time (in seconds) from addition of diluent (sterile water for injection) to the product vial until the lyophilized solid was completely dissolved, as determined visually.

[0180] TIFF2024506931000026.tif173170

[0181] The present disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics of the present disclosure. The above-described embodiments are therefore to be considered in all respects as illustrative and not limiting of the present disclosure. The scope of the present disclosure is therefore defined by the appended claims, rather than the above detailed description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

[0182] TIFF2024506931000027.tif232170

Claims

1. 1. A pharmaceutical formulation comprising a DR5 binding polypeptide, the formulation comprising 20 mg / mL to 70 mg / mL of the DR5 binding polypeptide, 5 mM to 20 mM histidine, 7% to 10% (w / v) sucrose and 0.1% to 0.8% poloxamer P188, at a pH of 5.3 to 6.7, and the DR5 binding polypeptide comprising at least one VHH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:1, a CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a CDR3 comprising the amino acid sequence of SEQ ID NO:

3.

2. 2. The pharmaceutical formulation of claim 1, comprising 30 mg / mL to 60 mg / mL of DR5 binding polypeptide, optionally comprising 50 mg / mL of DR5 binding polypeptide.

3. 3. The pharmaceutical formulation of claim 1 or 2, comprising 7 mM to 15 mM histidine, optionally comprising 10 mM histidine.

4. The pharmaceutical formulation according to any one of claims 1 to 3, wherein said histidine is histidine HCl.

5. A pharmaceutical formulation according to any one of claims 1 to 4, comprising 8% to 9% sucrose, optionally comprising 8% sucrose or 9% sucrose.

6. 6. A pharmaceutical formulation according to any one of claims 1 to 5, comprising 0.2% to 0.4% poloxamer P188, optionally comprising 0.2% poloxamer P188.

7. 7. A pharmaceutical formulation according to any one of claims 1 to 6 comprising from 1 mM to 10 mM, from 2 mM to 8 mM, from 3 mM to 7 mM or from 4 mM to 6 mM methionine, optionally comprising 5 mM methionine.

8. 8. The pharmaceutical formulation of any one of claims 1 to 7, wherein the pH of the formulation is 5.4 to 6.6, 5.5 to 6.5, 5.6 to 6.4, 5.7 to 6.3 or 5.8 to 6.2, optionally the pH of the formulation is about 6.

9. 9. The pharmaceutical formulation of any one of claims 1 to 8, wherein the formulation comprises 50 mg / mL DR5 binding polypeptide, 10 mM histidine HCl, 8% sucrose and 0.2% poloxamer P188, and the pH of the formulation is about 6.

10. 10. The pharmaceutical formulation of claim 9, wherein the formulation consists essentially of 50 mg / mL DR5 binding polypeptide, 10 mM histidine HCl, 8% sucrose, 0.2% poloxamer P188 and water, and the pH of the formulation is about 6.

11. The pharmaceutical formulation according to any one of claims 1 to 10, wherein the DR5 binding polypeptide comprises a VHH domain comprising the amino acid sequence of SEQ ID NO:

4.

12. The pharmaceutical formulation of any one of claims 1 to 11, wherein the DR5 binding polypeptide comprises an Fc region.

13. The pharmaceutical preparation of claim 12, wherein the Fc region comprises the amino acid sequence of SEQ ID NO:

6.

14. The pharmaceutical formulation according to any one of claims 1 to 13, wherein the DR5 binding polypeptide has the structure VHH-linker-VHH-linker-Fc.

15. The pharmaceutical formulation of claim 14, wherein the VHH-linker-VHH comprises the amino acid sequence of SEQ ID NO:

5.

16. The pharmaceutical formulation of any one of claims 1 to 15, wherein the DR5 binding polypeptide comprises the amino acid sequence of SEQ ID NO:

7.

17. The pharmaceutical formulation according to any one of claims 1 to 16, wherein the DR5 binding polypeptide consists of the amino acid sequence of SEQ ID NO:

7.

18. 1. A lyophilized formulation comprising a DR5 binding polypeptide, the DR5 binding polypeptide comprising at least one VHH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:1, a CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a CDR3 comprising the amino acid sequence of SEQ ID NO:3, wherein when the lyophilized formulation is reconstituted in water to form an aqueous formulation, the aqueous formulation comprises 20 mg / mL to 70 mg / mL of the DR5 binding polypeptide, 5 mM to 20 mM histidine, 7% to 10% sucrose, and 0.1% to 0.5% poloxamer P188, and has a pH of 5.3 to 6.

7.

19. The lyophilized formulation of claim 18, wherein when the lyophilized formulation is reconstituted in water to form an aqueous formulation, the aqueous formulation is a pharmaceutical formulation according to any one of claims 1 to 17.

20. A lyophilized formulation formed by lyophilizing the pharmaceutical formulation of any one of claims 1 to 17.

21. 21. The lyophilized formulation of any one of claims 18 to 20, wherein after storage at 2°C to 8°C for up to 3 months, up to 6 months, up to 9 months, up to 12 months or more than 12 months, the lyophilized formulation is an off-white, uniform and fine cake free of any visible impurities.

22. 22. The lyophilized formulation of any one of claims 18 to 21, wherein after storage at 2°C to 8°C for up to 3 months, up to 6 months, up to 9 months, up to 12 months, or more than 12 months, the aqueous formulation is substantially free of visible particles upon reconstitution of the formulation in water to form an aqueous formulation.

23. 23. The lyophilized formulation of any one of claims 18 to 22, wherein following storage at 2°C to 8°C for up to 3 months, up to 6 months, up to 9 months, up to 12 months or more than 12 months, upon reconstitution of the formulation in water to form an aqueous formulation, less than 3% or less than 2% of the DR5 binding polypeptide present in the aqueous formulation is aggregated as measured by size exclusion chromatography.

24. 24. The lyophilized formulation of claim 23, wherein less than 1% or less than 0.5% of the DR5 binding polypeptide present in the aqueous formulation is degraded as measured by size exclusion chromatography.

25. A pharmaceutical formulation formed by reconstituting the lyophilized formulation of any one of claims 18 to 24.

26. A pharmaceutical formulation according to any one of claims 1 to 17 and 25 for treating cancer.

27. 27. The pharmaceutical preparation of claim 26, wherein the cancer is chondrosarcoma, mesothelioma, colorectal cancer, Ewing's sarcoma, or pancreatic adenocarcinoma.