Formulation of DR5-binding polypeptide

Abstract

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

Description

[Technical Field]

[0001] [Cross-reference of related applications] This application claims priority to U.S. Provisional Application No. 63 / 151,131, filed on 19 February 2021, which in whole constitutes a part of this specification by reference for all purposes.

[0002] The present invention relates to a formulation of DR5-binding protein and a method for using the formulation. Such a method includes, but is not limited to, a method 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 the TNF-associated apoptosis-inducing ligand (TRAIL). TRAIL has evolved to play a crucial role in mammalian development and host defense by selectively removing unwanted, infected, and malignant cells from healthy cell populations. TRAIL induces cell death via caspase-dependent apoptosis by binding to DR4 or DR5, members of the TNF receptor family. DR5 appears to be the primary receptor on tumor cells that promotes the tumor-defining activity observed in the TRAIL pathway. DR5 is activated by its native ligand, TRAIL, and by bringing three DR5 receptors into proximity, it activates intracellular caspase-8, initiating the activation of other cell death-inducing caspases such as caspase-9 and caspase-3. Therefore, clustering of DR5 receptors for efficient cell death is required for the initiation of this cell death pathway. [Overview of the Initiative]

[0004] The use of stable liquid and lyophilized pharmaceutical formulations of polyvalent DR5-binding polypeptides capable of agonizing DR5 signaling, which mediates direct cell death, as well as pharmaceutical formulations for the treatment of cancer, is provided herein. [Means for solving the problem]

[0005] Embodiment 1. A pharmaceutical formulation comprising a DR5-binding polypeptide, wherein the formulation comprises 20 mg / mL to 70 mg / mL of the DR5-binding polypeptide, 5 mM to 20 mM of histidine, 7% to 10% (w / v) of sucrose, and 0.1% to 0.8% of poloxamer P188, and has a pH of 5.3 to 6.7, and the DR5-binding polypeptide comprises at least one VHH domain comprising CDR1 containing the amino acid sequence of SEQ ID NO: 1, CDR2 containing the amino acid sequence of SEQ ID NO: 2, and CDR3 containing the amino acid sequence of SEQ ID NO: 3. Embodiment 2. The pharmaceutical formulation of Embodiment 1, comprising a DR5-binding polypeptide in a concentration of 30 mg / mL to 60 mg / mL. Embodiment 3. The pharmaceutical formulation of Embodiment 1, comprising 50 mg / mL of a DR5-binding polypeptide. Embodiment 4. A pharmaceutical formulation according to any one of Embodiments 1 to 3, containing 7 mM to 15 mM histidine. Embodiment 5. A pharmaceutical formulation according to any one of Embodiments 1 to 3, comprising 10 mM histidine. Embodiment 6. A pharmaceutical formulation according to any one of Embodiments 1 to 5, wherein the histidine is histidine HCl. Embodiment 7. A pharmaceutical formulation according to any one of Embodiments 1 to 6, comprising 8% to 9% sucrose. Embodiment 8. A pharmaceutical formulation according to any one of Embodiments 1 to 7, comprising 8% sucrose or 9% sucrose. Embodiment 9. A pharmaceutical formulation according to any one of Embodiments 1 to 8, comprising 0.2% to 0.4% poloxamer P188. Embodiment 10. A pharmaceutical formulation according to any one of Embodiments 1 to 9, comprising 0.2% poloxamer P188. Embodiment 11. A pharmaceutical formulation according to any one of Embodiments 1 to 10, comprising methionine in a concentration of 1 mM to 10 mM, 2 mM to 8 mM, 3 mM to 7 mM, or 4 mM to 6 mM. Embodiment 12. A pharmaceutical formulation according to any one of Embodiments 1 to 11, comprising 5 mM methionine. Embodiment 13. A pharmaceutical formulation according to 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. A pharmaceutical formulation according to any one of Embodiments 1 to 13, wherein the pH of the formulation is approximately 6. Embodiment 15. A pharmaceutical formulation according to any one of Embodiments 1 to 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 approximately 6. Embodiment 16. The pharmaceutical formulation of Embodiment 15, wherein the formulation essentially consists of 50 mg / mL of DR5-binding polypeptide, 10 mM histidine HCl, 8% sucrose, 0.2% poloxamer P188, and water, and the pH of the formulation is approximately 6. Embodiment 17. A pharmaceutical formulation according to any one of Embodiments 1 to 16, wherein the DR5-binding polypeptide comprises a VHH domain containing the amino acid sequence of SEQ ID NO: 4. Embodiment 18. A pharmaceutical formulation according to any one of Embodiments 1 to 17, wherein the DR5-binding polypeptide includes an Fc region. Embodiment 19. The pharmaceutical formulation of Embodiment 18, wherein the Fc region contains the amino acid sequence of SEQ ID NO: 6. Embodiment 20. A pharmaceutical formulation according to 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 VHH-linker-VHH contains the amino acid sequence of SEQ ID NO: 5. Embodiment 22. A pharmaceutical formulation according to any one of Embodiments 1 to 21, wherein the DR5-binding polypeptide contains the amino acid sequence of SEQ ID NO: 7. Embodiment 23. A pharmaceutical formulation according to 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, wherein 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, and the lyophilized formulation is reconstituted in water to form an aqueous formulation, wherein the aqueous formulation contains 20 mg / mL to 70 mg / mL of 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. Embodiment 25. The lyophilized formulation of Embodiment 24, wherein when the formulation is reconstituted in water to form an aqueous formulation, the aqueous formulation contains 30 mg / mL to 60 mg / mL of DR5-binding polypeptide. Embodiment 26. The lyophilized formulation of Embodiment 24, wherein the formulation is reconstituted in water to form an aqueous formulation, and the aqueous formulation contains 50 mg / mL of DR5-binding polypeptide. Embodiment 27. A lyophilized formulation of any one of Embodiments 24 to 26, wherein when the formulation is reconstituted in water to form an aqueous formulation, the aqueous formulation contains 7 mM to 15 mM histidine. Embodiment 28. A lyophilized formulation of any one of Embodiments 24 to 26, wherein the formulation is reconstituted in water to form an aqueous formulation, the aqueous formulation containing 10 mM histidine. Embodiment 29. A lyophilized formulation according to any one of Embodiments 24 to 28, wherein histidine is histidine HCl. Embodiment 30. A lyophilized formulation according to any one of Embodiments 24 to 29, wherein when the lyophilized formulation is reconstituted in water to form an aqueous formulation, the aqueous formulation contains 8% to 9% sucrose. Embodiment 31. A lyophilized formulation of any one of Embodiments 24 to 30, wherein when the formulation is reconstituted in water to form an aqueous formulation, the aqueous formulation contains 8% or 9% sucrose. Embodiment 32. A lyophilized formulation of any one of Embodiments 24 to 31, wherein the formulation is reconstituted in water to form an aqueous formulation, the aqueous formulation containing 0.2% to 0.4% poloxamer P188. Embodiment 33. A lyophilized formulation according to any one of Embodiments 24 to 32, wherein when the formulation is reconstituted in water to form an aqueous formulation, the aqueous formulation contains 0.2% poloxamer P188. Embodiment 34. A lyophilized formulation according to any one of Embodiments 24 to 33, wherein when the formulation is reconstituted in water to form an aqueous formulation, the aqueous formulation contains methionine at 1 mM to 10 mM, 2 mM to 8 mM, 3 mM to 7 mM or 4 mM to 6 mM. Embodiment 35. A lyophilized formulation according to any one of Embodiments 24 to 33, wherein when the formulation is reconstituted in water to form an aqueous formulation, the aqueous formulation contains 5 mM methionine. Embodiment 36. A lyophilized formulation according to any one of Embodiments 24 to 35, wherein when the formulation is reconstituted in water to form an aqueous formulation, the pH of the aqueous 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 37. A lyophilized formulation according to any one of Embodiments 24 to 35, wherein when the formulation is reconstituted in water to form an aqueous formulation, the pH of the aqueous formulation is about 6. Embodiment 38. A lyophilized formulation according to any one of Embodiments 24 to 37, wherein when the formulation is reconstituted in water to form an aqueous formulation, the aqueous formulation contains 50 mg / mL DR5-binding polypeptide, 10 mM histidine HCl, 8% sucrose and 0.2% poloxamer P188, and has a pH of about 6. Embodiment 39. A lyophilized formulation according to Embodiment 28, wherein when the formulation is reconstituted in water to form an aqueous formulation, the aqueous 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. Embodiment 40. A lyophilized formulation according to any one of Embodiments 24 to 39, wherein the DR5-binding polypeptide contains a VHH domain comprising the amino acid sequence of SEQ ID NO: 4. Embodiment 41. A lyophilized formulation according to any one of Embodiments 24 to 40, wherein the DR5-binding polypeptide contains an Fc region. Formulation 42. The lyophilized formulation of Formulation 41, wherein the Fc region contains the amino acid sequence of SEQ ID NO: 6. Formulation 43. The lyophilized formulation according to any one of Formulations 24 to 42, wherein the DR5-binding polypeptide has a structure of VHH-linker-VHH-linker-Fc. Formulation 44. The lyophilized formulation of Formulation 43, wherein VHH-linker-VHH contains the amino acid sequence of SEQ ID NO: 5. Formulation 45. The lyophilized formulation according to any one of Formulations 24 to 44, wherein the DR5-binding polypeptide contains the amino acid sequence of SEQ ID NO: 7. Formulation 46. The lyophilized formulation according to any one of Formulations 24 to 44, wherein the DR5-binding polypeptide consists of the amino acid sequence of SEQ ID NO: 7. Formulation 47. The lyophilized formulation formed by lyophilizing any one of the pharmaceutical formulations of Formulations 1 to 23. Formulation 48. The lyophilized formulation according to any one of Formulations 24 to 47, which is an off-white, uniform, and elegant cake containing no visible impurities after being stored 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. Formulation 49. The lyophilized formulation according to any one of Formulations 24 to 48, wherein when the formulation is reconstituted in water to form an aqueous formulation after being stored 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 contains substantially no visible particles. Formulation 50. The lyophilized formulation according to any one of Formulations 24 to 49, wherein when the formulation is reconstituted in water to form an aqueous formulation after being stored 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, 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. Formulation 51. The lyophilized formulation of Formulation 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 one of the lyophilized formulations from Embodiments 24 to 51. Embodiment 53. A method for treating cancer, comprising administering one of the pharmaceutical formulations of Embodiments 1 to 23 and 52 to a subject having cancer. Embodiment 54. The method of Embodiment 53, wherein the cancer is chondrosarcoma, mesothelioma, Ewing's sarcoma, colorectal cancer, or pancreatic adenocarcinoma. [Modes for carrying out the invention]

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

[0007] Definitions and various embodiments The section headings used in this specification are for organizational purposes only and should not be construed as limiting the subjects described.

[0008] All references cited herein, including patent applications, patent publications, and Genbank accession numbers, are incorporated by reference in the same manner as each individual reference is specifically and individually indicated to be incorporated by reference as part of this specification.

[0009] The techniques and procedures described or referenced herein are generally well understood and typically refer to, 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 METHODS IN ENZYMOLOGY (Academic Press, Inc.) series; 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 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), as well as their latest editions, are used in accordance with the widely used methodologies and customary methodologies of those skilled in the art.

[0010] Unless otherwise specified, scientific and technical terms used in connection with this disclosure shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise required by context or explicitly indicated, singular nouns shall include the plural and plural nouns shall include the singular. In the event of any inconsistency in definitions between various sources or references, the definitions provided herein shall prevail.

[0011] Generally, the numbering of residues in immunoglobulin heavy chains is based on the EU index numbering, as described 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 described by Kabat" refers to the residue numbering of human IgG1 EU antibodies.

[0012] Embodiments of the Invention described herein are understood to include embodiments "consisting" and / or embodiments "consisting essentially of". Where used herein, the singular forms ("a", "an", and "the") include plural references unless otherwise indicated. The use of the term "or" herein is not construed as meaning that the options are mutually exclusive.

[0013] In this application, the use of “or” means “and / or” unless otherwise explicitly specified or understood by those skilled in the art. In the context of multiple dependent claims, the use of “or” refers to two or more prior independent or dependent claims.

[0014] The terms “reference sample,” “reference cells,” or “reference tissue” refer to a sample having at least one known characteristic that can be used for comparison with a sample having at least one unknown characteristic. In some embodiments, the reference sample may be used as a positive or negative indicator. By using the reference sample, the levels of proteins and / or mRNA present in healthy tissue, for example, can be established against the levels of proteins and / or mRNA present in the sample having the unknown characteristic. In some embodiments, the reference sample is a sample derived from the same subject but from a different part of the subject than the part being tested. In some embodiments, the reference sample is a sample derived from a tissue region surrounding or adjacent to the cancer. In some embodiments, the reference sample is not derived from the subject being tested but from a subject known to have or not have the disorder of interest (e.g., a specific cancer or DR5-related disorder). In some embodiments, the reference sample is derived from the same subject but from a time before the subject developed cancer. In some embodiments, the reference sample is a sample derived from a benign cancer sample from the same or a different subject. When a negative reference sample is used for comparison, the expression level or amount of the molecule in question in the negative reference sample indicates a level at which a person skilled in the art would recognize, considering this disclosure, that the molecule is absent and / or present at a low level. When a positive reference sample is used for comparison, the expression level or amount of the molecule in question in the positive reference sample indicates a level at which a person skilled in the art would recognize, considering this disclosure, that the molecule is present at a certain level.

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

[0016] The terms “nucleic acid molecule,” “nucleic acid,” and “polynucleotide” are used interchangeably and may refer to polymers of nucleotides. Such polymers of nucleotides may include, but are not limited to, natural and / or unnatural nucleotides, including DNA, RNA, and PNA. “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 the minimum length. Such polymers of amino acid residues may include, but are not limited to, natural or unnatural amino acid residues, peptides, oligopeptides, dimers, trimers, and polymers of amino acid residues. This definition includes both full-length proteins and their fragments. These terms also include post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, and phosphorylation. Furthermore, for the purposes of this disclosure, “polypeptide” refers to a protein that includes modifications (generally conserved in nature), such as deletions, additions, and substitutions of the natural sequence, as long as the protein maintains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or accidental, such as by mutations in the host producing the protein or errors in PCR amplification.

[0018] As used herein, the terms “DR5,” “cell death receptor 5,” and “TNFRSF10B” refer to any naturally occurring mature DR5 resulting from the processing of DR5 precursors within cells. Unless otherwise indicated, this 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). This term also includes naturally occurring variants of DR5, such as splice variants or allele variants. A non-limiting exemplary precursor human DR5 amino acid sequence is shown, for example, in NCBI accession number 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 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” when it reacts or associates with a particular cell or substance more frequently, more rapidly, for a longer duration and / or with higher affinity than it reacts or associates with other cells or substances. A single-domain antibody (sdAb) or VHH-containing polypeptide “specifically binds” or “preferentially binds” to a target when it binds with higher affinity, avidity, more easily, and / or for a longer duration than it binds to other substances. For example, an sdAb or VHH-containing polypeptide that specifically or preferentially binds to a DR5 epitope is an sdAb or VHH-containing polypeptide that binds to this epitope with higher affinity, avidity, more easily, and / or for a longer duration than it binds to other DR5 epitopes or non-DR5 epitopes. Furthermore, by interpreting this definition, it can be understood, for example, that 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. Therefore, "specific binding" or "preferential binding" does not necessarily require (though it may include) exclusive binding. Generally, though not always, references to binding imply preferential binding. "Specificity" refers to the ability of a binding protein to selectively bind to an antigen.

[0020] The terms “inhibit” or “inhibit” refer to a reduction or cessation of any phenotypic feature, or a reduction or cessation of the incidence, degree, or likelihood of such feature. “Reduce” or “inhibit” means to reduce, reduce, or cessate activity, function, and / or quantity compared to a reference. In some embodiments, “reduce” or “inhibit” means the ability to cause an overall reduction of 10% or more. In some embodiments, “reduce” or “inhibit” means the ability to cause an overall reduction of 50% or more. In some embodiments, “reduce” or “inhibit” means the ability to cause an overall reduction of 75%, 85%, 90%, 95% or more. In some embodiments, the above quantities are inhibited or reduced over a certain period of time compared 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 a chemically active surface configuration of a molecule such as an amino acid, polypeptide, or sugar side chain, and possess specific three-dimensional structural and charge characteristics. Epitopes can be formed from both continuous residues and / or juxtaposed discontinuous residues (e.g., amino acids, nucleotides, sugars, or lipid moieties) of the target molecule. Epitopes formed from continuous residues (e.g., amino acids, nucleotides, sugars, or lipid moieties) are usually retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are usually lost upon treatment with denaturing solvents. Epitopes may, but are not limited to, comprise at least three, at least five, or eight to ten 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 identified by a certain minimum distance from a CDR residue on the antigen-binding molecule. In some embodiments, an epitope may be identified by the above distance and further limited to those residues involved in the binding (e.g., hydrogen bonding) between residues of the antigen-binding molecule and antigen residues. Epitopes can also be identified by various scans. For example, an alanine scan or an arginine scan may indicate one or more residues with which the antigen-binding molecule can interact. Unless expressly indicated, a set of residues as an epitope does not exclude other residues from being part of an epitope for a particular antigen-binding molecule. Rather, the presence of such a set indicates a minimal sequence (or set of types) of epitopes. Therefore, in some embodiments, the set of residues identified as an epitope represents the smallest epitope associated with the antigen, rather than being an exclusive list of residues for epitopes on the antigen.

[0022] A “nonlinear epitope” or “stereotypic epitope” comprises a discontinuous polypeptide, amino acids, and / or sugars within an antigenic protein to which an antigen-binding molecule specific to the epitope binds. In some embodiments, at least one residue is discontinuous with other indicated residues of the epitope, but one or more residues may be continuous with other residues.

[0023] A "linear epitope" comprises a sequence of polypeptides, amino acids, and / or sugars within an antigenic protein to which an antigen-binding molecule specific to the epitope binds. Note that in some embodiments, not all residues within the linear epitope need to be directly bound (or involved in binding) by the antigen-binding molecule. In some embodiments, the linear epitope may originate from immunization with a peptide consisting of a sequence of linear epitopes, or from a structural segment of a protein relatively isolated from the rest of the protein (thus, the antigen-binding molecule may interact, at least primarily, with that very sequence segment).

[0024] The term “antibody” is used in its broadest sense and is not limited to, but encompasses a variety of polypeptides, including 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 comprising any of the aforementioned fragments insofar as they exhibit the desired antigen-binding activity. In some embodiments, antibodies include a dimerizing domain. Such dimerizing domains include, but are not limited to, a heavy chain constant domain (comprising CH1, hinge, CH2, and CH3, where CH1 typically pairs with the 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" is not limited to this, but can include chimeric antibodies, humanized antibodies, and antibodies from various species such as camels (including llamas), sharks, mice, humans, and cynomolgus monkeys.

[0026] As used herein, the term “antigen-binding domain” refers to a portion of the 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 including 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 including 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 including 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 terms "VHH," "VHH domain," or "VHH antigen-binding domain" refer to the antigen-binding portion of a single-domain antibody, such as a camel antibody or shark antibody. In some embodiments, the VHH comprises three CDRs and four framework regions, represented as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, the VHH may be cleaved at the N-terminus or C-terminus to contain only partial FR1 and / or FR4, or to lack one or both of those framework regions, as long as the VHH substantially maintains antigen-binding and specificity.

[0028] The terms "single-domain antibody" and "sdAb" are used herein without distinction to refer to antibodies containing at least one monomeric domain, such as a VHH domain without a light chain and Fc region. In some embodiments, an sdAb is a dimer of two polypeptides, each polypeptide containing at least one VHH domain and an Fc region. As used herein, the terms "single-domain antibody" and "sdAb" include polypeptides containing multiple VHH domains, for example, polypeptides having the structure VHH1-VHH2-Fc or VHH1-VHH2-VHH3-Fc, where VHH1, VHH2, and VHH3 may be the same or different.

[0029] The term "VHH-containing polypeptide" refers to a polypeptide containing at least one VHH domain. In some embodiments, a VHH polypeptide contains 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 contains 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 VHH1-Fc, VHH1-VHH2-Fc, and VHH1-VHH2-VHH3-Fc, where VHH1, VHH2, and VHH3 may be the same or different. In some embodiments of such structures, one VHH may be linked to another VHH by a linker, or one VHH may be linked to an Fc by a linker. In some such embodiments, the linker contains 1 to 20 amino acids, preferably 1 to 20 amino acids mainly composed of glycine and optionally serine. In some embodiments, if the VHH-containing polypeptide contains Fc, it forms a dimer. Thus, the structure VHH1-VHH2-Fc is considered tetravalent when it forms a dimer (i.e., the dimer has four VHH domains). Similarly, the structure VHH1-VHH2-VHH3-Fc is considered hexavalent when 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 polypeptides) from a substantially homogeneous population of antibodies. That is, the individual antibodies constituting the population are identical except for naturally occurring mutations that may exist in small amounts. Monoclonal antibodies are highly specific and target a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody is an antibody against a single determinant on an antigen. Therefore, samples of monoclonal antibodies can bind to the same epitope on an antigen. The modifying phrase "monoclonal" indicates the nature of antibodies obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies can be produced by the hybridoma method, first described in Kohler and Milstein, 1975, Nature 256:495, or by recombinant DNA methods, as described in U.S. Patent No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries prepared using techniques such as those described, for example, McCafferty et al., 1990, Nature 348:552-554.

[0031] The term "CDR" refers to a complementarity determination region defined by a specific form to at least one person skilled in the art. In some embodiments, a CDR may be defined according to any of the following: a Chothia numbering scheme, a Kabat numbering scheme, a combination of Kabat and Chothia, an AbM definition, and / or a contact definition. VHH includes three CDRs represented as CDR1, CDR2, and CDR3.

[0032] As used herein, the term “heavy chain constant region” means at least three heavy chain constant domains, i.e., C H 1. Hinge, C H 2, and C HThis refers to the region containing 3. Naturally, deletions and modifications within a domain that do not alter its function are included within the scope of the term "heavy chain constant region" unless otherwise specified. Non-limiting exemplary heavy chain constant regions include γ, δ, and α. Non-limiting exemplary heavy chain constant regions also include ε and μ. Each heavy chain constant region corresponds to one antibody isotype. For example, an antibody containing the γ constant region is an IgG antibody, an antibody containing the δ constant region is an IgD antibody, and an antibody containing the α constant region is an IgA antibody. Furthermore, an antibody containing the μ constant region is an IgM antibody, and an antibody containing the ε constant region is an IgE antibody. A particular isotype may be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 antibodies (including the γ1 constant region), IgG2 antibodies (including the γ2 constant region), IgG3 antibodies (including the γ3 constant region), and IgG4 antibodies (including the γ4 constant region); IgA antibodies include, but are not limited to, IgA1 antibodies (including the α1 constant region) and IgA2 antibodies (including the α2 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 the heavy chain constant region containing CH2 and CH3. In some embodiments, the Fc region includes a hinge, CH2, and CH3. In various embodiments, when the Fc region includes a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region may 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, “acceptor human framework” means heavy chain variable domains (V) derived from the human immunoglobulin framework or human consensus framework, as discussed herein. H) It is a framework comprising the amino acid sequence of the framework. An acceptor human framework derived from a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence or can comprise changes in the amino acid sequence. 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 total strength 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 molecule X for its partner Y is generally represented by the dissociation constant (K D ) or K D(見かけ) . Affinity can be measured by conventional methods known in the art, including the methods 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, methods requiring BIAcore (trademark), Octet (trademark), or flow cytometry.

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

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

[0038] The term "biological activity" refers to any one or more biological properties of a molecule, whether naturally occurring as observed in vivo, or provided or made possible by recombinant means. Examples of biological properties include, but are not limited to, ligand binding, induction or increase of cell proliferation, and induction or increase of cytokine expression.

[0039] An "agonist" antibody 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%, or 85% or more.

[0040] Antagonist antibodies, blocking antibodies, or neutralizing antibodies are antibodies that inhibit, reduce, and / or inactivate the biological activity of a target antigen. In some embodiments, neutralizing antibodies bind to an antigen and reduce its biological activity by at least about 20%, 40%, 60%, 80%, 85%, 90%, 95%, or 99% or more.

[0041] "Affinity-matured" sdAb or VHH-containing polypeptides refer to sdAb or VHH-containing polypeptides having one or more such modifications in one or more CDRs compared to a parent sdAb or VHH-containing polypeptide that does not have modifications that result in improved affinity of the sdAb or VHH-containing polypeptide to the antigen.

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

[0043] An "effector-positive Fc region" possesses the "effector function" of a naturally occurring 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, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Such effector functions generally require the combination of the Fc region with a binding domain (e.g., an antibody-variable domain) and can be evaluated using various assays.

[0044] The "natural Fc region" contains amino acid sequences identical to those found in naturally occurring Fc regions. Natural human Fc regions include natural human IgG1 Fc regions (non-A allotype and A allotype), natural human IgG2 Fc regions, natural human IgG3 Fc regions, and natural human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0045] A "mutant Fc region" includes an amino acid sequence that differs from the amino acid sequence of the Fc region of the natural sequence by at least one amino acid modification. In some embodiments, a "mutant Fc region" includes an amino acid sequence that differs from the amino acid sequence of the Fc region of the natural sequence by at least one amino acid modification, but retains at least one effector function of the Fc region of the natural sequence. In some embodiments, the mutant 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, compared to the Fc region of the natural sequence or the Fc region of the parent polypeptide. In some embodiments, the mutant Fc region described herein has at least about 80% sequence identity with the Fc region of the natural sequence and / or the Fc region of the parent polypeptide, at least about 90% sequence identity with them, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with them.

[0046] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, FcγR is a naturally occurring human FcR. In some embodiments, FcR is a receptor that binds to an IgG antibody (gamma receptor) and includes receptors of the FcγRI subclass, FcγRII subclass, and FcγRIII subclass, including allele 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. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The 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 have been reviewed, e.g., 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 to be identified in the future, are encompassed herein by the term “FcR”. 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 the regulation of immunoglobulin homeostasis.Methods for measuring binding to FcRn are known (see, for example, 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), and International Publication No. 2004 / 92219 (Hinton et al.)).

[0047] As used herein, the terms “substantially similar” or “substantially identical” indicate a sufficiently high degree of similarity between two or more numerical values ​​such that a person skilled in the art would consider the difference between them to have little or no biological significance and / or statistical significance in the context of the biological characteristics measured by those values. In some embodiments, two or more substantially similar values ​​differ by only one approximate number or less than 5%, 10%, 15%, 20%, 25%, or 50%.

[0048] A polypeptide "mutant" means a biologically active polypeptide that, after aligning its sequence and introducing gaps as necessary to achieve maximum percentage sequence identity, has at least about 80% amino acid sequence identity with the native polypeptide, without considering any conservative substitutions as part of the sequence identity. Such mutants 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, the mutant has at least about 80% amino acid sequence identity. In some embodiments, the mutant has at least about 90% amino acid sequence identity. In some embodiments, the mutant has at least about 95% amino acid sequence identity with the native polypeptide.

[0049] As used herein, “percent (%) amino acid sequence identity” and “homology” with respect to peptide sequences, polypeptide sequences, or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide sequence or polypeptide sequence, after the sequences have been aligned and gaps introduced as necessary to achieve maximum percentage sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for determining percentage amino acid sequence identity can be achieved in various ways within the scope of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN® (DNASTAR) software. A person skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0050] Amino acid substitutions may include, but are not limited to, replacing one amino acid in a polypeptide with another. Exemplary substitutions are shown in Table 1. Amino acid substitutions can be introduced into the target antibody, and the product can be screened for desired activity, such as retention / improvement of antigen binding, decreased immunogenicity, or improvement of ADCC or CDC.

[0051] TIFF0007894878000001.tif111170

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

[0053] Non-conservative substitution involves replacing one member of one of these classes with one of another.

[0054] The term "vector" is used to describe a polynucleotide that can be manipulated to contain one or more cloned polynucleotides that can be grown in a host cell. A vector may contain one or more of the following elements: an origin of replication, one or more regulatory sequences that regulate the expression of the target polypeptide (e.g., promoters and / or enhancers), and / or one or more selectable marker genes (e.g., antibiotic resistance genes and genes that can be used in colorimetric assays, e.g., β-galactosidase). The term "expression vector" refers to a vector used to express the target polypeptide in a host cell.

[0055] "Host cell" refers to a cell that may or may have been a recipient of a vector or isolated polynucleotide. A host cell may be a prokaryotic or eukaryotic cell. Exemplary eukaryotic cells include mammalian cells such as primate or non-primate animal cells, fungal cells such as yeast, plant cells, and insect cells. Non-exclusive exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), and 293 and CHO cells, as well as their derivatives, such as 293-6E cells, CHO-DG44 cells, CHO-K1 cells, CHO-S cells, and CHO-DS cells. Host cells include offspring of a single host cell, but due to spontaneous, accidental, or intentional mutations, the offspring are not necessarily identical (in morphology or genomic DNA complementarity) to the original parent cell. Host cells include cells transfected in vivo with the polynucleotide(s) provided herein.

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

[0057] The terms “individual” and “subject” are used herein without distinction to refer to animals, such as mammals. In some embodiments, but not limited to, methods are provided for treating mammals, including humans, rodents, monkeys, cats, dogs, horses, cattle, pigs, sheep, goats, laboratory mammals, mammalian livestock, mammalian sport animals, and mammalian pets. In some examples, “individual” or “subject” refers to an individual or subject that requires treatment for a disease or disorder. In some embodiments, a subject receiving treatment may be a patient who has been identified as having or being at sufficient risk of developing a disorder relevant to the treatment.

[0058] As used herein, “disease” or “disorder” means a condition that requires and / or is desired to be treated.

[0059] The terms “tumor cells,” “cancer cells,” “cancer,” “tumor,” and / or “neoplasm” are used herein without distinction unless otherwise specified, and refer to cells (or groups of cells) that exhibit uncontrolled proliferation and / or abnormal increased cell survival and / or inhibition of apoptosis that interfere with the normal functioning of organs and systems of the body. This definition includes benign and malignant cancers, polyps, hyperplasia, and latent tumors or micrometastases.

[0060] The terms "cancer" and "tumor" encompass solid tumors and hematological / lymphatic cancers, as well as malignant tumors, pre-malignant tumors, 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 cancer and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, chondrosarcoma, Ewing's sarcoma, colorectal cancer, connective tissue cancer, gastrointestinal cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, and gastric cancer. Cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatocellular carcinoma, carcinoma in situ, kidney cancer or renal 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 cancer, mesothelioma, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer This includes lymphomas such as testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary tract cancer, vulvar cancer, Hodgkin lymphoma and non-Hodgkin lymphoma, and B-cell lymphomas (low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocyte (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small undivided cell NHL, giant lesion NHL). This includes mantle cell lymphoma (including L, AIDS-associated lymphoma, and Waldenström 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 nevus disorders, edema (such as edema associated with brain tumors), and abnormal angiogenesis associated with Meigs syndrome.

[0061] In some embodiments, “increase” or “decrease” refers to a statistically significant increase or decrease, respectively. As will be apparent to those skilled in the art, “modulation” may also include causing a change (which may be either an increase or a decrease) in the affinity, avidity, specificity and / or selectivity of the target or antigen to one or more of its ligands, binding partners, partners with which it associates to homomultimeric or heteromultimeric forms, or substrates, compared to the same conditions except the presence of the test agent; causing a change (which may be either an increase or a decrease) in the sensitivity of the target or antigen to one or more conditions (such as pH, ionic strength, presence of cofactors, etc.) in the culture medium or environment in which the target or antigen is present; and / or cell proliferation or cytokine production. This may be determined, depending on the target involved, by any suitable method known or described herein and / or by any suitable assay.

[0062] As used herein, “treatment” refers to a method for obtaining a beneficial or desired clinical outcome. As used herein, “treatment” refers to any administration or application of a therapeutic agent for a disease in a mammal, including humans. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, relief of one or more symptoms, reduction of disease severity, 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, improvement of disease condition, inhibition of disease or disease progression, inhibition or slowing of disease or its progression, cessation of its development, and remission (whether partial or total). “Treatment” also includes mitigation of the pathological consequences of proliferative disorders. The methods provided herein are intended to achieve one or more of these aspects of treatment. Accordingly, the term treatment does not need to eliminate all aspects of the disorder 100 percent.

[0063] "Improvement" means that one or more symptoms are reduced or improved compared to when no treatment is administered. "Improvement" also includes a reduction or decrease in the duration of symptoms.

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

[0065] The term "biological sample" means any quantity of material from a living or formerly living organism. Such material includes, but is 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 terms “control” or “reference” refer to a composition known to not contain the analyte (“negative control”) or a composition known to contain the analyte (“positive control”). A positive control may contain the analyte at a known concentration.

[0067] As used herein, “delaying the onset of a disease” means delaying, preventing, slowing, stabilizing, suppressing, and / or prolonging the onset of a disease (such as cancer). This delay can be of varying lengths depending on the disease history and / or the individual being treated. As will be apparent to those skilled in the art, a sufficient or substantial delay may effectively encompass prevention in that the individual does not develop the disease. For example, it can delay the onset of terminal cancer, such as the development of metastases.

[0068] As used herein, “prevention” includes bringing about prevention of the onset 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” refer to or require prevention only for the period measured, and not for or require complete prevention over the entire period.

[0069] The "therapeutic effective dose" of a substance / molecule, agonist, or antagonist may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the substance / molecule, agonist, or antagonist's ability to induce the desired response in the individual. The therapeutic effective dose is also the amount in which the therapeutically beneficial effect outweighs any toxic or adverse effects of the substance / molecule, agonist, or antagonist. The therapeutic effective dose may be delivered in one or more doses. The therapeutic effective dose refers to the amount effective in achieving the desired therapeutic and / or prophylactic outcome over the required duration at the required dosage.

[0070] The terms “pharmaceutical preparation” and “pharmaceutical composition” are used interchangeably and refer to preparations that are in a form that enables the biological activity of the active ingredient(s) to be effective and that do not contain additional ingredients that are unacceptably toxic to the subject to whom the preparation is administered. Such preparations may be sterilized.

[0071] A "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier commonly used in the art for use with a therapeutic agent containing a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dose and concentration used and is compatible with the other components 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 additional therapeutic agents includes simultaneous (concurrent) administration and sequential administration in any order.

[0073] The term "simultaneously" is used herein to mean the administration of two or more therapeutic agents in which at least a portion of the administrations overlaps in time, or the administration of one therapeutic agent is included in a short period relative to the administration of another therapeutic agent, or the therapeutic effects of both therapeutic agents overlap for at least a certain period of time.

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

[0075] As used herein, “in combination with” means adding another treatment to the administration of one treatment. Therefore, “in combination with” means administering one treatment before, during, or after administering another treatment to an individual.

[0076] The term “package insert” is used to refer to instructions for use that are typically included in the market packaging of a therapeutic product, and which include information relating to the use of such therapeutic product, including instructions for use, method of use, dosage, administration, combination therapy, contraindications and / or warnings.

[0077] "Product" is any product (e.g., package or container) or kit comprising at least one reagent, for example, a pharmaceutical for the treatment of a disease or disorder (e.g., cancer), or a probe for the specific detection of a biomarker described herein. In some embodiments, the product or kit is advertised, distributed, or sold as a unit for carrying out the method described herein.

[0078] The terms “labeling” and “detectable labeling” refer to a portion that, for example, binds to an antibody or antigen to make the reaction (e.g., binding) between members of a specific binding pair detectable. A labeled member of a specific binding pair is referred to as “detectable labeling.” Thus, the term “labeled binding protein” refers to a protein into which a label has been incorporated that results in the identification of the binding protein. In some embodiments, the labeling is the incorporation of a detectable marker that can produce a signal detectable by visual or instrumental means, e.g., radiolabeled amino acids or the binding of a biotinyl moiety to a polypeptide that can be detected by a marked avidin (e.g., streptavidin containing enzyme activity detectable by a fluorescent marker or optical or colorimetric method). Examples of polypeptide labeling 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 These include Sm), chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorescent agents), 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. Typical examples of labels commonly used in immunoassays include photoluminescent moieties, e.g., acridinium compounds, and fluorescent moieties, e.g., fluorescein. In this regard, the moiety itself may not be detectably labeled, but may become detectable upon reaction with other moieties.

[0079] Exemplary DR5-binding polypeptide This specification provides formulations of DR5-binding polypeptides. In various embodiments, the DR5-binding polypeptide comprises at least one VHH domain comprising CDR1 comprising the sequence of SEQ ID NO: 1, CDR2 comprising the sequence of SEQ ID NO: 2, and 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 the dimerization of the DR5-binding polypeptide under physiological conditions, and the number of DR5-binding sites doubles as a result of the dimerization. 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, and thus the DR5-binding polypeptide exists as a tetravalent dimer under such conditions.

[0081] In some embodiments, the DR5-binding polypeptide comprises a VHH-linker-VHH-linker-Fc structure. 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, Fc comprises a hinge. In some such embodiments, 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 to DR5 can be humanized. Humanized antibodies (such as sdAbs or VHH-containing polypeptides) are useful as therapeutic molecules because they reduce or eliminate the human immune response to non-human antibodies that can lead to an immune response to antibody therapeutics and reduce the effectiveness of the therapeutic agent. Generally, humanized antibodies contain one or more variable domains in which the CDR (or a portion thereof) is derived from a non-human antibody and the FR (or a portion thereof) is derived from a human antibody sequence. Humanized antibodies also optionally contain at least a portion of the 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 residue originates) to restore or improve the specificity or affinity of the antibody, for example.

[0083] Humanized antibodies and their production methods have been reviewed, for example, in Almagro and Fransson, (2008) Front. Biosci. 13: 1619-1633, as well as 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, and Padlan, (1991) Mol. Immunol. Further details are provided in 28:489-498 (which describes "resurfacing"), Dall'Acqua et al., (2005) Methods 36:43-60 (which describes "FR shuffling"), and Osbourn et al., (2005) Methods 36:61-68 and Klimka et al., (2000) Br. J. Cancer, 83:252-260 (which describes the "guided selection" approach to FR shuffling).

[0084] Human framework regions that can 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 specific 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. This includes (see 272: 10678-10684 and Rosok et al., (1996) J. Biol. Chem. 271:22611-22618). Typically, humanized VHH is produced by replacing the FR region of VHH with a human FR region. In some embodiments, replacing certain FR residues of human FR improves 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 contained 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 in the DR5-binding polypeptide is derived from a human Fc region and contains three amino acid deletions corresponding to IgG1 E233, L234, and L235 in the lower hinge, and is referred to herein as "Fc xELL". The Fc xELL polypeptide does not bind to FcγR and is therefore referred to as "effector silent" or "effector null". However, in some embodiments, the xELL Fc region binds to FcRn, resulting in transcytosis associated with an 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 A nucleic acid molecule is provided that contains a polynucleotide encoding a DR5-binding polypeptide. In some embodiments, the nucleic acid molecule may also encode a leader sequence that directs the secretion of the DR5-binding polypeptide, which is typically cleaved so that it is not present in the secreted polypeptide. The leader sequence may be a native heavy chain (or VHH) leader sequence or another heterologous leader sequence.

[0088] Nucleic acid molecules can be constructed using recombinant DNA techniques conventional in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in selected host cells.

[0089] Vectors comprising nucleic acids encoding DR5-binding polypeptides described herein are provided. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like. In some embodiments, vectors optimized for polypeptide expression in desired cell types such as CHO cells or CHO-derived cells, or in NSO cells, are selected. 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 (e.g., yeast), plant cells, insect cells, and mammalian cells. Such expression may be carried out, for example, according to procedures known in the art. Exemplary eukaryotic cells that may be used for polypeptide expression 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, U.S. Patent Application Publication No. 2006 / 0270045. In some embodiments, specific eukaryotic host cells are selected based on their ability to perform desired post-translational modifications on the polypeptide. For example, in some embodiments, CHO cells produce polypeptides with higher levels of sialylation than the same polypeptide produced in 293 cells.

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

[0092] Host cells containing either 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 host cells can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include active agents that bind to the ROR1 ECD and Fc region. For example, DR5-binding polypeptides containing the Fc region can be purified by binding to the Fc region using a protein A, protein G, protein A / G, or antibody affinity column. Hydrophobic interaction chromatography, such as butyl or phenyl columns, may also be suitable for purifying some polypeptides, such as antibodies. Ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography) may 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.) may also be suitable for purifying some polypeptides, such as antibodies. Many methods for purifying polypeptides are known in the field.

[0093] In some embodiments, DR5-binding polypeptides are 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, DR5-binding polypeptides prepared by the above method are provided. In some embodiments, the DR5-binding polypeptides are prepared in host cells. In some embodiments, the DR5-binding polypeptides are prepared in a cell-free system. In some embodiments, the DR5-binding polypeptides are purified. In some embodiments, a cell culture medium containing the DR5-binding polypeptide is provided.

[0095] In some embodiments, compositions comprising antibodies prepared by the above method are provided. In some embodiments, the composition comprises a DR5-binding polypeptide prepared in host cells. In some embodiments, the composition comprises a DR5-binding polypeptide prepared 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 DR5-binding polypeptide pharmaceutical formulation is an aqueous liquid formulation. In other embodiments, the formulation is lyophilized. In all cases, the formulation contains the DR5-binding polypeptide. In some embodiments, the DR5-binding polypeptide contains 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 contains at least one VHH domain comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments, the DR5-binding polypeptide comprises a VHH-linker-VHH-linker-Fc structure. 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, Fc comprises a hinge. In some such embodiments, 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 includes two VHH domains and an Fc region.

[0097] In some embodiments, the concentration of the DR5-binding polypeptide in the aqueous formulation or the reconstitute of the 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 contains 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 contains 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, 5.9 to 6.1, 6 to 6.1, or 6 to 6.2. In some embodiments, the pH of the formulation is approximately 5.5, approximately 5.6, approximately 5.7, approximately 5.8, approximately 5.9, approximately 6, approximately 6.1, approximately 6.2, approximately 6.3, approximately 6.4, or approximately 6.5. In some embodiments, the pH of the formulation is approximately 6. In some embodiments, the pH of the formulation is 5.5 to 6.5.

[0099] In some embodiments, the formulation contains 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, or 7 mM to 12 mM. In some embodiments, the formulation contains 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 contains 10 mM histidine. In some embodiments, the histidine is histidine HCl.

[0100] In some embodiments, the formulation contains sucrose, the concentration of which may be, for example, 7% to 10% (w / v), for example, 7% to 9%, 8% to 10%, 8% to 9%, 7%, 8%, 9%, or 10% (w / v). In some embodiments, the formulation contains 8% sucrose. In some embodiments, the formulation contains 9% sucrose.

[0101] In some embodiments, the formulation contains poloxamer 188 (P188) in concentrations of, for example, 0.1% to 0.8%, 0.1% to 0.5%, 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 contains 0.2% poloxamer P188.

[0102] A non-limiting exemplary formulation comprises 50 mg / mL of DR5-binding polypeptide, 10 mM histidine HCl, 8% sucrose, and 0.2% poloxamer P188, with a pH of approximately 6. In some embodiments, the DR5-binding polypeptide comprises at least one VHH domain comprising CDR1 containing the amino acid sequence of SEQ ID NO: 1, CDR2 containing the amino acid sequence of SEQ ID NO: 2, and CDR3 containing 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 containing the amino acid sequence of SEQ ID NO: 4. In some embodiments, the DR5-binding polypeptide comprises a VHH-linker-VHH-linker-Fc structure. 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, Fc comprises a hinge. In some such embodiments, 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 includes 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 formulations provided herein are 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 and beautiful cake of off-white color, completely free of visible impurities.

[0104] In some embodiments, upon reconstitution of lyophilized formulations 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 polypeptides present in the aqueous formulation are aggregated, and / or less than 1% or less than 0.5% of the DR5-binding polypeptides present in the aqueous formulation are degraded. The presence of aggregated and / or degraded DR5-binding polypeptides can be measured, for example, by size exclusion chromatography.

[0105] The DR5-binding polypeptide pharmaceutical formulations disclosed herein may be provided in unit dosage forms or stored in a form suitable for supplying two or more unit doses. The pharmaceutical formulations must be adapted to their intended route of administration. Lyophilized formulations are typically reconstituted in solution before administration or use, while aqueous formulations may be, for example, “ready to use immediately,” meaning they are administered directly without initial dilution, or they may be diluted with physiological saline or another solution before use.

[0106] Pharmaceutical formulations are preferably sterilized. Sterilization can be achieved by any preferred method, such as filtration through a sterile filtration membrane. If the composition is lyophilized, filtration sterilization can be performed before or after lyophilization and reconstitution. In various embodiments, lyophilized formulations are provided, which can be reconstituted to form the liquid pharmaceutical formulations provided herein.

[0107] Exemplary methods for treating diseases using DR5-binding polypeptide formulations In some embodiments, a method is provided for treating a disease in an individual, comprising administering a pharmaceutical formulation containing a DR5-binding polypeptide. In some embodiments, a method is provided for treating cancer in an individual.

[0108] In some embodiments, the method comprises administering an effective amount of a pharmaceutical formulation containing a DR5-binding polypeptide provided herein to an individual. Such therapeutic methods may be therapeutic methods in humans or animals. In some embodiments, methods for treating humans are provided. Non-limiting exemplary cancers that can be treated with DR5-binding polypeptides provided herein include basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain cancer 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, carcinoma in situ, kidney cancer or renal 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. Examples include skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary tract cancer and vulvar cancer, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, B-cell lymphoma, low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocyte (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small undivided cell NHL, giant lesion NHL, mantle cell lymphoma, AIDS-associated lymphoma, Waldenström macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, and chronic myeloblastic leukemia.

[0109] The pharmaceutical preparation 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, and the overall health status of the subject being treated. In some embodiments, an effective dose of DR5-binding polypeptide is administered to the subject once or more times. In some embodiments, an effective dose of DR5-binding polypeptide is administered to the subject daily, twice a week, weekly, every two weeks, once a month, etc. An effective dose of DR5-binding polypeptide is administered to the subject at least once. In some embodiments, an effective dose of 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 effective dose to treat (including prevent cancer) cancer. The therapeutically effective dose typically depends on the body weight of the subject being treated, the subject's physical or health condition, the extent of the condition being treated, or the subject's age. Generally, antibodies may be administered in doses ranging from about 0.05 mg / kg (body weight) to about 100 mg / kg (body weight). In some embodiments, antibodies may be administered in doses ranging from about 10 μg / kg (body weight) to about 100 mg / kg (body weight). In some embodiments, antibodies may be administered in doses ranging from about 50 μg / kg (body weight) to about 5 mg / kg (body weight). In some embodiments, antibodies may be administered in doses ranging from about 100 μg / kg (body weight) to about 10 mg / kg (body weight). In some embodiments, antibodies may be administered in doses ranging from about 100 μg / kg (body weight) to about 20 mg / kg (body weight). In some embodiments, the antibody may be administered in amounts 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 amounts 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 amounts 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 amounts 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 amounts of about 5 mg / kg (body weight) or less, for example, less than 4 mg / kg, less than 3 mg / kg, less than 2 mg / kg, or less than 1 mg / kg.

[0111] In some embodiments, DR5-binding polypeptides may be administered in vivo via various routes, including, but not limited to, intramuscular, intravenous, intra-arterial, parenteral, intraperitoneal, or subcutaneous. Appropriate formulations and routes of administration can be selected depending on the intended use.

[0112] Combination therapy DR5-binding polypeptides may be administered alone or in combination with other therapeutic methods, such as other anticancer agents. DR5-binding polypeptides may be supplied before, substantially simultaneously with, or after other therapeutic methods (i.e., simultaneously or sequentially). In some embodiments, the therapeutic methods described herein may further include radiotherapy, chemotherapy, vaccination, targeted tumor therapy, CAR-T therapy, oncolytic virus therapy, cancer immunotherapy, cytokine therapy, surgical resection, chromatin modification, excision, cryotherapy, antisense agents against tumor targets, siRNA agents against tumor targets, microRNA agents or anticancer / antitemocyte agents against tumor targets, or biological agents such as antibodies, cytokines or receptor extracellular domain-Fc fusions.

[0113] In some embodiments, the DR5-binding polypeptide provided herein is given concurrently with a second therapeutic agent, such as a PD-1 / PD-L1 therapeutic agent. Examples of PD-1 / PD-L1 therapeutic agents 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 that act on programmed death-1 (PD-1) or programmed death ligand 1 (PD-L1).

[0114] In some embodiments, the DR5-binding polypeptides provided herein are given simultaneously with an immunostimulant, 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 immunostimulant TNFRSF members include OX40, GITR, 41BB, CD27, and HVEM. Non-limiting examples of B7 family members include CD28 and ICOS. Therefore, in some embodiments, the CD8-binding polypeptides provided herein are given 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 concurrently with CAR-T (chimeric antigen receptor T cell) therapeutic agents, oncolytic virus therapeutic agents, cytokine therapeutic agents, and / or active agents that target other checkpoint molecules such as VISTA, gpNMB, B7H3, B7H4, HHLA2, CTLA4, and TIGIT.

[0116] kit Products and kits comprising any of the formulations provided herein and appropriate packaging are also provided. In some embodiments, the present invention includes a kit comprising (i) a formulation comprising a DR5-binding polypeptide, and (ii) instructions for use for administering the formulation to an individual using the kit.

[0117] Suitable packaging for the compositions described herein is known in the art and includes, for example, vials (e.g., sealed vials), containers, ampoules, bottles, wide-mouthed bottles, flexible packaging (e.g., sealed Mylar or plastic bags), etc. These products 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-dose or multi-dose units and may also be further sterilized and sealed. Instructions for use provided in the kits of the present invention are typically instructions written on a label or accompanying document (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions stored on a magnetic or optical storage disk) are also acceptable. Instructions for use regarding the use of antibodies generally include information on dosage, administration schedule, and route of administration for the intended therapeutic or industrial use. The kit may further include instructions for selecting the appropriate individual treatment.

[0118] The container may be a unit dose, bulk packaging (e.g., multi-dose packaging), or subunit dose. Kits may also be provided containing a dose of the molecules disclosed herein sufficient to provide effective treatment to an individual over a long period of time, such as an approximate number of periods, such as one week, two weeks, three weeks, four weeks, six weeks, eight weeks, three months, four months, five months, six months, seven months, eight months, or nine months or more. The kit may also contain multi-dose unit dose molecules and instructions for use, and may be packaged in quantities sufficient for storage and use in pharmacies, such as hospital pharmacies and compounding pharmacies. In some embodiments, the kit includes a dried (e.g., lyophilized) composition that can generally form a stable aqueous solution of the antibody by reconstitution, resuspension, or rehydration. [Examples]

[0119] The embodiments discussed below are intended purely to illustrate the invention and should not be considered to limit it in any way. These embodiments are not intended to represent that the following experiments are all or only experiments performed. Efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., quantity, temperature, etc.), but some experimental error and deviation should be taken into account. Unless otherwise specified, parts are parts by weight, molecular weight is the average molecular weight, temperature is Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

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

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

[0122] The Phase 1 formulation contained 20 mM acetate, but because histidine was more suitable for the freeze-drying process, a lyophilized formulation containing histidine was developed and tested first. 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 lyophilized protective agents—sucrose, trehalose, or sucrose and mannitol—were tested at various %(w / v) concentrations. The formulations were filled into 2 ml glass vials. Changes in soluble aggregates after one freeze / thaw cycle, lyophilization, and storage of the lyophilized formulation at 40°C or 50°C for one month were determined by SEC analysis using a TSK gel G3000SWxl column (30 cm × 7.8 mm, 5 μm) and 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, and if visible particles were present, they were sterile filtered through a 0.22 μm PES membrane. A volume of 10 μL was injected into the HPLC column and detected at 280 nm. The results are shown in Table 1.

[0123] TIFF0007894878000002.tif254170

[0124] As shown in Table 1, sucrose was superior to trehalose as a lyophilization protective agent in this formulation (comparison of 1A-3A vs. 4A-6A; 1B-3B vs. 4B-6B), and stability increased with higher sucrose concentrations (comparison of 4A vs. 6A; 4B vs. 6B).

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

[0126] TIFF0007894878000003.tif155170

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

[0128] TIFF0007894878000004.tif54170

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

[0130] TIFF0007894878000005.tif124170

[0131] The appearance of all formulations was slightly milky white before and after lyophilization, and after one month of storage in the lyophilized state, and they did not contain any visible particles.

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

[0133] TIFF0007894878000006.tif120170

[0134] To semi-quantify the relative appearance of the cakes, a "visual appearance" score was developed. Here, a score of "0" represents a cake with a very poor appearance (collapse, cracking, meltback, etc.), and a score of "10" represents a perfectly solid cake with no undesirable appearance (a solid cake without cracking, meltback, collapse, etc.). Table 6 shows the appearance and visual appearance score of the cakes at time 0.

[0135] TIFF0007894878000007.tif103170

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

[0137] TIFF0007894878000008.tif60170

[0138] The trehalose-containing formulations (F1-F4) were found to be highly likely to form particles, showing visible particles after only 4 hours of storage (F1), and all trehalose-containing formulations showed visible particles after 24 hours of storage. Both F5 and F6 (0.8% poloxamer) showed visible particles after 4 hours, the same as F1 (0.2% poloxamer), indicating that the concentration of poloxamer does not alter the time to particle formation. Sucrose-containing formulations were superior to trehalose in terms of the time to particle formation. Formulation F7 (260 mM sucrose) formed particles in 24 hours compared to 4 hours for F1 (260 mM trehalose), and F9 (200 mM sucrose) did not contain particles after 30 hours, compared to F3 (200 mM trehalose), which did not contain particles until 18 hours.

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

[0140] TIFF0007894878000009.tif59170

[0141] TIFF0007894878000010.tif42170

[0142] As shown in Table 9, when stored at 25°C, these formulations remained particle-free for up to 3 days, indicating that histidine formulations using 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 formulation showed a higher aggregation rate compared to the 20 mg / ml formulation (bold; F11, F13, and F15). LMW = low molecular weight species.

[0144] TIFF0007894878000011.tif67170

[0145] Example 4: Sucrose and INBRX-109 at various concentrations To optimize the sucrose concentration and evaluate the protein concentration at 25 mg / ml, additional formulations were developed. These formulations also contained methionine, which can act as a stabilizer. The formulations are shown in Table 11.

[0146] TIFF0007894878000012.tif44170

[0147] Three formulations were assayed for their appearance before lyophilization, at point 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 milky white; FVP = no visible particles.

[0148] TIFF0007894878000013.tif86170

[0149] As shown in Table 12, all three formulations were free of visible particles when observed using standard testing procedures before and after freeze-drying, after 4 weeks of storage at 40°C in the freeze-dry state, after three freeze-thaw cycles, and after 2 weeks and 4 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 approximately 10 very small particles at time 0 after lyophilization, and all three formulations showed these small particles upon close examination after the longest stress condition of 4 weeks at 25°C in liquid form.

[0150] The visual appearance of all lyophilized formulations was acceptable, resembling a white or off-white solid cake with no signs of physical collapse, shrinkage, or cracking.

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

[0152] TIFF0007894878000014.tif60170

[0153] The formulations were also assayed using SEC to assess HMW aggregate formation. Measurements based on HMW aggregate formation revealed that all three formulations were stable before lyophilization, after lyophilization, and after 4 weeks of storage at 40°C as lyophilized formulations. The results are shown in Table 14.

[0154] TIFF0007894878000015.tif254170

[0155] As shown in Table 15, in liquid form, the 50 mg / ml formulation showed a slight increase in HMW aggregation as measured by SEC after 4 weeks at 25°C, and no changes were observed after 3 freeze / thaw cycles or 4 weeks of storage at 25°C.

[0156] TIFF0007894878000016.tif254170

[0157] Next, the formulations were assayed using High Accuracy Fluid Particle Counting (HIAC) to determine the presence and number of invisible particles per milliliter before lyophilization, at point 0 after lyophilization, and after 4 weeks of storage at 40°C. Particles were measured and counted using an HIAC device, and the reported value was the average of three consecutive measurements for each sample. The results are shown in Table 16.

[0158] TIFF0007894878000017.tif71170

[0159] All three formulations showed very low levels of invisible particles per milliliter after lyophilization and storage at 40°C for 4 weeks in the lyophilized state (the acceptable limit for invisible particles was less than 6,000 particles / container (1,000 particles / mL) for particles 10 μm or larger, and less than 600 particles / container (100 particles / mL) for particles 25 μm or larger).

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

[0161] TIFF0007894878000018.tif254170

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

[0163] The size distribution of proteins in the formulations (aggregates (HMW), non-reducing monomers (main), and fragments (LMW)) was measured by non-reducing CE-SDS (capillary electrophoresis using an 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] TIFF0007894878000019.tif70170

[0165] The protein also remained stable in liquid form in all three formulations, undergoing minimal physical degradation under the assay conditions, as shown in Table 19.

[0166] TIFF0007894878000020.tif254170

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

[0168] TIFF0007894878000021.tif86170

[0169] The formulation also did not show any significant changes in the protein charge profile in the liquid state under the tested conditions, as shown in Table 21. Therefore, the formulation is protected from chemical modification of the protein in the liquid state and is stable enough to enable the manufacturing process prior to lyophilization of the drug product.

[0170] TIFF0007894878000022.tif254170

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

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

[0173] TIFF0007894878000023.tif139170

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

[0175] Example 5: Stability of INBRX-109 drug product in formulation The stability of batches of lyophilized INBRX-109 drug products manufactured on a pilot scale was evaluated after storage at several temperatures. The lyophilized products were reconstituted with sterile water to form aqueous formulations (pH 6.0) containing 50 mg / ml of 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, charge variant profile by CE-SDS and iCIEF), and biological (potency) properties after 9 months of storage under the intended storage conditions of 2°C to 8°C, and for extended periods at 25°C and 40°C, temperatures used to accelerate product degradation. Under these storage conditions, the lyophilized drug product remained substantially free of visible particles. In Tables 23 and 24, "SVP" means invisible particles, "SEC" means size exclusion chromatography, "cIEF" means capillary isoelectric focusing, "CE-SDS-NR" means capillary electrophoresis-sodium dodecyl sulfate-non-reducing, "CE-SDS-R" means capillary electrophoresis-sodium dodecyl sulfate-reducing, "# / C" means particle count / 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] TIFF0007894878000024.tif254170

[0178] TIFF0007894878000025.tif90170

[0179] Table 25 shows the results of the analytical tests of the lyophilized product before reconstitution. This data indicates that the lyophilized drug product maintained its stability over a long period and at several temperatures, as only very slight changes were detected in the physical appearance, reconstitution time, and moisture content of the drug product. "RH" stands for relative humidity, "NT" means not tested, and "reconstitution time" is the time (in seconds) from the addition of diluent (sterile water for injection) to the product vial until the lyophilized solid is completely dissolved, as determined visually.

[0180] TIFF0007894878000026.tif173170

[0181] This disclosure may be embodied in other specific forms without departing from the spirit or essential features of this disclosure. Therefore, the embodiments described above should be considered illustrative in all respects and not limiting to this disclosure. Accordingly, the scope of this disclosure is indicated by the appended claims rather than the detailed description above, and all modifications within the meaning of the claims and their equivalents are intended to be incorporated herein.

[0182] TIFF0007894878000027.tif232170

Claims

1. A pharmaceutical formulation comprising a DR5-binding polypeptide, wherein the formulation comprises 20 mg / mL to 70 mg / mL of the DR5-binding polypeptide, 5 mM to 20 mM of histidine, 7% to 10% (w / v) of sucrose, and 0.1% to 0.8% of poloxamer P188, the pH of the formulation is 5.3 to 6.7, and the DR5-binding polypeptide comprises the amino acid sequence of SEQ ID NO:

7.

2. A pharmaceutical formulation according to claim 1, comprising 30 mg / mL to 60 mg / mL of a DR5-binding polypeptide.

3. The pharmaceutical formulation according to claim 1 or 2, comprising 50 mg / mL of a DR5-binding polypeptide.

4. A pharmaceutical preparation according to any one of claims 1 to 3, comprising 7 mM to 15 mM histidine.

5. A pharmaceutical preparation according to any one of claims 1 to 4, comprising 10 mM histidine.

6. The pharmaceutical preparation according to any one of claims 1 to 5, wherein the histidine is histidine HCl.

7. A pharmaceutical preparation according to any one of claims 1 to 6, comprising 8% to 9% sucrose.

8. A pharmaceutical preparation according to any one of claims 1 to 7, comprising 8% sucrose or 9% sucrose.

9. A pharmaceutical preparation according to any one of claims 1 to 8, comprising 0.2% to 0.4% poloxamer P188.

10. A pharmaceutical preparation according to any one of claims 1 to 9, comprising 0.2% poloxamer P188.

11. A pharmaceutical preparation according to any one of claims 1 to 10, comprising methionine in a concentration of 1 mM to 10 mM, 2 mM to 8 mM, 3 mM to 7 mM, or 4 mM to 6 mM.

12. A pharmaceutical preparation according to any one of claims 1 to 11, comprising 5 mM methionine.

13. The pharmaceutical preparation according to any one of claims 1 to 12, wherein the pH of the preparation 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.

14. The pharmaceutical preparation according to any one of claims 1 to 13, wherein the pH of the preparation is 6.

15. The pharmaceutical formulation according to any one of claims 1 to 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 6.

16. The pharmaceutical formulation according to claim 15, wherein the formulation essentially consists of 50 mg / mL of DR5-binding polypeptide, 10 mM histidine HCl, 8% sucrose, 0.2% poloxamer P188, and water, and the pH of the formulation is 6.

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

7.

18. A lyophilized formulation comprising a DR5-binding polypeptide, wherein the DR5-binding polypeptide comprises at least one VHH domain containing the amino acid sequence of SEQ ID NO: 7, and 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 of histidine, 7% to 10% of sucrose, and 0.1% to 0.5% of poloxamer P188, and the pH of the aqueous formulation is 5.3 to 6.

7.

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

20. A lyophilized preparation formed by freeze-drying a pharmaceutical preparation according to any one of claims 1 to 17.

21. The freeze-dried preparation according to any one of claims 18 to 20, wherein after being stored at 2°C to 8°C for a maximum of 3 months, 6 months, 9 months, 12 months, or more than 12 months, the freeze-dried preparation is a uniform and beautiful cake of off-white color that is completely free of visible impurities.

22. The lyophilized formulation according to any one of claims 18 to 21, wherein, after being stored at 2°C to 8°C for a maximum of 3 months, 6 months, 9 months, 12 months, or more than 12 months, the formulation is reconstituted in water to form an aqueous formulation, and the aqueous formulation is substantially free of visible particles.

23. The lyophilized formulation according to any one of claims 18 to 22, wherein, after being stored at 2°C to 8°C for a maximum of 3 months, 6 months, 9 months, 12 months, or more than 12 months, the formulation is reconstituted in water to form an aqueous formulation, and as measured by size exclusion chromatography, less than 3% or less than 2% of the DR5-binding polypeptide present in the aqueous formulation is aggregated.

24. The lyophilized formulation according to 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 preparation formed by reconstituting a lyophilized preparation according to any one of claims 18 to 24.

26. A pharmaceutical preparation according to any one of claims 1 to 17 and 25 for the treatment of cancer.

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

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