CD123-binding polypeptides and uses thereof

Abstract

To provide VHH-containing polypeptides that bind CD123, and a method for treating cancer that includes administration of the VHH-containing polypeptides.SOLUTION: Provided is a polypeptide comprising at least one VHH domain that binds to CD123, comprising CDR1, CDR2, and CDR3, each comprising a specific amino acid sequence.SELECTED DRAWING: Figure 3A

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 843,407, filed May 4, 2019, which is incorporated by reference in its entirety for all purposes.

[0002] The present invention relates to CD123 binding polypeptides and methods of using CD123 binding polypeptides to modulate the biological activity of CD123, including, but not limited to, methods of treating cancer. [Background technology]

[0003] CD123, or interleukin 3 receptor alpha chain (IL-3Rα), has been identified as a leukemia stem cell marker and is often upregulated in AML blasts, whereas its expression is low or absent in normal hematopoietic stem cells. CD123 may play a role in hematopoietic cell proliferation, differentiation, and survival. Poor prognosis, high blast count, and resistance to apoptotic cell death are associated with high expression of CD123 in blood cancers such as leukemia. Therefore, there is a therapeutic need for more potent treatments for cancers that express CD123. Summary of the Invention [Problem to be solved by the invention]

[0004] Provided herein are CD123 binding polypeptides and methods of using CD123 binding polypeptides to treat, for example, leukemia. In some embodiments, the CD123 binding polypeptide comprises at least one VHH domain. Some embodiments are provided below. [Means for solving the problem]

[0005] Embodiment 1. A polypeptide comprising at least one VHH domain that binds to CD123, comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:33, SEQ ID NO:42, 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:36, SEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, or SEQ ID NO:93, a CDR2 comprising the amino acid sequence of SEQ ID NO:34, SEQ ID NO:43, 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:37, SEQ ID NO:40, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, or SEQ ID NO:94, and a CDR3 comprising the amino acid sequence of SEQ ID NO:35, SEQ ID NO:44, 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:38, SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, or SEQ ID NO:95. Embodiment 2. A polypeptide of embodiment 1, wherein at least one VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 3, a CDR2 comprising the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 4, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 5. Embodiment 3. A polypeptide of embodiment 1 or embodiment 2, wherein at least one VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 42, SEQ ID NO: 19, or SEQ ID NO: 45, a CDR2 comprising the amino acid sequence of SEQ ID NO: 43, SEQ ID NO: 20, or SEQ ID NO: 46, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 44, SEQ ID NO: 21, or SEQ ID NO: 47. Embodiment 4. At least one VHH domain is selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 36. The polypeptide of any one of embodiments 1 to 3, comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 37, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 38. Embodiment 5. A polypeptide of any one of embodiments 1 to 4, wherein at least one VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 39, a CDR2 comprising the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 40, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 41. Embodiment 6. A polypeptide of any one of embodiments 1 to 5, wherein at least one VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 48, SEQ ID NO: 51, or SEQ ID NO: 93, a CDR2 comprising the amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 49, SEQ ID NO: 52, or SEQ ID NO: 94, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 50, SEQ ID NO: 53, or SEQ ID NO: 95. Embodiment 7. A polypeptide according to any one of embodiments 1 to 6, wherein at least one VHH domain comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35; SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:44; SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9; SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13; SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17; SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21; SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25; SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38; SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41; SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:47; SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50; SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53; or SEQ ID NO:93, SEQ ID NO:94 and SEQ ID NO:95. Embodiment 8. The polypeptide of any one of embodiments 1 to 7, wherein at least one VHH domain is humanized. Embodiment 9. A polypeptide of any one of embodiments 1 to 8, 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:32, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:92. Embodiment 10. A polypeptide of any one of embodiments 1 to 8, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NO: 32, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 92. Embodiment 11. A polypeptide of any one of embodiments 1 to 7, 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, or SEQ ID NO:22. Embodiment 12. A polypeptide of any one of embodiments 1 to 7, 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, or SEQ ID NO:22. Embodiment 13. A polypeptide of any one of embodiments 1 to 12, comprising two VHH domains. Embodiment 14. The polypeptide of any one of embodiments 1 to 12, comprising three VHH domains. Embodiment 15. The polypeptide of any one of embodiments 1 to 14, wherein the polypeptide comprises at least one binding domain that binds to an antigen other than CD123. Embodiment 16. The polypeptide of embodiment 15, wherein the polypeptide comprises at least one binding domain that binds to CD3, T cell receptor (TCR) alpha, TCR beta, CD28, CD16, CD32A, CD64, CD89, NKp46, or NKG2D. Embodiment 17. The polypeptide of embodiment 13 or 14, wherein each VHH domain binds to CD123. Embodiment 18. The polypeptide of embodiment 17, wherein each VHH domain comprises the same CDR1, CDR2, and CDR3 amino acid sequences. Embodiment 19. The polypeptide of embodiment 17, wherein each VHH domain comprises the same VHH sequence. Embodiment 20. A polypeptide of any one of embodiments 1 to 12, comprising one VHH domain. Embodiment 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises an Fc region. Embodiment 22. The polypeptide of embodiment 21, wherein the Fc region comprises an amino acid sequence selected from SEQ ID NO:54 to SEQ ID NO:89. Embodiment 23. The polypeptide of embodiment 21 or embodiment 22, which forms a dimer under physiological conditions. Embodiment 24 The polypeptide of any one of embodiments 1 to 23, wherein CD123 is human CD123. Embodiment 25. The polypeptide of embodiment 24, wherein human CD123 comprises the sequence of SEQ ID NO:1. Embodiment 26 The polypeptide of any one of the preceding embodiments, wherein the polypeptide blocks binding of CD123 to IL-3. Embodiment 27. An immunoconjugate comprising the polypeptide of any one of embodiments 1 to 26 and a cytotoxic agent. Embodiment 28. The immunoconjugate of embodiment 31, wherein the cytotoxic agent is selected from a calicheamicin, an auristatin, a dolastatin, a tubulysin, a maytansinoid, a cryptophycin, a duocarmycin, an esperamicin, a pyrrolobenzodiazepine, and an enediyne antibiotic. Embodiment 29. A pharmaceutical composition comprising a polypeptide of any one of embodiments 1 to 26 or an immunoconjugate of embodiment 27 or embodiment 28, and a pharma- ceutical acceptable carrier. Embodiment 30. An isolated nucleic acid encoding the polypeptide of any one of embodiments 1-26. Embodiment 31. A vector comprising the nucleic acid of embodiment 30. Embodiment 32. A host cell comprising the nucleic acid of embodiment 30 or the vector of embodiment 31. Embodiment 33. A host cell expressing a polypeptide of any one of embodiments 1 to 26. Embodiment 34. A method for producing a polypeptide of any one of embodiments 1 to 26, comprising incubating a host cell of embodiment 32 or embodiment 33 under conditions suitable for expression of the polypeptide. Embodiment 35 The method of embodiment 34, further comprising isolating the polypeptide. Embodiment 36. A method for treating cancer, comprising administering to a subject with cancer a pharma- ceutical effective amount of a polypeptide of any one of embodiments 1 to 26, an immunoconjugate of embodiment 27 or embodiment 28, or a pharmaceutical composition of embodiment 30. Embodiment 37. The method of embodiment 36, wherein the cancer is selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-dividing cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), hairy cell leukemia, and chronic myeloblastic leukemia. Embodiment 38. The cancer is acute myeloid leukemia (AML). How to. Embodiment 39. The method of any one of embodiments 36-38, further comprising administering an additional therapeutic agent. Embodiment 40. The method of embodiment 39, wherein the additional therapeutic agent is an anti-cancer agent. Embodiment 41. The method of embodiment 40, wherein the anti-cancer agent is selected from a chemotherapeutic agent, an anti-cancer biologic, radiation therapy, a CAR-T therapy, and an oncolytic virus. Embodiment 42. The method of any one of embodiments 36 to 41, wherein the cancer is a cancer that expresses CD123. [Brief description of the drawings]

[0006] [Figure 1A-1C]Figure 1 shows biolayer interferometry data for polypeptides comprising a VHH domain that binds to CD123. Figure 1A shows biolayer interferometry data for hzA5v2 compared to other CD123-binding sdAbs described herein. Figure 1B shows biolayer interferometry data for hzF3v22 compared to other CD123-binding sdAbs described herein. Figure 1C shows biolayer interferometry data for hz1B11v28 compared to hz4F2v3. [Figures 2A-2N] Figure 2 shows the binding of certain single domain antibodies (sdAbs) to CD123 expressed on HEK293 or Molm-13 cells. "CD123-FL" refers to HEK293 cells transfected with a plasmid encoding full-length CD123 as described in Example 2. "HEK293" or "parent HEK293" refers to non-transfected HEK293 cells. Figure 2A shows the binding of A5-IgG1 to CD123. Figure 2B shows the binding of hzA5v2-IgG1 to CD123. Figure 2C shows the binding of F3-IgG1 to CD123. Figure 2D shows the binding of hzF3v22-IgG1 to CD123. Figure 2E shows the binding of hzF3v26-IgG1 to CD123. Figure 2F shows the binding of 1F5-IgG1 to CD123. Figure 2G shows the binding of hz1F5v1-IgG1 to CD123. Figure 2H shows the binding of 1B11-IgG1 to CD123. Figure 2I shows the binding of hz1B11v28-IgG1 to CD123. Figure 2J shows the binding of 4F2-IgG1 to CD123. Figure 2K shows the binding of hz4F2v3-IgG1 to CD123. Figure 2L shows the binding of C5-IgG1 to CD123. Figure 2M shows the binding of hz1F5v2-IgG1 to CD123. Figure 2N shows the binding of hz1F5v6-IgG1 to CD123. [Figure 3A-3B] Figure 3 shows inhibition of CD123 binding to IL-3 by A5, C5 and F3 sdAbs (3A) and hz1F5v1 sdAb (3B). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] The embodiments presented herein relate to CD123 binding polypeptides and their use in various methods of treating cancer.

[0008] Definitions and Various Embodiments The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0009] All references cited in this specification, including patent applications, patent publications, and Genbank accession numbers, are incorporated herein by reference to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference in its entirety.

[0010] The techniques and procedures described or referenced herein are generally well understood and generally described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (2013). 01) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (2003)), the series METHODS IN ENZYMOLOGY (Academic Press, Inc.), PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)), Oligonucleotide Synthesis (M. J. Gait, ed., 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press, Animal Cell Culture (R. I. Freshney, ed., 1987), Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press, Cell and Tissue Culture Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell eds., 1993 - 8) J. Wiley and Sons, Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.), Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. 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), and their latest editions. It is used with a methodology that is consistent with the

[0011] Unless otherwise specified, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context or expressly indicated otherwise, singular terms shall include the plural and plural terms shall include the singular. In the event of discrepancies in definitions among various sources or references, the definitions set forth herein shall prevail.

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

[0013] It is understood that embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" embodiments. As used herein, the singular forms "a," "an," and "the" include plural references unless specifically indicated. The use of the term "or" herein is not to be construed as implying that alternatives are mutually exclusive.

[0014] In this application, the use of "or" means "and / or" unless expressly stated otherwise or understood by a person skilled in the art. In the context of a multiple dependent claim, the use of "or" refers to more than one preceding independent or dependent claim.

[0015] The phrases "reference sample," "reference cell," or "reference tissue" refer to a sample having at least one known characteristic that can be used as a comparison to a sample having at least one unknown characteristic. In some embodiments, a reference sample is used as a positive or negative indicator. The reference sample can be used to establish, for example, the level of protein and / or mRNA present in healthy tissue relative to the level of protein and / or mRNA present in a sample with unknown characteristics. In some embodiments, the reference sample is a sample from the same subject, but from a part of the subject different from the part being tested. In some embodiments, the reference sample is a sample from a tissue area surrounding or adjacent to the cancer. In some embodiments, the reference sample is not from the subject being tested, but from a subject known to have or not have the disorder of interest (e.g., a particular cancer or CD123-associated disorder). In some embodiments, the reference sample is from the same subject, but from a time point before the subject develops cancer. In some embodiments, the reference sample is a sample from a benign cancer sample from the same subject or a different subject. When a negative reference sample is used for comparison, the expression level or amount of the molecule of interest in the negative reference sample indicates a level at which the skilled artisan would recognize, in view of the present 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 of interest in the positive reference sample indicates the level at which one of skill in the art, in light of the present disclosure, would recognize that a certain level of the molecule is present.

[0016] The terms "benefit", "clinical benefit", "responsiveness" and "therapeutic response" as used herein in the context of benefiting from or responding to the administration of a therapeutic agent can be measured by assessing various endpoints, such as some degree of inhibition of disease progression, including slowing and complete halt, reduction in the number of disease episodes and / or symptoms, reduction in lesion size, inhibition (i.e., reduction, slowing, or complete halt) of disease cell invasion into adjacent peripheral organs and / or tissues, inhibition (i.e., reduction, slowing, or complete halt) of disease spread, some degree of relief of one or more symptoms associated with the disorder, disease-free presentation after treatment, such as increased length of progression-free survival, increased overall survival, higher response rate, and / or reduced mortality at a given time point after treatment. A "non-responsive" or "non-responsive" subject or cancer is one that does not meet the above criteria of "responding".

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

[0018] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues and may include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. This definition includes both full-length proteins and fragments thereof. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of this disclosure, "polypeptide" refers to proteins that contain modifications (generally conservative in nature), such as deletions, additions, and substitutions to the native sequence, so long as the protein maintains a desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as by mutations of the host that produces the protein or by errors due to PCR amplification.

[0019] As used herein, "CD123" refers to any naturally occurring mature CD123 resulting from processing of a CD123 precursor in a cell. The term includes CD123 from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. also includes naturally occurring variants of CD123, such as splice variants or allelic variants. A non-limiting exemplary mature human CD123 amino acid sequence is shown, for example, in UniProt Accession No. P26951-1. See SEQ ID NO:1.

[0020] The term "specifically binds" to an antigen or epitope is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, with a longer duration and / or with a higher affinity with a particular cell or substance than it reacts or associates with another cell or substance. A single domain antibody (sdAb) or VHH-containing polypeptide "specifically binds" or "preferentially binds" to a target if it binds with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other substances. For example, an sdAb or VHH-containing polypeptide that specifically or preferentially binds to a CD123 epitope is an sdAb or VHH-containing polypeptide that binds this epitope with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other CD123 epitopes or non-CD123 epitopes. It is also understood by interpreting this definition that, for example, an sdAb or VHH-containing polypeptide that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding. "Specificity" refers to the ability of a binding protein to selectively bind to an antigen.

[0021] The term "abrogate" or "abrogate" refers to the reduction or cessation of any phenotypic characteristic, or the reduction or cessation of the incidence, extent, or likelihood of that characteristic. "Reduce" or "abrogate" refers to the decrease, reduction, or cessation of an activity, function, and / or amount compared to a reference. In some embodiments, "reduce" or "abrogate" refers to the ability to cause an overall decrease of 10% or more. In some embodiments, "reduce" or "abrogate" refers to the ability to cause an overall decrease of 50% or more. In some embodiments, "reduce" or "abrogate" refers to the ability to cause an overall decrease of 75%, 85%, 90%, 95% or more. In some embodiments, the amount is abrogated or reduced over a period of time relative to a control over the same period of time. As used herein, the term "inhibit" with respect to the activity of CD123 refers to a decrease in an activity of CD123, such as binding to IL-3. In some embodiments, "inhibit" refers to a decrease in CD123 activity compared to CD123 activity in the absence of a modulator. In some embodiments, a CD123 binding polypeptide described herein inhibits binding of CD123 to IL-3.

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

[0023] A "non-linear epitope" or "conformational epitope" comprises non-contiguous polypeptides, amino acids and / or sugars within an antigenic protein to which an epitope-specific antigen-binding molecule binds. In some embodiments, at least one residue is non-contiguous with other represented residues of the epitope, although one or more residues may be contiguous with other residues.

[0024] A "linear epitope" comprises a continuous polypeptide, amino acid and / or sugar in an antigenic protein to which an antigen-binding molecule specific for the epitope binds. Note that in some embodiments, not all of the residues in a linear epitope need to be directly bound (or involved in binding) by an antigen-binding molecule. In some embodiments, a linear epitope can be derived from immunization with a peptide that essentially consists of the sequence of the linear epitope, or from a structural section of a protein that is relatively isolated from the rest of the protein (so that the antigen-binding molecule can interact, at least primarily, with just that sequence section).

[0025] The term "antibody" is used in the broadest sense and includes various polypeptides, including but not limited to conventional antibodies (typically comprising at least one heavy chain and at least one light chain), single domain antibodies (sdAbs comprising at least one VHH domain and an Fc region), VHH-containing polypeptides (polypeptides comprising at least one VHH domain), and antibody-like antigen-binding domains, including fragments of any of the above, so long as they exhibit the desired antigen-binding activity. In some embodiments, the antibody comprises a dimerization domain. Such dimerization domains include, but are not limited to, a heavy chain constant domain (comprising CH1, hinge, CH2, and CH3, where CH1 is typically paired with a light chain constant domain CL, while the hinge mediates dimerization) and an Fc region (comprising hinge, CH2, and CH3, where the hinge mediates dimerization).

[0026] The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species such as camel (including llama), shark, mouse, human, cynomolgus monkey, etc.

[0027] The term "antigen-binding domain" as used herein refers to a portion of an antibody sufficient to bind to an antigen. In some embodiments, the antigen-binding domain of a conventional antibody comprises three heavy chain CDRs and three light chain CDRs. Thus, in some embodiments, the antigen-binding domain comprises a heavy chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 required to maintain binding to the antigen, and a light chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 required to maintain binding to the antigen. In some embodiments, the antigen-binding domain of an sdAb or VHH-containing polypeptide comprises the three CDRs of a VHH domain. Thus, in some embodiments, an sdAb Alternatively, the antigen-binding domain of a VHH-containing polypeptide comprises a VHH domain comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 required to maintain binding to the antigen.

[0028] The term "VHH" or "VHH domain" or "VHH antigen-binding domain" as used herein refers to the antigen-binding portion of a single domain antibody, such as a camelid antibody or a shark antibody. In some embodiments, a VHH comprises three CDRs and four framework regions, designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, a VHH may be truncated at the N-terminus or C-terminus to include only partial FR1 and / or FR4, or to lack one or both of these framework regions, so long as the VHH substantially retains antigen binding and specificity.

[0029] The terms "single domain antibody" and "sdAb" are used interchangeably herein to refer to an antibody that comprises at least one monomer domain, such as a VHH domain without a light chain, and an Fc region. In some embodiments, an sdAb is a dimer of two polypeptides, each polypeptide comprising at least one VHH domain and an Fc region. As used herein, the terms "single domain antibody" and "sdAb" encompass polypeptides that comprise 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 that comprises at least one VHH domain. In some embodiments, a VHH polypeptide comprises two, three, or four or more VHH domains, where each VHH domain may be the same or different. In some embodiments, a VHH-containing polypeptide comprises an Fc region. In some such embodiments, a VHH-containing polypeptide may be referred to as an sdAb. Furthermore, in some such embodiments, a VHH polypeptide may form a dimer. Non-limiting structures of a VHH-containing polypeptide, also referred to as an sdAb, include VHH1-Fc, VHH1-VHH2-Fc, and VHH1-VHH2-VHH3-Fc, where VHH1, VHH2, and VHH3 may be the same or different. In some embodiments of such structures, a VHH may be linked to another VHH by a linker, or a VHH may be linked to an Fc by a linker. In some such embodiments, the linker comprises 1-20 amino acids, preferably 1-20 amino acids consisting primarily of glycine and optionally serine. In some embodiments, when the VHH-containing polypeptide comprises an Fc, it forms a dimer. Thus, the structure VHH1-VHH2-Fc is considered to be tetravalent when it forms a dimer (i.e., the dimer has four VHH domains). Similarly, the structure VHH1-VHH2-VHH3-Fc is considered to be 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 polypeptide) from a substantially homogeneous antibody population. That is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Thus, a sample of monoclonal antibodies is capable of binding to the same epitope on the antigen. The modifier "monoclonal" indicates the nature of the antibody being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies are the first described by Kohler and Milstein, 1975, Nature 256:495, The monoclonal antibodies may be made by the hybridoma method described in, or by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. The monoclonal antibodies may also be made using the techniques described in, for example, McCafferty et al., 1990, Nature 348:552-554. The vector can be isolated from a phage library prepared by the method described above.

[0032] The term "CDR" refers to a complementarity determining region defined by at least one specific manner to one skilled in the art. In some embodiments, the CDRs may be defined according to the Chothia numbering scheme, the Kabat numbering scheme, a combination of Kabat and Chothia, the AbM definition, and / or the contact definition. A VHH comprises three CDRs designated CDR1, CDR2, and CDR3.

[0033] The term "heavy chain constant region" as used herein refers to a region that includes at least three heavy chain constant domains, namely, CH1, hinge, CH2, and CH3. Of course, deletions and modifications that do not alter function within a domain are included within the scope of the term "heavy chain constant region" unless otherwise specified. Non-limiting exemplary heavy chain constant regions include gamma, delta, and alpha. Non-limiting exemplary heavy chain constant regions also include epsilon and mu. Each heavy chain constant region corresponds to one antibody isotype. For example, an antibody that includes a gamma constant region is an IgG antibody, an antibody that includes a delta constant region is an IgD antibody, and an antibody that includes an alpha constant region is an IgA antibody. Furthermore, an antibody that includes a mu constant region is an IgM antibody, and an antibody that includes an epsilon constant region is an IgE antibody. Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (containing a gamma 1 constant region), IgG2 (containing a gamma 2 constant region), IgG3 (containing a gamma 3 constant region), and IgG4 (containing a gamma 4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (containing an alpha 1 constant region) and IgA2 (containing an alpha 2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0034] As used herein, "Fc region" refers to a portion of a heavy chain constant region comprising CH2 and CH3. In some embodiments, the Fc region comprises a hinge, CH2, and CH3. In various embodiments, when the Fc region comprises a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region can be of any antibody heavy chain constant region isotype discussed herein. In some embodiments, the Fc region is IgG1, IgG2, IgG3, or IgG4.

[0035] As used herein, an "acceptor human framework" refers to a heavy chain variable domain (V) derived from a human immunoglobulin framework or a human consensus framework, as discussed herein. H) framework amino acid sequence. The acceptor human framework derived from a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence or can comprise amino acid sequence changes. In some embodiments, the number of amino acid changes is less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or less than 4, or less than 3 across all human frameworks within a single antigen-binding domain such as a VHH.

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

[0037] As used herein, "K d As used herein, the term "K" refers to the equilibrium dissociation constant of an antigen-binding molecule / antigen interaction. d When the term "K" is used, it includes d and K d(見かけ) Includes:

[0038] In some embodiments, the K of the antigen binding molecule dis measured by flow cytometry using antigen-expressing cell lines and fitting the mean fluorescence measured at each antibody concentration to a nonlinear one-site binding equation (Graphpad's Prism Software). In some such embodiments, K d is K d(見かけ) It is.

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

[0040] An "agonist" or "activating" antibody is an antibody that increases and / or activates the biological activity of a target antigen. In some embodiments, an agonist antibody binds to an antigen and increases its biological activity by at least about 20%, 40%, 60%, 80%, 85% or more.

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

[0042] An "affinity matured" sdAb or VHH containing polypeptide refers to an sdAb or VHH containing polypeptide which has one or more modifications in one or more CDRs which result in an improvement in the affinity of the sdAb or VHH containing polypeptide for its antigen compared to a parent sdAb or VHH containing polypeptide which does not possess such modifications.

[0043] As used herein, "humanized VHH" refers to a VHH in which one or more framework regions have been substantially replaced with human framework regions. In some cases, certain framework region (FR) residues of a human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized VHH may contain residues that are not found in the original VHH or in the human framework sequence, but are included to further improve and optimize the performance of the sdAb or VHH-containing polypeptide. In some embodiments, a humanized sdAb or VHH-containing polypeptide comprises a human Fc region. As will be appreciated, a humanized sequence may be identified by its primary sequence and does not necessarily indicate the process by which the antibody was made.

[0044] An "effector-positive Fc region" is a native sequence Fc region. Exemplary "effector functions" include Fc receptor binding, Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Such effector functions generally require combining the Fc region with a binding domain (e.g., an antibody variable domain) and can be assessed using a variety of assays.

[0045] A "native sequence Fc region" is an Fc region having an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native-sequence human Fc regions include native-sequence human IgG1 Fc regions (non-A and A allotypes), native-sequence human IgG2 Fc regions, native-sequence human IgG3 Fc regions, and native-sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0046] A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by at least one amino acid modification. In some embodiments, a "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by at least one amino acid modification, but that retains at least one effector function of the native sequence Fc region. In some embodiments, a variant Fc region has at least one amino acid substitution, e.g., about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, in the native sequence Fc region or the Fc region of the parent polypeptide compared to the native sequence Fc region or the Fc region of the parent polypeptide. In some embodiments, a variant Fc region herein has at least about 80% sequence identity, at least about 90% sequence identity, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the native sequence Fc region and / or the Fc region of the parent polypeptide.

[0047] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, the FcγR is a native human FcR. In some embodiments, the FcR binds IgG antibodies (gamma receptors), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences but differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein. For example, the term "Fc receptor" or "FcR" also includes the fetal receptor FcRn, which 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 G (Ig) (FcR). FcRn plays a role in regulating the homeostasis of FcRn. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004 / 92219 (Hinton et al.)).

[0048] As used herein, a "chimeric antigen receptor" refers to an engineered polypeptide that includes 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 the same" refer to a sufficiently high degree of similarity between two or more numerical values ​​such that one of skill in the art would consider the difference between the two or more values ​​to have little or no biological and / or statistical significance within the context of the biological characteristic measured by the values. In some embodiments, two or more substantially similar values ​​differ by no more than the approximate value of any one of 5%, 10%, 15%, 20%, 25%, or 50%.

[0050] A polypeptide "variant" refers to a biologically active polypeptide having at least about 80% amino acid sequence identity with a native sequence polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N-terminus or C-terminus of the polypeptide. In some embodiments, a variant has at least about 80% amino acid sequence identity. In some embodiments, a variant has at least about 90% amino acid sequence identity. In some embodiments, a variant has at least about 95% amino acid sequence identity with a native sequence polypeptide.

[0051] As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide, or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be easily performed by one of skill in the art using publicly available computer software, such as, for example, BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. This can be achieved in a variety of ways that are within the skill of the art. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0052] Amino acid substitutions can include, but are not limited to, replacing one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 1. Amino acid substitutions can be introduced into a subject antibody and the products screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0053] [Table 1]

[0054] Amino acids can be grouped according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe

[0055] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0056] The term "vector" is used to describe a polynucleotide that can be manipulated to include a cloned polynucleotide or polynucleotides that can be propagated in a host cell. A vector can include one or more of the following elements: an origin of replication, one or more regulatory sequences that regulate the expression of a polypeptide of interest (such as, for example, a promoter and / or enhancer), and / or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, such as, for example, β-galactosidase). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0057] "Host cell" refers to a cell that can be or has been the recipient of a vector or an isolated polynucleotide. A host cell can be a prokaryotic or eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells, fungal cells, such as yeast, plant cells, and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6™ cells (Crucell), and and 293 cells and CHO cells, and their derivatives, such as 293-6E cells, CHO-DG44 cells, CHO-K1 cells, CHO-S cells, and CHO-DS cells. A host cell includes the progeny of a single host cell, although the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) provided herein.

[0058] The term "isolated" as used herein refers to a molecule that is separated from at least some of the components that it is typically found or produced with in nature. For example, a polypeptide is referred to as "isolated" when it is separated from at least some of the components of the cell that produced it. If the polypeptide is secreted by the cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to "isolate" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" when it is not part of a larger polynucleotide that it is typically found in nature (e.g., in the case of a DNA polynucleotide, genomic DNA or mitochondrial DNA, etc.) or when it is separated from at least some of the components of the cell that produced it, for example, in the case of an RNA polynucleotide. Thus, a DNA polynucleotide contained in a vector within a host cell can be referred to as "isolated".

[0059] The terms "individual" and "subject" are used interchangeably herein to refer to animals, such as mammals. In some embodiments, the subject is a mammalian animal, including, but not limited to, humans, rodents, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets. Methods of treating animals are provided. In some instances, "individual" or "subject" refers to an individual or subject in need of treatment for a disease or disorder. In some embodiments, the subject receiving treatment may be a patient, which means the subject has been identified as having or at sufficient risk of suffering from a disorder relevant to the treatment.

[0060] As used herein, "disease" or "disorder" refers to a condition for which treatment is needed and / or desired.

[0061] The terms "tumor cell," "cancer cell," "cancer," "tumor," and / or "neoplasm" are used interchangeably herein, unless otherwise specified, to refer to a cell (or cells) that exhibit uncontrolled proliferation and / or abnormally increased cell survival and / or inhibited apoptosis that interferes with the normal functioning of bodily organs and systems. Included in this definition are benign and malignant cancers, blood cancers such as leukemia, lymphoma, and multiple myeloma, polyps, hyperplasias, and occult tumors or micrometastases.

[0062] The terms "cancer" and "tumor" encompass solid tumors and hematological / lymphatic cancers, as well as malignant, pre-malignant, and benign tumors, such as dysplasia. Exemplary cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, 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, intraepithelial neoplasia, renal or kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), melanoma, myeloma, neuroblastoma, oral cancer (lips, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, cancer), testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancer, vulvar cancer, lymphomas including Hodgkin lymphoma and non-Hodgkin lymphoma, and B-cell lymphomas including low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-dividing cell NHL, bulky disease NHL, mammary gland lymphoma, myeloma, ovarian cancer ... These include: 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 abnormal blood vessel growth associated with phacomas, edema (such as edema associated with brain tumors), and Meigs' syndrome.

[0063] The term "non-tumor cell" or "non-cancer cell" as used herein refers to a normal cell or tissue. 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 synoviocytes, osteoblasts, and cells located in breast, skeletal muscle, pancreas, stomach, ovary, small intestine, placenta, uterus, testes, kidney, lung, heart, brain, liver, prostate, colon, lymphoid organs, bone, and bone-derived mesenchymal stem cells. The term "peripherally located cell or tissue" as used herein refers to a non-tumor cell that is 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 vasculature, tumor-infiltrating lymphocytes, fibroblastic 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, bone marrow-derived cells, and / or tumor-associated lymphocytes. These include tumor suppressor cells (MDSCs) and other immune cells located in the vicinity of the tumor. Methods for identifying tumor cells and / or cells / tissues located within the tumor microenvironment are well known in the art, as described herein below.

[0065] In some embodiments, "increase" or "decrease" refers to a statistically significant increase or decrease, respectively. As will be clear to the skilled artisan, "modulation" can also include causing a change (which can be either an increase or a decrease) in the affinity, avidity, specificity and / or selectivity of a target or antigen to one or more of its ligands, binding partners, partners that associate into homo- or heteromultimeric forms or substrates, causing a change (which can be either an increase or a decrease) in the sensitivity of the target or antigen to one or more conditions (pH, ionic strength, presence of cofactors, etc.) in the medium or environment in which the target or antigen is present, and / or cell proliferation or cytokine production, compared to the same conditions except in the presence of the test agent. This can be determined in any suitable manner and / or using any suitable assay known per se or described herein, depending on the target involved.

[0066] As used herein, an "immune response" is intended to encompass a cellular and / or humoral immune response that is sufficient to inhibit or prevent the onset of or ameliorate a symptom of a disease (e.g., cancer or cancer metastasis). An "immune response" can encompass aspects of both the innate and adaptive immune systems.

[0067] As used herein, "treatment" is an approach to obtain beneficial or desired clinical results. As used herein, "treatment" covers any administration or application of a therapeutic agent for a disease in a mammal, including a human. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, reduction in the extent of the disease, prevention or delay of disease progression (e.g., metastasis, e.g., to the lungs or lymph nodes), prevention or delay of disease recurrence, delay or slowing of disease progression, amelioration of the disease state, arrest of the disease or disease progression, arrest or slowing of the disease or its progression, arrest of its development, and remission (whether partial or total). "Treatment" also includes reduction of the pathological consequences of a proliferative disease. The methods provided herein contemplate any one or more of these aspects of treatment. In accordance with the above, the term treatment does not require 100 percent elimination of all aspects of the disorder.

[0068] By "ameliorate" is meant that one or more symptoms are lessened or improved compared to when the therapeutic agent is not administered. "Amelioration" also includes a shortening or reduction in the duration of the symptoms.

[0069] The term "anti-cancer agent" is used herein in its broadest sense to refer to an agent used to treat one or more cancers. Exemplary classes of such agents include, but are not limited to, chemotherapeutic agents, anti-cancer biologics (such as cytokines, receptor extracellular domain-Fc fusions, and antibodies), radiation therapy, CAR-T therapy, therapeutic oligonucleotides (such as antisense oligonucleotides and siRNAs), and oncolytic viruses.

[0070] The term "biological sample" refers to a quantity of material from a living or formerly living organism, including, but not limited to, blood (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

[0071] The term "control" or "reference" in an experimental or comparative context refers to a composition known to be free of the analyte (a "negative control") or a composition known to contain the analyte. A control or reference may refer to a composition of matter (a "positive control") that is a known concentration of the analyte. A positive control may include a known concentration of the analyte. A control or reference may also refer to a control agent that is known to lack the activity of the agent being tested, such as an antibody.

[0072] As used herein, "delaying the onset of disease" means to delay, prevent, slow down, retard, stabilize, inhibit, and / or prolong the onset of a disease (such as cancer). This delay can be of varying lengths of time depending on the history of the disease and / or the individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, late-stage cancer, such as the onset of metastases, can be delayed.

[0073] "Prevention," as used herein, includes providing prophylaxis against the occurrence or recurrence of a disease in a subject who may have a predisposition to the disease, but has not yet been diagnosed with the disease. Unless otherwise indicated, the terms "reduce," "inhibit," or "prevent" do not indicate or require complete prevention over the entire period of time, but only prevention over the period of time being measured.

[0074] A "therapeutically effective amount" of a substance / molecule, agonist or antagonist may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule, agonist or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the substance / molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects. A therapeutically effective amount may be delivered in one or more administrations. A therapeutically effective amount refers to an amount effective to achieve a desired therapeutic and / or prophylactic result at the required dosages for the required time.

[0075] The terms "pharmaceutical formulation" and "pharmaceutical composition" are used interchangeably and refer to a preparation that is in a form that allows the biological activity of the active ingredient(s) to be effective and does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered. Such formulations may be sterile.

[0076] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation aid, or carrier conventional in the art used with therapeutic agents that together comprise a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to a recipient at the dosage and concentration used and is compatible with other ingredients of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation in which it is used.

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

[0078] The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents where at least a portion of the administration overlaps in time, or where the administration of one therapeutic agent falls within a short period of time relative to the administration of the other therapeutic agent, or where the therapeutic effects of both therapeutic agents overlap for at least some period of time.

[0079] The term "sequentially" is used herein to refer to the administration of two or more therapeutic agents that do not overlap in time or where the therapeutic effects of the therapeutic agents do not overlap.

[0080] As used herein, "in combination with" refers to the administration of one therapy in addition to another. Thus, "in combination with" refers to the administration of one therapy before, during, or after the administration of another therapy to an individual.

[0081] The term "package insert" is used to refer to instructions typically included in commercial packaging for a therapeutic product, which contain information regarding directions, usage, dosage, administration, concomitant therapy, contraindications and / or warnings regarding the use of such therapeutic product.

[0082] An "article of manufacture" is any manufacture (e.g., package or container) or kit that contains at least one reagent, e.g., a pharmaceutical agent for treating a disease or disorder (e.g., cancer), or a probe that specifically detects a biomarker described herein. In some embodiments, the article of manufacture or kit is advertised, delivered, or sold as a unit for performing a method described herein.

[0083] The terms "label" and "detectable label" refer to a moiety that, for example, when attached to an antibody or antigen, renders the reaction (e.g., binding) between members of a specific binding pair detectable. A labeled member of a specific binding pair is said to be "detectably labeled." Thus, the term "labeled binding protein" refers to a protein that incorporates a label that provides for the identification of the binding protein. In some embodiments, the label is a detectable marker that can generate a signal that is detectable visually or by instrumental means, for example, the incorporation of a radiolabeled amino acid or the attachment of a biotinyl moiety to the polypeptide that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or an enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C. 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153Sm), chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorophores), enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), and magnetic agents such as gadolinium chelates. Representative examples of labels commonly used in immunoassays include moieties that emit light, e.g., acridinium compounds, and moieties that emit fluorescence, e.g., fluorescein. In this regard, the moiety itself may not be detectably labeled, but may become detectable upon reaction with yet another moiety.

[0084] Exemplary CD123 Binding Polypeptides Provided herein are CD123 binding polypeptides. In various embodiments, the CD123 binding polypeptides comprise at least one VHH domain that binds to CD123. In some embodiments, the CD123 is human CD123. In some embodiments, the CD123 binding polypeptides block the binding of CD123 to IL-3. In some embodiments, the CD123 binding polypeptides provided herein comprise one, two, three, four, five, six, seven, or eight VHH domains that bind to CD123. In some embodiments, the CD123 binding polypeptides provided herein comprise one, two, three, or four VHH domains that bind to CD123. The CD123 binding polypeptides may comprise one or more VHH domains that bind to one or more target proteins other than CD123. Such polypeptides may be referred to as "multispecific" polypeptides.

[0085] In some embodiments, a CD123 binding polypeptide comprises at least one VHH domain that binds to CD123 and an Fc region. In some embodiments, a CD123 binding polypeptide provided herein comprises one, two, three, or four VHH domains and an Fc region. In some embodiments, the Fc region mediates dimerization of the CD123 binding polypeptide under physiological conditions, such that a dimer is formed. This doubles the number of CD123-binding sites. For example, a CD123-binding polypeptide comprising three VHH domains that bind to CD123 and an Fc region is trivalent as a monomer, but under physiological conditions, the Fc region can mediate dimerization such that the CD123-binding polypeptide exists as a hexavalent dimer under such conditions.

[0086] In some embodiments, a CD123 binding polypeptide comprises at least two VHH domains, where a first VHH domain binds to a first epitope of CD123 and a second VHH domain binds to a second epitope of CD123. When a CD123 binding polypeptide comprises a VHH domain that binds to a first epitope of CD123 and a VHH domain that binds to a second epitope of CD123, the CD123 binding polypeptide may be referred to as "dual epitopic" or "dual specific". In some embodiments, a CD123 binding polypeptide comprises at least two VHH domains, where a first VHH domain binds to CD123 and a second VHH domain binds to an antigen other than CD123. Such a polypeptide may be referred to as "dual specific" or "multispecific".

[0087] Non-limiting exemplary CD123 binding polypeptides are shown in Table 2. Sequences for the indicated single domain antibodies are shown in certain sequence tables herein. Polypeptide names beginning with "hz" indicate that they are humanized versions of the corresponding parent polypeptide.

[0088] [Table 2]

[0089] CD123-binding polypeptides In various embodiments, the VHH domain that binds to CD123 comprises a CDR1 sequence selected from 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:33, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, and SEQ ID NO:93; a CDR2 sequence selected from 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:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, and SEQ ID NO:94; and a CDR3 sequence selected from 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:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, and SEQ ID NO:95. In various embodiments, the VHH domains that bind to CD123 are selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5; SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13; SEQ ID NO:15, SEQ ID NO:16, and and SEQ ID NO:17; SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21; SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25; SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35; SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38; SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41; SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44; SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47; SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50; SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53; and SEQ ID NO:93, SEQ ID NO:94, and SEQ ID NO:95. In various embodiments, the VHH domain is humanized.

[0090] In some embodiments, the VHH domain that binds CD123 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to an amino acid sequence selected from 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:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:92. In some embodiments, the VHH domain that binds CD123 comprises an amino acid sequence selected from 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:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:92.

[0091] In various embodiments, the CD123 binding polypeptide comprises one, two, three, or four VHH domains that bind to CD123.

[0092] In various embodiments, a CD123 binding polypeptide comprises at least one VHH domain that binds CD123 and at least one VHH domain that binds a natural killer cell antigen or a T cell antigen, in some such embodiments, a CD123 binding polypeptide may be referred to as a multispecific antibody.

[0093] In some embodiments, the CD123 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 NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89.

[0094] In some embodiments, the VHH domain that binds CD123 is humanized. Humanized antibodies (such as sdAbs or VHH-containing polypeptides) are useful as therapeutic molecules because they reduce or eliminate human immune responses to non-human antibodies that may result in an immune response to antibody therapeutics and reduce the efficacy of the therapeutics. Generally, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. The humanized antibody also optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0095] Humanized antibodies and methods for producing them are described, for example, in Almagro and Fransson, (2008) Front. Bios ci. 13: 1619-1633, and see, for example, Riechmann et al., (1988) Nature 332:323-329, Queen et al., (1989) Proc. Natl Acad. Sci. USA 86: 10029-10033, U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409, Kashmiri et al., (2005) Methods 36:25-34, Padlan, (1991) Mol. Immunol. 28:489-498 (describing "resurfacing"), Dall'Acqua et al., (2005) Methods 36:43-60 (describing "FR shuffling"), as well as Osbourn et al., (2005) Methods 36:61-68 and Klimka et al., (2000) Br. J. Cancer, 83:252-260 (describing a "guided selection" approach to FR shuffling).

[0096] Human framework regions that may be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (e.g., Sims (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285, and Presta et al. (1993) J. Immunol. 151:2623), framework regions derived from the consensus sequence of human antibodies of a particular subgroup of heavy chain variable regions (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285, and Presta et al. (1993) J. Immunol. 151:2623), human mature (somatically mutated) framework regions, or human germline Framework regions (see, e.g., Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633), and framework regions obtained from screening FR libraries (see, e.g., Baca et al., (1997) J. Biol. Chem. 272: 10678-10684, and Rosok et al., (1996) J. Biol. Chem. 271:22611-22618). Typically, The FR region is replaced with a human FR region to generate a humanized VHH. In some embodiments, certain FR residues of human FR are replaced to improve one or more properties of the humanized VHH. The VHH domain with such replaced residues is also referred to herein as "humanized".

[0097] In various embodiments, the Fc region included in the CD123 binding polypeptide is a human Fc region or is derived from a human Fc region.

[0098] In some embodiments, the Fc region included in the CD123 binding polypeptide is derived from a human Fc region and contains a three amino acid deletion in the lower hinge corresponding to IgG1 E233, L234, and L235, and is referred to herein as "Fc xELL". Fc xELL polypeptides do not bind FcγR and are therefore referred to as "effector silent" or "effector null", however, in some embodiments, the xELL Fc region binds to FcRn, with associated extended half-life and transcytosis associated with FcRn mediated recycling.

[0099] In some embodiments, the Fc region included in the CD123 binding polypeptide is derived from a human Fc region and contains the mutations M252Y and M428V, referred to herein as "Fc-YV". In some embodiments, such mutations enhance binding to FcRn at the acidic pH of the endosome (near 6.5) while losing detectable binding at neutral pH (about 7.2), allowing for enhanced FcRn-mediated recycling and extended half-life.

[0100] In some embodiments, the Fc region included in the CD123 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 a mutation such as S354C on the first member of the heterodimeric Fc pair. which forms an asymmetric disulfide with the corresponding mutation Y349C on the second member of the heterodimeric Fc pair. In some embodiments, one member of the heterodimeric Fc pair contains the modification H435R or H435K to prevent Protein A binding while maintaining FcRn binding. In some embodiments, one member of the heterodimeric Fc pair contains the modification H435R or H435K, while the second member of the heterodimeric Fc pair is not modified at H435. In various embodiments, the hole Fc region contains the modification H435R or H435K (sometimes referred to as "hole-R" when the modification is H435R), while the knob Fc region does not. In some cases, the hole-R mutation improves purification of the heterodimer relative to a homodimeric hole Fc region that may exist.

[0101] Non-limiting exemplary Fc regions that can be used in CD123 binding polypeptides include Fc regions comprising the amino acid sequences of SEQ ID NO:54-SEQ ID NO:89.

[0102] Chimeric receptors and engineered cells Provided herein is a chimeric antigen receptor (CAR) having an extracellular domain comprising one or more CD123-binding VHH domains provided herein. Provided herein is a CAR construct comprising an extracellular domain comprising one or more CD123-binding VHH domains, a transmembrane domain, and an intracellular signaling region. One or more CD123-binding VHH domains forming the antigen-binding unit of the CAR bind or can bind to, i.e. target, CD123 binding with sufficient affinity that the CAR is therapeutically useful in targeting cells or tissues expressing CD123 binding.

[0103] CARs are synthetic receptors that typically contain an extracellular targeting / binding moiety associated with one or more signaling domains in a single fusion molecule expressed on the surface of cells such as T cells. Thus, CARs combine antigen specificity and T cell activation properties in a single fusion molecule. First generation CARs typically contained the cytoplasmic region of CD3ζ or 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 cancer, lymphoma, and neuroblastoma, where they induced moderate responses (reviewed in Sadelain et al., Curr Opin Immunol, 21 (2): 215-223, 2009). Signaling of costimulatory molecules such as CD28 and CD3ζ Second generation CARs, which contain multiple signaling domains, provide dual signaling for a combination of activating and costimulatory signals. Third generation CARs are more complex, with 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 CAR provided comprises a CD123-binding VHH domain. In some embodiments, the CAR comprises at least two VHH domains that target one or more antigens. In one embodiment, the antigen-binding domain of the CAR comprises two or at least two CD123-binding VHH domains, thus providing a bivalent molecule. In one embodiment, the antigen-binding domain comprises two or at least two CD123-binding VHH domains, but binds to different epitopes on CD123. In such cases, the antigen-binding domain comprises a first CD123-binding VHH domain that binds to a first epitope on CD123 and a second VHH domain that binds to a second epitope on CD123. 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 comprises two CD123-binding VHH domains that bind to the same epitope on CD123.

[0105] The transmembrane domain of the CAR provided herein typically spans or is in the form of a plasma membrane. A transmembrane domain is a domain that can cross or span the plasma membrane, which is directly or indirectly (e.g., via a spacer such as an immunoglobulin hinge sequence) linked to an endoplasmic portion that includes an extracellular antigen-binding domain and an intracellular signaling domain. In one embodiment, 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 comprises a CD3 zeta domain or a CD28 transmembrane domain. Other transmembrane domains will be apparent to one of skill in the art and can be used in conjunction with the CAR embodiments provided herein.

[0106] The intracellular signaling region of the CAR provided herein comprises one or more intracellular signaling domains that transmit a signal to the T cell upon binding of the antigen binding domain of the CAR, e.g., upon binding of an antigen. In some embodiments, the intracellular region comprises an intracellular signaling domain that is or comprises an ITAM signaling domain. Exemplary intracellular signaling domains include signaling domains derived from, for example, the zeta chain of the T cell receptor complex or any of its homologs (e.g., the eta chain, the FcsRIy chain and the beta chain, the MB1 (Iga) chain, the B29 (Ig) chain, etc.), human CD3 zeta 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 signaling, such as CD2, CD5, OX40, and CD28. In certain embodiments, the intracellular signaling region comprises an intracellular signaling domain derived from the human CD3 zeta chain.

[0107] In some embodiments, the intracellular signaling region of the CAR may further comprise an intracellular signaling domain derived from a costimulatory molecule. In such instances, such a signaling domain may enhance CAR-T cell activity, e.g., via enhanced memory cell proliferation, survival and / or development, following antigen-specific binding, as compared to a CAR that comprises only an ITAM-containing signaling domain, e.g., CD3ζ. In some embodiments, the costimulatory 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 costimulatory signaling domain is derived from or obtained from a human protein. In some aspects, the costimulatory signaling domain is derived from or obtained from human CD28 or human CD137(4-IBB).

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

[0109] In certain embodiments, the CAR further comprises a hinge or spacer region that connects the extracellular antigen binding domain and the transmembrane domain. This hinge or spacer region can be used to achieve various lengths and flexibility of the resulting CAR. Examples of hinge or spacer regions that can be used include, but are not limited to, an Fc fragment of an antibody, or a fragment or derivative thereof, an antibody hinge region, or a fragment or derivative thereof, an antibody CH2 region, an antibody CH3 region, an artificial spacer sequence, such as a peptide sequence, or a combination thereof. Other hinge or spacer regions will be apparent to those skilled in the art and can 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 the hinge and transmembrane domains derived from CD8.

[0111] Also provided herein is an isolated nucleic acid construct comprising at least one nucleic acid encoding a CAR provided herein. In some embodiments, the construct is an expression vector for expressing a CAR in a cell. The expression vector can be a viral vector. Viral vector technology is well known in the art 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 viral-based systems have been developed, for example using retroviruses such as adenoviral vectors, in one embodiment lentiviral vectors are used.

[0112] In a further aspect, an isolated cell or cell population comprising one or more nucleic acid constructs as described above is also provided. Also provided is an isolated cell or cell population that is genetically modified to express a CAR provided herein. Thus, provided herein is a genetically engineered cell that comprises, e.g., stably expresses, a CAR provided herein. In one embodiment, the cell is 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 cell is a T cell, such as a CD4 T cell and / or a CD8 T cell. In some embodiments, the cell is autologous to the subject. For example, in some embodiments, T cells can be isolated from a patient (also referred to as primary T cells) for engineering, e.g., transfection or transduction, with a CAR nucleic acid construct.

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

[0114] CAR-engineered T cells can be assayed for appropriate function by various means. In some cases, in vitro cytotoxicity, proliferation, or cytokine assays (e.g., IFNγ expression) can be used to assess the functionality of engineered T cells. Exemplary standard endpoints are the percentage of lysis of tumor lines, proliferation of engineered T cells, or expression of IFNγ protein in culture supernatants. In some cases, the ability to stimulate T cell activation upon stimulation of the CAR, for example, via an antigen, can be assessed by monitoring expression of activation markers, such as CD69, CD44, or CD62L, proliferation, and / or cytokine production.

[0115] Polypeptide Expression and Production Nucleic acid molecules are provided that include a polynucleotide encoding a CD123 binding polypeptide. In some embodiments, the nucleic acid molecule can also encode a leader sequence that directs secretion of the CD123 binding polypeptide, and the leader sequence is typically cleaved so that it is not present in the secreted polypeptide. The leader sequence can be the native heavy chain (or VHH) leader sequence, or can be another heterologous leader sequence.

[0116] The nucleic acid molecule can be constructed using recombinant DNA techniques routine in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0117] Vectors are provided that contain a nucleic acid encoding a CD123 binding polypeptide as described herein. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like. In some embodiments, a vector is selected that is optimized for expression of the polypeptide in a desired cell type, such as a CHO cell or CHO-derived cell, or an NSO cell. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0118] In some embodiments, CD123 binding polypeptides may be expressed in prokaryotic cells, such as bacterial cells, or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression may be carried out, for example, according to procedures known in the art. Exemplary eukaryotic cells that may be used to express the polypeptides include, but are not limited to, COS cells, including COS7 cells, 293 cells, including 293-6E cells, CHO cells, including CHO-S, DG44, Lec13 CHO cells, and FUT8 CHO cells, PER.C6™ cells (Crucell), and NSO cells. In some embodiments, CD123 binding polypeptides may be expressed in prokaryotic cells, such as bacterial ... In embodiments, CD123 binding polypeptides can be expressed in yeast. See, for example, US Patent Application Publication No. 2006 / 0270045. In some embodiments, a particular eukaryotic host cell is selected based on its ability to perform desired post-translational modifications on a polypeptide. For example, in some embodiments, CHO cells produce polypeptides with higher levels of sialylation than the same polypeptides produced in 293 cells.

[0119] Introduction of one or more nucleic acids (e.g., vectors) into a desired host cell can be accomplished by any method, including, but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, etc. Non-limiting exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3 rd ed. Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be prepared according to any suitable method. , may be transiently or stably transfected into the desired host cell.

[0120] Also provided are host cells comprising any of the nucleic acids or vectors described herein. In some embodiments, host cells are provided that express the CD123 binding polypeptides described herein. The CD123 binding polypeptides expressed in the host cells can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include agents that bind to the ROR1 ECD and Fc region. For example, Protein A, Protein G, Protein A / G, or antibody affinity columns can be used to purify CD123 binding polypeptides that include an Fc region by binding to the Fc region. Hydrophobic interaction chromatography, e.g., butyl or phenyl columns, can also be suitable for purifying some polypeptides, such as antibodies. Ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography) can also be suitable for purifying some polypeptides, such as antibodies. Mixed-mode chromatography (e.g., reversed-phase / anion exchange, reversed-phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, etc.) may also be suitable for purifying some polypeptides, such as antibodies. Many methods of purifying polypeptides are known in the art.

[0121] In some embodiments, the CD123 binding polypeptide is produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009), Spirin, Trends Biotechnol. 22: 538-45 (2004), and Endo et al., Biotechnol. Adv. 21: 695-713 (2003).

[0122] In some embodiments, a CD123 binding polypeptide produced by the above method is provided. In some embodiments, the CD123 binding polypeptide is produced in a host cell. In some embodiments, the CD123 binding polypeptide is produced in a cell-free system. In some embodiments, the CD123 binding polypeptide is purified. In some embodiments, a cell culture medium comprising the CD123 binding polypeptide is provided.

[0123] In some embodiments, a composition is provided comprising an antibody produced by the above method. In some embodiments, the composition comprises a CD123 binding polypeptide produced in a host cell. In some embodiments, the composition comprises a CD123 binding polypeptide produced in a cell-free system. In some embodiments, the composition comprises a purified CD123 binding polypeptide.

[0124] Exemplary Methods of Treating Disease Using CD123 Binding Polypeptides In some embodiments, a method of treating a disease in an individual is provided, comprising administering a CD123 binding polypeptide or a cell expressing a CD123 binding polypeptide. In some embodiments, a method of treating a cancer in an individual is provided. In some embodiments, a method of treating a CD123 expressing or CD123 positive cancer in an individual is provided. The method comprises administering to the individual an effective amount of a CD123 binding polypeptide or a cell expressing a CD123 binding polypeptide provided herein. In some embodiments, the CD123 binding polypeptide blocks binding of CD123 to IL-3. In some embodiments, the CD123 binding polypeptide is used to introduce a cytotoxic agent into a cell expressing CD123. In some such embodiments, the CD123 binding polypeptide comprises a binding domain that binds to a cytotoxic T cell or a NK cell. In some such embodiments, the binding domain binds to CD3, T cell receptor (TCR) alpha, TCR beta, CD28, CD16, CD32A, CD64, CD89, NKp46, or NKG2D. The binding domain may in some embodiments be a VHH domain, or an antibody binding domain comprising a heavy chain variable region and a light chain variable region, such as a VH / VL, scFv, Fab fragment, etc.

[0125] In some embodiments, the CD123 binding polypeptide is linked to a cytotoxic agent to form an immunoconjugate. A variety of cytotoxic agents for use in immunoconjugates are known in the art and include, but are not limited to, calicheamicins, auristatins, dolastatins, tubulysins, maytansinoids, cryptophycins, duocarmycins, esperamicins, pyrrolobenzodiazepines, and enediyne antibiotics.

[0126] In some embodiments, the CD123 binding polypeptide is a chimeric antigen receptor expressed on a cytotoxic cell, such as a chimeric antigen receptor expressed on a T cell (CAR-T) or a chimeric antigen receptor expressed on a NK cell (CAR-NK). Such a method of treatment can be a method of treatment in a human or an animal. In some embodiments, a method of treatment in a human is provided.

[0127] Non-limiting exemplary cancers that can be treated with the CD123 binding polypeptides or cells expressing CD123 binding polypeptides provided herein include, but are not limited to, However, the cancer may be lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-dividing cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's macroglobulinemia, 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 cancer that expresses CD123 (i.e., CD123 positive).

[0128] The CD123 binding polypeptide or cells expressing a CD123 binding polypeptide may be administered to the subject as needed. The frequency of administration may be determined by a person skilled in the art, such as the attending physician, based on considerations such as the condition being treated, the age of the subject being treated, the severity of the condition being treated, and the general health of the subject being treated. In some embodiments, an effective dose of the CD123 binding polypeptide or cells expressing a CD123 binding polypeptide is administered to the subject one or more times. In some embodiments, an effective dose of the CD123 binding polypeptide or cells expressing a CD123 binding polypeptide is administered to the subject daily, twice a week, weekly, every two weeks, monthly, etc. An effective dose of the CD123 binding polypeptide or cells expressing a CD123 binding polypeptide is administered to the subject at least once. In some embodiments, an effective dose of a CD123 binding polypeptide or cells expressing a CD123 binding polypeptide may be administered multiple times, including multiple times 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 amount effective to treat (including prevent) cancer. The therapeutically effective amount typically depends on the weight of the subject being treated, the physical or health condition of the subject, the extent of the condition being treated, or the age of the subject being treated. In general, the antibody may be administered in an amount ranging from about 0.05 mg / kg body weight to about 100 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 10 μg / kg body weight to about 100 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 50 μg / kg body weight to about 5 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 100 μg / kg body weight to about 10 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 100 μg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 0.5 mg / kg body weight to about 10 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 0.05 mg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 0.05 mg / kg body weight to about 10 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 5 mg / kg body weight or less, e.g., less than 4 mg / kg, less than 3 mg / kg, less than 2 mg / kg, or less than 1 mg / kg of antibody.

[0130] In some embodiments, CD123 binding polypeptides or cells expressing CD123 binding polypeptides may be administered in vivo by a variety of routes, including, but not limited to, intravenous, intraarterial, parenteral, intraperitoneal, or subcutaneous. Depending on the intended use, appropriate formulations and administration routes can be selected.

[0131] In some embodiments, therapeutic treatment using CD123 binding polypeptides is accomplished by targeting cytotoxic agents to cells expressing CD123, such as cancer cells expressing CD123. The synthetic polypeptide is a chimeric antigen receptor expressed on a cytotoxic cell, such as a T cell or a NK cell.

[0132] Pharmaceutical Compositions In some embodiments, compositions comprising CD123 binding polypeptides are provided in formulations that include a wide variety of pharma- ceutically acceptable carriers (see, 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 ed., Pharmaceutical Press (2000). A variety of pharma- ceutically acceptable carriers, including adjuvants and diluents, can be used. In addition, a variety of pharma-ceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, etc., can also be used. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0133] In some embodiments, the pharmaceutical composition comprises a CD123 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 CD123 binding polypeptides or engineered cells of the present disclosure may be administered alone or in combination with other therapeutic modalities, such as other anti-cancer agents. The CD123 binding polypeptides or engineered cells may be provided prior to, substantially simultaneously with, or subsequent to (i.e., simultaneously or sequentially with) other therapeutic modalities. In some embodiments, the therapeutic methods described herein may further include administering a biologic, such as radiation therapy, chemotherapy, vaccination, targeted tumor therapy, CAR-T therapy, oncolytic virus therapy, cancer immunotherapy, cytokine therapy, surgical resection, chromatin modification, resection, cryotherapy, antisense agents against tumor targets, siRNA agents against tumor targets, microRNA agents against tumor targets, or anti-cancer / anti-tumor agents, or antibodies, cytokines, or receptor extracellular domain-Fc fusions.

[0135] In some embodiments, the CD123 binding polypeptides provided herein are given concomitantly with one or more chemotherapeutic agents, CAR-T (chimeric antigen receptor T cell) therapies, oncolytic virus therapies, cytokine therapies, and / or agents that target other checkpoint molecules, such as VISTA, gpNMB, B7H4, HHLA2, CD73, CTLA4, TIGIT, etc.

[0136] In some embodiments, the CD123 binding polypeptides or engineered cells of the disclosure are administered in combination with other anti-tumor agents, such as, for example, 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™)), PD-1 inhibitors (e.g., anti-PD1 antibody, BMS-936558), PDL1 inhibitors (e.g., anti-PDL1 antibody, MPDL3280A), PDL2 inhibitors (e.g., anti-PDL2 antibody), cytokines, targets of ErbB2 receptor, ErbB3 receptor, ErbB4 receptor, PDGFRβ receptor, BlyS receptor, APRIL receptor, BCMA receptor, PD-1 receptor, PDL1 receptor, PDL2 receptor In some embodiments, the therapeutic agents may be used in combination with antagonists (eg, neutralizing antibodies) that bind to one or more of the VEGF receptors, CTLA4 receptors, or VEGF receptors, TRAIL / Apo2, and other bioactive and organic chemical agents.

[0137] In some embodiments, the CD123 binding polypeptides or engineered cells provided herein are given simultaneously with a PD-1 / PD-L1 therapy. Examples of PD-1 / PD-L1 therapy include nivolumab (BMS), pidilizumab (CureTech, CT-011). , pembrolizumab (Merck), durvalumab (Medimmune / AstraZeneca), atezolizumab (Genentech / Roche), avelumab (Pfizer), AMP-224 (Amplimmune), B MS-936559, AMP-514 (Amplimmune), MDX-1105 (Merck), T SR-042 (Tesaro / AnaptysBio, ANB-011), STI-A1010 (Sorrento Therapeutics), STI-A1110 (Sorrento Therapeutics), and Programmed Death- These include other agents directed against the cytokine activator 1 (PD-1) or programmed death-ligand 1 (PD-L1).

[0138] In some embodiments, the CD123 binding polypeptides or engineered cells of the present disclosure may be used in combination with chemotherapeutic agents, including, but not limited to, alkylating agents such as thiotepa and CYTOXAN™ cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; benzodopa, carboquone, meturedopa, and Aziridines such as altretamine, triethylenemelamine, triethylenediamine, Ethylenimines and methylamelamines, including triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including the synthetic analogues KW-2189 and CB1-TM1); elatherobin; pancratistatin; sarcodictiin; spongistatins; chlorambucil, chlornaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trophospha nitrogen mustards, such as mustard mide, and uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; enediyne antibiotics, such as the calicheamicins, particularly calicheamicin γ1I and calicheamicin ω11 (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN™ doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin). Antibiotics such as epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as cistabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, drostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenals such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide; doglycosides;aminolevulinic acid;eniluracil;amsacrine;bestrabulil;bisantrene;edatrexate;defofamine;demecolcine;diaziquone;eflornithine;elliptinium acetate;epothilone;e Toglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; rosoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK™ polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; schizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as TAXOL™ paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE™ Cremophor-free albumin engineered nanoparticle formulation of paclitaxel from American Pharmaceutical Partners (Schaumburg, Ill.), and TAXOTERE™ doxetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine in GEMZAR®; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues 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 treatment regimens of irinotecan with 5-FU and leucovorin); topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); inhibitors of PKCα, Raf, H-Ras, EGFR (e.g., erlotinib (TARCEVA™)) and VEGF-A that reduce cell proliferation, as well as pharmacologic acceptable salts, acids, or derivatives of any of the above.

[0139] Further non-limiting exemplary chemotherapeutic agents include anti-hormonal agents that act to regulate or inhibit hormone action on cancer, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including tamoxifen in NOLVADEX™), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxyfene, ketoxifene, LY117018, onapristone, and toremifene in FARESTON™; aromatase inhibitors, which inhibit the enzyme aromatase that regulates estrogen production in the adrenal glands, such as semestane, formestane, fadrozole, vorozole in RIVISOR™, letrozole in FEMARA™, and anastrozole in ARIMIDEX™; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction 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; rIL-2 in PROLEUKIN™ (aldesleukin); topoisomerase 1 inhibitors in LURTOTECAN™; GnRH agonists in ABARELIX™; and pharmaceutical acceptable salts, acids, or derivatives of any of the above.

[0140] In some embodiments, the CD123 binding polypeptide and the additional agent are formulated into a single therapeutic composition, and the CD123 binding polypeptide and the additional agent are administered simultaneously. Alternatively, the CD123 binding polypeptide or engineered cells and the additional agent are separate from each other, e.g., each is formulated into a separate therapeutic composition, and the CD123 binding polypeptide or engineered cells and the additional agent are administered simultaneously, or the CD123 binding polypeptide or engineered cells and the additional agent are administered at different times during the treatment regimen. For example, the CD123 binding polypeptide or engineered cells are administered before the administration of the additional agent, the CD123 binding polypeptide or engineered cells are administered after the administration of the additional agent, or the CD123 binding polypeptide or engineered cells and the additional agent are administered in alternation. The CD123 binding polypeptide and the additional agent may be administered in a single dose or multiple doses.

[0141] In some embodiments, the CD123 binding polypeptide or engineered cells and the additional agent(s) are administered simultaneously. For example, the CD123 binding polypeptide and the additional agent(s) may be formulated in a single composition or may be administered as two or more separate compositions. In some embodiments, the CD123 binding polypeptide or engineered cells and the additional agent(s) are administered sequentially, or the CD123 binding polypeptide or engineered cells and the additional agent(s) are administered at different times during a treatment regimen.

[0142] Non-Limiting Exemplary Methods of Diagnosis and Treatment In some embodiments, the methods described herein are useful for evaluating a subject and / or a sample from a subject (e.g., a cancer patient). In some embodiments, the evaluation is one or more of diagnosis, prognosis, and / or response to treatment.

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

[0144] In some embodiments, the evaluation may prescribe treatment, including treatment with an antibody described herein. In some embodiments, the evaluation may prescribe the use or forgoing of adjuvant therapy following resection. Adjuvant therapy, also called adjuvant therapy, is treatment given in addition to the primary, main, or initial treatment. As a non-limiting example, adjuvant therapy may be an additional treatment, usually given after surgery, when all detectable disease has been removed but there remains a statistical risk of relapse due to latent disease. In some embodiments, the polypeptides are used as adjuvant therapeutic agents in the treatment of cancer. In some embodiments, the polypeptides are used as the sole adjuvant therapeutic agents in the treatment of cancer. In some embodiments, the polypeptides described herein are used as adjuvant therapeutic agents in the treatment of cancer. Drugs are withheld. For example, if a patient is unlikely to respond to the antibodies described herein or has only a minimal response, treatment may be withheld for quality of life and to avoid unnecessary toxicity from ineffective chemotherapy. In such cases, palliative care may be used.

[0145] In some embodiments, the polypeptide is administered as a neoadjuvant therapy before resection. In some embodiments, neoadjuvant therapy refers to a therapy that shrinks and / or downgrades a tumor before any surgery. In some embodiments, neoadjuvant therapy refers to a chemotherapy drug administered to a cancer patient before surgery. In some embodiments, neoadjuvant therapy refers to an antibody administered to a cancer patient before surgery. Cancer types for which neoadjuvant chemotherapy is usually considered include, for example, breast cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, and lung cancer. In some embodiments, the polypeptide is used as a neoadjuvant therapy in the treatment of cancer. In some embodiments, the use is before resection.

[0146] In some embodiments, the tumor microenvironment contemplated by the methods described herein is one or more of tumor vasculature, tumor-infiltrating lymphocytes, fibroblastic 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 lymphoid cells, neutrophils, and other immune cells located in proximity to the tumor.

[0147] kit Also provided are articles of manufacture and kits comprising any of the CD123 binding polypeptides described herein and suitable packaging. In some embodiments, the invention includes a kit comprising (i) a CD123 binding polypeptide and (ii) instructions for using the kit to administer the CD123 binding polypeptide to an individual.

[0148] Suitable packaging for the compositions described herein are known in the art and include, for example, vials (e.g., sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles of manufacture may be further sterilized and / or sealed. Unit dosage forms comprising the compositions described herein are also provided. These unit dosage forms may be stored in suitable packaging in single or multiple unit dosage forms, and may also be further sterilized and sealed. Instructions provided in the kits of the invention are typically written instructions on a label or insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. Instructions for use of the antibody generally include information regarding dosage, administration schedule, and route of administration for the intended therapeutic or industrial use. The kit may further include instructions for selecting an appropriate individual treatment.

[0149] The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Kits may also be provided that contain a sufficient dose of the molecules disclosed herein to provide effective treatment to an individual for an extended period of time, such as, for example, any approximate period of 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months or more. Kits may also contain multiple unit doses of the molecules and instructions for use, and may be packaged in an amount sufficient for storage and use in pharmacies, such as hospital pharmacies and compounding pharmacies. In some embodiments, the kits include a dried (e.g., lyophilized) composition that can be reconstituted, resuspended, or rehydrated to form a generally stable aqueous suspension of the antibody. EXAMPLES

[0150] The examples discussed below are intended to be purely illustrative of the present invention and in no way limit the scope of the present invention. It should not be construed as limiting. These examples are not intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0151] Example 1: CD123 single domain antibody Single domain antibodies targeting human CD123 were generated through immunization of llamas and alpacas with a recombinant form of the human CD123 extracellular domain.

[0152] After specific anti-CD123 antibody titers were developed, llama / alpaca peripheral blood mononuclear cells (PBMCs) were isolated from 500 mL of blood from immunized animals, and total mRNA was isolated using Qiagen RNeasy Maxi kit and subsequently converted to first-strand cDNA using Thermo Superscript IV reverse transcriptase and oligo-dT priming. This cDNA was used as a template to specifically amplify VHH sequences via PCR and cloned into yeast surface display vectors as VHH-Fc-AGA2 fusion proteins.

[0153] The recombinant form of CD123 was used to enrich for yeast libraries displaying VHH-Fc-AGA2 fusion proteins via magnetic bead separation followed by fluorescence-activated cell sorting (FACS). Sorted yeast were plated out and isolated colonies were picked into 96-well blocks and grown in medium that switches expression from surface-displayed VHH-Fc to secretion into the medium. Supernatants from 96-well yeast secretion cultures were applied to 293F cells transiently transfected with CD123 (CD123 positive) or non-transfected 293F cells (CD123 negative), washed, treated with a fluorophore-labeled anti-human IgG1 Fc secondary antibody, and analyzed by 96-well flow cytometry.

[0154] Nucleic acid sequences encoding VHHs that bind to CD123-positive cells but not to CD123-negative cells were cloned in frame with the human Fc coding region into a mammalian expression vector and expressed by transient transfection using polyethylenimine in HEK293 Freestyle cells (293F cells) or CHO cells. Supernatants were harvested after 3 to 7 days, and secreted recombinant proteins were purified by Protein A chromatography, and concentrations were calculated from the absorbance at 280 nm and the extinction coefficient.

[0155] The epitopes of single domain antibodies (sdAbs) containing VHH domains that bind to CD123 were compared using biolayer interferometry. 5 μg / mL of histidine-tagged human CD123 was immobilized on a capture sensor coated with nickel-nitrilotriacetic acid (Ni-NTA). 100 nM of one sdAb was then loaded onto the CD123 antigen and allowed to equilibrate. The sensor was then transferred into 100 nM of the second sdAb. An increase in assay signal represents binding and indicates that the second sdAb targets an epitope different from that of the first sdAb.

[0156] The human VH3-23 germline was used as a scaffold to humanize the Camelidae-derived CD123 VHH. The remainder of the Camelidae that contribute to solubility, specificity, stability, and / or affinity was left unmodified. Furthermore, where possible and necessary, amino acid sequences that would be detrimental to potential developability were modified to mitigate this risk. Additionally, all humanized variants contained the Leu11Glu (L11E) modification described in US Patent Application Publication No. 2016 / 0207981.

[0157] These results show that four different epitopes were found among the humanized versions of A5, F3, C5, 1B11, 1F5, and 4F2 sdAbs. As shown in Figure 1A, none of the tested sdAbs have the same epitope as the hzA5v2 sdAb. As shown in Figure 1B, the C5 and hz1F5v1 sdAbs have the same epitope as the hzF3v22 sdAb and different epitopes from the hz1B11v28 and hz4F2v3 sdAbs. As shown in Figure 1C, the hz4F2v3 sdAb has a different epitope from the hz1B11v28 sdAb. A summary of the results is shown in Table 3 below.

[0158] [Table 3]

[0159] Example 2: Binding of Polypeptides to CD123 Binding of sdAbs to human CD123 was assessed by flow cytometry. Each sdAb contained the VHH domains shown in Table 4 below and the Fc region of human IgG1 xELL with deletion of amino acids Glu233, Leu234, and Leu235 according to EU numbering (SEQ ID NO: 55). HEK293 cells were transiently transfected with a plasmid encoding full-length CD123 (UniProt accession number P26951-1; mature form, SEQ ID NO: 1) and used as the positive cell line in Figures 2A-M, and untransfected HEK293 cells were used as the CD123 negative cell line in Figure 2N, where Molm-13 cells expressing CD123 were used as the positive cell line. Each cell type was incubated in 96-well round-bottom plates in FACS buffer (PBS Cells were seeded at 30,000 cells / well in 1% BSA, 0.1% NaN3 pH 7.4). sdAbs were diluted in 11-point 3-fold serial dilutions in FACS buffer. sdAb dilutions were added to the seeded cells and the assay plate was incubated for 30 min at 4° C. After washing twice in 150 μL FACS buffer, 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 for 30 min at 4° C. After washing the cells two more times, bound antibodies were detected by flow cytometry.

[0160] Flow cytometry analysis was performed on Intellicyte's iQue Plus, and fluorescence was The apparent affinity (K d , nM) were determined using one-site binding nonlinear regression in PRISM graph software.

[0161] As shown in Figures 2A-N, the sdAbs tested demonstrated CD123 binding and did not bind to untransfected HEK293 cells which do not express CD123. The apparent binding affinities are shown in Table 4 below.

[0162] [Table 4]

[0163] Example 3: Inhibition of CD123 Binding to IL-3 Certain CD123-binding sdAbs were tested for their ability to block CD123 binding to IL-3 by titrating the sdAbs against HEK-293 cells expressing CD123 in the presence of a fixed amount of recombinant IL-3-mFc. Bound IL-3 was detected by flow cytometry using a fluorophore-conjugated anti-mouse IgG specific secondary antibody.

[0164] The results of that experiment are shown in Figure 3. The A5, C5, and F3 sdAbs were all able to block CD123 binding to IL-3 (Figure 3A). hz1F5v1 and the anti-CD123 antibody analog CSL362 (see US Patent Application Publication No. 2013 / 0137855) were also able to block CD123 binding to IL-3 (Figure 3B).

[0165] The present disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics of the present disclosure. The above-described embodiments are therefore to be considered in all respects as illustrative and not limiting of the present disclosure. The scope of the present disclosure is therefore defined by the appended claims, rather than the above detailed description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

[0166] [Table 5] TIFF2025060941000007.tif244170TIFF2025060941000008.tif243170TIFF2025060941000009.tif247170TIFF2025060941000010.tif247170TIFF2025060941000011.tif247170TIFF2025060941000012.tif102170

Claims

1. A polypeptide comprising at least one VHH domain that binds to CD123, wherein the at least one VHH domain comprises CDR1, CDR2, and CDR3, and CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44; SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25; SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5; SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21; or SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively.

2. The polypeptide of claim 1, wherein at least one VHH domain comprises CDR1, CDR2, and CDR3, and CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, respectively.

3. 3. The polypeptide of claim 1 or 2, wherein at least one VHH domain comprises CDR1, CDR2, and CDR3, and CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, respectively.

4. A polypeptide described in any one of claims 1 to 3, wherein at least one VHH domain comprises CDR1, CDR2, and CDR3, and CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively.

5. A polypeptide described in any one of claims 1 to 4, wherein at least one VHH domain comprises CDR1, CDR2, and CDR3, and CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively.

6. A polypeptide described in any one of claims 1 to 5, wherein at least one VHH domain comprises CDR1, CDR2, and CDR3, and CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively.

7. The polypeptide of any one of claims 1 to 6, wherein at least one VHH domain is humanized.

8. The polypeptide of any one of claims 1 to 7, wherein at least one VHH domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:18, or SEQ ID NO:

6.

9. The polypeptide of any one of claims 1 to 7, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NO: 29, SEQ ID NO: 31, or SEQ ID NO:

26.

10. A polypeptide according to any one of claims 1 to 9, comprising two VHH domains.

11. A polypeptide according to any one of claims 1 to 9, comprising three VHH domains.

12. The polypeptide according to any one of claims 1 to 9, wherein the polypeptide comprises at least one binding domain that binds to an antigen other than CD123.

13. 13. The polypeptide of claim 12, wherein the polypeptide comprises at least one binding domain that binds to CD3, T cell receptor (TCR) alpha, TCR beta, CD28, CD16, CD32A, CD64, CD89, NKp46, or NKG2D.

14. A polypeptide according to claim 12 or 13, wherein each VHH domain binds to CD123.

15. The polypeptide of claim 14 , wherein each VHH domain comprises the same CDR1, CDR2, and CDR3 amino acid sequences.

16. The polypeptide of claim 14, wherein each VHH domain comprises the same VHH sequence.

17. A polypeptide according to any one of claims 1 to 9, comprising one VHH domain.

18. The polypeptide of any one of claims 1 to 17, wherein the polypeptide comprises an Fc region.

19. The polypeptide of claim 18, wherein the Fc region comprises an amino acid sequence selected from SEQ ID NO: 54 to SEQ ID NO:

89.

20. 20. The polypeptide of claim 18 or 19, which forms a dimer under physiological conditions.

21. The polypeptide according to any one of claims 1 to 20, wherein the CD123 is human CD123.

22. The polypeptide of claim 21, wherein the human CD123 comprises the sequence of SEQ ID NO:

1.

23. The polypeptide of any one of claims 1 to 22, wherein the polypeptide blocks the binding of CD123 to IL-3.

24. An immunoconjugate comprising a polypeptide according to any one of claims 1 to 23 and a cytotoxic agent.

25. 25. The immunoconjugate of claim 24, wherein the cytotoxic agent is selected from a calicheamicin, an auristatin, a dolastatin, a tublysin, a maytansinoid, a cryptophycin, a duocarmycin, an esperamicin, a pyrrolobenzodiazepine, and an enediyne antibiotic.

26. A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 23 or the immunoconjugate of claim 24 or 25, and a pharmaceutically acceptable carrier.

27. An isolated nucleic acid encoding a polypeptide according to any one of claims 1 to 23.

28. A vector comprising the nucleic acid of claim 27.

29. 29. A host cell comprising the nucleic acid of claim 27 or the vector of claim 28.

30. A host cell expressing a polypeptide according to any one of claims 1 to 23.

31. A method for producing a polypeptide according to any one of claims 1 to 23, comprising incubating a host cell according to claim 29 or 30 under conditions suitable for expression of said polypeptide.

32. 32. The method of claim 31, further comprising isolating the polypeptide.

33. A pharmaceutical composition for cancer treatment, comprising the polypeptide according to any one of claims 1 to 23, or the immunoconjugate according to claim 24 or 25.

34. 34. The pharmaceutical composition of claim 33, wherein the cancer is selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-dividing cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), hairy cell leukemia, and chronic myeloblastic leukemia.

35. 35. The pharmaceutical composition of claim 33 or 34, wherein the cancer is acute myeloid leukemia (AML).

36. A pharmaceutical composition according to any one of claims 33 to 35 for administration together with an additional therapeutic agent.

37. 37. The pharmaceutical composition of claim 36, wherein the additional therapeutic agent is an anti-cancer agent.

38. 38. The pharmaceutical composition of claim 37, wherein the anti-cancer agent is selected from a chemotherapeutic agent, an anti-cancer biologic, radiation therapy, a CAR-T therapeutic agent, and an oncolytic virus.

39. The pharmaceutical composition according to any one of claims 33 to 38, wherein the cancer is a cancer that expresses CD123.

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