CD33-binding polypeptide and its use

Abstract

CD33 offers a more powerful antagonist. [Solution] A CD33-binding polypeptide and a method for treating, for example, leukemia using the CD33-binding polypeptide are provided. The CD33-binding polypeptide comprises at least one VHH domain.

Description

[Technical Field]

[0001] [Cross-reference of related applications] This application claims priority to U.S. Provisional Application No. 62 / 843,408, filed 4 May 2019, and U.S. Provisional Application No. 62 / 844,359, filed 7 May 2019, the latter both of which are incorporated herein by reference for all purposes.

[0002] The present invention relates to CD33-binding polypeptides and methods for modulating the biological activity of CD33 using CD33-binding polypeptides. Such methods include, but are not limited to, methods for treating cancer. [Background technology]

[0003] CD33, also known as p67, SIGLEC3, or SIGLEC-3, binds to sialic acid and modulates the inflammatory and immune responses of hematopoietic cells. CD33 can also influence the initiation, growth, and progression of various hematological malignancies. For example, over 85% of acute myeloid leukemia (AML) cases express the CD33 antigen. Thus, CD33 is a suitable tumor-associated antigen for targeted therapy of hematological malignancies such as leukemia. Therefore, there is a therapeutic need for more potent antagonists of CD33. [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] This specification provides CD33-binding polypeptides and methods for treating, for example, leukemia using CD33-binding polypeptides. In some embodiments, the CD33-binding polypeptide comprises at least one VHH domain. Some embodiments are described below. [Means for solving the problem]

[0005] Embodiment 1. A CDR1 containing the amino acid sequence of SEQ ID NO: 47, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 68, or SEQ ID NO: 71, and SEQ ID NO: 48, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 51, SEQ ID NO: 54, SEQ ID NO: 57, A polypeptide comprising a CD33-binding VHH domain, comprising a CDR2 containing the amino acid sequence of SEQ ID NO: 60, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 69, or SEQ ID NO: 72, and a CDR3 containing the amino acid sequence of SEQ ID NO: 49, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 70, or SEQ ID NO: 73. Embodiment 2. The polypeptide of Embodiment 1, wherein at least one VHH domain comprises CDR1 containing the amino acid sequence of SEQ ID NO: 47 or SEQ ID NO: 3, CDR2 containing the amino acid sequence of SEQ ID NO: 48 or SEQ ID NO: 4, and CDR3 containing the amino acid sequence of SEQ ID NO: 49 or SEQ ID NO: 5. Embodiment 3. A polypeptide of Embodiment 1 or Embodiment 2, wherein at least one VHH domain comprises CDR1 containing the amino acid sequence of SEQ ID NO: 7, CDR2 containing the amino acid sequence of SEQ ID NO: 8, and CDR3 containing the amino acid sequence of SEQ ID NO: 9. Embodiment 4. At least one VHH domain comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 50, and a C containing the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 51. A polypeptide according to any one of Embodiments 1 to 3, comprising DR2 and CDR3 containing the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 52. Embodiment 5. A polypeptide from any one of Embodiments 1 to 4, wherein at least one VHH domain comprises CDR1 containing the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 53, or SEQ ID NO: 56, CDR2 containing the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 54, or SEQ ID NO: 57, and CDR3 containing the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 55, or SEQ ID NO: 58. Embodiment 6. A polypeptide from any one of Embodiments 1 to 5, wherein at least one VHH domain comprises CDR1 containing the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 59, CDR2 containing the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 60, and CDR3 containing the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 61. Embodiment 7. A polypeptide from any one of Embodiments 1 to 6, wherein at least one VHH domain comprises CDR1 containing the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 62, CDR2 containing the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 63, and CDR3 containing the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 64. Embodiment 8. A polypeptide from any one of Embodiments 1 to 7, wherein at least one VHH domain comprises CDR1 containing the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 65, CDR2 containing the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 66, and CDR3 containing the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 67. Embodiment 9. A polypeptide from any one of Embodiments 1 to 8, wherein at least one VHH domain comprises CDR1 containing the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 68, CDR2 containing the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 69, and CDR3 containing the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 70. Embodiment 10. A polypeptide from any one of Embodiments 1 to 9, wherein at least one VHH domain comprises CDR1 containing the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 71, CDR2 containing the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 72, and CDR3 containing the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 73. Embodiment 11. At least one VHH domain is sequence number 47, 48, and 49; sequence number 3, 4, and 5; sequence number 7, 8, and 9; sequence number 11, 12, and 13; sequence number 15, 16, and 17; sequence number 19, 20, and 21; sequence number 23, 24, and 25; sequence number 27, 28, and 29; sequence number 31, 32, and 33; sequence number 35, 36, and 35 37; A polypeptide comprising CDR1, CDR2, and CDR3, which comprises the amino acid sequences of SEQ ID NOs. 50, 51, and 52; SEQ ID NOs. 53, 54, and 55; SEQ ID NOs. 56, 57, and 58; SEQ ID NOs. 59, 60, and 61; SEQ ID NOs. 62, 63, and 64; SEQ ID NOs. 65, 66, and 67; SEQ ID NOs. 68, 69, and 70; or SEQ ID NOs. 71, 72, and 73, comprising any one polypeptide of Embodiments 1 to 10. Embodiment 12. A polypeptide from any one of Embodiments 1 to 11, wherein at least one VHH domain is humanized. Embodiment 13. A polypeptide of any one of Embodiments 1 to 12, wherein at least one VHH domain comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 38, SEQ ID NO: 114, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122. Embodiment 14. At least one VHH domain is represented by Sequence ID No. 38, Sequence ID No. 114 A polypeptide according to any one of Embodiments 1 to 12, comprising the amino acid sequence of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122. Embodiment 15. A polypeptide of any one of Embodiments 1 to 12, wherein at least one VHH domain comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131. Embodiment 16. A polypeptide from any one of Embodiments 1 to 12, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131. Embodiment 17. A polypeptide according to any one of Embodiments 1 to 16, comprising two VHH domains. Embodiment 18. A polypeptide according to any one of Embodiments 1 to 16, comprising three VHH domains. Embodiment 19. A polypeptide according to any one of Embodiments 1 to 18, wherein the polypeptide includes at least one binding domain that binds to an antigen other than CD33. Embodiment 20. The polypeptide of Embodiment 19, wherein the polypeptide comprises at least one binding domain that binds to CD3, T cell receptor (TCR) α, TCR β, CD28, CD16, CD32A, CD64, CD89, NKp46, or NKG2D. Embodiment 21. Each VHH domain is a polypeptide of Embodiment 17 or 18 that binds to CD33. Embodiment 22. The polypeptide of Embodiment 21, wherein each VHH domain contains the same CDR1, CDR2, and CDR3 amino acid sequences. Embodiment 23. Each VHH domain is the polypeptide of Embodiment 21, containing the same VHH sequence. Embodiment 24. A polypeptide according to any one of Embodiments 1 to 16, comprising one VHH domain. Embodiment 25. The polypeptide is one of the polypeptides from Embodiments 1 to 24, including an Fc region. Embodiment 26. The polypeptide of Embodiment 25, wherein the Fc region comprises an amino acid sequence selected from SEQ ID NOs: 74 to 109. Embodiment 27. A polypeptide of Embodiment 25 or Embodiment 26 that forms a dimer under physiological conditions. Embodiment 28. CD33 is human CD33, and is a polypeptide from any one of Embodiments 1 to 27. Embodiment 29. Human CD33 is the polypeptide of Embodiment 28, comprising the sequence of Sequence ID No. 1. Embodiment 30. An immune complex comprising one polypeptide from any one of Embodiments 1 to 29 and a cytotoxic substance. Embodiment 31. The immune complex of Embodiment 30, wherein the cytotoxic agent is selected from calicheamicin, auristatin, dorastatin, tubulicin, meitansinoid, cryptophycin, duocalmycin, esperamycin, pyrrolobenzodiazepine, and enediyne antibiotic. Embodiment 32. Any one polypeptide from Embodiments 1 to 29 or Embodiment 3 A pharmaceutical composition comprising an immune complex of embodiment 0 or 31 and a pharmaceutically acceptable carrier. Embodiment 33. An isolated nucleic acid encoding one polypeptide from any one of Embodiments 1 to 29. Embodiment 34. A vector containing the nucleic acid of Embodiment 33. Embodiment 35. A host cell comprising the nucleic acid of Embodiment 33 or the vector of Embodiment 34. Embodiment 36. A host cell that expresses any one polypeptide of Embodiments 1 - 29. Embodiment 37. A method for producing any one polypeptide of Embodiments 1 - 29, the method comprising incubating the host cell of Embodiment 35 or Embodiment 36 under conditions suitable for the expression of the polypeptide. Embodiment 38. The method of Embodiment 37, further comprising isolating the polypeptide. Embodiment 39. A method for treating cancer, the method comprising administering to a subject with cancer any one polypeptide of Embodiments 1 - 29, the immune complex of Embodiment 30 or Embodiment 31, or the pharmaceutical composition of Embodiment 32 in a pharmaceutically effective amount. Embodiment 40. The method of Embodiment 39, wherein the cancer is selected from 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 non - cleaved cell NHL, large - lesion NHL, mantle cell lymphoma, AIDS - related lymphoma, Waldenström macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), hairy cell leukemia, and chronic myelogenous leukemia. Embodiment 41. The method of Embodiment 39 or 40, wherein the cancer is acute myeloid leukemia (AML). Embodiment 42. The method of any one of Embodiments 39 - 41, further comprising administering an additional therapeutic agent. Embodiment 43. The method of Embodiment 42, wherein the additional therapeutic agent is an anti - cancer agent. Embodiment 44. The method of Embodiment 43, wherein the anti - cancer agent is selected from chemotherapeutic agents, anti - cancer biologics, radiotherapy, CAR - T therapy drugs, and oncolytic viruses. Embodiment 45. The method of any one of Embodiments 39 - 44, wherein the cancer is a cancer that expresses CD33. [Brief explanation of the drawing]

[0006] [Figure 1] This figure shows biolayer interferometry data for Hz1E4v2 compared to other CD33-binding sdAbs described herein. [Figure 2A-2M] This figure shows the binding of a specific single-domain antibody (sdAb) to CD33 expressed on MOLM-13 cells or HEK293 cells. "MOLM-13" refers to MOLM-13 cells transfected with a plasmid encoding CD33, as described in Example 2. "CD33m" refers to the shortened CD33 protein (SEQ ID NO: 113). "CD33M" refers to the full-length CD33 protein (SEQ ID NO: 112). "HEK293" or "Parent HEK293" refers to untransfected HEK293 cells. Figure 2A shows the binding of 1E4-IgG1 to CD33. Figure 2B shows the binding of hz1E4v2-IgG1 to CD33. Figure 2C shows the binding of 1H9-IgG1 to CD33. Figure 2D shows the binding of hz1H9v2-IgG1 to CD33. Figure 2E shows the binding of hz1G3v3-IgG1 to CD33. Figure 2F shows the binding of 1C7-IgG1 to CD33. Figure 2G shows the binding of hz1C7v1-IgG1 to CD33. Figure 2H shows the binding of hz1C7v11-IgG1 to CD33. Figure 2I shows the binding of hzA07v4-IgG1 to CD33. Figure 2J shows the binding of hzB07v7-IgG1 to CD33. Figure 2K shows the binding of hzG11v2-IgG1 to CD33. Figure 2L shows the binding of F02-IgG1 to CD33. Figure 2M shows the binding of hzF02v18-IgG1 to CD33. [Modes for carrying out the invention]

[0007] The embodiments described herein relate to CD33-binding polypeptides and their use in various methods for treating cancer.

[0008] 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.

[0009] 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.

[0010] 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 series METHODS IN ENZYMOLOGY (Academic Press, Inc.); PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor eds. (1995)); Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL; and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press, Animal Cell Culture (RI Freshney, ed., 1987), Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell eds., 1993-8) J. Wiley and Sons, Handbook of Experimental Immunology (DM Weir and C.C. Blackwell, 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 widely used methodologies and practices of those skilled in the art described in their latest editions. It is used using a specific methodology.

[0011] 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.

[0012] Generally, the numbering of residues in immunoglobulin heavy chains is as described by Kabat et al., Sequences of The EU index numbering is as in Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) "The EU index, like in Kabat," is a numbering system for human IgG1 EU antibodies. It refers to.

[0013] The embodiments of the present invention described herein are understood to include embodiments that "consist" and / or embodiments that "consist 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.

[0014] 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 preceding independent or dependent claims.

[0015] 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 a sample having an 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 CD33-related disorder). In some embodiments, the reference sample is derived from the same subject but from a point in 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.

[0016] As used herein in the context of benefiting from or responding to the administration of a therapeutic agent, the terms “benefit,” “clinical benefit,” “responsiveness,” and “therapeutic responsiveness” 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.”

[0017] The terms “nucleic acid molecule,” “nucleic acid,” and “polynucleotide” are used interchangeably and may refer to polymers of nucleotides. Such polymers of nucleotides include, but are not limited to, natural and / or unnatural nucleotides, including DNA, RNA, and May contain PNA. "Nucleic acid sequence" refers to the linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0018] 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, peptides, oligopeptides, dimers, trimers, and polymers of amino acid residues, and may include natural or non-natural 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 through site-directed mutagenesis, or accidental, such as due to mutations in the host producing the protein or errors in PCR amplification.

[0019] As used herein, “CD33” refers to any naturally occurring mature CD33 resulting from the processing of CD33 precursors within cells. Unless otherwise specified, this term includes CD33 from any vertebrate origin, including mammals such as primates (e.g., humans and cynomolgus or rhesus macaques) and rodents (e.g., mice and rats). This term also includes naturally occurring variants of CD33, such as splice variants or allele variants. A non-limiting, exemplary mature human CD33 amino acid sequence is shown, for example, in UniProt accession number P20138. Mature forms of CD33 may lack signal peptides (e.g., mature forms of CD33 may lack amino acids 1-17 of UniProt accession number P20138). See Sequence ID No. 1.

[0020] The term “specifically binding” to an antigen or epitope is well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit “specific binding” or “preferential binding” if 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” or “preferentially” binds to a target if 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 CD33 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 CD33 epitopes or non-CD33 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.

[0021] The terms “deterrence” or “deterrence” refer to a reduction or cessation of any phenotypic feature, or a reduction or cessation of the occurrence, degree, or possibility of such feature. “Reduction” or “deterrence” means to reduce, reduce, or cessate the activity, function, and / or amount compared to a reference. In some embodiments, “reduction” or “deterrence” means the ability to cause an overall reduction of 10% or more. In some embodiments, “reduction” or “deterrence” means the ability to cause an overall reduction of 50% or more. In some embodiments, “reduction” or “deterrence” means 75%, 8% This refers to the ability to cause an overall reduction of 5%, 90%, 95% or more. In some embodiments, the above amounts are suppressed or reduced over a certain period compared to a control over the same period. As used herein, the term “inhibits” with respect to CD33 activity refers to a reduction in CD33 activity, such as binding to sialic acid. In some embodiments, “inhibits” refers to a reduction in CD33 activity compared to CD33 activity in the absence of the modifier. In some embodiments, the CD33-binding polypeptides described herein inhibit the binding of CD33 to sialic acid.

[0022] 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.

[0023] 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.

[0024] 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).

[0025] The term "antibody" is used in its broadest sense and is not limited to, but conventional antibodies (typically containing at least one heavy chain and at least one light chain), single The present invention encompasses a variety of polypeptides, including heavy-domain antibodies (sdAbs containing at least one VHH domain and an Fc region), VHH-containing polypeptides (polypeptides containing at least one VHH domain), and antibody-like antigen-binding domains containing any of the aforementioned fragments, insofar as they exhibit the desired antigen-binding activity. In some embodiments, the antibody contains a dimerization domain. Such a dimerization domain includes, but is not limited to, a heavy-chain constant domain (containing 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 (containing hinge, CH2, and CH3, where the hinge mediates dimerization).

[0026] 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.

[0027] 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.

[0028] 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 a 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.

[0029] The terms “single-domain antibody” and “sdAb” are used herein without distinction to refer to antibodies comprising at least one monomeric domain, such as a VHH domain without a light chain, and an Fc region. In some embodiments, an sdAb is a dimer of two polypeptides, each polypeptide comprising at least one VHH domain and an Fc region. As used herein, the terms “single-domain antibody” and “sdAb” include polypeptides comprising 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.

[0030] The term "VHH-containing polypeptide" refers to a polypeptide containing at least one VHH domain. In some embodiments, the VHH polypeptide contains two, three, or four or more VHH domains, where each VHH domain may be the same or different. In some embodiments, the VHH-containing polypeptide contains an Fc region. In some such embodiments, the VHH-containing polypeptide may be referred to as sdAb. Furthermore, in some such embodiments, the VHH polypeptide may form dimers. Non-limiting structures of the VHH-containing polypeptide, also referred to as sdAb, include VHH1-Fc, VHH1-VHH2-Fc, and VHH1-VHH2-VHH3-Fc. Here, VHH1, VHH2, and VHH3 may be the same or different. In some embodiments of such a structure, one VHH may be linked to another VHH by a linker, or one VHH may be linked to Fc by a linker. In some such embodiments, the linker comprises one to twenty amino acids, preferably consisting mainly of glycine and optionally of 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).

[0031] 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 variations 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 were first described by Kohler and Milstein, 1975, Nature 256:495. Monoclonal antibodies can be produced by the hybridoma method described, or by recombinant DNA methods such as those described in U.S. Patent No. 4,816,567. Monoclonal antibodies can also be produced using techniques such as those described, for example, McCafferty et al., 1990, Nature 348:552-554. It can be isolated from a prepared phage library.

[0032] 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.

[0033] As used herein, the term “heavy chain constant region” refers to a region containing at least three heavy chain constant domains, namely CH1, hinge, CH2, and CH3. 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. Certain isotypes may be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (including the γ1 constant region), IgG2 (including the γ2 constant region), IgG3 (including the γ3 constant region), and IgG4 (including the γ4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (including the α1 constant region) and IgA2 (including the α2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0034] As used herein, the “Fc region” refers to a portion of the heavy chain steady region that includes CH2 and CH3. In some embodiments, the Fc region includes the hinge, CH2, and CH3. In some embodiments, if the Fc region includes a hinge, the hinge mediates the dimerization between two Fc-containing polypeptides. The Fc region may be any antibody heavy chain constant region isotype discussed herein. In some embodiments, the Fc region is IgG1, IgG2, IgG3, or IgG4.

[0035] As used herein, “acceptor human framework” refers to heavy chain variable domains (V) derived from the human immunoglobulin framework or human consensus framework, as discussed herein. H The framework includes the amino acid sequence of the framework. Acceptor human frameworks derived from human immunoglobulin frameworks or human consensus frameworks may include the same amino acid sequence or may include 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 VHH.

[0036] "Affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody, e.g., an sdAb or VHH-containing polypeptide) and its binding partner (e.g., an antigen). The affinity or apparent affinity of molecule X to its partner Y is generally expressed by the dissociation constant (K). d ) or K d(見かけ) Affinity can be expressed by conventional methods known in the art, including the methods described herein (e.g., ELISA K d It can be measured by methods such as KinExA, flow cytometry, and / or surface plasmon resonance devices. Such methods include, but are not limited to, BIAcore®, Octet®, or methods requiring flow cytometry.

[0037] The term "K" used in this specification dThe term "K" refers to the equilibrium dissociation constant of an antigen-binding molecule / antigen interaction. As used herein, when the term "K d " is used, it includes K d and K d(見かけ) .

[0038] 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, K d is K d(見かけ) .

[0039] The term "biological activity" refers to any one or more biological properties of a molecule (whether naturally occurring as seen in vivo, or provided or made possible by recombinant means).

[0040] 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%, 85% or more.

[0041] An "antagonist" antibody, "blocking" antibody, or "neutralizing" antibody is an antibody that inhibits, reduces and / or inactivates the biological activity of a target antigen. In some embodiments, a neutralizing antibody binds to an antigen and reduces its biological activity by at least about 20%, 40%, 60%, 80%, 85%, 90%, 95%, 99% or more.

[0042] An "affinity matured" sdAb or VHH-containing polypeptide refers to an sdAb or VHH-containing polypeptide having one or more such modifications in one or more CDRs compared to a parental sdAb or VHH-containing polypeptide that does not have modifications that result in an improvement in the affinity of the sdAb or VHH-containing polypeptide for an antigen. ​

[0043] 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.

[0044] The "effector-positive Fc region" is the Fc region in the natural sequence. It possesses "effector functions." 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 combining the Fc region with a binding domain (e.g., an antibody-variable domain) and can be evaluated using various assays.

[0045] 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.

[0046] 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, for example, about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, in the Fc region of the natural sequence or the Fc region of the parent polypeptide, 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.

[0047] "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 activating 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)). FcR is, for example, Ravetch a These have been reviewed in nd 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 terms “Fc receptor” or “FcR” also include the fetal receptor FcRn, which is involved 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 immunoglobulin It plays a role in regulating the homeostasis of the steroid. 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.)).

[0048] As used herein, “chimeric antigen receptor” refers to an engineered polypeptide comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the extracellular antigen recognition domain includes a VHH domain.

[0049] 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 less than or equal to 5%, 10%, 15%, 20%, 25%, or 50%.

[0050] 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.

[0051] 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 performed using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN® (DNASTAR) software, for example, by those skilled in the art. This can be achieved in various ways within the scope of one's skills. A person skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve the maximum alignment over the entire length of the arrays being compared.

[0052] 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.

[0053] [Table 1]

[0054] 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

[0055] Non-conservative substitution involves replacing one member of one of these classes with one of another.

[0056] 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.

[0057] "Host cell" refers to a cell that may or may have been a recipient of the vector or isolated polynucleotide. The 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 This includes 293 cells 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. The host cells include offspring of a single host cell, but due to spontaneous, accidental, or intentional mutations, the offspring are not necessarily completely identical (in morphology or genomic DNA complementarity) to the original parent cell. The host cells include cells transfected in vivo with the polynucleotides (which may be more) provided herein.

[0058] 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 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.”

[0059] The terms “individual” and “subject” are used herein without distinction to refer to animals, such as mammals. In some embodiments, but not limited to, humans, rodents, monkeys, cats, dogs, horses, cattle, pigs, sheep, goats, mammalian laboratory animals, mammalian livestock, mammalian sport animals, and mammalian pets. Methods for treating animals are provided. In some examples, “individual” or “subject” refers to an individual or subject that requires treatment for a disease or disorder. In some embodiments, the subject receiving treatment may be a patient who has been identified as having or being at sufficient risk of developing a disorder related to the treatment.

[0060] As used herein, “disease” or “disorder” refers to a condition that requires and / or is desired to be treated.

[0061] 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, hematological malignancies such as leukemia, lymphoma and multiple myeloma, polyps, hyperplasia, and latent tumors or micrometastases.

[0062] 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. Examples of 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, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric 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, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, and stomach cancer. This includes cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary tract cancer, vulvar cancer, lymphomas including 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 This includes small, undivided cell NHL (including giant lesion NHL), mantle cell lymphoma, AIDS-associated lymphoma, and Waldenström macroglobulinemia, acute myeloid leukemia (AML), 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.

[0063] As used herein, the terms “non-tumor cells” or “non-cancerous cells” refer to normal cells or tissues. Exemplary non-tumor cells include, but are not limited to, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, monocytes, macrophages, epithelial cells, fibroblasts, hepatocytes, stromal kidney cells, fibroblast-like synovial cells, osteoblasts, and cells located in the breast, skeletal muscle, pancreas, stomach, ovaries, small intestine, placenta, uterus, testes, kidneys, lungs, heart, brain, liver, prostate, colon, lymphoid organs, bone, and bone-derived mesenchymal stem cells. As used herein, the terms “peripherally located cells or tissues” refer to non-tumor cells that are not located near tumor cells and / or within the tumor microenvironment.

[0064] As used herein, the term “cells or tissues within the tumor microenvironment” refers to cells, molecules, extracellular matrix, and / or blood vessels that surround and / or nourish tumor cells. Exemplary cells or tissues within the tumor microenvironment include, but are not limited to, tumor vascular systems, tumor-infiltrating lymphocytes, reticular cells, endothelial progenitor cells (EPCs), cancer-associated fibroblasts, pericytes, other stromal cells, components of the extracellular matrix (ECM), dendritic cells, antigen-presenting cells, T cells, regulatory T cells (Treg cells), macrophages, neutrophils, myeloid-derived suppressor cells (MDSCs), and other immune cells located proximal to the tumor. Methods for identifying tumor cells and / or cells / tissues located within the tumor microenvironment are well known in the art, as described below herein.

[0065] 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.

[0066] As used herein, “immune response” is intended to encompass cellular and / or humoral immune responses that are sufficient to suppress or prevent the development of a disease (e.g., cancer or cancer metastasis) or to improve its symptoms. “Immune response” may encompass aspects of both the innate and adaptive immune systems.

[0067] 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 status, suppression of disease or disease progression, suppression or slowing of disease or its progression, inhibition of its development, and remission (whether partial or total). “Treatment” also includes mitigation of the pathological consequences of proliferative disorders. The methods provided herein aim to address one or more of these aspects of treatment. Accordingly, the term treatment does not require the complete elimination of all aspects of the disorder.

[0068] "Improvement" means that one or more symptoms are reduced or improved compared to when the therapeutic agent is not administered. "Improvement" also includes a reduction or decrease in the duration of symptoms.

[0069] 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, CAR-T therapy agents, therapeutic oligonucleotides (antisense oligonucleotides and siRNAs, etc.), and oncolytic viruses.

[0070] The term "biological sample" means any amount 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.

[0071] In the context of experiments or comparisons, 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. A control or reference may also refer to a control active substance, such as an antibody, that is known to lack the activity of the active substance being tested.

[0072] As used herein, “delaying the onset of 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.

[0073] 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,” “suppress,” or “prevent” refer to or require prevention only for the period measured, and not for complete prevention over the entire period.

[0074] 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.

[0075] 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. Eel preparations can be sterilized.

[0076] 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.

[0077] Administration "in combination" with one or more additional therapeutic agents includes simultaneous (concurrent) administration and sequential administration in any order.

[0078] 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.

[0079] In this specification, the term "consecutively" is used to refer to the administration of two or more therapeutic agents that do not overlap in time or in which the therapeutic effects of the therapeutic agents do not overlap.

[0080] 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.

[0081] 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.

[0082] "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.

[0083] 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 153These include Sm), chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorescent dyes), 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: The material includes light-emitting parts, such as acridinium compounds, and fluorescent parts, such as fluorescein. While these parts themselves may not be detectably labeled, they can become detectable upon reaction with other parts.

[0084] Exemplary CD33-binding polypeptide CD33-binding polypeptides are provided herein. In various embodiments, the CD33-binding polypeptide comprises at least one VHH domain that binds to CD33. In some embodiments, CD33 is human CD33. In some embodiments, the CD33-binding polypeptide blocks the binding of CD33 to sialic acid. In some embodiments, the CD33-binding polypeptides provided herein comprise one, two, three, four, five, six, seven, or eight VHH domains that bind to CD33. In some embodiments, the CD33-binding polypeptides provided herein comprise one, two, three, or four VHH domains that bind to CD33. The CD33-binding polypeptide may comprise one or more VHH domains that bind to one or more target proteins other than CD33. Such polypeptides may be referred to as “multispecific” polypeptides.

[0085] In some embodiments, the CD33-binding polypeptide comprises at least one VHH domain that binds to CD33 and an Fc region. In some embodiments, the CD33-binding polypeptide provided herein comprises one, two, three, or four VHH domains and an Fc region. In some embodiments, the Fc region mediates the dimerization of the CD33-binding polypeptide under physiological conditions, thereby doubling the number of CD33-binding sites. For example, a CD33-binding polypeptide comprising three VHH domains that bind to CD33 and an Fc region is trivalent as a monomer, but under physiological conditions, the Fc region can mediate dimerization, so that the CD33-binding polypeptide exists as a hexavalent dimer under such conditions.

[0086] In some embodiments, the CD33-binding polypeptide comprises at least two VHH domains, one of which binds to a first epitope of CD33 and the other to a second epitope of CD33. When the CD33-binding polypeptide comprises a VHH domain that binds to a first epitope of CD33 and a VHH domain that binds to a second epitope of CD33, the CD33-binding polypeptide may be referred to as “dual-epitoped” or “dual-specific.” In some embodiments, the CD33-binding polypeptide comprises at least two VHH domains, one of which binds to CD33 and the other to an antigen other than CD33. Such polypeptides may be referred to as “dual-specific” or “multi-specific.”

[0087] Table 2 shows non-exclusive exemplary CD33-binding polypeptides. The sequences for the single-domain antibodies shown are given in the table of specific sequences in this specification. Polypeptide names beginning with "hz" indicate that they are humanized versions of the corresponding parent polypeptide.

[0088] [Table 2]

[0089] CD33-binding polypeptide In various embodiments, the VHH domain bound to CD33 is a CDR1 sequence selected from SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 47, 50, 53, 56, 59, 62, 65, 68, and 71, and a sequence from SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, and It includes a CDR2 sequence selected from sequence number 36, sequence number 48, sequence number 51, sequence number 54, sequence number 57, sequence number 60, sequence number 63, sequence number 66, sequence number 69, and sequence number 72, and a CDR3 sequence selected from sequence number 5, sequence number 9, sequence number 13, sequence number 17, sequence number 21, sequence number 25, sequence number 29, sequence number 33, sequence number 37, sequence number 49, sequence number 52, sequence number 55, sequence number 58, sequence number 61, sequence number 64, sequence number 67, sequence number 70, and sequence number 73. In various embodiments, the VHH domains that bind to CD33 are: SEQ ID NOs: 3, 4, and 5; SEQ ID NOs: 7, 8, and 9; SEQ ID NOs: 11, 12, and 13; SEQ ID NOs: 15, 16, and 17; SEQ ID NOs: 19, 20, and 21; SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 27, 28, and 29; SEQ ID NOs: 31, 32, and 33; SEQ ID NOs: 35, 36, and 37; SEQ ID NOs: 4 The sequence includes CDR1, CDR2, and CDR3 sequences selected from 7, SEQ ID NOs. 48 and 49; SEQ ID NOs. 50, 51 and 52; SEQ ID NOs. 53, 54 and 55; SEQ ID NOs. 56, 57 and 58; SEQ ID NOs. 59, 60 and 61; SEQ ID NOs. 62, 63 and 64; SEQ ID NOs. 65, 66 and 67; SEQ ID NOs. 68, 69 and 70; and SEQ ID NOs. 71, 72 and 73. In various embodiments, the VHH domain is humanized.

[0090] In some embodiments, the VHH domain that binds to CD33 is represented by SEQ ID NOs. 2, SEQ ID NOs. 6, SEQ ID NOs. 10, SEQ ID NOs. 14, SEQ ID NOs. 18, SEQ ID NOs. 22, SEQ ID NOs. 26, It includes an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to an amino acid sequence selected from sequence number 30, sequence number 34, sequence number 38, sequence number 39, sequence number 40, sequence number 41, sequence number 42, sequence number 43, sequence number 44, sequence number 45, sequence number 46, sequence number 114, sequence number 115, sequence number 116, sequence number 117, sequence number 118, sequence number 119, sequence number 120, sequence number 121, sequence number 122, sequence number 123, sequence number 124, sequence number 125, sequence number 126, sequence number 127, sequence number 128, sequence number 129, sequence number 130, and sequence number 131. In some embodiments, the VHH domain that binds to CD33 includes an amino acid sequence selected from SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 39, 40, 41, 42, 43, 44, 45, 46, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, and 131.

[0091] In various embodiments, the CD33-binding polypeptide comprises one, two, three, or four VHH domains that bind to CD33.

[0092] In various embodiments, the CD33-binding polypeptide comprises at least one VHH domain that binds to CD33 and at least one VHH domain that binds to a natural killer cell antigen or a T cell antigen. In some such embodiments, the CD33-binding polypeptide may be referred to as a multispecific antibody.

[0093] In some embodiments, the CD33-binding polypeptide comprises at least one VHH domain described herein fused to an Fc region. In some embodiments, the Fc region has a sequence selected from SEQ ID NOs: 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, and 109.

[0094] In some embodiments, the VHH domain that binds to CD33 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.

[0095] Humanized antibodies and methods for producing them have been reviewed, for example, in Almagro and Fransson, (2008) Front. Biosci. 13: 1619-1633, and also in, for example, Riechmann et al., (1988) Nature 332:323-329, Queen et al., (1989) Proc. Natl Acad. Sci. USA 86: 10029-10033, U.S. Patent No. 5,821,337, U.S. Patent No. 7,527,791, U.S. Patent No. 6, U.S. Patent No. 982,321, and U.S. Patent No. 7,087,409, Kashmiri et al., (2005) Methods 36:25-34, Padlan, (1991) Mol. Immunol. 28:489-498 (described as "resurfacing"), Dall'Acqua et al., (2005) Methods 36:43-60 (described as "FR shuffling"), and Osbourn et al., (2005) This is further described in Methods 36:61-68 and Klimka et al., (2000) Br. J. Cancer, 83:252-260 (which describes the "guided selection" approach to FR shuffling).

[0096] Human framework domains that can be used for humanization are not limited to, but include framework domains selected using the "best-fit" method (e.g., Sims). See 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, for example, 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 cells. Framework regions (see, for example, Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633), and framework regions obtained from screening of FR libraries (see, for example, Baca et al., (1997) J. Biol. Chem. 272: 10678-10684, and Rosok et al., (See (1996) J. Biol. Chem. 271:22611-22618) which typically includes VHH Humanized VHH is produced by replacing the FR region 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".

[0097] In various embodiments, the Fc region contained in the CD33-binding polypeptide is a human Fc region or is derived from a human Fc region.

[0098] In some embodiments, the Fc region of the CD33-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.

[0099] In some embodiments, the Fc region contained in the CD33-binding polypeptide is derived from the human Fc region and includes mutations M252Y and M428V, hereafter referred to as "Fc-YV". In some embodiments, such mutations enhance binding to FcRn at the acidic pH of endosomes (nearly 6.5), while undetectable binding is lost at the neutral pH (approximately 7.2), thereby enabling enhanced FcRn-mediated recycling and extension of the half-life.

[0100] In some embodiments, the Fc region contained in the CD33-binding polypeptide is derived from a human Fc region and includes mutations designed for heterodimerization, referred to herein as “knob” and “hole.” In some embodiments, the “knob” Fc region includes the mutation T366W. In some embodiments, the “hole” Fc region includes the mutations T366S, L368A, and Y407V. In some embodiments, the Fc region used for heterodimerization includes an additional mutation, such as the mutation S354C, on the first member of the heterodimer Fc pair, which forms an asymmetric disulfide with the corresponding mutation Y349C on the second member of the heterodimer Fc pair. In some embodiments, one member of the heterodimer Fc pair includes a modification H435R or H435K, thereby forming FcRn It maintains binding while preventing the binding of protein A. In some embodiments, one member of the heterodimer Fc pair contains modified H435R or H435K, while the second member of the heterodimer Fc pair is not modified with H435. In various embodiments, the whole Fc region contains modified H435R or H435K (sometimes referred to as "hole-R" when the modification is H435R), but the knob Fc region does not. In some cases, the hole-R mutation improves the purification of heterodimers to any homodimer whole Fc region that may exist.

[0101] Non-limiting exemplary Fc regions that may be used in CD33-binding polypeptides include Fc regions containing the amino acid sequences of SEQ ID NOs. 74 to 109.

[0102] Chimeric receptors and manipulated cells This specification provides chimeric antigen receptors (CARs) having an extracellular domain containing one or more CD33-binding VHH domains. The CAR constructs provided herein include an extracellular domain containing one or more CD33-binding VHH domains, a transmembrane domain, and an intracellular signaling region. The one or more CD33-binding VHH domains that form the antigen-binding unit of the CAR bind to or can bind to CD33 binding with sufficient affinity to be useful for therapeutic purposes in targeting cells or tissues that express CD33 binding, i.e., target it.

[0103] CARs are synthetic receptors that typically contain one or more signaling domains in a single fusion molecule expressed on the surface of cells such as T cells, along with associated extracellular targeting / binding moieties. Therefore, CARs possess both antigen specificity and T cell activation properties in a single fusion molecule. First-generation CARs typically contained the cytoplasmic region of CD3ζ or the Fcl receptor γ chain as their signaling domain. First-generation CARs have been tested in Phase I clinical trials in patients with ovarian cancer, renal cell carcinoma, lymphoma, and neuroblastoma, and they induced moderate responses (reviewed in Sadelain et al., Curr Opin Immunol, 21 (2): 215-223, 2009). Signaling of co-stimulatory molecules such as CD28 and CD3ζ. Second-generation CARs, which include domains, provide dual signaling toward a combination of activating and co-stimulatory signals. Third-generation CARs are more complex, involving three or more signaling domains (reviewed in Sadelain et al., Cancer Discovery (3), 388-398, 2013 and Dotti et al, Immuno. Rev, 257 (1), 1-36, 2014).

[0104] In some embodiments, the provided CAR includes a CD33-binding VHH domain. In some embodiments, the CAR includes at least two VHH domains targeting one or more antigens. In one embodiment, the antigen-binding domain of the CAR includes two or at least two CD33-binding VHH domains, thus providing a bivalent molecule. In one embodiment, the antigen-binding domain includes two or at least two CD33-binding VHH domains, but binds to different epitopes on CD33. In such a case, the antigen-binding domain includes a first CD33-binding VHH domain that binds to a first epitope on CD33 and a second VHH domain that binds to a second epitope on CD33. The epitopes may overlap. Thus, in some embodiments, the antigen-binding domain is biparatopic, and the CAR is a biparatopic CAR. In yet another embodiment, the antigen-binding domain includes two CD33-binding VHH domains that bind to the same epitope on CD33.

[0105] The transmembrane domains of CARs provided herein are typically domains that cross or can cross the plasma membrane, or can span the plasma membrane, and these domains are directly or indirectly linked (e.g., via spacers such as immunoglobulin hinge sequences) to an endoplasmic portion containing an extracellular antigen-binding domain and an intracellular signaling domain. In embodiments, the transmembrane domain of the CAR is a transmembrane region of a transmembrane protein (e.g., a type I transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. In one embodiment, the transmembrane domain includes a CD3ζ domain or a CD28 transmembrane domain. Other transmembrane domains will be apparent to those skilled in the art and may be used in connection with embodiments of CAR provided herein.

[0106] The intracellular signaling domain of a CAR provided herein includes one or more intracellular signaling domains that transmit a signal to a T cell upon binding of the antigen-binding domain of the CAR, for example, upon binding of an antigen. In some embodiments, the intracellular domain is an ITAM signaling domain or includes an intracellular signaling domain containing an ITAM signaling domain. Exemplary intracellular signaling domains include, for example, signaling domains derived from any of the ζ chains of the T cell receptor complex or its homologs (e.g., η chains, FcsRIy chains and β chains, MB1(Iga) chain, B29(Ig) chain, etc.), human CD3ζ chain, CD3 polypeptides (Δ, δ, and ε), syk family tyrosine kinases (Syk, ZAP70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transmission such as CD2, CD5, OX40, and CD28. In certain embodiments, the intracellular signaling domain includes an intracellular signaling domain derived from a human CD3ζ chain.

[0107] In some embodiments, the intracellular signaling domain of a CAR may further include an intracellular signaling domain derived from a co-stimulatory molecule. In such examples, such a signaling domain may enhance CAR-T cell activity, for example, after antigen-specific binding, by enhancing the proliferation, survival, and / or development of memory cells compared to a CAR containing, for example, an ITAM-containing signaling domain, such as CD3ζ alone. In some embodiments, the co-stimulatory domain is a functional signaling domain obtained from a protein selected from CD28, CD137(4-IBB), CD134(OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1(CD11a / CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, or a combination thereof. In certain embodiments, the co-stimulatory signaling domain is derived from or obtained from a human protein. In some embodiments, the co-stimulatory signaling domain is derived from or obtained from human CD28 or human CD137(4-IBB).

[0108] In some embodiments, the co-stimulatory signaling domain is derived from CD28 or 41BB.

[0109] In certain embodiments, the CAR further includes a hinge region or spacer region connecting the extracellular antigen-binding domain and the transmembrane domain. This hinge region or spacer region can be used to achieve various lengths and flexibility of the resulting CAR. Examples of hinge regions or spacer regions that may be used include, but are not limited to, Fc fragments or fragments or derivatives thereof of antibodies, hinge regions or fragments or derivatives thereof of antibodies, CH2 regions of antibodies, CH3 regions of antibodies, artificial spacer sequences, such as peptide sequences, or combinations thereof. Other hinge regions or spacer regions will be apparent to those skilled in the art and may be used. In one embodiment, the hinge is an IgG4 hinge or a CD8A hinge.

[0110] In some embodiments, the spacer and transmembrane domains are hinges and transmembrane domains derived from CD8.

[0111] Also provided herein are isolated nucleic acid constructs comprising at least one nucleic acid encoding a CAR provided herein. In some embodiments, the construct The expression vector is for the expression of CAR in cells. The expression vector can be a viral vector. Viral vector technology is well known in this field and is described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2013). For gene transfer into mammalian cells Many virus-based systems have been developed. For example, retroviruses such as adenovirus vectors are used. In one embodiment, lentiviral vectors are used.

[0112] In a further embodiment, isolated cells or cell populations containing one or more of the above-described nucleic acid constructs are also provided. Also provided are isolated cells or cell populations genetically modified to express the CARs provided herein. Thus, genetically engineered cells containing, for example, the CARs provided herein, that stably express them are provided herein. In one embodiment, the cells are selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, hematopoietic stem cells, and / or pluripotent embryonic stem cells / pluripotent induced stem cells. In some cases, the cells are T cells such as CD4 T cells and / or CD8 T cells. In some embodiments, the cells are autologous to the subject. For example, in some embodiments, T cells (also referred to as primary T cells) may be isolated from a patient for manipulation with the CAR nucleic acid construct, e.g., transfection or transduction.

[0113] For example, primary T cells can be purified ex vivo (CD4 cells or CD8 cells or both) and stimulated with a TCR / CD28 agonist, such as beads coated with anti-CD3 / anti-CD28. After a 2 or 3 day activation process, a recombinant expression vector encoding the CAR can be stably introduced into primary T cells via a standard lentiviral or retroviral transduction protocol or a plasmid electroporation strategy. For example, CAR expression can be monitored by flow cytometry using an anti-epitope tag or an antibody that cross-reacts with the native parent molecule. T cells expressing the CAR can be enriched by sorting with an anti-epitope tag antibody, or enriched for high or low expression depending on the application.

[0114] CAR-mediated T cells can be assayed for appropriate function using various methods. In some cases, the function of engineered T cells can be evaluated using in vitro cytotoxicity, proliferation, or cytokine assays (e.g., IFNγ expression). Exemplary standard endpoints include the percentage of tumor lineage lysis, the proliferation of engineered T cells, or the expression of IFNγ protein in the culture supernatant. In some cases, for example, the ability to stimulate T cell activation upon antigen-mediated CAR stimulation can be evaluated by monitoring the expression of activation markers such as CD69, CD44, or CD62L, proliferation, and / or cytokine production.

[0115] Polypeptide expression and production A nucleic acid molecule is provided comprising a polynucleotide encoding a CD33-binding polypeptide. In some embodiments, the nucleic acid molecule may also encode a leader sequence that directs the secretion of the CD33-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.

[0116] 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.

[0117] Vectors comprising nucleic acids encoding CD33-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).

[0118] In some embodiments, CD33-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. Morphologically, CD33-binding polypeptides can 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 polypeptides. For example, in some embodiments, CHO cells produce polypeptides with higher levels of sialylation than the same polypeptides produced in 293 cells.

[0119] 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 are obtained according to any appropriate method. The cells can be transiently or stably transfected into the desired host cells.

[0120] Host cells containing either the nucleic acids or vectors described herein are also provided. In some embodiments, host cells expressing the CD33-binding polypeptides described herein are provided. The CD33-binding polypeptide expressed in the host cell 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, CD33-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, e.g., 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.

[0121] In some embodiments, CD33-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), Endo et al., This is described in Biotechnol. Adv. 21: 695-713 (2003).

[0122] In some embodiments, CD33-binding polypeptides prepared by the above method are provided. In some embodiments, the CD33-binding polypeptides are prepared in host cells. In some embodiments, the CD33-binding polypeptides are prepared in a cell-free system. In some embodiments, the CD33-binding polypeptides are purified. In some embodiments, a cell culture medium containing the CD33-binding polypeptide is provided.

[0123] In some embodiments, compositions comprising antibodies prepared by the above method are provided. In some embodiments, the composition comprises a CD33-binding polypeptide prepared in host cells. In some embodiments, the composition comprises a CD33-binding polypeptide prepared in a cell-free system. In some embodiments, the composition comprises a purified CD33-binding polypeptide.

[0124] Exemplary methods for treating diseases using CD33-binding polypeptides In some embodiments, methods are provided for treating diseases in an individual, comprising administering a CD33-binding polypeptide or cells expressing a CD33-binding polypeptide. In some embodiments, methods are provided for treating cancer in an individual. In some embodiments, methods are provided for treating CD33-expressing cancer or CD33-positive cancer in an individual. The methods comprise administering an effective amount of a CD33-binding polypeptide or cells expressing a CD33-binding polypeptide, as provided herein, to an individual. In some embodiments, the CD33-binding polypeptide blocks the binding of CD33 to sialic acid. In some embodiments, a cytotoxic agent is introduced into CD33-expressing cells using the CD33-binding polypeptide. In some such embodiments, the CD33-binding polypeptide includes a binding domain that binds to cytotoxic T cells or NK cells. In some such embodiments, the binding domain binds to CD3, T cell receptor (TCR)α, TCRβ, CD28, CD16, CD32A, CD64, CD89, NKp46, or NKG2D. In some embodiments, the binding domain may be an antibody-binding domain comprising a VHH domain, or a heavy chain variable region and a light chain variable region such as a VH / VL, scFv, or Fab fragment.

[0125] In some embodiments, CD33-binding polypeptides are linked to cytotoxic agents to form immune complexes. Various cytotoxic agents used in immune complexes are known in the art and include, but are not limited to, calicheamicin, auristatin, dorastatin, tubulicin, meitansinoids, cryptophycin, duocalmycin, esperamycin, pyrrolobenzodiazepines, and enediyne antibiotics.

[0126] In some embodiments, the CD33-binding polypeptide is a chimeric antigen receptor expressed on cytotoxic cells, such as a chimeric antigen receptor (CAR-T) expressed on T cells or a chimeric antigen receptor (CAR-NK) expressed on NK cells. Such therapeutic methods may be used in humans or animals. In some embodiments, methods for treating humans are provided.

[0127] Non-limiting exemplary cancers that can be treated with CD33-binding polypeptides or cells expressing CD33-binding polypeptides provided herein include, but are not limited to, 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, Examples include high-grade small, undivided cell NHL, giant lesion NHL, mantle cell lymphoma, AIDS-associated lymphoma, Waldenström macroglobulinemia, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, and chronic myeloblastic leukemia. In some embodiments, the cancer is a CD33-expressing (i.e., CD33-positive) cancer.

[0128] CD33-binding polypeptides or cells expressing CD33-binding polypeptides may be administered to a 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 CD33-binding polypeptides or cells expressing CD33-binding polypeptides is administered to the subject once or more times. In some embodiments, an effective dose of CD33-binding polypeptides or cells expressing CD33-binding polypeptides is administered to the subject daily, twice a week, weekly, every two weeks, once a month, etc. An effective dose of CD33-binding polypeptides or cells expressing CD33-binding polypeptides is administered to the subject at least once. In some embodiments, an effective dose of CD33-binding polypeptides or cells expressing CD33-binding polypeptides may be administered multiple times, including multiple doses over at least one month, at least six months, or at least one year.

[0129] In some embodiments, the pharmaceutical composition 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.

[0130] In some embodiments, CD33-binding polypeptides or cells expressing CD33-binding polypeptides may be administered in vivo via various routes, including, but not limited to, intravenous, intra-arterial, parenteral, intraperitoneal, or subcutaneous. Appropriate formulations and routes of administration can be selected depending on the intended use.

[0131] In some embodiments, therapeutic treatment using CD33-binding polypeptides is achieved by targeting CD33-expressing cells, such as CD33-expressing cancer cells, with cytotoxic agents. In some such embodiments, the CD33-binding polypeptide is a chimeric antigen receptor expressed on cytotoxic cells, such as T cells or NK cells.

[0132] Pharmaceutical composition In some embodiments, compositions comprising CD33-binding polypeptides are provided in formulations comprising a wide variety of pharmaceutically acceptable carriers (e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003), Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7 th ed., Lippencott Williams and Wilkins (2004), Kibbe et al., Handbook of Pharmaceutical Excipients, 3 rd See ed., Pharmaceutical Press (2000). Excipients, A A variety of pharmaceutically acceptable carriers, including juvants and diluents, are available. Furthermore, a variety of pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffers, tonicity adjusters, stabilizers, and wetting agents, are also available. Non-limiting exemplary carriers include physiological saline, buffered physiological saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0133] In some embodiments, the pharmaceutical composition contains a CD33-binding polypeptide at a concentration of at least 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 125 mg / mL, 150 mg / mL, 175 mg / mL, 200 mg / mL, 225 mg / mL, or 250 mg / mL.

[0134] Combination therapy The CD33-binding polypeptides or engineered cells of this disclosure may be administered alone or in combination with other therapeutic methods, such as other anticancer agents. The CD33-binding polypeptides or engineered cells 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 / antitemoma agents against tumor targets, or biological agents such as antibodies, cytokines or receptor extracellular domain-Fc fusions.

[0135] In some embodiments, the CD33-binding polypeptides provided herein are given simultaneously with one or more chemotherapeutic agents, CAR-T (chimeric antigen receptor T cell) therapeutic agents, oncolytic virus therapeutic agents, cytokine therapeutic agents, and / or other checkpoint molecule-targeting agents such as VISTA, gpNMB, B7H4, HHLA2, CD73, CTLA4, TIGIT, etc.

[0136] In some embodiments, the CD33-binding polypeptide or manipulated cells of the present disclosure may be used in conjunction with other antitumor agents, such as anti-HER-2 antibodies, anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib (TARCEVA®)), platelet-derived growth factor inhibitors (e.g., GLEEVEC® (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, CTLA4 inhibitors (e.g., anti-CTLA antibody ipilimumab (YERVOY®)), and PD-1 inhibitors. It is used in combination with agents (e.g., anti-PD1 antibody, BMS-936558), PDL1 inhibitors (e.g., anti-PDL1 antibody, MPDL3280A), PDL2 inhibitors (e.g., anti-PDL2 antibody), cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following target ErbB2 receptors, ErbB3 receptors, ErbB4 receptors, PDGFRβ receptors, BlyS receptors, APRIL receptors, BCMA receptors, PD-1 receptors, PDL1 receptors, PDL2 receptors, CTLA4 receptors, or VEGF receptors, TRAIL / Apo2, and other biological and organic chemical agents.

[0137] In some embodiments, the CD33-binding polypeptide or manipulated cells provided herein are given concurrently with a PD-1 / PD-L1 therapeutic agent. Examples of PD-1 / PD-L1 therapeutic agents include nivolumab (BMS) and 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 program death It contains other agents that act on -1 (PD-1) or programmed death ligand 1 (PD-L1).

[0138] In some embodiments, the CD33-binding polypeptides or manipulated cells of this disclosure may be used in combination with chemotherapeutic agents. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide of CYTOXAN®; alkyl sulfonates such as busulfan, improsulfan, and pigosulfan; benzodopa, carbocone, meturedopa, and Aziridines such as uredopa; altoretamine, triethylenemelamine, tri Ethyleneimines and methylamelamines, including ethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially bratacin and bratacinone); camptothecin (including its synthetic analog topotecan); bryostatin; callistatin; CC-1065 (including its synthetic analogs adzeresin, karzeresin, and bizeresin); cryptophycin (especially cryptophycin 1 and c Liptophycin 8); Dorastatin; Duocalmycin (including synthetic analogs KW-2189 and CB1-TM1); Eloiterobin; Pancratistatin; Sarcodictiin; Spongestatin; Chlorambucil, Chlornafadin, Cyclophosphamide, Estramustine, Ifosfamide, Mechloretamine, Mechloretamine Oxide Hydrochloride, Melphalan, Novembitin, Fenesterine, Prednimustine, Trophosphamide Nitrogen mustards such as uracil mustard; nitrosoureas such as carmustine, chlorozotosine, fotemustine, lomustine, nimustine, and ranimustine; engine antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ωI1 (see Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)) ); Dynemycin including Dynemycin A; Bisphosphonates such as clodronate; Esperamycin; and neocardinostatin chromophore and related pigment protein enediin antibiotic chromophore); Acrasinomycin, Actinomycin, Anthramycin, Azaserin, Bleomycin, Kakutinomycin, Carabicin, Carminomycin, Cardinophilin, Chromomycin, Dactinomycin, Daunorubicin, Detorubicin, 6-Diazo-5-Oxo-L-Norleucine, Doxorubicin of ADRIAMYCIN (trademark) (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), Epirubicin, Esolubicin, Idarubicin, Marcelomycin, Mitomycin such as Mitomycin C, Mycophenolic acid, Nogaramycin, Olibomycin Antibiotics such as peplomycin, porphyromycin, promycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, and zolubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimethrexate; fludarabine, 6-fluorouracil Purine analogs such as lucaptopurine, thiamipurine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and phloxuridine; androgens such as carsterone, drostanolone propionate, epithiostanol, mepithiostan, and testotractone; and antiprotozoal agents such as aminoglutethimide, mitotane, and trilostane. Kidney (anti-adrenals); folic acid supplements such as fluorophosphate; acegraton; aldofosphamide Glycosides; aminolevulinic acid; enyluracil; amsacrin; bestlovesil; bistolene; edatrexate; defofamine; demecolsin; diazicone; eflornithine; elliptinium acetate; epotilon; eto Gluside; gallium nitrate; hydroxyurea; lentinan; ronidamine; meitansinoids such as meitansin and anthamitosin; mitogwazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK (trademark) polysaccharide complex (JHS Natural) Products, Eugene, Oregon); Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic Acid; Triadicone; 2,2',2''-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, verracurin A, loridine A, and anguidin); Urethanes; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, e.g., Paclitaxel of TAXOL (Bristol-Myers Squibb Oncology, Princeton, New Jersey), ABRAXANE (Trademark) Cremofor-free albumin-modified paclitaxel nanoparticle formulation (American Pharmaceutical Partners, Schaumburg, Illinois), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); Chlorambucil; Gemcitabine in GEMZAR®; 6-Thiogunine; Mercaptopurine; Methotrexate; Platinum analogs such as cisplatin, oxaliplatin, and carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; Vinorelbine in NAVELBINE®; Novantrone; Teniposide; Edatrexate; Daunomycin; Aminopterin; Xeloda; Ibandronate; Irinotecan (Camptosar, CPT-11) (including therapeutic regimens with irinotecan, 5-FU, and leucovorin); Topoisomerase inhibitor RFS Examples include: difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatins, including oxaliplatin therapeutic regimens (FOLFOX); inhibitors of PKCα, Raf, H-Ras, EGFR (e.g., erlotinib (TARCEVA®)) and VEGF-A that reduce cell proliferation, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0139] Further non-limiting exemplary chemotherapeutic agents include anti-estrogens and selective estrogen receptor modulators (SERMs) that modulate or inhibit the hormonal effects on cancer, such as tamoxifen (including tamoxifen from NOLVADEX®), raloxifen, droloxifen, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and toremifene from FARESTON®; for example, 4(5)-imidazole, aminoglutethimide, megestrol acetate from MEGASE®, exemestane, formestan, fadrozol from AROMASIN®, borozol from RIVISOR®, letrozole from FEMARA®, and anastrozole from ARIMIDESEX®. Aromatase inhibitors that inhibit aromatase, an enzyme that regulates estrogen production in the adrenal glands; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, in particular, antisense oligonucleotides that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as PKCα, Ralf, and H-Ras; ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME® ribozyme) and HER2 expression inhibitors; gene therapy vaccines, such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® (AL Examples include rIL-2 of desleukin; topoisomerase 1 inhibitors of LURTOTECAN®; GnRH agonists of ABARELIX®; and any pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0140] In some embodiments, the CD33-binding polypeptide and additional active agents are formulated into a single therapeutic composition and administered simultaneously. Alternatively, the CD33-binding polypeptide or engineered cells and the additional active agents may be separate, for example, each formulated into a separate therapeutic composition, and the CD33-binding polypeptide or engineered cells and the additional active agents may be administered simultaneously, or at different times in the therapeutic regimen. For example, the CD33-binding polypeptide or engineered cells may be administered before the administration of the additional active agent, the CD33-binding polypeptide or engineered cells may be administered after the administration of the additional active agent, or the CD33-binding polypeptide or engineered cells and the additional active agent may be administered alternately. The CD33-binding polypeptide and additional active agents may be administered as a single dose or in multiple doses.

[0141] In some embodiments, the CD33-binding polypeptide or engineered cells and additional active agents (or more) are administered simultaneously. For example, the CD33-binding polypeptide and additional active agents (or more) may be formulated in a single composition or administered as two or more separate compositions. In some embodiments, the CD33-binding polypeptide or engineered cells and additional active agents (or more) are administered sequentially, or they are administered at different times in the treatment regimen.

[0142] Non-exclusive illustrative methods for diagnosis and treatment In some embodiments, the methods described herein are useful for evaluating subjects and / or specimens from subjects (e.g., cancer patients). In some embodiments, the evaluation is one or more of the following: diagnosis, prognosis, and / or response to treatment.

[0143] In some embodiments, the methods described herein include evaluating the presence, absence, or level of a protein. In some embodiments, the methods described herein include evaluating the presence, absence, or level of expression of a nucleic acid. The compositions described herein may be used for these measurements. For example, in some embodiments, the methods described herein include contacting a tumor specimen or cells cultured from a tumor with the therapeutic agent described herein.

[0144] In some embodiments, evaluation may indicate the use or withholding of treatment (including treatment with antibodies described herein). In some embodiments, evaluation may indicate the use or withholding of adjuvant therapy after resection. Adjuvant therapy, also called adjuvant treatment, is treatment given in addition to first-line, primary, or initial treatment. In non-limiting examples, adjuvant therapy may be additional treatment, usually given after surgery, when all detectable disease has been eliminated but a statistical risk of relapse remains due to the underlying disease. In some embodiments, polypeptides are used as adjuvant therapy agents in the treatment of cancer. In some embodiments, polypeptides are used as monoadjuvant therapy agents in the treatment of cancer. In some embodiments, polypeptides described herein are withheld as adjuvant therapy agents in the treatment of cancer. For example, treatment may be withheld for the sake of quality of life and to avoid unnecessary toxicity from ineffective chemotherapy if a patient is unlikely to respond to the antibodies described herein or has only a minimal response. In such cases, palliative care may be used.

[0145] In some embodiments, polypeptides are administered as neoadjuvant therapy agents before resection. In some embodiments, neoadjuvant therapy agents refer to therapeutic agents that reduce and / or downgrade tumors before any surgery. In some embodiments, neoadjuvant therapy agents mean chemotherapeutic agents administered to cancer patients before surgery. In some embodiments, neoadjuvant therapy agents mean antibodies administered to cancer patients before surgery. Cancer types for which neoadjuvant chemotherapeutic agents are typically considered include, for example, breast cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, and lung cancer. In some embodiments, polypeptides are used as neoadjuvant therapy agents in the treatment of cancer. In some embodiments, their use is pre-resection.

[0146] In some embodiments, the tumor microenvironment intended by the methods described herein includes the tumor vascular system, tumor-infiltrating lymphocytes, reticular cells, endothelial progenitor cells (EPCs), cancer-associated fibroblasts, pericytes, other stromal cells, components of the extracellular matrix (ECM), dendritic cells, antigen-presenting cells, T cells, regulatory T cells, macrophages, other lymphocytes, neutrophils, and one or more other immune cells located proximal to the tumor.

[0147] kit Products and kits comprising any of the CD33-binding polypeptides described herein and appropriate packaging are also provided. In some embodiments, the present invention includes a kit comprising (i) a CD33-binding polypeptide and (ii) instructions for use for administering the CD33-binding polypeptide to an individual using the kit.

[0148] 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.

[0149] 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 be reconstituted, resuspended, or rehydrated to generally form a stable aqueous suspension of the antibody. [Examples]

[0150] 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.

[0151] Example 1: CD33 single-domain antibody Single-domain antibodies targeting human CD33 were produced by immunizing llamas and alpacas using recombinant human CD33 extracellular domains.

[0152] After specific anti-CD33 antibody titers were generated, llama / alpaca peripheral blood mononuclear cells (PBMCs) were isolated from 500 mL of blood from immunized animals. Total mRNA was isolated using the Qiagen RNeasy Maxi kit and subsequently converted to first-strand cDNA using Thermo Superscript IV reverse transcriptase and oligo-dT priming. Using this cDNA as a template, the VHH sequence was specifically amplified via PCR and cloned into a yeast surface display vector as a VHH-Fc-AGA2 fusion protein.

[0153] Yeast libraries displaying the VHH-Fc-AGA2 fusion protein were enriched using recombinant CD33 ECD via magnetic bead separation followed by fluorescence-activated cell sorting (FACS). The selected yeast was plated out, isolated colonies were collected and placed in 96-well blocks, and grown in a medium that switched expression from surface-presented VHH-Fc to secretion into the culture medium. The supernatant from the 96-well yeast secretion cultures was applied to 293F cells transiently transfected with CD33 (CD33-positive) or untransfected 293F cells (CD33-negative), washed, treated with a fluorescently labeled anti-human IgG1 Fc secondary antibody, and analyzed by 96-well flow cytometry.

[0154] The nucleic acid sequence encoding VHH, which binds to CD33-positive cells but not to CD33-negative cells, was cloned in-frame with the human Fc coding region into a mammalian expression vector. This vector was then expressed in HEK293 Freestyle cells (293F cells) or CHO cells by transient transfection using polyethyleneimine. The supernatant was collected after 3 to 7 days, and the secreted recombinant protein was purified by protein A chromatography. The concentration was calculated from the absorbance and extinction coefficient at 280 nm.

[0155] The epitopes of single-domain antibodies (sdAbs) containing a VHH domain that binds to CD33 were compared using biolayer interferometry. Human CD33 tagged with 7 μg / mL AviTag® histidine was immobilized on a streptavidin-coated capture sensor. A 100 nM single sdAb was then loaded onto the CD33 antigen and equilibrated. The sensor was then transferred to a 100 nM second sdAb. An increase in assay signal indicated binding, showing that the second sdAb targeted a different epitope than the first sdAb.

[0156] We humanized CD33 VHH derived from camelids using the human VH3-23 germline as a scaffold. The camelid residues contributing to solubility, specificity, stability, and / or affinity were left unmodified. Furthermore, where possible and where necessary, we mitigated the risk of potential development disadvantages by modifying amino acid sequences. In addition, all humanized variants contained the Leu11Glu(L11E) modification described in U.S. Patent Application Publication 2016 / 0207981.

[0157] These results indicate that a single epitope was found among the humanized forms of sdAb 1E4, 1H9, 1G3, and 1C7. As shown in Figure 1, hz1E4v2, hz1H9v2, hz1G3v3, and hz1C7v1 share a common epitope.

[0158] Example 2: Binding of polypeptide to CD33 The binding of sdAbs to human CD33 was evaluated by flow cytometry. Each sdAb contained the VHH domain shown in Table 3 below, and the Fc region of human IgG1 xELL with deletions of amino acids Glu233, Leu234, and Leu235 according to EU numbering (SEQ ID NO: 75). MOLM-13 cells were transiently transfected with a plasmid encoding a sequence containing "CD33M" (SEQ ID NO: 112) or a plasmid encoding a sequence containing the abbreviated "CD33m" (SEQ ID NO: 113). The sequence encoded by the CD33M plasmid includes a leader sequence, a complete extracellular domain, a transmembrane domain, and a cytoplasmic domain. The sequence encoded by the CD33m plasmid includes a leader sequence, a membrane-proximal IgC2 domain, and a transmembrane domain. Untransfected HEK293 cells were used as CD33-negative cells. Each cell type was seeded at 30,000 cells / well in FACS buffer (PBS 1% BSA, 0.1% NaN3 pH 7.4) in a 96-well round-bottom plate. sdAb was diluted in FACS buffer in 11 3-fold serial dilutions. The sdAb dilution was added to the seeded cells, and the assay plate was incubated at 4°C for 30 minutes. After washing twice in 150 μL of FACS buffer, the cells in each well were resuspended in 100 μL of a 1:2000 dilution of Alexa Fluor 647-conjugated secondary anti-human IgG in FACS buffer and incubated at 4°C for 30 minutes. After washing the cells two more times, the conjugated antibody was detected by flow cytometry.

[0159] Flow cytometry analysis was performed using Intellicyte's iQue Plus, and fluorescence was detected. The central fluorescence intensity was plotted. Apparent affinity (K d The nM values ​​were determined using one-site coupled nonlinear regression in PRISM graph software.

[0160] As shown in Figures 2A to 2M, the tested sdAb showed CD33 binding but did not bind to untransfected cells that do not express CD33. The apparent binding affinity is shown in Table 3 below.

[0161] [Table 3]

[0162] 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.

[0163] [Table 4] TIFF2026086490000006.tif241170TIFF2026086490000007.tif244170TIFF2026086490000008.tif245170TIFF2026086490000009.tif24617 0TIFF2026086490000010.tif245170TIFF2026086490000011.tif245170TIFF2026086490000012.tif245170TIFF2026086490000013.tif49170

Claims

1. CDR1 containing the amino acid sequence of SEQ ID NO: 47, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 68, or SEQ ID NO: 71, and SEQ ID NO: 48, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 51, SEQ ID NO: 54, SEQ ID NO: 57, A polypeptide comprising CD33 and at least one VHH domain, comprising CDR2 containing the amino acid sequence of SEQ ID NO: 60, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 69, or SEQ ID NO: 72, and CDR3 containing the amino acid sequence of SEQ ID NO: 49, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 70, or SEQ ID NO:

73.

2. The polypeptide according to claim 1, wherein at least one VHH domain comprises CDR1 containing the amino acid sequence of SEQ ID NO: 47 or SEQ ID NO: 3, CDR2 containing the amino acid sequence of SEQ ID NO: 48 or SEQ ID NO: 4, and CDR3 containing the amino acid sequence of SEQ ID NO: 49 or SEQ ID NO:

5.

3. The polypeptide according to claim 1 or 2, wherein at least one VHH domain comprises CDR1 containing the amino acid sequence of SEQ ID NO: 7, CDR2 containing the amino acid sequence of SEQ ID NO: 8, and CDR3 containing the amino acid sequence of SEQ ID NO:

9.

4. The polypeptide according to any one of claims 1 to 3, wherein at least one VHH domain comprises CDR1 containing the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 50, CDR2 containing the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 51, and CDR3 containing the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO:

52.

5. The polypeptide according to any one of claims 1 to 4, wherein at least one VHH domain comprises CDR1 containing the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 53, or SEQ ID NO: 56, CDR2 containing the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 54, or SEQ ID NO: 57, and CDR3 containing the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 55, or SEQ ID NO:

58.

6. The polypeptide according to any one of claims 1 to 5, wherein at least one VHH domain comprises CDR1 containing the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 59, CDR2 containing the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 60, and CDR3 containing the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO:

61.

7. The polypeptide according to any one of claims 1 to 6, wherein at least one VHH domain comprises CDR1 containing the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 62, CDR2 containing the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 63, and CDR3 containing the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO:

64.

8. The polypeptide according to any one of claims 1 to 7, wherein at least one VHH domain comprises CDR1 containing the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 65, CDR2 containing the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 66, and CDR3 containing the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO:

67.

9. At least one VHH domain contains the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 68 A polypeptide according to any one of claims 1 to 8, comprising CDR1 containing the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 69, and CDR3 containing the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO:

70.

10. The polypeptide according to any one of claims 1 to 9, wherein at least one VHH domain comprises CDR1 having the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 71, CDR2 having the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 72, and CDR3 having the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO:

73.

11. At least one VHH domain corresponds to sequence numbers 47, 48, and 49; sequence numbers 3, 4, and 5; sequence numbers 7, 8, and 9; sequence numbers 11, 12, and 13; sequence numbers 15, 16, and 17; sequence numbers 19, 20, and 21; sequence numbers 23, 24, and 25; sequence numbers 27, 28, and 29; sequence numbers 31, 32, and 33; sequence numbers 35, 36, and 3 7; A polypeptide according to any one of claims 1 to 10, comprising CDR1, CDR2, and CDR3, each comprising the amino acid sequence of SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52; SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO: 55; SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58; SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61; SEQ ID NO: 62, SEQ ID NO: 63, and SEQ ID NO: 64; SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67; SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70; or SEQ ID NO: 71, SEQ ID NO: 72, and SEQ ID NO:

73.

12. The polypeptide according to any one of claims 1 to 11, wherein at least one VHH domain is humanized.

13. The polypeptide according to any one of claims 1 to 12, wherein at least one VHH domain comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 38, SEQ ID NO: 114, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO:

122.

14. The polypeptide according to any one of claims 1 to 12, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NO: 38, SEQ ID NO: 114, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO:

122.

15. The polypeptide according to any one of claims 1 to 12, wherein at least one VHH domain comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, and SEQ ID NO:

131.

16. At least one VHH domain contains the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131 A polypeptide according to any one of claims 1 to 12, including

17. A polypeptide according to any one of claims 1 to 16, comprising two VHH domains.

18. A polypeptide according to any one of claims 1 to 16, comprising three VHH domains.

19. The polypeptide according to any one of claims 1 to 18, wherein the polypeptide comprises at least one binding domain that binds to an antigen other than CD33.

20. The polypeptide according to claim 19, wherein the polypeptide comprises at least one binding domain that binds to CD3, T cell receptor (TCR) α, TCR β, CD28, CD16, CD32A, CD64, CD89, NKp46, or NKG2D.

21. Each VHH domain binds to CD33, the polypeptide according to claim 17 or 18.

22. The polypeptide according to claim 21, wherein each VHH domain contains the same CDR1, CDR2, and CDR3 amino acid sequences.

23. The polypeptide according to claim 21, wherein each VHH domain contains the same VHH sequence.

24. A polypeptide according to any one of claims 1 to 16, comprising one VHH domain.

25. The polypeptide is the polypeptide according to any one of claims 1 to 24, wherein the polypeptide includes an Fc region.

26. The polypeptide according to claim 25, wherein the Fc region comprises an amino acid sequence selected from SEQ ID NOs: 74 to 109.

27. The polypeptide according to claim 25 or 26, which forms a dimer under physiological conditions.

28. The polypeptide according to any one of claims 1 to 27, wherein the CD33 is human CD33.

29. The polypeptide according to claim 28, wherein the human CD33 comprises the sequence of sequence number 1.

30. An immune complex comprising a polypeptide according to any one of claims 1 to 29 and a cytotoxic substance.

31. The immune complex according to claim 30, wherein the cytotoxic substance is selected from calicheamicin, auristatin, dorastatin, tubulicin, meitansinoid, cryptophycin, duocalmycin, esperamycin, pyrrolobenzodiazepine, and enediyne antibiotic.

32. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 29 or an immune complex according to claim 30 or 31, and a pharmaceutically acceptable carrier.

33. An isolated nucleic acid encoding a polypeptide according to any one of claims 1 to 29.

34. A vector comprising the nucleic acid described in claim 33.

35. A host cell comprising the nucleic acid described in claim 33 or the vector described in claim 34.

36. A host cell expressing the polypeptide described in any one of claims 1 to 29.

37. A method for producing a polypeptide according to any one of claims 1 to 29, comprising incubating a host cell according to claim 35 or 36 under conditions suitable for the expression of the polypeptide.

38. The method according to claim 37, further comprising isolating the polypeptide.

39. A method for treating cancer, comprising administering to a subject with cancer a pharmaceutically effective amount of a polypeptide according to any one of claims 1 to 29, an immune complex according to claim 30 or 31, or a pharmaceutical composition according to claim 32.

40. The method according to claim 39, wherein the cancer is selected from 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), acute myeloid leukemia (AML), hairy cell leukemia, and chronic myeloblastic leukemia.

41. The method according to claim 39 or 40, wherein the cancer is acute myeloid leukemia (AML).

42. The method according to any one of claims 39 to 41, further comprising administering an additional therapeutic agent.

43. The method according to claim 42, wherein the additional therapeutic agent is an anticancer agent.

44. The method according to claim 43, wherein the anticancer agent is selected from chemotherapeutic agents, anticancer biological agents, radiotherapy, CAR-T therapy agents, and oncolytic viruses.

45. The method according to any one of claims 39 to 44, wherein the cancer is a cancer that expresses CD33.

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